Molecular and biochemical mechanisms of bile duct injury after liver transplantation Buis, Carlijn Ineke

Transcription

1 University of Groningen Molecular and biochemical mechanisms of bile duct injury after liver transplantation Buis, Carlijn Ineke IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below. Document Version Publisher's PDF, also known as Version of record Publication date: 2008 Link to publication in University of Groningen/UMCG research database Citation for published version (APA): Buis, C. I. (2008). Molecular and biochemical mechanisms of bile duct injury after liver transplantation. Groningen: s.n. Copyright Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons). Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Downloaded from the University of Groningen/UMCG research database (Pure): For technical reasons the number of authors shown on this cover page is limited to 10 maximum. Download date:

2 Molecular and biochemical mechanisms of bile duct injury after liver transplantation Carlijn I. Buis

3 This thesis is funded by:. Different parts of this thesis were funded by grants from the Jan Kornelis de Cock Foundation and the Groningen Graduate School for Drug Exploration GUIDE. The financial support of the following institutions and companies in the publication of this thesis is highly appreciated: Buis, C.I. Molecular and biochemical mechanisms of bile duct injury after liver transplantation. Thesis, University of Groningen, The Netherlands ISBN: Copyright 2008 Carlijn I. Buis, The Netherlands All rights reserved. No part of this book may be reproduced, stored in a retrieval system or transmitted in any form or by any means, without prior permission of the author. Cover: ICO-Communucations & Carlijn Buis Lay-out: Gildeprint drukkerijen, Enschede, the Netherlands Printed by: Gildeprint drukkerijen, Enschede, the Netherlands

4 Molecular and biochemical mechanisms of bile duct injury after liver transplantation Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de Rijksuniversiteit Groningen op gezag van de Rector Magnificus, dr. F. Zwarts, in het openbaar te verdedigen op maandag 8 december 2008 om 13:15 uur door Carlijn Ineke Buis geboren op 29 december 1978 te Vught

5 Promotor: Prof. dr. R. J. Porte Beoordelingscommissie: Prof. dr. H.J. Metselaar Prof. dr. M.J.H. Slooff Prof. dr. H.J. Verkade

6 Paranimfen: Marieke de Boer Mark-Hugo Maathuis

7 The cover shows an old advertisement of ossegalzeep by Jawson Wood, dated in This soap made from ox bile was especially used in the twentieth century to clean clothes with fatty stains. The bile salts acts as detergents and thereby enables fatty stains to dissolve in water by formation of micelles. A similar pattern can be found in human bile after transplantation. Bile salts form micelles with phospholipids in the bile. In case phospholipids are relatively reduced compared to bile salts, in other words if there is a low biliary phospholipids-to-bile salt ratio, bile can act as a detergent for the bile ducts by recruiting phospholipids from the membrane. After liver transplantation the bile formation is altered and in some patients, a detergent toxic bile composition, with a low phospholipids-tobile salt ratio, is observed. This toxic bile is found to contribute to the development of bile duct injury after liver transplantation.

8 Contents Chapter 1 Introduction and outline of this thesis 9 Chapter 2 Causes and consequences of ischemic type biliary 15 lesions after liver transplantation. Journal of HPB surgery 2006; 13: Part I. Non-anastomotic biliary complications after liver transplantation Chapter 3 Non-anastomotic biliary strictures after adult liver 37 transplantation part I: radiological features and risk factors for early versus late presentation Liver Transpl 2007; 13: Chapter 4 Non-anastomotic biliary strictures after adult liver 61 transplantation part 2: Management, outcome and risk factors for disease progression Liver Transpl 2007; 13: Part II. Bile physiology after liver transplantation Chapter 5 The role of bile salt toxicity in the pathogenesis of bile duct 81 injury after non heart-beating porcine liver transplantation Transplantation 2008; 85: Chapter 6 Altered bile composition after liver transplantation is associated with the development of Nonanastomotic biliary strictures J of Hepatol, in press. 99

9 Chapter 7 Polymorphisms of hepatobiliary phospholipid transporter 123 MDR-3 associated with non anastomotic strictures after human liver transplantation submitted Part III. HO-1 and hepatobiliary injury after liver transplantation Chapter 8 Expression of Heme oxygenase -1 in human livers before 139 transplantation correlates with graft injury and function after transplantation Am J Transplant. 2005; 5: Chapter 9 Heme oxygenase-1 genotype of the donor is associated 167 with graft survival after liver transplantation. Am J Transplant. 2008; 8: Chapter 10 Summary, discussion and future perspectives 191 Nederlandse samenvatting 203 List of contributing authors 211 List of publications 217 Dankwoord 221 Curriculum Vitae 229 List of abbreviations

10 1 Introduction and outline of this thesis

11 Introduction and outline Introduction and outline of this thesis Liver transplantation is the ultimate treatment for end-stage liver disease. Survival following liver transplantation has improved substantially over the years due to better pre-transplant care, improved anesthesia and surgical techniques, enhanced intensive care medicine, and more effective immunosuppressant medications. Currently, 1-year patient survival rate is almost 90% and 5-year patient survival rate is 75% (1). The first attempt to transplant a liver in a human was reported by Starzl in 1963 (2). In the Netherlands, the first liver was transplanted in Groningen in 1979 (3). Nowadays, around 120 livers are transplanted annually in the Netherlands. In the Netherlands, around 135 patients are currently on the waiting list for liver transplantation. Although transplantation accounts for 77% of the outflow from the waiting list, unfortunately still 12% of the patients die whilst on the waiting list. Worldwide, around patients are on a waiting list for liver transplantation, while the estimated number of liver transplants performed in 2008 will be less then (4). The focus on the recruitment of organ donors therefore remains of vital importance in order to continue and improve the success of transplantation. Posttransplant-related complications can grossly be classified into primary graft dysfunction, vascular complications, graft rejection, recurrent disease, and biliary complications. Reconstruction of biliary drainage is historically considered as the technical Achilles heel of liver transplantation (5). Although the surgical technique of biliary reconstruction has emerged and is now a more or less standardized technique, complications arising from the bile duct and its reconstruction remain a serious source of morbidity. The resulting biliary complications comprise leakage and strictures. Depending on the localization, strictures are classified as anastomotic or non-anastomotic. Non-anastomotic strictures (NAS) are considered to be the most troublesome biliary complication after liver transplantation. NAS are defined as any stricture, dilatation or irregularity of the intra- or extrahepatic bile ducts detected on imaging studies of the biliary tree after liver transplantation. Approximately one in seven patients suffers from NAS after liver transplantation. In patients with NAS graft loss is reported in up to 50% after 2 years (6). Accepted risk factors for NAS are hepatic artery thrombosis, chronic ductopenic rejection, and ABO blood group incompatibility. In 1991 it was first described that NAS may 10

12 Chapter 1 also occur in the absence of these known risk factors (7). Because of the resemblance of intrahepatic biliary strictures occurring after hepatic artery thrombosis, NAS that appeared despite occlusion of the hepatic artery were also called ischemic type biliary lesions (ITBL). The two names NAS and ITBL are still both used in the literature. A relationship between NAS and the duration of cold ischemia time was discovered soon after. Ever since, research in this area has focussed on identifying pathophysiological mechanisms and implementing therapeutic strategies. Nevertheless, NAS still occur in many patients and in most cases no apparent clinical risk factor can be identified. Therefore, the aim of this thesis was to perform a more fundamental analysis, using genetic, molecular and biochemical methods in an attempt to identify the underlying mechanisms of these biliary complications. This thesis is divided in three parts, focusing on I) Clinical risk factors for the development and progression of NAS, II) The role of bile salt toxicity in the development of bile duct injury and NAS after liver transplantation, III) The role of heme oxygenase-1 (HO-1) in the protection of liver grafts from ischemia / reperfusion (I/R) injury. The three parts are preceded by a general overview of the causes and consequences of nonanastomotic biliary strictures (chapter 2). Part I. Non-anastomotic biliary complications after liver transplantation. The specific aims of this section were to describe the various forms of NAS and the accompanying clinical risk factors as well as to study clinical risk factors for progression of NAS. Chapter 3 describes the non-anastomotic biliary strictures in the Groningen cohort of liver transplant recipients. All imaging studies of the biliary tree were reviewed. Localization and severity of NAS at first presentation were categorized using a newly developed classification. Time interval between transplantation and the initial presentation of NAS were recorded. The purpose of this study was to identify risk factors for the clinical and radiological presentation of NAS, as well as for the timing of NAS after liver transplantation. Chapter 4 concerns the cohort of patients identified with NAS in chapter 3. This chapter focuses mostly on the consequences of NAS. We defined a number of serious complications of NAS, studied their prevalence and risk factors, and evaluated the effects of therapeutic measures. 11

13 Introduction and outline Part II. Bile physiology after liver transplantation. The specific aims of this section were to evaluate the contribution of bile composition to the development of bile duct injury. Bile salts have potent detergent properties and may damage cells of the biliary tract by affecting the integrity of the membranes. The detergent properties of bile salts are normally counteracted by phospholipids. By forming mixed micelles of bile salts, phospholipids and cholesterol, phospholipids neutralize bile salts thereby protecting against cellular injury. In a previous study our group has shown that bile produced early after transplantation has an abnormal composition characterized by a low phospholipids-to-bile salt ratio (8). Based on these findings we hypothesized that bile salt toxicity early after liver transplantation contributes to the formation of NAS. NAS are a frequently encountered complication after non-heart-beating (NHB) liver transplantation. Aim of chapter 5 was to study the role of bile salt toxicity in the pathogenesis of bile duct injury after NHB liver transplantation. We hypothesized that NHB liver transplantation is associated with increased bile salt toxicity early after liver transplantation depending on the length of the warm ischemia time in the donor. To test this hypothesis we studied bile composition, graft survival and the degree of bile duct injury in a porcine liver transplant model. Chapter 6 describes the role of altered bile composition in the development of NAS after human liver transplantation. In a large clinical study in 111 patients bile composition and the development of NAS were studied in a prospective fashion. The aim was to test whether bile composition is involved in the pathogenesis of NAS. Chapter 7 concerns the genetic variations in hepatobiliary transporters. These transporter proteins are responsible for bile secretion. The bile salt export pump (BSEP, official name ATP binding cassette, subfamily B, member 11. ABCB11) mediates ATP-dependent secretion of bile salts across the canalicular membrane of hepatocytes. Multidrug resistant protein 3 (MDR3, official name ATP binding cassette, subfamily B, member 4. ABCB4) acts as a primary active phospholipid flippase and translocates phosphatidylcholine from the inner to the outer leaflet of the canalicular membrane. Multidrug resistant related protein 2 (MRP-2, official name ATP binding cassette, subfamily C, member 2. ABCC2 is a multispecific organic anion transporter that mediates biliary excretion of a broad spectrum of divalent organic anions, including bilirubin and glutathione. Via the subsequent passive diffusion of water into the bile, this process is the most significant contributor to the bile salt independent bile flow. Aim of this study was to assess whether genetic variations in the above described transporters, present in the donor liver, are associated with the occurrence of NAS in the recipient after transplantation. 12

14 Chapter 1 Part III. HO-1 and hepatobiliary injury after liver transplantation. HO-1 has been proposed as a graft survival gene. Upregulation of HO-1 is considered to be one of the most critical cellular protection mechanisms during cellular stress such as ischemia and reperfusion occurring during a transplant procedure. The specific aim of this section was to study the role of HO-1 expression in relation to postoperative hepatobiliary injury and graft function. Chapter 8 concerns endogenous HO-1 expression levels in human liver transplants. We studied changes in HO-1 expression levels during liver transplantation and correlated this with immediate postoperative hepatobiliary injury and graft function after transplantation. Chapter 9 describes two genetic polymorphisms in the promoter influencing the inducebility of HO-1: a (GT) n polymorphism and a single nucleotide polymorphism (SNP), A(-413)T. We analyzed these two functional HO-1 promoter polymorphisms in donor genomic DNA in relation to hepatobiliary injury and outcome after human liver transplantation. Furthermore, we studied the functional relevance of these polymorphisms by measuring hepatic messenger ribonucleic acid (mrna) expression. Finally, in Chapter 10 the results as described in this thesis are summarized and future perspectives are discussed. References Starzl TE, Marchioro TL, Vonkaulla KN, Hermann G, Brittain RS, Waddell WR. Homotransplantation of the liver in humans. Surg Gynecol Obstet 1963; 117: Krom RA, Gips CH, Houthoff HJ, Newton D, van der Waaij D, Beelen J, Haagsma EB, Slooff MJ. Orthotopic liver transplantation in Groningen, The Netherlands ( ). Hepatology 1984; 4:61S-65S O Leary JG, Lepe R, Davis GL. Indications for liver transplantation. Gastroenterology. 2008;134: Calne RY. A new technique for biliary drainage in orthotopic liver transplantation utilizing the gall bladder as a pedicle graft conduit between the donor and recipient common bile ducts. Ann Surg 1976; 184: Guichelaar MM, Benson JT, Malinchoc M, Krom RA, Wiesner RH, Charlton MR. Risk factors for and clinical course of non-anastomotic biliary strictures after liver transplantation. Am J Transplant 2003;3: Sanchez-Urdazpal L, Gores GJ, Ward EM, Maus TP, Wahlstrom HE, Moore SB, et al. Ischemic-type biliary complications after orthotopic liver transplantation. Hepatology 1992;16: Geuken E, Visser D, Kuipers F, Blokzijl H, Leuvenink HG, de Jong KP, et al. Rapid increase of bile salt secretion is associated with bile duct injury after human liver transplantation. J Hepatol 2004;41:

15

16 2 Causes and Consequences of ischemic type biliary lesions after liver transplantation Jounal of HPB surgery 2006; 13: Carlijn I Buis Harm H Hoekstra Robert C Verdonk Robert J Porte

17 Causes and consequences of ITBL after liver transplantation Abstract Biliary complications are a major source of morbidity, graft loss and even mortality after liver transplantation. The most troublesome are the so called ischemic type biliary lesions (ITBL), with an incidence varying between 5-15%. ITBL is a radiological diagnosis, characterized by intrahepatic strictures and dilatations on a cholangiogram in the absence of hepatic artery thrombosis. Several risk factors of ITBL have been identified, strongly suggesting a multifactorial origin. Main categories of risk factors for ITBL include ischemia related injury, immunological induced injury and cytotoxic injury by bile salts. However, in many cases no specific risk factor can be identified. Ischemia related injury comprises prolonged ischemic times and disturbance in blood flow through the peribiliary vascular plexus. Immunological injury is assumed as risk factor based on the relationship of ITBL with ABO incompatibility, polymorphism in genes coding for chemokines, and pre-existing immunologically mediated diseases as primary sclerosing cholangitis and autoimmune hepatitis. The clinical presentation of patients with ITBL is often not specific, symptoms may include fever, abdominal complaints and increased cholestatic liver function tests. Diagnosis is made by imaging studies of the bile ducts. Treatment starts with relieving symptoms of cholestasis and dilatation by endoscopic retrograde cholangiopancreaticography (ERCP) or percutaneous transhepatic cholangiodrainage (PTCD) followed by stenting if possible. Eventually up to 50% of the patients with ITBL will require a re-transplantation or may die. In selected cases, a re-transplantation can be avoided or delayed by resection of the extra hepatic bile ducts and construction of a hepatico-jejunostomy. More research on the pathogenesis of ITBL is needed before more specific preventive or therapeutic strategies can be developed. 16

18 Chapter 2 Introduction Biliary complications have since long been recognized as a major cause of morbidity and graft failure in patients after orthotopic liver transplantation (OLT) (1-3). Bile leakage and bile duct strictures are the most common complications. According to the localization, strictures can be classified as anastomotic or non-anastomotic. Non-anastomotic intrahepatic strictures (NAS) are considered to be the most troublesome biliary complication. NAS were first described in OLT associated with hepatic artery thrombosis, where the biliary tree becomes ischemic and eventually necrotic, resulting in a typical cholangiographic picture of biliary strictures, dilatations and intraductal cast formation (4). However, these cholangiographic abnormalities of strictures and dilatations can also be seen in patients who do not have an hepatic artery thrombosis (5,6), so the term ischemic-type biliary lesions (ITBL) emerged (figure 1). The reported incidence of ITBL differs greatly between different series, ranging from 1-19% (7,8). Variations in the definitions of ITBL used in different studies as well as the reporting of only symptomatic patients can at least partly explain these differences. In the majority of series an incidence of 5 to 15% is reported (9-16). A B Figure 1. Cholangiogram 4 months after OLT. (A) normal, (B) ischemic type biliary lesions (ITBL). 17

19 Causes and consequences of ITBL after liver transplantation Etiology and risk factors The exact pathophysiological mechanism of ITBL is still unknown. However, several risk factors of this often cumbersome complication have been identified, strongly suggesting a multifactorial origin (Table 1). In general, risk factors of ITBL can be divided in three different categories: ischemia related injury to the biliary epithelium, imunologically mediated injury and cytotoxic injury induced by bile salts. These categories may point towards different etiological mechanisms of ITBL, as will be described below. Table 1. Risk factors for the development of ITBL Ischemic injury Warm ischemia in the donor Prolonged cold ischemia Reperfusion injury Warm ischemia during implantation Disturbed blood flow in the peribiliary plexus Immunological injury ABO incompatibility Pre-existing disease with auto immune component Auto-immune hepatitis Primary sclerosing cholangitis Cytomegalovirus infection Chronic rejection Chemokine polymorphism CCR5 delta 32 Bile salt induced injury Hydrophilic bile salts are cytoprotective Hydrophobic bile salts are cytotoxic 18

20 Chapter 2 A. Ischemic injury The similarities between the radiological abnormalities of ITBL and the bile duct lesions seen in the presence of hepatic artery thrombosis strongly suggest an ischemic factor in the origin of ITBL. The quest for pathogenic mechanisms, therefore, started with factors associated with ischemia. A.1. Cold ischemic and reperfusion injury Multiple studies have indicated that prolonged cold ischemia time (CIT) predisposes the graft to the development of ITBL (6,15,17-20). In 1992, Sanchez-Urdazpal et al, reported an incidence of ITBL of 2% in livers with a CIT < 11.5h, rising to 35% in livers with a CIT between 11.5h and <13h and even up to 52% in grafts with a CIT > 13h (6). Nowadays many centers therefore try to keep the CIT below 10h. However, even with a CIT shorter then 10h, Guichelaar et al have shown that the duration of cold storage is still a risk factor for the development of ITBL (17). The strong positive correlation between CIT and ITBL can be explained by either direct ischemic injury of the biliary epithelium, increased susceptibility of the biliary epithelium for a second factor such as reoxygenation injury, or secondary ischemia of the biliary epithelium due to damage to the peribiliary arterial plexus (6). The hypothesis that reperfusion injury during OLT contributes to bile duct injury is supported by data provided by the experimental work of Noack et al (21). Using cell cultures, these investigators have shown that biliary epithelial cells are more susceptible to reperfusion / reoxygenation injury than hepatocytes. In an anoxic environment bile duct epithelial cells and hepatocytes show equally reduced levels of ATP. However, the rate of cell death after reoxygenation was significantly higher in the bile duct epithelial cells, compared to hepatocytes. Increased production of reactive oxygen species by bile duct epithelial cells as well as a lower intracellular concentration of glutathione as antioxidant, may explain this difference (21). Clinical evidence for a contributing role of preservation injury is provided in a clinical study by Li et al. These investigators have shown that the incidence of ITBL is significantly increased in livers with increased preservation injury, as reflected postoperative peaks in serum aspartate aminotransferase and alanine aminotransferase (20). 19

21 Causes and consequences of ITBL after liver transplantation A.2. Injury of the peribiliary vascular plexus Preservation injury results in increased arterial resistance and may cause circulatory disturbances in small capillaries, such as the biliary plexus (20). Since the blood supply to the biliary tract is solely dependant on arterial inflow, disturbances in the blood flow through the peribiliary plexus may result in insufficient preservation and subsequent damage of the biliary epithelium. Several studies have indicated that the viscosity of preservation solutions may play a role in the development of ITBL (22,23). The highly viscous University of Wisconsin (UW) preservation solution, now routinely used in most centers, might not completely flush out the small donor peribiliary arterial plexus. Microcirculatory disturbances in the peribiliary plexus may lead to obstruction and subsequently result in insufficient bile duct preservation (23). Strengthening the evidence that insufficient perfusion of the peribiliary plexus might contribute to the development of ITBL is provided in a study by Moench et al (24). These investigators have shown that additional flushing of the peribiliary plexus by controlled arterial back-table pressure perfusion is associated with a considerable reduction in ITBL after preservation with UW solution (24). Apart from this, a proper harvesting technique of the liver and the extra hepatic bile duct is critically important to preserve the viability and vasculature of the bile duct. Although, never studied in a clinical trial, it is accepted by every surgeon that the extra hepatic bile duct should be left covered with as much tissue as possible. Stripping of the bile duct should be avoided in order not to injure the microcirculatory blood supply. A.3. Warm ischemic Injury Two periods of warm ischemia can be distinguished during the transplant procedure. The first warm ischemia time (WIT), during harvesting and before cold preservation, and the second WIT during graft implantation and before complete reperfusion. The first WIT is especially a major concern in grafts from non heart-beating (NHB) donors. Several studies have shown that liver grafts form NHB donors are at increased risk of developing ITBL (25-27). Concern exists that harvesting time, extending the first WIT, in addition to subsequent CIT and ischemia-reperfusion injury may result in damage to the biliary epithelium (25). Despite plausible reasoning, no direct clinical evidence has directly linked prolonged harvesting time with ITBL, and the literature concerning this item is not conclusive (25-29). To reduce the incidence of ITBL, attempts have been made to reduce the second WIT. During revascularization of the graft the most common technique is initial reperfusion via the 20

22 Chapter 2 portal vein with subsequent reconstruction and reperfusion of the hepatic artery. Bile ducts, solely dependant on the hepatic artery for their blood supply, are exposed to warm ischemia during reperfusion via the portal vein alone. This situation has been hypothesized to increase damage of the biliary epithelium. To overcome this potential harmful situation, Sankary et al. (18) have studied the impact of simultaneous versus sequential reperfusion of the portal vein and hepatic artery on the incidence of ITBL. These investigators have observed a significant reduction of ITBL when livers were reperfused simultaneously via the portal vein and hepatic artery (18). However, in a more recent study, we were not able to demonstrate a favorable effect of simultaneous arterial and portal reperfusion on the incidence of ITBL (30). In an attempt to reduce the second WIT further, some investigators have introduced retrograde perfusion of the liver graft via the inferior vena cava, after completing its anastomosis and during construction of the portal vein anastomosis (31). Although this technique certainly results in an earlier reperfusion of the graft, the central venous blood it is reperfused with has a lower oxygen pressure than the portal or arterial blood. In a randomized controlled clinical trial, Heidenhain et al. (32) have recently observed a higher incidence of ITBL in livers that were reperfused in a retrograde fashion, compared to antegrade reperfusion via the portal vein. The low perfusion pressure obtained during retrograde perfusion via the caval anastomosis may be an explanation for this. This low venous pressure may result in poor flush out and reperfusion of the peribiliary plexus, causing more ischemic biliary injury (J. Langrehr, personal communication, 2005). B. Immunological injury Several papers have provided evidence for an immunological component in the pathogenesis of ITBL (15,17,33). ITBL has been associated with various immunologically mediated processes, such as ABO incompatible liver transplantation, pre-existing diseases with a presumed autoimmune component (such as primary sclerosing cholangitis (PSC) and autoimmune hepatitis (AIH)), cytomegalovirus (CMV) infection, chronic rejection, and finally with genetic polymorphism of chemokines. B.1. ABO incompatibility ABO blood type mismatched liver transplantation has since long been recognized to give rise to multiple complications (5,34). The incidence of ITBL in ABO-incompatible OLT varies 21

23 Causes and consequences of ITBL after liver transplantation from 20-82% (15). An explanation for this could be the fact that the antigens of the blood type system are not only expressed on the vascular endothelium, but also on the biliary epithelial cells, making them a target for preformed ABO blood group antibodies (5,15). Because of this high rate of complications and reduced graft survival rates, transplantation across the ABO border is nowadays discouraged. B.2. Association with pre-existing disease It has been well described in several studies that patients who are transplanted for PSC have a higher incidence of ITBL after transplantation (13,14,17,35,36). The association between ITBL and AIH has only been described recently (17). PSC and AIH share a similar genetic predisposition to autoimmunity (17). All together, these findings strengthen the hypothesis that ITBL may have an underlying (auto) immune component. B.3. Cytomegalovirus In patients suffering from acquired immunodeficiency syndrome (AIDS), infection with CMV has been shown to contribute to biliary problems, like cholangitis (37). After OLT, CMV infection has been associated with an increased incidence of anastomotic strictures and biliary leaks (38). CMV inclusions have been demonstrated histopathologically in the extra-hepatic bile duct specimen in a liver transplant patient developing a biliary stricture during CMV infection (38,39). A clear association between CMV and ITBL, however, has never been demonstrated (17). In a recent large study of 1714 liver transplant recipients, Heidenhain et al. (40) could not find a higher incidence of ITBL in patients who had suffered from CMV infection versus those who had not. The role for CMV infection in the pathogenesis of ITBL, therefore, remains unclear. B.4. Chronic rejection Chronic rejection has been implicated as a potential cause of biliary strictures (12,41,42). This effect is thought to be modulated not via direct injury to the biliary epithelium, but rather via the arteriopathy accompanying chronic rejection, leading to narrowing of the medium-sized arteries. The resulting ischemia of the bile duct wall seems to play an important role in the loss of small bile ducts (15,43,44). Although chronic rejection has been identified as a risk factor for the development of ITBL in several series (15,20,41,45), this could not always be confirmed by others (13,46). Therefore the role of chronic rejection in the pathogenesis of ITBL remains to be elucidated. 22

24 Chapter 2 B.5. Chemokines Chemokines play a key role in the postoperative immunomodulation, especially during rejection as well as in post-ischemic injury. Evidence for a role of chemokines in the pathogenesis of ITBL after OLT has been provided by a genetic association study focusing on CC-chemokine receptor 5 (CCR5). CCR5 is a receptor for CC-chemokine ligand (CCL) 3 (macrophage inflammatory protein 1 alpha) and CCL4 (macrophage inflammatory protein 1 beta), which are over-expressed in infiltrating leukocytes (47). Biliary epithelial cells have been shown to produce CC-chemokines that may bind specifically to CCR5 (48). CCR5 32 polymorphism is a nonfunctional mutant allele of CCR5 with an internal deletion of 32 base pairs. A study on this polymorphism showed no differences in patient survival, rejection rates, re-transplantation rates, and survival in OLT patients with CCR5 32 compared with patients with wild-type CCR5 (49). Interestingly however, Moench et al recently found a very strong association between the presence of the CCR5 32 polymorphism in recipients and the development of ITBL after OLT (33). These findings add to the existing evidence that immunological factors play a role in the pathogenesis of ITBL. C. Bile salt induced injury Another potential factor in the pathogenesis of bile duct injury after liver transplantation is bile salt toxicity. Bile salts have potent detergent properties towards cellular membranes of hepatocytes and biliary epithelial cells. Normally, the toxic effects of bile salts are prevented by complex (mixed micelle) formation with phospholipids. Evidence for a pivotal role of bile salt-mediated hepatotoxicity in the pathogenesis of I/R injury of liver grafts, has gradually emerged during the last decade. Using experiments in pigs, Hertl et al. (50) have shown that bile salts can seriously amplify preservation injury of the biliary epithelium. When porcine livers are flushed at the time of procurement with saline containing hydrophobic bile salts, intrahepatic bile ducts are more seriously injured after even short periods of ischemia, compared to control livers which are flushed with saline (50-52). Injury of the biliary tree can be prevented when an infusion of hydrophilic, instead of hydrophobic, bile salts are given to the donor animals prior to liver procurement (50). Moreover, it has been demonstrated that morphological characteristics of human common bile ducts alter significantly when livers are perfused with UW solution mixed with gallbladder bile, compared to livers which are preserved with normal UW solution (53). Of interest, we recently found that microscopic 23

25 Causes and consequences of ITBL after liver transplantation bile duct injury occurring early after human liver transplantation correlates with the formation of toxic bile, characterized by a high bile salt / phospholipids ratio (54). Whether an increased bile salt / phospholipids ratio contributes to hepatic injury or is an epiphenomenon, however, could not be identified in this clinical study. Therefore, we recently initiated a study, using a model of arterialized liver transplantation in mice that are heterozygous for the disruption of the gene encoding for the transporter of phospholipids into the bile, the Mdr2 gene (Multidrug resistance protein 2) (55). These mice disclose approximately half of the normal phospholipid concentration in bile, leading to an abnormally high bile salt/phospholipid ratio, but have a normal liver histology under normal conditions. When Mdr2+/- livers were transplanted after, a short period of cold storage, into wild-type recipients serious biliary injury developed. These findings provide evidence that endogenous bile salts act synergistically to I/R in the origin of bile duct injury in vivo. In addition, these data indicate that intrahepatic cholestasis and intracellular bile salt retention may be critical mechanisms triggering hepatobiliary injury after liver transplantation. Even when the primary insult occurs to the bile ducts, hepatocellular injury is an invariable feature of cholestasis, associated with accumulation of bile salts in the liver and blood (56). Current evidence indicates that bile salt retention is a key early event that contributes to hepatocellular and biliary injury after OLT. Until more specific strategies become available, great care should be taken to avoid exposure of bile duct epithelium to toxic bile salts during the cold storage. Careful retrograde flushing of the bile ducts with preservation solution is therefore considered to be critical to remove residual bile salts. Furthermore, the extra-hepatic bile duct should not be ligated during organ procurement in order to ensure the flush out of bile and bile salts during organ procurement and cold storage. Clinical presentation The clinical presentation of ITBL is often not specific; symptoms may include fever, abdominal complaints and cholestatic liver function tests. In many patients, asymptomatic elevation of serum gamma glytamyl transferase and/or alkaline phosphatase is the first sign of biliary complications, prompting initiation of further examinations, such as cholangiography (16). Most patients with ITBL present with symptoms within the first 6 months after OLT (7,12,13,17,57). 24

26 Chapter 2 Diagnostic work-up The appropriate diagnostic workup has been discussed in several recent review papers (58-60). Direct visualization of the bile ducts by endoscopic retrograde cholangiopancreaticography (ERCP), percutaneous transhepatic cholangiodrainage (PTCD) or drain-cholangiography remains the gold standard for making the diagnosis ITBL (7,12,13,17,24,61). Magnetic resonance cholangiopancreaticography (MRCP) is becoming increasingly important as a diagnostic test, with high positive and negative predictive values (62-64). Cholangiographic imaging can show mucosal irregularities, narrowing of the lumen, and ductal dilatations (65). A classification of ITBL has been proposed based on the localization of the abnormalities, distinguishing type I (extrahepatic lesions), type II (intrahepatic lesions), and type III (intra- and extra-hepatic alterations) (66,67). However, this classification has not been widely accepted and used. In all cases of non-anastomotic biliary strictures, patency of the hepatic artery should be carefully studied and confirmed before the diagnosis of ITBL can be made. The presence of ITBL can be suggested by biliary abnormalities in a liver biopsy, such as ductular proliferation and cholestasis (13). However, ITBL remains a macroscopic and not a microscopic entity. No studies have been conducted correlating histological abnormalities in liver biopsies and the presence of ITBL. Treatment More than in any other biliary complication, treatment of ITBL has to be individualized. Direct treatment of strictures should be attempted via endoscopy or percutaneous dilatations and stenting. With prolonged and intensive endoscopic or radiological treatment, over 50% of patients can be treated successfully (7,12,17,20,68,69) some centers even reporting success in over 70% (70). In many other cases, re-transplantation may at least be postponed by using this strategy. Success will depend mainly on the severity of strictures and their localization, with extra-hepatic strictures responding better to therapy. In patients with successful radiological treatment, liver tests may improve, but often remain disturbed (14,69). Many physicians will provide medical treatment with ursodeoxycholic to their patients in order improve bile flow and to obtain a more favorable composition of the bile (68,71,72). However, the efficacy of this strategy in influencing the incidence or outcome of ITBL has never been properly evaluated in a randomized controlled clinical trial. 25

27 Causes and consequences of ITBL after liver transplantation If non-operative techniques are unsuccessful, surgery may be appropriate in selected cases. Especially when lesions are predominantly present at the level of the bile duct bifurcation, resection of the extrahepatic bile ducts and Roux-en-Y hepatico-jejunostomy should be considered. Schlitt et al. (73) have reported clinical and biochemical improvement in 14 out of 16 patients with hilar ITBL, who were treated by a hepatico-jejunostomy or portoenterostomy. If all other treatment options have failed, retransplantation may be the only therapy left. Especially in the presence of secondary biliary cirrhosis, recurrent cholangitis, or progressive cholestasis due to extensive intrahepatic ITBL, retransplantation is mostly unavoidable. The presence of ITBL is associated with a marked decrease in graft survival. Ultimately, up to 50% of patients with ITBL either die or need a re-transplantation, however mortality rates differ markedly amongst studies (12,15,17). Conclusion Since the introduction of liver transplantation, biliary drainage has formed the so called Achilles heel of this procedure. Early studies have reported disabling complications of the biliary tract in over 30% of the patients (74). Fortunately, much has changed during the last decades. Liver transplantation is nowadays a standard treatment for patients with end stage liver disease and survival is excellent, with one-year patient survival rates of 80 to 90%. Multiple improvements in patient selection, perioperative management, as well as changes in surgical technique have contributed to the success of OLT today. Unfortunately, despite these important improvements and enormous gain in experience, biliary complications can still be regarded as the Achilles heel. The most incomprehensible type of biliary complications is ITBL. Although several risk factors for ITBL have been identified in recent years, the direct cause of ITBL can often not be identified in an individual patient. Although it is most likely that the pathogenesis of ITBL is multifactorial, several studies have strongly suggested a critical role for ischemic injury of the peribiliary vascular plexus. In addition, studies have provided evidence for the involvement of immunological processes, as well as bile salt induced injury of the biliary epithelium. Despite the important progress that has been made in the understanding of the pathogenesis of ITBL, the actual cause remains unidentified in many patients suffering from this troublesome complication after OLT. Therefore, more research will be needed in this area to better identify and understand the mechanism of ITBL. Only in this way, more specific preventive and therapeutic strategies can developed, which may further improve patient and graft survival after OLT 26

28 Chapter 2 Reference List 1. Starzl TE, Marchioro TL, Vonkaulla KN, Hermann G, Brittain RS, Waddell WR. Homotransplantation of the liver in humans. Surg Gynecol Obstet 1963; 117: Lerut J, Gordon RD, Iwatsuki S, Esquivel CO, Todo S, Tzakis A Starzl,TE Biliary tract complications in human orthotopic liver transplantation. Transplantation 1987; 43: Calne RY. A new technique for biliary drainage in orthotopic liver transplantation utilizing the gall bladder as a pedicle graft conduit between the donor and recipient common bile ducts. Ann Surg 1976; 184: Zajko AB, Campbell WL, Logsdon GA, Bron KM, Tzakis A, Esquivel CO Starzl,TE. Cholangiographic findings in hepatic artery occlusion after liver transplantation. AJR Am J Roentgenol 1987; 149: Sanchez-Urdazpal L, Sterioff S, Janes C, Schwerman L, Rosen C, Krom RA. Increased bile duct complications in ABO incompatible liver transplant recipients. Transplant Proc 1991; 23: Sanchez-Urdazpal L, Gores GJ, Ward EM, Maus TP, Wahlstrom HE, Moore SB Wiesner RH, Krom RA. Ischemic-type biliary complications after orthotopic liver transplantation. Hepatology 1992; 16: Sanchez Urdazpal L. Diagnostic features and clinical outcome of ischemic-type biliary. Hepatology 1993; 17: Thethy S, Thomson BN, Pleass H, Wigmore SJ, Madhavan K, Akyol M Akyol M, Forsythe JL, James Garden O. Management of biliary tract complications after orthotopic liver transplantation. Clin Transplant 2004; 18: Sawyer RG, Punch JD. Incidence and management of biliary complications after 291 liver transplants following the introduction of transcystic stenting. Transplantation 1998; 66: Turrion VS, Alvira LG, Jimenez M, Lucena JL, Nuno J, Pereira F, Vicente E, Ardaiz J. Management of the biliary complications associated with liver transplantation: 13 years of experience. Transplant Proc 1999; 31(6): Rizk RS, McVicar JP, Emond MJ, Rohrmann CA, Jr., Kowdley KV, Perkins J, Carithers RL Jr, Kimmey MB. Endoscopic management of biliary strictures in liver transplant recipients: effect on patient and graft survival. Gastrointest Endosc 1998; 47: Ward EM, Kiely MJ, Maus TP, Wiesner RH, Krom RA. Hilar biliary strictures after liver transplantation: cholangiography and percutaneous treatment. Radiology 1990; 177: Campbell WL, Sheng R, Zajko AB, Abu-Elmagd K, Demetris AJ. Intrahepatic biliary strictures after liver transplantation. Radiology 1994; 191: Feller RB, Waugh RC, Selby WS, Dolan PM, Sheil AG, McCaughan GW. Biliary strictures after liver transplantation: clinical picture, correlates and outcomes. J Gastroenterol Hepatol 1996; 11:

29 Causes and consequences of ITBL after liver transplantation 15. Rull R, Garcia Valdecasas JC, Grande L, Fuster J, Lacy AM, Gonzalez FX, Rimola A, Navasa M, Iglesias C, Visa J. Intrahepatic biliary lesions after orthotopic liver transplantation. Transpl Int 2001; 14: Pascher A, Neuhaus P. Bile duct complications after liver transplantation. Transpl Int 2005; 18: Guichelaar MM, Benson JT, Malinchoc M, Krom RA, Wiesner RH, Charlton MR. Risk factors for and clinical course of non-anastomotic biliary strictures after liver transplantation. Am J Transplant 2003; 3: Sankary HN, McChesney L, Frye E, Cohn S, Foster P, Williams J. A simple modification in operative technique can reduce the incidence of nonanastomotic biliary strictures after orthotopic liver transplantation. Hepatology 1995; 21: Torras J, Llado L, Figueras J, Ramos E, Lama C, Fabregat, J Rafecas A, Escalante E, Dominguez J, Sancho C, Jaurrieta E. Biliary tract complications after liver transplantation: type, management, and outcome. Transplant Proc 1999; 31: Li S, Stratta RJ, Langnas AN, Wood RP, Marujo W, Shaw BW, Jr. Diffuse biliary tract injury after orthotopic liver transplantation. Am J Surg 1992; 164: Noack K. The greater vulnerability of bile duct cells to reoxygenation injury than to anoxia. Transplantation 1993; 56: Canelo R, Hakim NS, Ringe B. Experience with hystidine tryptophan ketoglutarate versus University Wisconsin preservation solutions in transplantation. Int Surg 2003; 88: Pirenne J, Van Gelder F, Coosemans W, Aerts R, Gunson B, Koshiba T, Fourneau I, Mirza D, Van Steenbergen W, Fevery J, Nevens F, McMaster P. Type of donor aortic preservation solution and not cold ischemia time is a major determinant of biliary strictures after liver transplantation. Liver Transpl 2001; 7: Moench C, Moench K, Lohse AW, Thies J, Otto G. Prevention of ischemic-type biliary lesions by arterial back- table pressure perfusion. Liver Transpl 2003; 9: Abt P, Crawford M, Desai N, Markmann J, Olthoff K, Shaked A. Liver transplantation from controlled non-heart- beating donors: an increased incidence of biliary complications. Transplantation 2003; 75: D alessandro AM, Hoffmann RM, Knechtle SJ, Odorico JS, Becker YT, Musat A, Pirsch JD, Sollinger HW, Kalayoglu M. Liver transplantation from controlled non-heart-beating donors. Surgery 2000; 128: Otero A, Gomez-Gutierrez M, Suarez F, Arnal F, Fernandez-Garcia A, Aguirrezabalaga J, Garcia-Buitron J, Alvarez J, Manez R. Liver transplantation from Maastricht category 2 non-heart-beating donors. Transplantation 2005; 15: Foley DP, Fernandez L, Leverson G, Chin LT, Kreiger N, Cooper JT et al. Donation After Cardiac Death: The University of Wisconsin Experience With Liver Transplantation. Ann.Surg 2005; 242:

30 Chapter Manzarbeitia CY, Ortiz JA, Jeon H, Rothstein KD, Martinez O, Araya VR, Munoz SJ, Reich DJ. Long-term outcome of controlled, non-heart-beating donor liver transplantation. Transplantation 2004; 78: Polak WG, Miyamoto S, Nemes BA, Peeters PM, de Jong KP, Porte RJ, Slooff MJH. Sequential and simultaneous revascularization in adult orthotopic piggyback liver transplantation. Liver Transpl 2005; 11: Kniepeiss D, Iberer F, Grasser B, Schaffellner S, Stadlbauer V, Tscheliessnigg KH. A single-center experience with retrograde reperfusion in liver transplantation. Transpl Int 2005; 16 : Heidenhain C., Heise M, Jonas S, Neuhaus P, Langrehr J. Retrograde reperfusion via the vena cava lowers the risk of initial non function but increases the risk of ischemic-type biliary lesions in human liver transplantation. A prospective, controlled, randomised clinical trial. [abstract] Transpl.Int 2005; 18[S1], Moench C, Uhrig A, Lohse AW, Otto G. CC chemokine receptor 5delta32 polymorphism-a risk factor for ischemic-type biliary lesions following orthotopic liver transplantation. Liver Transpl 2004; 10 : Gugenheim J, Samuel D, Reynes M, Bismuth H. Liver transplantation across ABO blood group barriers. Lancet 1990; 336 : Sankary HN, McChesney L, Hart M, Foster P, Williams J. Identification of donor and recipient risk factors associated with nonanastomotic biliary strictures in human hepatic allografts. Transplant Proc 1993; 25: Brandsaeter B, Schrumpf E, Bentdal O, Brabrand K, Smith HJ, Abildgaard A, Clausen OP, Bjoro K. Recurrent primary sclerosing cholangitis after liver transplantation: A magnetic resonance cholangiography study with analyses of predictive factors. Liver Transpl 2005; 11: Dolmatch BL, Laing FC, Ferderle MP, Jeffrey RB, Cello J. AIDS-related cholangitis: radiographic findings in nine patients. Radiology 1987; 163: Halme L, Hockerstedt K, Lautenschlager I. Cytomegalovirus infection and development of biliary complications after liver transplantation. Transplantation 2003; 75: Kowdley KV, Fawaz KA, Kaplan MM. Extrahepatic biliary stricture associated with cytomegalovirus in a liver transplant recipient. Transpl Int 1996; 9: Heidenhain C., Heise M, Jonas S, Schmitt S., Neuhaus P, Langrehr J. Incidence and risk factors for ischemic- type biliary lesions following orthotopic liver transplantation. A retrospective analysis of 1714 patients. [abstract] Transpl.Int 2005; 18[s1]: Scotte M, Dousset B, Calmus Y, Conti F, Houssin D, Chapuis Y. The influence of cold ischemia time on biliary complications following liver transplantation. J Hepatol 1994; 21:

31 Causes and consequences of ITBL after liver transplantation 42. Lerut J, Demetris AJ, Stieber AC, Marsh JW, Gordon RD, Esquivel CO, Iwatsuki S, Starzl TE. Intrahepatic bile duct strictures after human orthotopic liver transplantation. Recurrence of primary sclerosing cholangitis or unusual presentation of allograft rejection? Transpl Int 1988; 1: Ludwig J, Wiesner RH, Batts KP, Perkins JD, Krom RA. The acute vanishing bile duct syndrome (acute irreversible rejection) after orthotopic liver transplantation. Hepatology 1987; 7: Oguma S, Belle S, Starzl TE, Demetris AJ. A histometric analysis of chronically rejected human liver allografts: insights into the mechanisms of bile duct loss: direct immunologic and ischemic factors. Hepatology 1989; 9: Lewis WD. Biliary strictures after liver transplantation. The Surgical clinics of North America 1994; 74:967. Colonna JO, Shaked A, Gomes AS, Colquhoun SD, Jurim O, McDiarmid SV, Millis JM, Goldstein LI, Busuttil RW. Biliary strictures complicating liver transplantation. Incidence, pathogenesis, management, and outcome. Ann Surg 1992; 216: Moench C, Uhrig A, Wunsch A, Thies J, Otto G. Chemokines: reliable markers for diagnosis of rejection and inflammation following orthotopic liver transplantation. Transplant Proc 2001; 33: Morland CM, Fear J, McNab G, Joplin R, Adams DH. Promotion of leukocyte transendothelial cell migration by chemokines derived from human biliary epithelial cells in vitro. Proc Assoc Am Physicians 1997; 109: Schroppel B, Fischereder M, Ashkar R, Lin M, Kramer BK, Mardera B, Schiano T, Murphy B. The impact of polymorphisms in chemokine and chemokine receptors on outcomes in liver transplantation. Am J Transplant 2002; 2: Hertl M, Harvey PR, Swanson PE, West DD, Howard TK, Shenoy S, Strasberg SM. Evidence of preservation injury to bile ducts by bile salts in the pig and. Hepatology 1995; 21: Hertl M, Hertl MC, Kluth D, Broelsch CE. Hydrophilic bile salts protect bile duct epithelium during cold. Liver transplantation 2000; 6: Knoop M, Schnoy N, Keck H, Neuhaus P. Morphological changes of human common bile ducts after extended cold preservation. Transplantation 1993; 56: Doctor R, ahl R, alter K, ouassier. ATP depletion in rat cholangiocytes leads to marked internalization of membrane proteins. Hepatology 2000; 31: Geuken E, Visser D, Kuipers F, Blokzijl H, Leuvenink HG, de Jong KP, Peeters PM, Jansen PL, Slooff MJH, Gouw AS, Porte RJ. Rapid increase of bile salt secretion is associated with bile duct injury after human liver transplantation. J Hepatol 2004; 41: Hoekstra H, Porte RJ, Tian Y, Jochum W, Stieger B, Moritz W, et al. Bile salt toxicity aggravates cold ischemic injury of bile ducts after liver transplantation in Mdr2+/- mice. Hepatology ;43:

32 Chapter Palmeira CM, Rolo AP. Mitochondrially-mediated toxicity of bile acids. Toxicology 2004; 203 :1-15. Sanchez-Urdazpal L, Gores GJ, Ward EM, Hay E, Buckel EG, Wiesner RH, Krom RA. Clinical outcome of ischemic-type biliary complications after liver transplantation. Transplant Proc 1993; 25 : Holbert BL, Campbell WL, Skolnick ML. Evaluation of the transplanted liver and postoperative complications. Radiol Clin North Am 1995; 33: Bowen A, Hungate RG, Kaye RD, Reyes J, Towbin RB. Imaging in liver transplantation. Radiol Clin North Am 1996; 34: Keogan MT, McDermott VG, Price SK, Low VH, Baillie J. The role of imaging in the diagnosis and management of biliary complications after liver transplantation. AJR Am J Roentgenol 1999; 173: Kok T, Van der Sluis A, Klein JP, Van der Jagt EJ, Peeters PM, Slooff MJ, Bijleveld CM, Haagsma EB. Ultrasound and cholangiography for the diagnosis of biliary complications after orthotopic liver transplantation: a comparative study. J Clin Ultrasound 1996; 24: Boraschi P, Donati F, Gigoni R, Urbani L, Femia M, Cossu MC, Filipponi F, Falaschi F. Ischemic-type biliary lesions in liver transplant recipients: evaluation with magnetic resonance cholangiography. Transplant Proc 2004; 36: Boraschi P, Braccini G, Gigoni R, Sartoni G, Neri E, Filipponi F, Mosca F, Bartolozzi C. Detection of biliary complications after orthotopic liver transplantation with MR cholangiography. Magn Reson Imaging 2001; 19: Ward J, Sheridan MB, Guthrie JA, Davies MH, Millson CE, Lodge JP, Pollard SG, Prasad KR, Toogood GJ, Robinson PJ. Bile duct strictures after hepatobiliary surgery: assessment with MR cholangiography. Radiology 2004; 231: Malcolm S CP-A. Biliary complications following liver transplantation. Medical care of the liver transplant patient. Massachusetts: Blackwell Science; 1997., 2005: Hintze RE, Adler A, Veltzke W, Abou-Rebyeh H, Felix R, Neuhaus P. Endoscopic management of biliary complications after orthotopic liver transplantation. Hepatogastroenterology 1997; 44: Theilmann L, Kuppers B, Kadmon M, Roeren T, Notheisen H, Stiehl A, Otto G. Biliary tract strictures after orthotopic liver transplantation: diagnosis and management. Endoscopy 1994; 26: Gopal DV, Pfau PR, Lucey MR. Endoscopic Management of Biliary Complications After Orthotopic Liver Transplantation. Curr Treat Options Gastroenterol 2003; 6: Rerknimitr R, Sherman S, Fogel EL, Kalayci C, Lumeng L, Chalasani N, Kwo P, Lehman GA. Biliary tract complications after orthotopic liver transplantation with choledochocholedochostomy anastomosis: endoscopic findings and results of therapy. Gastrointest Endosc 2002; 55:

33 Causes and consequences of ITBL after liver transplantation 70. Pfau PR, Kochman ML, Lewis JD, Long WB, Lucey MR, Olthoff K, Shaked A, Ginsberg GG. Endoscopic management of postoperative biliary complications in orthotopic liver transplantation. Gastrointest Endosc 2000; 52: Farouk M, Branum GD, Watters CR, Cucchiaro G, Helms M, McCann R, Bollinger R, Meyers W C. Bile compositional changes and cholesterol stone formation following orthotopic liver transplantation. Transplantation 1991; 52: Gong Y, Gluud C. Colchicine for primary biliary cirrhosis. Cochrane Database Syst Rev 2004;:CD Schlitt HJ, Meier PN, Nashan B, Oldhafer KJ, Boeker K, Flemming P, Raab R, Manns MP, Pichlmayr R. Reconstructive surgery for ischemic-type lesions at the bile duct bifurcation after liver transplantation. Ann Surg 1999; 229: Calne RY. A new technique for biliary drainage in orthotopic liver transplantation utilizing the gall bladder as a pedicle graft conduit between the donor and recipient common bile ducts. Ann Surg 1976; 184:

34

35

36 Part I Non-anastomotic biliary complications after liver transplantation

37

38 3 Non-anastomotic biliary strictures after adult liver transplantation: part I: radiological features and risk factors for early versus late presentation Liver Transpl 2007; 13: Carlijn I Buis Robert C Verdonk Eric J Van der Jagt Christian S van der Hilst Maarten JH Slooff Elizabeth B Haagsma Robert J Porte

39 NAS after liver transplantation: risk factors for early versus late presentation Abstract Non-anastomotic biliary strictures (NAS) are a serious complication after orthotopic liver transplantation (OLT). The exact pathogenesis is unclear. The purpose of this study was to identify risk factors for the clinical and radiological presentation of NAS, as well as for the period of presentation of NAS after OLT. A total of 487 adult liver transplants performed between 1986 and 2003 were studied. All imaging studies of the biliary tree were reviewed, cholangiography was routinely performed between postoperative day and later on demand. Localization of NAS at first presentation was categorized into 4 anatomical zones of the biliary tree. Severity of NAS was semi-quantified as mild, moderate or severe. A large number of donor, recipient and surgical variables were analyzed to identify risk factors for NAS. NAS developed in 81 (16.6%) of the livers. Thirty-seven (7.3%) were graded as moderate to severe. In 85% of the cases, anatomical localization of NAS was around or below the bifurcation of the common bile duct. A large variation was observed in the time interval between OLT and first presentation of NAS (median 4.1 months; range months). NAS presenting early ( 1 year) after OLT was strongly associated with preservation-related risk factors (Cold ischemia time Early NAS 694 min ( ), Late NAS 490 min ( ) (p=0.01)) and most frequently located in the central bile ducts. NAS presenting late (> 1 year) after OLT was found more frequently in the periphery of the liver and associated with immunological risk factors (PSC as indication for OLT Early NAS n=12 (24%), Late NAS n=14 (45%) (p< 0.05)). In conclusion, by separating cases of NAS based on the time of presentation after transplantation, we were able to identify significant differences in risk factors, indicating different pathogenic mechanisms depending on the time of initial presentation. Introduction Biliary complications are a major cause of morbidity and graft failure in patients after orthotopic liver transplantation (OLT) (1-3). Non-anastomotic biliary strictures (NAS) are considered to be the most troublesome biliary complication (4,5). NAS were first described in association with bile duct ischemia due to hepatic artery thrombosis after OLT (6). However, intrahepatic biliary lesions, such as strictures and dilatations, can also be seen in patients without hepatic artery thrombosis (7,8). Another name that is frequently used to describe this type of complication is 38

40 Chapter 3 ischemic-type biliary lesions based on the radiological resemblance with biliary abnormalities that can be seen after hepatic artery occlusion (8). The reported incidence of NAS varies greatly between different series, ranging from 1-19% (9,10). This variation can, at least partly, be explained by differences in the definition of NAS used in different studies, as well as the reporting of only symptomatic patients and variations in the length of follow up after OLT. In the majority of series an incidence between 5 to 15% has been reported for NAS (11-18). The exact pathogenic mechanisms of NAS occurring in the absence of hepatic artery thrombosis are still unknown. However, previous studies have strongly suggested two major groups of risk factors: a) preservation (ischemia / reperfusion) injury-related factors and b) variables related to immunological processes (4,19-21). In addition, recent studies have indicated that hydrophobic bile salts are involved in the pathogenesis of biliary injury after OLT (22-25). In most previous studies, all patients with NAS were considered as one group, independent from the time of occurrence after OLT and the anatomical localization (8,17,19,21,26-29), In some studies only NAS occurring within 6 months after OLT were analyzed (20). However, the time of presentation of NAS after OLT varies widely among different patients. In addition, the severity and anatomical localization of biliary abnormalities at initial presentation may vary considerably. We therefore performed a analysis of the anatomical localization and the severity of NAS at the time of initial presentation in a large group of liver transplant recipients with longterm follow-up. By separating cases based on the time of presentation after transplantation, we were able to identify significant differences in risk factors for NAS, suggesting different pathogenic mechanisms depending on the time of initial presentation. Progression of the disease after initial presentation as well as long-term outcome of NAS in the same cohort of liver transplants are presented separately (30). Patients and Methods Patients Between January 1986 and May 2003 a total number of 717 liver transplants were performed in 639 patients at the University Medical Center Groningen. After exclusion of children (<18 years), and patients with NAS based on hepatic artery thrombosis, 487 transplants in 428 adult patients were included in this study. Follow-up was until November 1, 2005 and median 39

41 NAS after liver transplantation: risk factors for early versus late presentation follow-up was 7.9 years (interquartile range years). Clinical information was obtained from a prospectively collected database. If necessary the original patient notes were reviewed for missing information. Retrospective studies were approved by the institutional ethical committee. Surgical Procedure ABO blood group identical or compatible grafts from brain-death donors with normal or near normal liver function tests were used for all patients. Organ procurement was performed according to standard techniques, using either university of Wisconsin (UW) preservation fluid, histidine-tryptophane-ketoglutarate (HTK) solution, or Euro-Collins (EC) solution (before 1989) (31). On the back table, bile ducts were thoroughly flushed with preservation solution. A standardized technique was used for implantation, as has been described previously (32,33). In our institution a duct-to-duct bile duct anastomosis is preferred, including in patients with primary sclerosing cholangitis (PSC) if the recipient bile duct is suitable (34). A straight, open tip silicon drain was placed transanastomotically in the bile duct, independent from the type of bile duct anastomosis (duct-to-duct or Roux-en-Y hepatico-jejunostomy). Postoperative Management Two types of immunosuppressive scheme was used during the study period. For patients with autoimmune diseases like autoimmune hepatitis, primary biliary cirrhosis, and primary sclerosing cholangitis a triple immunosuppressive scheme [prednisolon, azathioprine and cyclosporine A (CyA)]. All other patients received a double immunosuppressive scheme, consisting of prednisolon together with either tacrolimus or CyA. In patients with compromised renal function calcineurin inhibitors were withheld until creatinine clearance was over 50 ml/ min. If postoperative renal insufficiency was anticipated, induction therapy with basiliximab was started. Biopsy-proven acute rejection was treated, when clinically indicated, with a bolus of methylprednisolone on three consecutive days. Steroid-resistant rejections were treated either by conversion to tacrolimus in patients on cyclosporine A, or by giving 5 doses of antithymocyte globulin (4 mg/kg i.v.) on alternating days. When the cytomegalovirus (CMV) status of the donor/recipient combination was positive/negative, prophylaxis with oral ganciclovir was started at postoperative day 10 and continued for three months. 40

42 Chapter 3 Doppler ultrasound was performed routinely at postoperative days 1, 3, and 7 and on demand, to rule out vascular or biliary complications or parenchymal lesions. Cholangiography via the bile drain was routinely performed between postoperative day and later on demand (i.e. for rising cholestatic parameters or dilatation of bile ducts on ultrasound). The drain was clamped when no anastomotic leakage or biliary complications were found at cholangiography. The timing of bile drain removal has increased during the study period from one to currently six months after transplantation. When a biliary complication was suspected after removal of the bile drain, the preferred method for further imaging and or treatment was endoscopic retrograde cholangiopancreaticography (ERCP). This technique has been available in our center since the early 1980 s. In case of a hepatico-jejunostomy, percutaneous transhepatic cholangiographic drainage (PTCD) was used to treat biliary complications. In recent years, magnetic resonance cholangiopancreaticography (MRCP) has been used more frequently as a diagnostic tool. Diagnosis and Radiological Classification of NAS For the purpose of this study, NAS were defined as any stricture, dilatation or irregularity of the intra- or extrahepatic bile ducts of the liver graft, either with or without biliary sludge formation, after exclusion of hepatic artery thrombosis by either Doppler ultrasound or conventional angiography. Isolated strictures at the bile duct anastomosis were, by definition, excluded from this analysis and have been described elsewhere (35). The time of first presentation of NAS was recorded for all patients. For the purpose of this study, all imaging studies of the biliary tree (cholangiography via the biliary drain, PTCD, MRCP, or ERCP) of patients diagnosed with NAS were reviewed by a single radiologist (EJ), who was blinded to the clinical information. The localization of biliary lesions at the time of initial presentation was categorized according to predefined criteria, based on the region and side of the liver. For this purpose we developed a schematic presentation of the biliary tree in 4 different zones: the extrahepatic common bile duct (CBD) including the hilar bifurcation (Zone A), the bile ducts between 1st and 2nd order branches (Zone B), the bile ducts between 2nd and 3rd order branches (Zone C), and bile ducts in the periphery of the liver (Zone D). In addition, the location of the stricture(s) was/were categorized as left or right-sided, or bilateral (Figure 1). 41

43 NAS after liver transplantation: risk factors for early versus late presentation Figure 1. Schematic presentation of the anatomical zones of biliary tree used to define the localization of NAS after liver transplantation The severity of biliary strictures was categorized based on an arbitrary severity index in which strictures were scored per area as mild, moderate or severe. Severity scoring was based on number of strictures in total, the severity according to the degree of narrowing, pre-stenotic dilatation and mucosal irregularity and finally the extensiveness of the strictures per area. (fig. 2) Risk Factors for NAS A large number of potential risk factors for NAS were studied by comparing the group of patients with NAS with those who did not develop NAS. In addition, patients with NAS within the first year after transplantation were compared with those who developed NAS after the first year. Risk factors were grouped as donor-related variables (age, gender), recipient-related variables (age, gender, indication for transplantation and Child-Pugh score), surgical variables (preservation solution, cold ischemia time, warm ischemia time, revacularization time, type of graft and bile duct reconstruction) and postoperative outcome variables (anastomotic leakage, serum aspartate amino-transferase (AST), type of immunosuppression, length of stay in ICU, CMV infection, and acute rejection). 42

44 Chapter 3 Figure 2. Cholangiography of patients presenting with different severities of NAS. (A) Example of mild NAS. Central bile duct stenosis without more peripheral intrahepatic strictures and dilatations. (B) Example of moderate NAS. Central stenosis and a stenosis in the left hepatic duct, with intrahepatic dilatations. (C) Example of severe NAS. Diffuse strictures and irregularities of both the extra- and intrahepatic bile ducts on both sides of the liver. 43

45 NAS after liver transplantation: risk factors for early versus late presentation Statistical Methods Continuous variables were presented as medians with interquartile range (IQR) and categorical variables as numbers with percentages. Time to occurrence of NAS was calculated according to the Kaplan-Meier method. Categorical variables were compared using Pearson s chisquare test or Fisher exact test where appropriate. Comparison of continuous variables was performed using the Mann-Whitney U test. The level of significance was set at Statistical analysis was performed using the SPSS/PC+ Advanced Statistics Package, Version (SPSS, Chicago, IL). Results Initial Clinical and Radiological Presentation of NAS Clinical characteristics of donor and recipients for the entire series are presented in Table 1. Out of the total of 487 liver grafts, NAS was found in 81 (16.6%) livers, transplanted in 77 patients. Within the group with NAS, 71 were first transplants and 10 were retransplants. Four patients developed NAS in both a first and a second graft. The majority of patients with NAS presented with either elevated serum liver enzymes (n=49, 60%), and/or an episode of cholangitis (n=24, 30%). In 13 (16%) cases, the diagnosis of NAS was based on coincidental findings on routine cholangiography in an otherwise asymptomatic patient. The radiological modality, which led to the diagnosis of NAS, was cholangiography via ERCP in 29, bile drain cholangiography in 24, MRCP in 23, and PTCD in 5 patients. According to the inclusion criteria, all patients had a patent hepatic artery as confirmed by Doppler ultrasonography or angiography. The anatomical distribution of biliary lesions at the time of presentation is shown in Table 2. Imaging studies for radiological evaluation was present in 78 of the 81 (96%) transplants. 44

46 Chapter 3 Table 1. Clinical Characteristics of Donor and Recipient for the Entire Series of Liver Transplants (n=487)* Donor variables Age (years) 40 (25-50) Gender (male/female) 251 / 236 (52% / 48%) Recipient variables Age (Years) 45 (33-53) Gender (male/female) 236 / 251 (48% / 52%) Disease PSC 82 (17%) PBC + SBC 72 (15%) Post viral cirrhosis 79 (16%) Auto-immune hepatitis 47 (10%) Alcoholic cirrhosis 38 (8%) Cryptogenic cirrhosis 55 (11%) Other 114 (23%) Child Pugh Classification (A/ B/ C) 62 / 235 / 190 (13% / 48% / 39%) Re-transplantation 60 (12%) Surgical variables Preservation solution Low viscosity (EC or HTK) / High viscosity (UW) 44 / 443 (9% / 91%) Cold ischemia time (minutes) 599 ( ) Warm ischemia time (minutes) 56 (47-65) Bile duct reconstruction (dtd / Roux-Y) 410 / 71 (84% / 15%) Type of graft (whole / reduced size) 472 / 15 (97% / 3%) Postoperative variables Anastomotic bile leakage 22 (5%) serum AST postoperative day 2 (U/L) 351 ( ) Postoperative immunosuppressive treatment Azathioprine / Tacrolimus / Cyclosporine 13 / 246 / 124 (3% / 50% / 25%) ICU length of stay (days) 4 (2-8) CMV infection 190 (49%) Acute rejection 174 (36%) * Continuous variables are presented as median and interquartile range, categorical variables as numbers with percentage. Rejection: BANFF grade II - III or grade I and treated. 45

47 NAS after liver transplantation: risk factors for early versus late presentation Biliary lesions were observed around or below the bifurcation of the CBD (Zone A) in 66 (85%) cases. Biliary abnormalities became less frequent towards the periphery of the liver. The right and left system, however, were equally affected in all zones of the biliary tree. The severity of biliary strictures was classified as mild in 43 (55%) and as moderate to severe in 35 (45%) of the cases. The cumulative incidence of moderate to severe NAS in the entire population of liver transplant recipients was 7.3%. Table 2. Anatomical Localization of NAS at Time of First Presentation. Localization Number (%) * Extrahepatic or Bifurcation Zone A 66 (81%) left 9 right 8 both 46 CBD Only 3 Intrahepatic Zone B 52 (67%) left 8 right 9 both 35 Zone C 33 (42%) left 6 right 6 both 21 Zone D 15 (19%) 1 left 2 right 12 both 1 *) More than one area could be involved in one patient. 46

48 Chapter 3 When Are NAS First Detected After Liver Transplantation? A large variation was observed in the time interval between transplantation and the initial presentation of NAS. The median time from transplantation to diagnosis of NAS was 4.1 months (IQR months). More than 50% of the cases of NAS presented within the first year after transplantation (Figure 2). However, more long-term follow-up showed that the number of grafts that develop NAS gradually continued to increase up to 12 years after transplantation. This resulted in a sharp initial rise of the curve representing the cumulative incidence of NAS during the first year after OLT, followed by a smaller increment beyond the first year (Figure 2). The cumulative incidence was 14%, 15% and 16% at 3, 5 and 10 years after OLT, respectively. 20 Cumulative incidence of NAS (%) Years after Transplantation Numbers at risk Figure 3. Cumulative incidence of NAS after liver transplantation in the time period

49 NAS after liver transplantation: risk factors for early versus late presentation Which Risk Factors Are Associated with NAS? A comparison of demographic and clinical variables between grafts without NAS and the entire group of livers that developed NAS, independent from the time of occurrence after OLT, is presented in Table 3. The only significantly different variables between the two groups were PSC as the indication for transplantation, type of preservation solution (high-viscosity (UW-solution) versus low-viscosity solution (EC and HTK)), the type of bile duct reconstruction (duct-to-duct versus Roux-Y hepatico-jejunostomy), and postoperative CMV infection. However, standard testing whether a patient suffered from a CMV infection became only routine clinical practice in our center around 1992, and was therefore available in only a subset of 383 patients. In this first analysis, ischemia times did not emerge as a risk factor for NAS. This was surprising, because cold and warm ischemia time have both been associated with NAS in previous studies (8,20,26). However, as we have noted above, a change in the pattern of the cumulative incidence could be observed after one year and about half of the cases of NAS in our series were detected beyond the first year after transplantation. It is not likely that the length of warm or cold ischemia still has an impact on the development of NAS at such a long interval after transplantation. Therefore, we next examined radiological characteristics and potential risk factors for early (< 1 year) versus late (> 1 year) initial presentation of NAS after OLT. 48

50 Chapter 3 Table 3. Comparison of Donor and Recipient Characteristics of Liver Grafts With and Without Non Anastomotic Biliary Strictures. NAS no NAS (n = 81) (n = 406) P-value Donor variables Age (years) 41 (32-50) 39 (24-50) 0.16 Gender (male/female) 40 / 41 (49% / 51%) 211 / 195 (52% / 48%) 0.67 Gender match (donor/recipient) 0.75 M/M 23 (28%) 104 (27%) F/F 21 (26%) 106 (26%) M/F 17 (21%) 107 (26%) F/M 20 (25%) 89 (21%) Recipient variables Age (Years) 46 (37-54) 45 (32-53) 0.21 Gender (male/female) 43 / 38 (53% / 47%) 193 / 213 (48% / 52%) 0.36 Disease PSC 26 (32%) 56 (14%) <0.01 PBC + SBC 10 (12%) 62 (15%) 0.50 Post viral cirrhosis 9 (11%) 70 (17%) 0.17 Auto-immune hepatitis 7 (9%) 40 (10%) 0.74 Alcoholic cirrhosis 6 (7%) 32 (8%) 0.88 Cryptogenic cirrhosis 8 (10%) 47 (12%) 0.66 Other 15 (19%) 99 (24%) 0.26 Child Pugh Classification (A/ B/ C) 13 / 39 / 29 (16% / 48% / 36%) 49 / 196 / 161 (12% / 48% / 40%)) 0.58 Re-transplantation 10 (12%) 49 (12%)

51 NAS after liver transplantation: risk factors for early versus late presentation NAS no NAS (n = 81) (n = 406) P-value Time of transplant Quartile (1st/2nd/3rd/4th) 15 / 21 / 22 / 23 (19%/26%/27%/28%) 106 / 100 / 101 / 99 (26%/25%/25%/24%) 0.53 Surgical variables Preservation solution Low viscosity (EC or HTK) / High viscosity (UW) 2 / 79 (2% / 98%) 42 / 359 (10% / 90%) 0.02 Cold ischemia time (minutes) 609 ( ) 594 ( ) 0.54 Warm ischemia time (minutes) 55 (46-63) 56 (47-65) 0.42 Bile duct reconstruction (dtd / Roux-Y) 62 / 19 (77% / 23%) 384 / 52 (86% / 13%) 0.02 Type of graft (whole / reduced size) 80 / 1 (99% / 1%) 329 / 14 (97% / 3%) 0.29 Postoperative variables Anastomotic bile leakage 5 (6%) 17 (5%) 0.43 serum AST postoperative day 2 (U/L) 329 ( ) 370 ( ) 0.34 Postoperative immunosuppressive treatment Azathioprine / Tacrolimus / Cyclosporine 2 / 54 / 24 (3%/66%/30%) 11 / 195 / 102 (3%/48%/25%) 0.81 ICU length of stay (days) 4 (2-7) 4 (2-9) 0.23 CMV infection 44 / 29 (60%) 146 / 164 (47%) 0.04 Acute rejection 23 (28%) 151 (37%) 0.13 Continuous variables are presented as median and interquartile range, categorial variables as numbers with percentage. 50

52 Chapter 3 Is There a Difference in Radiological Presentation of Early Versus Late NAS? Differences in the anatomical localization of NAS presenting early (< 1 year) versus late (> 1 year) after transplantation are shown in Table 4. In contrast to the group with early presentation of NAS in which the vast majority of lesions were found around the bifurcation and the CBD (Zone A), biliary abnormalities in the group with late presentation of NAS were more frequently identified in the periphery of the liver, at a level which reached statistical significance (table 4). There were no significant differences in the severity of biliary strictures occurring early or late after OLT. In the group of livers presenting with NAS early after OLT lesions were classified as mild in 31 (64%) and as moderate to severe in 17 (36%) of the cases. In the group with late presentation of NAS, lesions were classified as mild in 15 (50%) and moderate to severe in 15 (50%). Moreover, when severity of NAS at time of presentation was studied per zone of the biliary tree, also no differences were found. Table 4. Anatomical Localization of NAS Presentating Early ( 1year) Versus Late (> 1 year). Localization Early NAS Late NAS Number (%)* Number (%)* p-value Extrahepatic or Bifurcation Zone A total 41 (85%) 25 (81%) 0.87 Intrahepatic Zone B 30 (63%) 22 (73%) 0.72 bilateral unilateral 9 8 Zone C 17 (35%) 16 (53%) 0.40 bilateral unilateral 7 5 Zone D 4 (4%) 11 (37%) 0.04 bilateral 3 9 unilateral 1 2 *) More than one area could be involved in one patient. 51

53 NAS after liver transplantation: risk factors for early versus late presentation Are Early and Late NAS Associated With Different Risk Factors? When comparing all potential risk factors for NAS between the livers with early or late presentation important differences were noted. Relevant variables with a p-value 0.1 are presented in Table 5. The cold ischemia time was significantly longer for the group with early NAS, compared to late NAS. In addition, the warm ischemia time was longer in the group with early NAS, although this did not reach statistical significance. Furthermore, all cases of anastomotic bile leakage, a condition generally associated with local bile duct ischemia, were observed in the group with early NAS. In contrast, significantly more patients transplanted for PSC, as well as more female/male gender matches and Roux-Y bile duct reconstructions, were observed in the group with a late presentation of NAS, compared to the group with early NAS. These findings indicate that different mechanisms are involved in the pathogenesis of NAS depending on the time of presentation after transplantation. Table 5. Relevant Characteristics of Liver Grafts Presenting with NAS Early ( 1year) Versus Late (> 1 year) After OLT * Early NAS Late NAS (n = 50) (n = 31) P-value Donor variables Gender match (donor/recipient) <0.01 M/M 16 (32%) 7 (23%) F/F 15 (30%) 6 (19%) M/F 13 (26%) 4 (13%) F/M 6 (12%) 14 (45%) Recipient variables Age 49 (37-57) 42 (36-51) 0.06 PSC as indication for OLT 12 (24%) 14 (45%) < 0.05 Surgical variables Cold ischemia time (minutes) 694 ( ) 490 ( ) 0.01 Warm ischemia time (minutes) 57 (48-65) 53 (45-57) < 0.05 Bile duct reconstruction (dtd / Roux-Y) 43 / 7 (86% / 14%) 19 / 12 (61% / 39%) 0.01 Postoperative variables Bile leakage 5 (10%) * Only variables with p value 0.1 are presented in this table. 52

54 Chapter 3 Discussion Strictures of the bile ducts are a serious complication after OLT, causing increased morbidity and graft loss (9,20). Although the exact pathogenesis of this type of biliary complication remains unknown, both preservation-related factors and immunological processes have been suggested to play a role (4,19,20). Results from previous clinical studies focusing on potential risk factors of NAS, however, are not unequivocal and conflicting data have been found (20,36,37) In this study we were able to identify differences in the anatomical localization as well as differences in risk factors for NAS depending on the time of first presentation after OLT. While ischemia and preservation-related variables were most prominent in the group with early presentation, a late presentation of NAS was more frequently associated with immunological factors. These findings provide new insights in the pathophysiological mechanisms of NAS. In the current series, the cumulative incidence of NAS at 15 years after transplantation was almost 17%, and 7% of all liver grafts were radiological graded as having moderate or severe biliary strictures. These figures are in line with most previous studies (11,15,18,20). However, lower percentages have also been reported in some series, which may be explained by differences in the definition and diagnostics used, as well as differences in the duration of follow up (17,38). The routine use of a biliary drain and postoperative cholangiography allowed us not only to carefully identify and localize all biliary abnormalities, but also to include minor or single strictures in otherwise asymptomatic patients. Routine cholangiography has not always been used in previous studies. These factors may largely explain the differences in the incidence of NAS reported in different series (9-18,20). In addition, we had a long followup in our series with a median of almost 8 years. However, a limitation of this retrospective single centre study could be different imaging modalities over the years and should be kept in mind. When all cases with NAS were studied as one group, independent from the severity and time of occurrence after OLT, only PSC as the indication for transplantation, the type of preservation solution and postoperative CMV infection could be identified as risk factors for NAS. The higher incidence of NAS in patients transplanted for PSC (15,20,21) and patients who suffered CMV infection postoperatively are in accordance with previous studies (19,36,37,39). Also the 53

55 NAS after liver transplantation: risk factors for early versus late presentation more frequent occurrence of NAS in patients with a Roux-Y hepatico-jejunostomy has been reported before and this can be explained by the more frequent use of this type of bile duct reconstruction in patients transplanted for PSC, compared to patients transplanted for other indications (20). The lower incidence of NAS in our series in livers which were preserved with a low viscosity preservation solution is in agreement with previous reports (40). It has been suggested that the peribiliary vascular plexus is better flushed out and better preserved when low viscosity fluids are used compared with high viscosity fluids. These observations, however, have not yet been confirmed to our knowledge in randomized controlled trials (41,42). In contrast with previous studies, we were not able to identify an association between the lengths of warm or cold ischemia time and the development of NAS when we analyzed all grafts with NAS as one group, regardless the time of occurrence after OLT. Most previous studies, however, had a short postoperative follow-up of less than one year, whereas in our series the median follow-up was 7.9 years with an interquartile range of 4.2 to 12.6 years. Although the cumulative incidence of NAS increased sharply within the first year, almost 50% of all cases were detected beyond the first year after transplantation. Biologically it is not plausible that preservation-related factors are still responsible for NAS that first present more than one year after OLT. We therefore performed a second analysis comparing patients with early (< 1 year after OLT) versus late (> 1 year after OLT) presentation of NAS. This analysis showed significant associations between preservation-related risk factors, such as the length of cold ischemia time and bile duct anastomotic leakage, and the occurrence of NAS early after OLT. Ischemia reperfusion- and preservation injury-related variables are well described risk factors for NAS, and include prolonged cold ischemia time (> 12 hours) or warm ischemia time (> 60 min) and variables related to the efficacy of preservation of the peribiliary plexus, such as viscosity and perfusion pressure of the preservation fluid (20,26,40). Moreover, the higher incidence of NAS in liver transplantation from donors after cardiac death (non-heart-beating donors) also strongly suggests an ischemia-related factor in the pathogenesis of NAS (43-45). For many years, it has been policy in our center to keep the cold ischemia time as short as possible and recipient operations usually start before the donor liver has arrived and as soon as the liver has been judged transplantable by the surgical team performing the procurement operation. In addition, the use of the piggyback technique has allowed us to shorten the warm ischemia time during implantation, in comparison with conventional implantation (33,46). With this policy we were able to keep the median cold and warm ischemia time below 12 hours 54

56 Chapter 3 and 60 min, respectively. Nevertheless, we could still identify cold ischemia time as one of the most important discriminators of NAS occurring early after OLT. Risk factors for the development of NAS late (> 1 year) after transplantation were a female to male donor/recipient match, and PSC as the indication for transplantation. These variables are not associated with preservation injury and suggest a more immunological pathogenesis of NAS presenting late after OLT. An immunological origin of NAS has been suggested by other investigators based on the relationship between NAS and ABO incompatibility, the strong association with pre-existing diseases with a presumed autoimmune component (such as PSC and autoimmune hepatitis), CMV infection, chronic rejection, but also with genetic polymorphism of chemokines (17,20,28). It is very likely that recurrent PSC may have been accountable for the occurrence of biliary lesions in some of the patients presenting with NAS late after OLT. Based on radiological evaluation, however, recurrent PSC cannot be distinguished from a late presentation of NAS. Although some of our patients fit well within the definition of recurrent PSC as proposed by Graziadei et al. (47), more than half of our patients who presented with NAS late after OLT were not transplanted for PSC. Several studies have shown a lower survival rate for grafts from female donors transplanted in male recipients. (48-51). Although some investigators have tried to explain this by differences in estrogen receptor expression (49), reduced outcome for the female to male donor/ recipient match has also been described after OLT in children below 10 years of age (48). This observation makes it less likely that a sex hormone-related pathogenesis is the only explanation for the worse outcome of female livers into male recipients and immunological processes have been suggested to play a role as well in other transplant settings (52). Immunologically-mediated injury of the bile ducts resulting in NAS may be a direct result of activated proinflammatory cytokines and influx of inflammatory cells. However, it cannot be deducted from a clinical study like this whether this type of bile duct injury is (at least partially) also caused by relative ischemia of the biliary epithelium due to immune-mediated obliterative arteriopathy of the peribiliary vascular plexus (8,9,19). Further research in this area seems warranted. Very few studies to our knowledge have focused on the anatomical localization of NAS at the time of first presentation. In the current series, over 80% of the NAS were localized around or below the bifurcation of the CBD and less than 20% presented in the peripheral branches of the biliary tree. Livers presenting with NAS more than one year after OLT had more frequently 55

57 NAS after liver transplantation: risk factors for early versus late presentation involvement of the smaller and peripheral bile ducts of the liver. These differences in the anatomical localization between NAS presenting early or late after OLT provide additional support for differences in the pathogenesis of NAS depending on the time of presentation after OLT. The critical relevance of arterial blood supply for the viability of the larger and extrahepatic bile ducts is well described (53). This part of the biliary tree depends entirely on the arterial peribiliary plexus which is perfused via the gastroduodenal artery and the hepatic artery. During OLT, blood supply via the pancreatic head and the gastro-duodenal artery, supplying the peribiliary plexus, is interrupted and the bile ducts become entirely depended on arterial blood from the hepatic artery, making them more prone to hypoperfusion and ischemia. This may explain the central localization of NAS presenting early after OLT. In addition, previous studies have shown a large morphological and functional heterogeneity of different sized intrahepatic bile ducts, providing an explanation why biliary lesions predominates in specific sized bile ducts in various types of diseases affecting the biliary tree (54). This could also be an explanation why immunologically-mediated NAS, presenting late after OLT, is more pronounced at the level the smaller bile ducts. In summary, by separating cases of NAS based on the time of presentation after transplantation, we were able to identify significant differences in risk factors, indicating different pathogenic mechanisms depending on the time of initial presentation. NAS presenting within the first year after transplantation is strongly correlated with ischemia related risk factors, whereas NAS presenting late, more than one year after OLT, is more associated with immunologically related risk factors. These finding have implications for the development of new strategies to prevent or treat NAS. 56

58 Chapter 3 Reference List 1. Starzl TE, Marchioro TL, Vonkaulla KN, Hermann G, Brittain RS, Waddell WR. Homotransplantation of the liver in humans. Surg Gynecol Obstet 1963; 117: Lerut J, Gordon RD, Iwatsuki S, Esquivel CO, Todo S, Tzakis A et al. Biliary tract complications in human orthotopic liver transplantation. Transplantation 1987; 43: Calne RY. A new technique for biliary drainage in orthotopic liver transplantation utilizing the gall bladder as a pedicle graft conduit between the donor and recipient common bile ducts. Ann Surg 1976; 184: Buis CI, Hoekstra H, Verdonk RC, Porte RJ. Causes and consequences of ischemic-type biliary lesions after liver transplantation. J Hepatobiliary Pancreat Surg 2006; 13: Verdonk RC, Buis CI, Porte RJ, Haagsma EB. Biliary complications after liver transplantation: a review. Scand J Gastroenterol Suppl 2006; Zajko AB, Campbell WL, Logsdon GA, Bron KM, Tzakis A, Esquivel CO et al. Cholangiographic findings in hepatic artery occlusion after liver transplantation. AJR Am J Roentgenol 1987; 149: Sanchez-Urdazpal L, Sterioff S, Janes C, Schwerman L, Rosen C, Krom RA. Increased bile duct complications in ABO incompatible liver transplant recipients. Transplant Proc 1991; 23(1 Pt 2): Sanchez-Urdazpal L, Gores GJ, Ward EM, Maus TP, Wahlstrom HE, Moore SB et al. Ischemic-type biliary complications after orthotopic liver transplantation. Hepatology 1992; 16: Sanchez Urdazpal L, Gores GJ, Ward EM, Maus TP, Buckel EG, Steers JL et al. Diagnostic features and clinical outcome of ischemic-type biliary. Hepatology 1993; 17: Thethy S, Thomson BN, Pleass H, Wigmore SJ, Madhavan K, Akyol M et al. Management of biliary tract complications after orthotopic liver transplantation. Clin Transplant 2004; 18: Sawyer RG, Punch JD. Incidence and management of biliary complications after 291 liver transplants following the introduction of transcystic stenting. Transplantation 1998; 66: Turrion VS, Alvira LG, Jimenez M, Lucena JL, Nuno J, Pereira F et al. Management of the biliary complications associated with liver transplantation: 13 years of experience. Transplant Proc 1999; 31: Rizk RS, McVicar JP, Emond MJ, Rohrmann CA, Jr., Kowdley KV, Perkins J et al. Endoscopic management of biliary strictures in liver transplant recipients: effect on patient and graft survival. Gastrointest Endosc 1998; 47: Ward EM, Kiely MJ, Maus TP, Wiesner RH, Krom RA. Hilar biliary strictures after liver transplantation: cholangiography and percutaneous treatment. Radiology 1990; 177: Campbell WL, Sheng R, Zajko AB, Abu-Elmagd K, Demetris AJ. Intrahepatic biliary strictures after liver transplantation. Radiology 1994; 191:

59 NAS after liver transplantation: risk factors for early versus late presentation 16. Feller RB, Waugh RC, Selby WS, Dolan PM, Sheil AG, McCaughan GW. Biliary strictures after liver transplantation: clinical picture, correlates and outcomes. J Gastroenterol Hepatol 1996; 11: Rull R, Garcia Valdecasas JC, Grande L, Fuster J, Lacy AM, Gonzalez FX et al. Intrahepatic biliary lesions after orthotopic liver transplantation. Transpl Int 2001; 14: Pascher A, Neuhaus P. Bile duct complications after liver transplantation. Transpl Int 2005; 18: Colonna JO, Shaked A, Gomes AS, Colquhoun SD, Jurim O, McDiarmid SV et al. Biliary strictures complicating liver transplantation. Incidence, pathogenesis, management, and outcome. Ann Surg 1992; 216: Guichelaar MM, Benson JT, Malinchoc M, Krom RA, Wiesner RH, Charlton MR. Risk factors for and clinical course of non-anastomotic biliary strictures after liver transplantation. Am J Transplant 2003; 3: Sankary HN, McChesney L, Hart M, Foster P, Williams J. Identification of donor and recipient risk factors associated with nonanastomotic biliary strictures in human hepatic allografts. Transplant Proc 1993; 25: Geuken E, Visser D, Kuipers F, Blokzijl H, Leuvenink HG, de Jong KP et al. Rapid increase of bile salt secretion is associated with bile duct injury after human liver transplantation. J Hepatol 2004; 41: Hertl M, Hertl MC, Kluth D, Broelsch CE. Hydrophilic bile salts protect bile duct epithelium during cold. Liver transplantation 2000; 6: Hertl M, Harvey PR, Swanson PE, West DD, Howard TK, Shenoy S et al. Evidence of preservation injury to bile ducts by bile salts in the pig and its prevention by infusions of hydrophilic bile salts. Hepatology 1995; 21: Hoekstra H, Porte RJ, Tian Y, Jochum W, Stieger B, Moritz W et al. Bile salt toxicity aggravates cold ischemic injury of bile ducts after liver transplantation in Mdr2+/- mice. Hepatology 2006; 43: Moench C, Moench K, Lohse AW, Thies J, Otto G. Prevention of ischemic-type biliary lesions by arterial back- table pressure perfusion. Liver Transpl 2003; 9: Li S, Stratta RJ, Langnas AN, Wood RP, Marujo W, Shaw BW, Jr. Diffuse biliary tract injury after orthotopic liver transplantation. Am J Surg 1992; 164: Moench C, Uhrig A, Lohse AW, Otto G. CC chemokine receptor 5delta32 polymorphism-a risk factor for ischemic-type biliary lesions following orthotopic liver transplantation. Liver Transpl 2004; 10: Graziadei IW, Schwaighofer H, Koch R, Nachbaur K, Koenigsrainer A, Margreiter R et al. Long-term outcome of endoscopic treatment of biliary strictures after liver transplantation. Liver Transpl 2006; 12: Verdonk RC, Buis CI, van der Jagt EJ, Gouw ASH, Limburg AJ, Slooff MJH, Kleibeuker JH, Porte RJ, Haagsma EB. Non-anastomotic biliary strictures after adult liver transplantation part two: Management, outcome and risk 58

60 Chapter 3 factors for disease progression. Liver Transpl. 2007;13: Starzl TE, Hakala TR, Shaw BW, Jr., Hardesty RL, Rosenthal TJ, Griffith BP et al. A flexible procedure for multiple cadaveric organ procurement. Surg Gynecol Obstet 1984; 158: Miyamoto S, Polak WG, Geuken E, Peeters PM, de Jong KP, Porte RJ et al. Liver transplantation with preservation of the inferior vena cava. A comparison of conventional and piggyback techniques in adults. Clin Transplant 2004; 18: Polak WG, Miyamoto S, Nemes BA, Peeters PM, de Jong KP, Porte RJ et al. Sequential and simultaneous revascularization in adult orthotopic piggyback liver transplantation. Liver Transpl 2005; 11: Feith MP, Klompmaker IJ, Maring JK, Peeters PM, van den Berg AP, de Jong KP et al. Biliary reconstruction during liver transplantation in patients with primary sclerosing cholangitis. Transplant Proc 1997; 29: Verdonk RC, Buis CI, Porte RJ, Van der Jagt EJ, Limburg AJ, van den Berg AP et al. Anastomotic biliary strictures after liver transplantation: Causes and consequences. Liver Transpl 2006; 12: Halme L, Hockerstedt K, Lautenschlager I. Cytomegalovirus infection and development of biliary complications after liver transplantation. Transplantation 2003; 75: Kowdley KV, Fawaz KA, Kaplan MM. Extrahepatic biliary stricture associated with cytomegalovirus in a liver transplant recipient. Transpl Int 1996; 9: Torras J, Llado L, Figueras J, Ramos E, Lama C, Fabregat J et al. Biliary tract complications after liver transplantation: type, management, and outcome. Transplant Proc 1999; 31: Dolmatch BL, Laing FC, Ferderle MP, Jeffrey RB, Cello J. AIDS-related cholangitis: radiographic findings in nine patients. Radiology 1987; 163: Pirenne J, Van Gelder F, Coosemans W, Aerts R, Gunson B, Koshiba T et al. Type of donor aortic preservation solution and not cold ischemia time is a major determinant of biliary strictures after liver transplantation. Liver Transpl 2001; 7: Canelo R, Hakim NS, Ringe B. Experience with hystidine tryptophan ketoglutarate versus University Wisconsin preservation solutions in transplantation. Int Surg 2003; 88: Cavallari A, Cillo U, Nardo B, Filipponi F, Gringeri E, Montalti R et al. A multicenter pilot prospective study comparing Celsior and University of Wisconsin preserving solutions for use in liver transplantation. Liver Transpl 2003; 9: Abt P, Crawford M, Desai N, Markmann J, Olthoff K, Shaked A. Liver transplantation from controlled non-heart- beating donors: an increased incidence of biliary complications. Transplantation 2003; 75: D alessandro AM, Hoffmann RM, Knechtle SJ, Odorico JS, Becker YT, Musat A et al. Liver transplantation from controlled non-heart-beating donors. Surgery 2000; 128:

61 45. Otero A, Gomez-Gutierrez M, Suarez F, Arnal F, Fernandez-Garcia A, Aguirrezabalaga J et al. Liver transplantation from Maastricht category 2 non-heart-beating donors. Transplantation 15[76], Ref Type: Abstract Miyamoto S, Polak WG, Geuken E, Peeters PM, de Jong KP, Porte RJ et al. Liver transplantation with preservation of the inferior vena cava. A comparison of conventional and piggyback techniques in adults. Clin Transplant 2004; 18: Graziadei IW, Wiesner RH, Batts KP, Marotta PJ, Larusso NF, Porayko MK et al. Recurrence of primary sclerosing cholangitis following liver transplantation. Hepatology 1999; 29: Francavilla R, Hadzic N, Heaton ND, Rela M, Baker AJ, Dhawan A et al. Gender matching and outcome after pediatric liver transplantation. Transplantation 1998; 66: Kahn D, Gavaler JS, Makowka L, van Thiel DH. Gender of donor influences outcome after orthotopic liver transplantation in adults. Dig Dis Sci 1993; 38: Brooks BK, Levy MF, Jennings LW, Abbasoglu O, Vodapally M, Goldstein RM et al. Influence of donor and recipient gender on the outcome of liver transplantation. Transplantation 1996; 62: Marino IR, Doyle HR, Aldrighetti L, Doria C, McMichael J, Gayowski T et al. Effect of donor age and sex on the outcome of liver transplantation. Hepatology 1995; 22: Sato M, Gutierrez C, Kaneda H, Liu M, Waddell TK, Keshavjee S. The effect of gender combinations on outcome in human lung transplantation: the International Society of Heart and Lung Transplantation Registry experience. J Heart Lung Transplant 2006; 25: Deltenre P, Valla DC. Ischemic cholangiopathy. J Hepatol 2006; 44: Marzioni M, Glaser SS, Francis H, Phinizy JL, Lesage G, Alpini G. Functional heterogeneity of cholangiocytes. Semin Liver Dis 2002; 22:

62 4 Non-anastomotic biliary strictures after liver transplantation part 2: Management, outcome and risk factors for disease progression Liver Transpl 2007; 13: Robert C Verdonk Carlijn I Buis Eric J Van der Jagt Annette SH Gouw Abraham J Limburg Maarten JH Slooff Jan H Kleibeuker Robert J Porte Elizabeth B Haagsma

63 NAS after liver transplantation: risk factors for disease progression Abstract Non-anastomotic biliary strictures (NAS) after orthotopic liver transplantation (OLT) are associated with high retransplant rates. The aim of the present study was to describe the treatment, and identify risk factors for radiological progression of bile duct abnormalities, recurrent cholangitis, biliary cirrhosis and retransplantation in patients with NAS. We retrospectively studied 81 cases of NAS. Strictures were classified according to severity and location. Management of strictures was recorded. Possible prognostic factors for bacterial cholangitis, radiological progression of strictures, development of severe fibrosis/cirrhosis and graft and patient survival were evaluated. Median follow up after OLT was 7.9 years. NAS were most prevalent in the extrahepatic bile duct. Twenty-eight patients (35%) underwent some kind of interventional treatment, leading to a significant improvement in biochemistry. Progression of disease was noted in 68% of cases with radiological follow-up. Radiological progression was more prevalent in patients with early NAS and one or more episodes of bacterial cholangitis. Recurrent bacterial cholangitis (> 3 episodes) was more prevalent in patients with a hepaticojejunostomy. Severe fibrosis or cirrhosis developed in 23 cases, especially in cases with biliary abnormalities in the periphery of the liver. Graft but not patient survival was influenced by the presence of NAS. Thirteen patients (16%) were re-transplanted for NAS. In conclusion, especially patients with a hepatico-jejunostomy, those with an early diagnosis of NAS, and those with NAS presenting at the level of the peripheral branches of the biliary tree, are at risk for progressive disease with severe outcome. 62

64 Chapter 4 Introduction Biliary complications are common after orthotopic liver transplantation (OLT). Biliary strictures and leakage of bile are most frequently encountered. Strictures are often referred to as anastomotic or non-anastomotic. Non-anastomotic strictures (NAS) are generally considered to be the most troublesome type of biliary complications after liver transplantation, with a graft loss rate of up to 46% after two years (1). In a separate study we have analyzed the radiological characteristics of NAS at the time of diagnosis and risk factors for the development of NAS (2). In this study, we were able to identify significant differences in risk factors for the development of NAS depending on the time of initial presentation. In addition, large variations in anatomical localization and severity of NAS at the time of presentation were found, indicating that NAS is not a single disease, but rather a group of biliary abnormalities with different pathogenesis. It is unknown whether the different subtypes of NAS are also associated with difference in outcome and prognosis. Previous studies concerning the treatment and outcome of NAS have not considered different types of NAS as relevant subgroups and risk factors for radiological and clinical progression once the diagnosis has been established have not been identified so far. The aim of the present work was to study NAS in a large cohort of liver transplant recipients with long-term follow up and to describe the results of treatment. In addition, we aimed to identify risk factors for radiological progression of bile duct abnormalities, recurrent cholangitis, biliary cirrhosis and re-transplantation. Patients and Methods Patients Between January 1986 and May 2003 a total number of 717 liver transplants were performed in 639 patients at the University Medical Center Groningen. After exclusion of children (<18 years), and patients with NAS caused by hepatic artery thrombosis, 487 transplants in 428 adult patients were included in this study. Follow-up was until November 1st 2005, allowing a minimal follow up time after transplantation of 2.5 years. Eighty-one grafts with NAS were identified in 77 patients as described previously (2). In short, all post-transplant radiological material of the biliary tree was reviewed by a radiologist blinded to the clinical data (EJ). Anatomical extent 63

65 NAS after liver transplantation: risk factors for disease progression and severity of the biliary abnormalities were classified using a standardized scoring system. The scheme used to classify anatomical localization and extension of NAS is depicted in Figure 1. Severity was arbitrarily scored as mild, moderate or severe, according to the degree of narrowing, pre-stenotic dilatation, and mucosal irregularity. Patient characteristics as well as anatomical localization and severity of NAS are summarized in Table 1. Figure 1. Schematic presentation of the anatomical zones of biliary tree used to define the localization of NAS after liver transplantation Study Endpoints Clinical variables. Clinical information was obtained from the original patient notes, operation notes and endoscopy reports. Records were reviewed for patient characteristics, indication for liver transplantation, type of biliary reconstruction and outcome. Laboratory values of alkaline phosphatase (APh), gamma glutamyltransferase (GGT), alanine-aminotransferase (ALT) and total bilirubin (bili) were studied for the following time points: at the time of presentation, at the beginning of treatment, and to study the effect of treatment, at a stable level within 3 months after the last intervention. 64

66 Chapter 4 Management of NAS. Information about interventions was obtained from the patient notes. Endoscopic retrograde cholangiopancreaticography (ERCP), percutaneous transhepatic cholangiodrainage (PTCD), surgery, and medical therapies (ursodeoxycholic acid, antibiotics) were noted. When ERCP or PTCD with interventions had been performed, the number of sessions was registered, as well as technical details of the procedure. In case of surgical treatment, the type of surgical procedure was recorded. Complications of treatment were registered. Radiological progression. To study radiological progression of NAS all cholangiograms (drain cholangiography, PTCD, MRCP, ERCP) that were performed after transplantation were reviewed by a single radiologist (EJ), blinded to clinical information, and using the same scoring system as described above. Bacterial cholangitis. Bacterial cholangitis episodes were noted. Bacterial cholangitis was defined as an episode of liver test abnormalities combined with fever for which antibiotic treatment was given. Recurrent cholangitis was defined as three or more episodes of cholangitis. Table 1. Patient Characteristics and Possible Prognostic Factors Characteristic Age at time of transplantation (median, range) Gender (M/F) Primary liver disease: PSC / Other Biliary reconstruction: Duct-to-duct / Roux-en-Y Re-transplant graft IBD before OLT IBD after OLT Early NAS (<1 year after OLT) Extent of NAS at presentation* Zone A Zone B Zone C Zone D Severity of NAS at presentation* Mild / Moderate / Severe Type of immunosuppresion Prednisone / azathioprine / cyclosporine Prednisone / tacrolimus Prednisone / tacrolimus / azathioprine Other N (% or range) 46 (18-66) 40 / (31%) / 56 (69%) 62 (77%) / 19 (23%) 10 (12%) 13 (16%) 14 (17%) 50 (62%) 66 (81%) 52 (67%) 33 (42%) 15 (19%) 43 / 28 / 7 51 (63%) 8 (10%) 6 (7%) 16 (20%) * Data on patients with radiological material available (n=78) 65

67 NAS after liver transplantation: risk factors for disease progression Pathology. To see whether NAS led to biliary fibrosis of cirrhosis, the most recent available pathology specimen of the liver of all patients was retrieved and scored by a liver pathologist (AG) blinded to the clinical context. Liver fibrosis was scored as absent, minimal, moderate or severe, with severe being either extensive bridging fibrosis or cirrhosis. Survival. Graft and patient survival were analyzed by comparing patients with NAS to controls matched for age and period of transplantation. Controls also had to be alive at the time of diagnosis of NAS in the patients with NAS. Causes of death and graft failure were noted. Prognostic factors. Possible prognostic factors for several outcome parameters are listed in Table 1. The definition of inflammatory bowel disease (IBD) after liver transplantation was an episode of abdominal pain and/or diarrhea, with inflammation seen during endoscopy, confirmed pathologically and after exclusion of infectious causes. In addition the following factors were included in the analysis: (type of) interventional treatment, the presence of radiological progression, the occurrence of bacterial cholangitis, the maintenance use of antibiotics, and the use of ursodeoxycholic acid. Statistical Methods Data were analyzed using SPSS 12.0 software. Comparison between groups was made using the Chi-square test for categorical variables and the Mann-Whitney U test for continuous variables. When indicated, a risk estimate was made calculating the relative risk (RR) and confidence intervals using a Chi-Square test. Comparison of survival between groups was made using Kaplan-Meier statistics with a log-rank test. A p-value of 0.05 or less was considered to indicate statistical significance. Ethical statement Retrospective studies are approved by the institutional ethical committee. Results NAS were present in 81 grafts of 77 patients. In four patients NAS occurred in both a first and second graft. Apart from NAS, a concomitant anastomotic stricture was diagnosed at some point in the postoperative course in 21 patients. Median follow-up after the diagnosis of NAS was 6.0 years ( ). Median follow-up after OLT was 7.9 years (range ). The biliary reconstruction was duct-to-duct in 62 cases (77%), and a hepaticojejunostomy with Roux-en-Y deviation in 19 cases (23%). 66

68 Chapter 4 Which Modalities Were Used for Treatment of NAS? Interventions. Twenty eight patients (35%) were treated with ERCP, PTCD, surgery, or a combination of these. Thirteen patients underwent one or more sessions of ERCP. Dilatation was performed in all cases; in 12 also one or more stents were placed. Complications occurred in 7, mostly cholangitis. No severe complications were observed. The median number of therapeutic ERCP s in these patients was 3 (range 1-11). Seven patients underwent PTCD. Four patients underwent both ERCP and PTCD. In patients treated with PTCD dilatation and stenting was performed in all cases. In two cases an expandable metal stent was placed. The median number of therapeutic PTCD sessions in these patients was 3 (range 1-6). A minor complication occurred in 2 cases. In the end, eight patients underwent surgery for NAS, four after previous ERCP or PTCD. The surgical procedure was conversion of duct-to-duct anastomosis to a hepatico-jejunostomy in five patients, and revision of a previous hepatico-jejunostomy in three. Patients with a dilated biliary tree were treated surgically more often than those without dilatation (20% vs. 2%, p=0.01). All concomitant anastomotic strictures were successfully treated with success by ERCP (n=13), PTCD (n=5), surgery (n=1) or a combination of these (n=2). Ursodeoxycholic acid. Seventy-one patients (88%) were treated with long-term ursodeoxycholic acid, mostly at a dose of 600 mg b.i.d. Biochemical response to interventions. When the biochemical response within 3 months after completion of interventional treatment was studied, significant improvements in serum ALT (median 65 U/l vs. 36 U/l, p=0.015), bilirubin (median 46 µmol/l vs. 23 µmol/l, p<0.000) and GGT (median 360 U/l vs. 125 U/l, p=0.014) was noted, compared to pretreatment values. No significant improvement in APh was seen. In 8 of the 28 patients no biochemical response to treatment was seen. Is NAS a Progressive Disease? Radiological progression. Material for retrospective radiological evaluation of NAS at presentation was available in 78 of the 81 transplants (96%). In 59 cases (80%) follow-up cholangiography was performed and available for determination of progression of the biliary abnormalities. The median time between the diagnostic and last cholangiography was 1.7 years (range ). Progression of the severity of biliary abnormalities was observed in 28 (42%) of the 59 grafts with follow up cholangiography. At the time of diagnosis, the 67

69 NAS after liver transplantation: risk factors for disease progression severity of NAS was scored as mild in 32 (54%), moderate in 22 (37%) and severe in 5 (9%) cases. At the end of follow up the severity of NAS was scored as mild in 17 (29%), moderate in 22 (37%) and severe in 20 (34%) cases. Progression of the anatomical extent of the biliary abnormalities was seen in 36 (61%) of the patients with follow up cholangiography. The details are listed in Table 2. Progression was seen at all levels of the biliary tree. Table 2. Radiological Progression of NAS in Patients With Follow-up Cholangiography (n=59)* Localization Extrahepatic Zone A all Intrahepatic Zone B left right both Presentation N (%) 50 (58) 5 (8.5) 7 (11.9) 27 (45.8) End of follow up N (%) 56 (97) 4 (6.8) 8 (13.6) 40 (67.8) Zone C left right both 5 (8.5) 5 (8.5) 15 (25.4) 4 (6.8) 3 (5.1) 25 (42.4) Zone D left right both 1 (1.7) 2 (3.4) 7 (11.9) * More than one area could be involved in one patient. 4 (6.8) 0 (0) 13 (22) Casts and sludge were seen at some time point after transplantation in 21 (27%) and 18 (23%) patients respectively. Cholangitis episodes. Thirty-nine subjects (48%) had at least one episode of cholangitis. Nineteen had to be admitted repeatedly for recurrent bacterial cholangitis (defined as three or more episodes). The median number of cholangitis episodes in these 19 was 5 (range 3-17). Thirty patients were put on maintenance use of antibiotics for some time, mostly ciprofloxacin. Liver pathology. Pathology specimens were available from 63 livers. The mean time from transplantation to biopsy was 3.7 years (range ). At the end of follow up, pathologically proven biliary cirrhosis or severe bridging fibrosis had developed in 17 cases (25%). In an additional six patients the diagnosis of cirrhosis was made on clinical grounds: these patients were known with severe NAS, and developed ascites, abnormal coagulation or varices with radiological evidence of cirrhosis while the portal vein was open. Thus, in the end severe fibrosis or cirrhosis developed in 23 (28%) of the livers with NAS. 68

70 Chapter 4 Are Patient and Graft Survival Affected by NAS? Graft survival. Graft survival of the patients with NAS after one, five and ten years was 91% (3.1), 73% (5.0) and 63% (6.1) respectively (standard error in parentheses). Graft survival was significantly lower in the patients with NAS, compared to matched controls without NAS (p=0.001, fig. 3). Thirteen patients (16%) underwent re-transplantation of the liver for NAS after a median of 0.9 years (mean 3.9 years, range ). At the end of this study, two patients were awaiting liver re-transplantation for NAS. Patient survival. Compared to matched controls, patient survival was lower in patients with NAS, although this did not reach statistical significance (fig. 3). At the end of the study 17 patients had died. In 5 cases, the cause of death was related to NAS. In four patients the cause of death was multi-organ failure after sepsis due to cholangitis, in one case liver failure due to biliary cirrhosis. Two patients had been offered a re-transplantation, but refused. Which Factors Are Predictive for Progression of NAS? An overview of the analyses of prognostic factors is presented in Table 3. Table 3. Prognostic Factors for Progression and Outcome of NAS Outcome parameter Prognostic factor RR (95% CI), p-value Radiological progression Early NAS (< 1 year) One or more episodes of cholangitis 1.9 ( ), ( ), Recurrent cholangitis Biliary cirrhosis/bridging fibrosis Severe outcome ** Roux-en-Y hepaticojejunostomy Abnormalities at Zone B* Abnormalities at Zone C* Abnormalities at Zone C* Radiological progression during follow up 3.6 ( ), ( ), ( ), ( ), ( ), Asymptomatic course*** Mild abnormalities* 1.9 ( ), * at presentation ** defined as: death due to NAS, cirrhosis/fibrosis, retransplantation *** defined as: no cholangitis, no fibrosis or cirrhosis, no re-transplantation, no need for treatment 69

71 NAS after liver transplantation: risk factors for disease progression Patients diagnosed with NAS (N=81) Treatment N=28 No treatment N=53 ERCP: 11 PTCD: 5 ERCP+PTCD: 4 Surgery 4 ERCP+Surgery: 2 PTCD+Surgery: 2 FOLLOW UP Outcome Rec. Cholangitis 5 (18%) * Cirrhosis 8 (29%) Re-transplantation 5 (18%) Death d/t NAS 2 (7%) Outcome Rec. Cholangitis 5 (10%) Cirrhosis 13 (25%) Re-transplantation 8 (15%) Death d/t NAS 3 (6%) Figure 2. Clinical course and outcome in patients with NAS. * A total of 14 patients experienced recurrent cholangitis (3 or more episodes). A total of 5 patients experienced recurrent cholangitis after treatment was finished. Predictors of radiological progression. When patients with progression of radiological abnormalities were compared with patients in whom the severity and extent of abnormalities was not progressive, two risk factors for progression were identified: early NAS presenting within 1 year after transplantation and one or more episodes of cholangitis. Patients presenting with early NAS were also at increased risk for both casts (RR 1.6, 95%CI , p=0.008) and sludge (RR 1.8, 95%CI , p=0.001). Predictors of bacterial cholangitis. The only risk factor for recurrent cholangitis, defined as 3 or more episodes, was a biliary reconstruction with a Roux-en-Y hepatico-jejunostomy. Predictors of progression of fibrosis. Radiological abnormalities at the intrahepatic level were identified as risk factors for development to severe bridging fibrosis or cirrhosis. These concerned Zone B and Zone C. 70

72 Chapter 4 Figure 3. Patient and graft survival in patients with NAS (n=81) and matched controls (n=81). N.s.: not significant Predictors of an asymptomatic course. In 23 cases (28%) the NAS were completely asymptomatic, defined as no episodes of cholangitis, no biliary fibrosis or cirrhosis, and no need for interventional treatment. When these patients were compared with the other 58 patients, the radiological findings at presentation were predictive of an asymptomatic course: patients with abnormalities that were scored as mild at the time of diagnosis had a significantly higher chance of an asymptomatic course (44% if mild vs. 11% not mild, p=0.002). Predictors of severe outcome. To analyze risk factors for NAS with severe outcome, we identified three markers of severe outcome: death due to NAS, re-transplantation, and biliary cirrhosis or severe bridging fibrosis. Patients experiencing one or more of these were compared with the remaining group of patients. Two risk factors for severe outcome were identified: NAS presenting at the intrahepatic Zone C, and NAS that showed radiological progression during follow up. With respect to severe outcome there was no difference between the 28 patients who received interventional treatment (ERCP, PTCD, surgery) versus the 53 patients who did not (severe outcome 46% versus 37%, p=

73 NAS after liver transplantation: risk factors for disease progression Discussion NAS or intrahepatic biliary strictures are a common and often troublesome complication after liver transplantation. Although previous studies on this subject differ markedly concerning methodology and results, a high incidence of retransplantation has been reported almost uniformly, as well as the need for frequent biliary interventions and admissions (3-5). In the present study, we found a relatively high incidence of NAS compared to previous reports. Whereas most large series report an incidence of NAS of 5-10% (6-8), we found that 17% of patients were diagnosed with NAS at some point after OLT. Most likely, this difference is due to the fact that we defined any type of biliary stricture other than anastomotic strictures as NAS. In adition, postoperative cholangiography via the biliary drain has been routine practice in our center. This allowed us to identify a large number of cases without any persisting clinical signs of biliary disease (23 cases, 28%). This is also reflected by the rather low number of retransplantations (16%) compared to previous reports from other centers. Another possible explanation is the large number of patients transplanted for PSC (17%) and relatively low number transplanted for viral disease (16%) in our center. PSC is a known risk factor for the development of NAS (9-12). From the present material it becomes clear that NAS after liver transplantation is not just one disease, but a spectrum of abnormalities, ranging from slight, localized mucosal irregularity to extensive and diffuse biliary strictures. Possibly, not all areas of non-anastomotic bile duct narrowing are due to a fibrotic type of stricture (13). We thus aimed to identify from this diverse group of NAS those cases that would progress to a clinically relevant or progressive disease. We found radiological progression in 68% of our patients with cholangiographic follow-up. Most likely, this number is lower for the entire group of patients with NAS, since those patients without radiological follow-up probably did not have marked progression. Interestingly, NAS presenting early after transplantation had a higher risk of progression. Previous investigators have also mentioned a more severe course of disease in patients with NAS presenting early after transplantation (14-16). Besides a higher risk of radiological progression, these cases also showed a significantly higher risk for the development of casts and sludge. These differences are probably due to a different pathogenesis of NAS presenting early or late after transplantation, as has been described by Buis et al. earlier in this journal. The most critical clinical consequences of NAS are recurrent cholangitis and biliary cirrhosis. 72

74 Chapter 4 Both may necessitate re-transplantation. We found recurrent cholangitis (arbitrarily defined as three or more episodes) in 19 of our patients, despite treatment with maintenance therapy with antibiotics in most patients. The only risk factor for recurrent cholangitis that was identified was the presence of a hepaticojejunostomy instead of a duct-to-duct anastomosis. Most likely, this type of biliary reconstruction leads to reflux of bacteria into the biliary tree, as has been shown in animal models (17). In a patient that is immunosuppressed and has diminished flow of bile due to NAS, it is foreseeable that this situation will lead to bacterial colonization of the bile ducts and repeated episodes of cholangitis. Biliary cirrhosis is the end-point of long-standing NAS. We found biliary cirrhosis or severe fibrosis in 23 of our cases (28%). At the end of follow up, nine of these 23 (39%) were re-transplanted. Interestingly, the two risk factors for development of biliary cirrhosis were strictures at the level of the segmental (Zone B) and sub-segmental (Zone C) branches. Apparently, these strictures cause more long-term damage to the liver than more centrally located lesions. This may be due to a number of factors. Perhaps, this type of NAS has a different pathogenesis than the more proximal type of NAS, leading to ongoing biliary damage. Another possible explanation is that these abnormalities are less amenable to treatment. Knowing that strictures at the site of the segmental and sub-segmental branches are a risk factor for biliary cirrhosis, one can make an estimate of the risk for progressive disease at the time of diagnosis. When biliary cirrhosis, retransplantation and death due to NAS were combined to define serious disease, strictures at the level of the subsegmental branches (Zone C) and radiological progression of strictures were identified as significant risk factors. Thus, one can use these characteristics to define patients with a higher risk of serious disease in the future. Although we did see NAS-related mortality in our series, overall patient survival was not significantly affected. This corresponds to previous studies on this subject (18-21). Graft survival however was impaired compared to matched controls (73% vs. 94% after five years). This is not a surprising finding. It is not possible from our results to conclude whether or not treatment for NAS prevented re-transplantation in a number of cases. Although the number of re-transplantations was similar in patients with and without endoscopic, percutaneous or surgical therapy; we do not know what these numbers would have been like without treatment. Previously, others have described successful treatment of NAS with a number of modalities (22-26). We did not study the success of treatment in these patients, since the group of patients treated with ERCP, PTCD or surgery is rather small (28 cases), heterogeneous concerning 73

75 NAS after liver transplantation: risk factors for disease progression location and severity of abnormalities, and several types of treatment modalities were used. However, in the majority of patients an improvement in liver test was seen, although this is not synonymous with uneventful long-term outcome. To date, practically all studies on the treatment of NAS are retrospective and descriptive in nature. Definitive answer on the best treatment modality for NAS should come from a multi-center, prospective, randomized study. However, practical difficulties in such a study would be the large variability in the timing of presentation and the progression of the biliary abnormalities. Our study on risk factors for the occurrence of NAS (2), combined with the current study on outcome and prognostic risk factors for disease progression, facilitates in the identification of important subgroups and clinical variables that can be used for stratification in a prospective study (see also figure 4). Roux- en-y anastomosis Preservation injury (Ischaemia) Immunology (PSC, Gender mismatch) Early NAS Intrahepatic NAS Late NAS Bacterial Cholangitis Radiological Progression of NAS Severe Outcome Fibrosis/cirrhosis Figure 4. Schematic representation of risk factors and prognostic factors for the development of early and late NAS, bacterial cholangitis, progressive radiological abnormalities and severe outcome. Each connection represents a statistical correlation (present study and work by Buis et al (2)). NAS: non-anastomotic strictures, PSC: primary sclerosing cholangitis. 74

76 Chapter 4 In conclusion, non-anastomotic biliary strictures are a common complication after orthotopic liver transplantation. The radiological and clinical picture of NAS shows a spectrum ranging from minor abnormalities without any symptoms to severe strictures eventually leading to re-transplantation. Graft survival is significantly reduced in patients suffering from NAS. Especially patients with a hepatico-jejunostomy, those with an early diagnosis of NAS, and those with NAS presenting at the level of the peripheral branches of the biliary tree, are at risk for the development of recurrent cholangitis, radiological progression, development of cirrhosis and eventually retransplantation. 75

77 NAS after liver transplantation: risk factors for disease progression References 1. Guichelaar MM, Benson JT, Malinchoc M, Krom RA, Wiesner RH, Charlton MR. Risk factors for and clinical course of non-anastomotic biliary strictures after liver transplantation. Am J Transplant 2003 Jul;3: Buis CI, Verdonk RC, van der Jagt EJ, van der Hilst CS, Slooff MJH, Haagsma EB, Porte RJ. Non-anastomotic biliary strictures after adult liver liver transplantation part one: Radiological features and risk factors for early versus late presentation. Liver Transpl 2007; 13: Guichelaar MM, Benson JT, Malinchoc M, Krom RA, Wiesner RH, Charlton MR. Risk factors for and clinical course of non-anastomotic biliary strictures after liver transplantation. Am J Transplant 2003 Jul;3: Hintze RE, Adler A, Veltzke W, Abou-Rebyeh H, Felix R, Neuhaus P. Endoscopic management of biliary complications after orthotopic liver transplantation. Hepatogastroenterology 1997 Jan;44: Rull R, Garcia Valdecasas JC, Grande L, Fuster J, Lacy AM, Gonzalez FX, et al. Intrahepatic biliary lesions after orthotopic liver transplantation. Transpl Int 2001 Jun;14: Campbell WL, Sheng R, Zajko AB, Abu-Elmagd K, Demetris AJ. Intrahepatic biliary strictures after liver transplantation. Radiology 1994 Jun;191: Guichelaar MM, Benson JT, Malinchoc M, Krom RA, Wiesner RH, Charlton MR. Risk factors for and clinical course of non-anastomotic biliary strictures after liver transplantation. Am J Transplant 2003 Jul;3: Rull R, Garcia Valdecasas JC, Grande L, Fuster J, Lacy AM, Gonzalez FX, et al. Intrahepatic biliary lesions after orthotopic liver transplantation. Transpl Int 2001 Jun;14: Campbell WL, Sheng R, Zajko AB, Abu-Elmagd K, Demetris AJ. Intrahepatic biliary strictures after liver transplantation. Radiology 1994 Jun;191: Guichelaar MM, Benson JT, Malinchoc M, Krom RA, Wiesner RH, Charlton MR. Risk factors for and clinical course of non-anastomotic biliary strictures after liver transplantation. Am J Transplant 2003 Jul;3: Sankary HN, McChesney L, Hart M, Foster P, Williams J. Identification of donor and recipient risk factors associated with nonanastomotic biliary strictures in human hepatic allografts. Transplant Proc 1993 Apr;25: Sawyer RG, Punch JD. Incidence and management of biliary complications after 291 liver transplants following the introduction of transcystic stenting. Transplantation 1998 Nov 15;66: Campbell WL, Sheng R, Zajko AB, Abu-Elmagd K, Demetris AJ. Intrahepatic biliary strictures after liver transplantation. Radiology 1994 Jun;191: Kuo PC, Lewis WD, Stokes K, Pleskow D, Simpson MA, Jenkins RL. A comparison of operation, endoscopic retrograde cholangiopancreatography, and percutaneous transhepatic cholangiography in biliary complications after hepatic transplantation. J Am Coll Surg 1994 Aug;179:

78 Chapter Sanchez-Urdazpal L, Gores GJ, Ward EM, Maus TP, Buckel EG, Steers JL, et al. Diagnostic features and clinical outcome of ischemic-type biliary complications after liver transplantation. Hepatology 1993 Apr;17: Sanchez-Urdazpal L, Gores GJ, Ward EM, Hay E, Buckel EG, Wiesner RH, et al. Clinical outcome of ischemic- type biliary complications after liver transplantation. Transplant Proc 1993 Feb;25(1 Pt 2): Chuang JH, Chen WJ, Lee SY, Chang NK. Prompt colonization of the hepaticojejunostomy and translocation of bacteria to liver after bile duct reconstruction. J Pediatr Surg 1998 Aug;33: Guichelaar MM, Benson JT, Malinchoc M, Krom RA, Wiesner RH, Charlton MR. Risk factors for and clinical course of non-anastomotic biliary strictures after liver transplantation. Am J Transplant 2003 Jul;3: Moench C, Uhrig A, Lohse AW, Otto G. CC chemokine receptor 5delta32 polymorphism-a risk factor for ischemic-type biliary lesions following orthotopic liver transplantation. Liver Transpl 2004 Mar;10: Otto G, Roeren T, Golling M, Datsis K, Hofmann WJ, Herfarth C, et al. [Ischemic type lesions of the bile ducts after liver transplantation: 2 years results]. Zentralbl Chir 1995;120: Sanchez-Urdazpal L, Gores GJ, Ward EM, Hay E, Buckel EG, Wiesner RH, et al. Clinical outcome of ischemic- type biliary complications after liver transplantation. Transplant Proc 1993 Feb;25(1 Pt 2): Hintze RE, Adler A, Veltzke W, Abou-Rebyeh H, Felix R, Neuhaus P. Endoscopic management of biliary complications after orthotopic liver transplantation. Hepatogastroenterology 1997 Jan;44: Schlitt HJ, Meier PN, Nashan B, Oldhafer KJ, Boeker K, Flemming P, et al. Reconstructive surgery for ischemic- type lesions at the bile duct bifurcation after liver transplantation. Ann Surg 1999 Jan;229: Sung RS, Campbell DA, Jr., Rudich SM, Punch JD, Shieck VL, Armstrong JM, et al. Long-term follow-up of percutaneous transhepatic balloon cholangioplasty in the management of biliary strictures after liver transplantation. Transplantation 2004 Jan 15;77: Theilmann L, Kuppers B, Kadmon M, Roeren T, Notheisen H, Stiehl A, et al. Biliary tract strictures after orthotopic liver transplantation: diagnosis and management. Endoscopy 1994 Aug;26: Zajko AB, Sheng R, Zetti GM, Madariaga JR, Bron KM. Transhepatic balloon dilation of biliary strictures in liver transplant patients: a 10-year experience. J Vasc Interv Radiol 1995 Jan;6:

79

80 Part II Bile physiology after liver transplantation

81

82 5 The role of bile salt toxicity in the pathogenesis of bile duct injury after non heart-beating porcine liver transplantation Transplantation 2008; 85: Marit J Yska Carlijn I Buis Diethard Monbaliu Theo A Schuurs Annette SH Gouw Olivier NH Kahman Dorien S Visser Jacques Pirenne Robert J Porte

83 Bile salt toxicity and bile duct injury after NHB porcine liver transplantation Abstract Background. Intrahepatic bile duct strictures are a serious complication after non-heartbeating (NHB) liver transplantation. Bile salt toxicity has been identified as an important factor in the pathogenesis of bile duct injury and cholangiopathies. The role of bile salt toxicity in the development of biliary strictures after NHB liver transplantation is unclear. Methods. In a porcine model of NHB liver transplantation, we studied the effect of different periods of warm ischemia in the donor on bile composition and subsequent bile duct injury after transplantation. After induction of cardiac arrest in the donor, liver procurement was delayed for 0 min (group A), 15 min (group B) or > 30 min (group C). Livers were subsequently transplanted after four hours of cold preservation. In the recipients, bile flow was measured and bile samples were collected daily to determine the bile salt / phospholipid ratio. Severity of bile duct injury was semi-quantified by using a histological grading scale. Results. Posttransplant survival was directly related to the duration of warm ischemia in the donor. The bile salt / phospholipid ratio in bile produced early after transplantation was significantly higher in group C, compared to group A and B. Histopathology showed the highest degree of bile duct injury in group C. Conclusion. Prolonged warm ischemia in NHB donors is associated with the formation of toxic bile after transplantation, with a high biliary bile salt / phospholipid ratio. These data suggest that bile salt toxicity contributes to the pathogenesis of bile duct injury after NHB liver transplantation. 82

84 Chapter 5 Introduction The success of orthotopic liver transplantation as a therapy for patients with end-stage liver disease has resulted in an increasing demand for donor livers. In many parts of the world this created a growing shortage of organs from brain death or deceased donors. A possible solution to reduce shortage of donor organs is expansion of the donor pool by accepting donation after cardiac death (DCD) or non-heart-beating (NHB) donors. Patient survival after transplantation of livers from NHB donors has shown to be comparable to survival after transplantation of livers from brain death donors (1-4). Graft survival after NHB liver transplantation, however, is about 10-15% lower due to a higher rate of primary non-function and other graft-related complications. Intrahepatic bile duct strictures, also known as non-anastomotic strictures or ischemic-type biliary lesions, are a serious cause of morbidity and a leading cause of graft failure after NHB liver transplantation (5-7). Although the exact pathogenesis is unknown, it is generally believed that warm ischemia in the NHB donor due to hypotension before cardiac arrest, as well as during the time period between cardiac and organ procurement, is a critical factor in the pathogenesis of these biliary strictures (5-7). Although hepatocytes may recover from the warm ischemic insult in donor, bile duct epithelial cells have a poor tolerance towards ischemia and regeneration of cellular ATP is much slower than in hepatocytes (8-12). Apart from this direct detrimental effect of ischemia on the bile duct epithelium (13), there is accumulating evidence that bile salt toxicity contributes to bile duct injury after liver transplantation (14-16). Although secretion of bile salts by hepatocytes is the main driving force of bile flow, bile salts can act as detergents injuring cellular phospholipid membranes. Under physiological circumstances, bile salts are therefore neutralized in bile by phospholipids after formation of mixed micelles (17). Experimental studies in mice as well as clinical studies in humans have indicated that bile formation early after liver transplantation may be disturbed, resulting in the formation of more toxic bile with a relatively high bile salt / phospholipid ratio. A high bile salt / phospholipid ratio has been associated with more severe bile duct injury after transplantation (14-16). The role of bile salts in the pathogenesis of bile duct injury after NHB liver transplantation has not been studied before. We hypothesized that bile salt toxicity acts in concert with warm ischemia injury in the pathogenesis of intrahepatic bile duct injury after NHB liver transplantation. To study the role of bile salts in the development of bile duct injury after NHB liver transplantation, we 83

85 Bile salt toxicity and bile duct injury after NHB porcine liver transplantation have used a well established model of NHB liver transplantation in pigs (18,19). The specific aim was to study whether increasing length of warm ischemia in NHB donors is associated with more toxic bile formation after transplantation, as indicated by the bile salt / phospholipid ratio, and subsequently more severe injury of the intrahepatic bile ducts. Materials and Methods Animals and NHB Liver Transplant Model Inbred female Landrace pigs, weighing 18 to 37 kg, were used as donors and recipients. In donors, cardiac arrest was induced by ventricular fibrillation, followed by standardized periods of warm ischemia before cold preservation and procurement of the liver, to mimic NHB donation. The pigs were divided in 5 groups (n=6 each) with different periods of donor warm ischemia time (WIT): 0 min (controls; group A), 15 min (group B), and 30, 45 or 60 minutes. Because of a low rate of survivors in the latter three subgroups (2/6, 2/6, and 0/6 at postoperative day 4, respectively) we regarded these as one group for analysis (group C, > 30 min WIT). In general, there were no major differences in outcome parameters between these three groups. After the period of warm ischemia, the liver was flushed with ice cold histidine tryptophan ketoglutarate (HTK) preservation solution. During cold perfusion, a cholecystectomy was performed and the common bile duct was transected and flushed out with cold saline solution. Subsequently, livers were stored at 4 C for four hours until transplantation. In the recipients, a midline laparotomy was performed, the native liver was removed, and the donor liver was implanted in an orthotopic position. No veno-venous bypass was used. After completing the anastomosis between the suprahepatic inferior vena cava of the recipient and donor, the portal vein was reconstructed and the liver was reperfused. Subsequently, the infrahepatic vena cava was reconstructed. Arterial recirculation was established by an end-toside anastomosis between the donor aorta (left in continuity with the hepatic artery) and the recipient aorta. There were no significant differences in the duration of the anhepatic time or the time interval between portal reperfusion and restoration of arterial blood low among the three groups. Mean (range) duration of the anhepatic phase in group A, B and C was 23 min (20-26 min), 25 min (20-30 min), and 24 min (18-30 min), respectively. Time interval between portal and arterial reperfusion was 38 min (30-45 min), 42 min (28-68 min), and 37 min (20-84

86 Chapter 5 68 min), respectively. Flow probes (Transonic Systems, Ithaca, NY, USA) were implanted around the hepatic artery and portal vein and connected to a dual channel ultrasonic transittime volume flow meter (T206, Transonic). Blood flow was measured continuously during the first 3 hours after reperfusion and twice daily thereafter. In addition, patency of the vascular anastomoses was macroscopically inspected during necropsy. All hepatic artery anastomoses were found to be patent. Perioperatively, arterial blood pressure was monitored via an arterial line in the left common carotid artery. Central venous pressure was monitored via a catheter in the left external jugular vein. Infusion of intravenous fluids was individually guided by clinical signs of hypovolemia, hemodynamic parameters, and laboratory blood analysis. Although moderate hypotension up to a period of 30 min was well tolerated during the anhepatic phase, 500 ml of oxyplatin was administered IV during the anhepatic phase to avoid severe hypotension (20). In general, there were no major differences in hemodynamics among the groups. During transplantation a catheter was inserted in the common bile duct and externalized via the abdominal wall. Daily bile production was completely diverted into a collecting bag. To maintain the enterohepatic circulation of bile salts, bile was readministered via a jejunostomy catheter. Antibiotic prophylaxis was provided by IV Ceftazidime, 500 mg. Postoperatively, animals received tacrolimus (0.05 mg/kg bid) as immunosuppressant. All animals had free access to water and food. All surviving animals were able to feed themselves normally as of postoperative day 2 and there were no apparent differences between the groups. The postoperative observation period was limited to four days to minimize confounding effects caused by sepsis and other late-onset phenomena. After four days the pigs were killed and animals surviving less than four days were autopsied to identify the cause of death (19). Experiments were performed in accordance with the Belgian law regarding animal welfare. Biochemical serum analyses Serum levels of aspartate aminotransferase (AST) and bilirubin were determined using routine chemical methods. Collection of Bile and Determination of Bile Composition Bile production was measured 3 hours after reperfusion and daily thereafter to calculate bile flow (bile production / kg body weight of the donor). Bile samples were collected daily to 85

87 Bile salt toxicity and bile duct injury after NHB porcine liver transplantation examine bile composition and determination of the biliary bile salt / phospholipid ratio. Total biliary bile salt concentration was measured spectrophotometrically using 3α-hydroxysteroid dehydrogenase (21). Biliary phospholipid concentration was analysed using a commercially available enzymatic method (Wako Chemicals GmbH, Neuss, Germany). Hepatic Gene Expression of Bile Transporters In parallel with the measurement of bile composition, we measured hepatic mrna expression of the bile salt transporter (bile salt export pump; BSEP or Abcb11) and the phospholipid translocator (multidrug resistance protein; MDR3 or Abcb4). The gene sequence of porcine MDR3 was not known and, therefore, determined for this experiment (NCBI Accession #: EF067318). Wedge biopsies were taken one hour after reperfusion and on postoperative day (POD) four in surviving animals. Biopsies were snap frozen and stored at -80 C until analysis. RNA isolation from liver biopsies was performed using TRIzol (Invitrogen Life Technologies, Breda, The Netherlands), Chloroform (Merck, Darmstadt, Germany) and the DNAse-kit from Sigma (Sigma-Aldrich, Zwijndrecht, The Netherlands). RNA integrity was quantified by electrophoresis using agarose-gel (Sphaero Q, Leiden, The Netherlands) and ethidiumbromide (Sigma- Aldrich). The enzyme M-MLV reverse transcriptase (Sigma-Aldrich) was used to convert RNA (1µg in a final volume of 21 µl) in copy-dna (cdna). Taq polymerase (Invitrogen Life Technologies, Breda, The Netherlands) and conventional PCR were used to multiply the cdna and make it detectable with DNA electrophoresis by an UV-transilluminator. For quantitative real-time detection, sense, anti-sense porcine primers (Invitrogen, Paisley, Scotland) and fluorogenic probes (Eurogentec, Herstal, Belgium) were designed for the hepatobiliary transporters BSEP and MDR3, using Primer Express software (PE Aplied Biosystems, Foster City, CA, USA). All probes were 5 labeled by a 6-carboxy-fluorescein (FAM) reporter and 3 labeled with a 6-carboxy-tetramethyl-rhodamine (TAMRA) quencher (table 1). In each PCR reaction duplicate samples of 5 µl cdna (25x) (2 ng RNA / µl) were used in a final volume of 20 µl (qpcr Core Kit Eurogentec, Seraing, Belgium). Every PCR sample was duplicated in triplo, in a real-time RT PCR 384 wells plate (Applied Biosystems). mrna copy numbers of transporter genes were normalized to those of porcine β-actine mrna. The ABI PRISM 7700 sequence detector (Applied Biosystems) was used for quantitative real-time RT PCR according to the manufacturer s instructions. 86

88 Chapter 5 Histopathological Grading of Bile Duct Injury Bile duct injury in biopsies taken during and after transplantation was semiquantified by calculating a modified bile duct injury severity score (BDISS) as described previously (21), and based on the following two components: bile duct epithelial damage (graded as 0 = absent, 1 = mild, 2 = moderate, 3 = severe; modified from the Banff criteria for acute rejection (22)) and ductular reaction (graded as 0 = absent, 1 = mild, 2 = moderate, 3 = severe). This resulted in a minimal BDISS of 0 and a maximum score of 6 points. All histological assessments were performed by a single pathologist (ASG) who was unaware of the study group of the animals and of the other study data. Statistics Values are expressed as mean ± standard error of the mean (SEM). Data were analyzed using SPSS software version 14.0 for Windows (SPSS Inc., Chicago, Il, USA). Differences within and between groups were compared using a paired and non-paired Student-T test, respectively. Total course of biochemical variables during the first week was compared by calculating the area under the curve (AUC, using the trapezium rule). All p-values were twotailed and considered statistically significant at a level of less than Results Survival Analysis Postoperative survival of animals was directly related to the duration of warm ischemia in the donor (figure 1). In the group A (0 min donor WIT), four days survival rate was 100%, compared to 90% in the group B (15 min donor WIT) and 20% in group C (> 30 min donor WIT). In group B, one recipient was found death on POD 1, despite a good initial recovery from the transplant procedure. On necropsy, death was contributed to hypoxia resulting from severe pulmonary edema. In group C, one animal was awake and recovering from the procedure, but could not be weaned from the ventilator, due to lack of spontaneous respiratory activity, possibly as a result of brain stem injury. In accordance with the international guidelines on animal welfare, this animal was sacrificed 12 hours after surgery. One animal recovered from the procedure, but was found death on POD 1. A subsequent necropsy did not reveal a clear cause of death (normal aspect of all thoraco-abdominal organs, no ascites, and no 87

89 Bile salt toxicity and bile duct injury after NHB porcine liver transplantation other indications of liver failure). The remaining animals that died were diagnosed with early postoperative liver failure or primary graft non-function. Usually, these animals displayed an incorrectable metabolic acidosis with increasing levels of lactate and severe coagulopathy after reperfusion, and could not be weaned from the ventilator. On necropsy, typically, large amounts of hemorrhagic ascites were found as a result of the severe coagulopathy and portal hypertension due to congestion in failing liver. In parallel with the clinical course, serum levels of AST at 3 hrs after reperfusion were significantly higher in group C, compared to group B and A ( , and U/L, respectively; p<0.05). However, there were no significant differences in serum AST levels among the three groups in the surviving animals at POD 4 (237+57, and U/L, respectively). 100 animal survival (%) WI = 0min WI = 15min WI > 30min postoperative days Figure 1. Survival after porcine NHB liver transplantation in relation to various time periods of warm ischemia in the donor. 88

90 Chapter 5 Early Recovery of Bile Flow and Bile Composition Animals which died immediately postoperative from early graft failure (mainly in group C) displayed very minimal or no bile production and, therefore, were not included in the bile analyses. Bile flow at 3 hr and 24 hr after graft reperfusion was significantly lower in the surviving animals in group C (> 30 min WIT) compared to the control group A, while there was no significant difference in bile flow recovery between the group B (15 min WIT) and the control group A (figure 2). The composition of bile produced by livers with a WIT > 30 min (group C) was also more cytotoxic, as expressed by a significantly higher bile salt / phospholipid ratio early after transplantation (figure 3). Serum bilirubin levels increased during the postoperative course in all groups and there were no significant differences between the groups at POD 4. 8 WI = 0 min bileflow (ml/kg/day) 6 4 P = 0.019* P = 0.010* WI = 15 min WI > 30 min time after transplantation (hours) Figure 2. Bile flow after NHB liver transplantation in relation to various time periods of warm ischemia in the donor. 89

91 Bile salt toxicity and bile duct injury after NHB porcine liver transplantation biliary BS / PL ratio WI = 0min WI = 15min WI > 30min P=0,012 P=0, postoperative days Figure 3. Mean biliary bile salts / phospholipid (BS / PL) ratio during four days after NHB liver transplantation. AUC, area under the curve. Histological Evaluation of Bile Duct Injury In the surviving animals, histological analysis of postoperative liver biopsies showed a higher degree of bile duct injury in livers with prolonged warm ischemia in the donor. There were no differences in the mean BDISS in the group of livers with a WIT of 0 min (group A), compared to the group with WIT of 15 min (group B). However, the BDISS was significantly higher in the group with a WIT > 30 min (group C) compared to the groups A and B together ( versus ; p= 0.013). Representative examples of histology of liver biopsies with a low, intermediate or high BDISS are presented in figure 4. 90

92 Chapter 5 A B C Figure 4. Representative examples of histology of liver biopsies (Masson Trichrome staining). A) Low BDISS: a portal tract showing a bile duct with mild epithelial damage, loss of nuclei and infiltration by a neutrophilic granulocyte. B) Intermediate BDISS: a portal tract containing inflammatory cells. A hepatic artery is shown on the left and a bile duct on the right side. The damaged bile duct shows epithelial desquamation (lumen), infiltration by inflammatory cells and disruption of the basement membrane (arrows). There is nuclear atypia, stratification and loss of biliary epithelial cells. C) High BDISS: a portal tract showing severely damaged and malformed bile ducts (arrows). There is loss of and diffuse damage to epithelial cells, disrupted basement membrane and heavy infiltration by inflammatory cells. 91

93 Bile salt toxicity and bile duct injury after NHB porcine liver transplantation Gene Expression of Bile Transporters Hepatic expression of BSEP and MDR3 mrna decreased after transplantation in all three groups (figure 5). However, there were no statistically significant differences between the three groups. WI = 0 min 2.00 WI = 15 min WI > 30 min 2.00 BSEP mrna levels MDR3 mrna levels time after transplantation (hours) A time after transplantation (hours) B Figure 5. Relative BSEP (A) and MDR3 (B) mrna levels in porcine liver grafts after 0 min, 15 min or > 30 min WIT. Biopsies were taken at 0 min, 60 min and 4 days after transplantation. Genes of interest were standardized for β-actin mrna. Discussion The aim of this study was to investigate whether prolonged warm ischemia in NHB donors is associated with the production of more toxic bile early after transplantation, which may subsequently contribute to the development of intrahepatic biliary strictures after NHB liver transplantation. In a porcine model of NHB liver transplantation we have shown that livers obtained from donors who suffered > 30 min of warm ischemia produced bile with a significantly 92

94 Chapter 5 higher bile salt / phospholipid ratio after transplantation than livers from donors with 0 or 15 min warm ischemia in the donor. In addition, bile duct injury was more severe and the survival rate was lower the group with > 30 min of warm ischemia in the donor. These findings indicate that prolonged warm ischemia in the NHB donor is associated with the posttransplant production of cytotoxic bile, characterized by a high biliary bile salt / phospholipid ratio, and suggest that these changes in bile composition contribute to the pathogenesis of bile duct injury after NHB liver transplantation. The current findings are in accordance with previous experimental and clinical studies, which indicated that bile salts contribute to the development of bile duct injury after liver transplantation (15,23,24). Although the secretion of bile salts by hepatocytes into the bile canaliculus is the main driving force behind the generation bile flow, bile salts are also potentially cytotoxic due to their detergent activity (17). Under normal conditions, bile salts form mixed micelles with phospholipids and cholesterol, which prevents bile salt toxicity. In case of excess of bile salts, either due to increased bile salt secretion or reduced secretion of phospholipids, free non-micellar bile salts may cause cholangiocyte injury, pericholangitis and periductal fibrosis (17,25). In human liver transplantation, it has been shown that bile salt secretion recovers more rapidly after liver transplantation than phospholipid secretion, resulting in a cytotoxic bile composition (14). A high bile salt / phospholipid ratio early after transplantation is correlated with the histological degree of bile duct injury. In an experimental mouse model of liver transplantation it was recently shown that livers from Mdr2 +/- mice, which secrete only 50% of the normal amount of phospholipids into the bile, develop severe bile duct injury after transplantation, as reflected by enlarged portal tracts with cellular damage, ductular proliferation, bile stasis and a dense inflammatory infiltrate (16). In contrast, no such abnormalities were seen in transplanted wild-type mouse livers. In addition to these studies, which focussed on the detrimental effects of endogenous bile salts, others have shown similar deleterious effects of exogenous administered bile salts. Experimental studies in pigs have shown that infusion of hydrophobic bile salts before liver procurement results in significantly increased intrahepatic biliary injury after transplantation, compared to control livers flushed with saline (15). The observed high biliary bile salt / phospholipid ratio early after NHB liver transplantation in the current study, suggest that bile salt toxicity is a contributing factor in the development of bile duct injury after NHB liver transplantation. The bile salt / phospholipid ratio correlated well with the length of warm ischemia due to cardiac arrest in the donor. It 93

95 Bile salt toxicity and bile duct injury after NHB porcine liver transplantation is likely that the ischemic insult to the biliary epithelium remains a key determinant in the pathogenesis bile duct injury after NHB liver transplantation, however, bile salt toxicity could aggravate the degree of injury. Hepatobiliary secretion of bile salts and phospholipids is an active process which is determined by the hepatic transporters BSEP and MDR3, respectively. Various molecular changes of hepatocellular-transport systems have been described in patients with cholangiopathy or cholestatic disorders (17), illustrating the importance of these transporter functions. Decreased activity of the MDR3 (26-28) or BSEP (29) transporters, due to a gene mutation for example, is associated with decreased bile formation and cholestasis. In our study we observed a reduction in the expression of BSEP and MDR3 mrna after transplantation. However, no significant differences were noted between the three groups. These data are in accordance with a recent study in human livers from heart-beating (deceased) donors where also no differences were found in BSEP and MDR3 mrna expression at three hours after graft reperfusion (13). In this human study, however, a small, but significant, increase in BSEP expression was found at one week after transplantation (14). Follow-up in our study was limited to only four days and further studies will be needed to determine whether similar changes also occur in this porcine model of NHB donor liver transplantation. In general, current findings suggest that the observed differences in bile composition are caused by posttranscriptional processes or changes in transporter activity rather than a direct effect on gene transcription. It is increasingly recognized that changes in protein levels of BSEP and NTCP are largely determined by the subapical storage or mobilization of these transporter proteins and to a lesser degree by changes in gene expression (30). Unfortunately, we were unable to perform immunohistochemistry or western blot analyses, due to the lack of adequate antibodies against porcine BSEP and MDR3. The accumulating evidence supporting the concept of bile salt toxicity as an important determinant in the pathogenesis of bile duct injury after liver transplantation opens new avenues for preventive and therapeutic measures. One obvious option would be the exogenous administration of hydrophilic bile salts, such as ursodeoxycholic acid, which lack the detergent properties of hydrophobic bile salts. Daily oral administration of ursodeoxycholic acid is a well known therapy to reduce bile salt toxicity by replacement of the hydrophobic bile salts in the bile salt pool (31,32). In addition, hydrophilic bile salts have been shown to possess more direct cytoprotective properties which are independent from the reduction in hydrophobic bile salts, 94

96 Chapter 5 and involve inhibition of apoptotic pathways (15,31). Another interesting therapeutic target could be MDR3, given the key role of biliary phospholipids in protecting bile duct epithelium from potentially toxic, aggressive biliary content (30). Therapeutic strategies aimed at reducing bile toxicity through stimulation of MDR3 expression and function may be an important future therapeutic approach to prevent bile duct injury after liver transplantation. Administration of fibrates, statins or peroxisome proliferators, have been shown to stimulate biliary phospholipid secretion by the induction of MDR3 (or its rodent homolog mdr2), making bile less toxic (33-35). However, more research in this area, including assessment of potential side effects of these compounds will be needed before clinical application of these compounds to prevent bile duct strictures can be advised. Currently, there is not an established animal model of bile duct injury after NHB-donor liver transplantation. Development of such a model, however, is of great relevance to facilitate studies on the pathogenesis and development of biliary strictures in liver grafts from NHB donors. In the current study we have focussed on injury of the small (microscopic) bile ducts in the liver parenchyma. In clinical practice, bile duct lesions in livers from NHB donors are typically found in the larger (macroscopic) bile ducts (9). More research using the current porcine model with more longterm follow-up will be needed to determine whether bile salt toxicity is also involved in the development of bilary strictures in the larger bile ducts. The ideal model of NHB liver donation is one in which the time period of cardiac arrest results in a timely recoverable hepatocellular injury (an thus animal and graft survival), but yet the developement of enough biliary damage to develop biliary strictures more longterm after transplantation. In this respect, 30 minutes of donor warm ischemia appeared to be a useful model for further research. In summary, we investigated the role of toxic bile composition in the pathogenesis of bile duct injury after NHB liver transplantation, using a well established porcine model. Our data indicate that the length of warm ischemia due to cardiac arrest in the NHB donor correlates with the formation of toxic bile, characterized by a high biliary bile salt / phospholipid ratio, after transplantation. These findings suggest that bile salt toxicity contributes to the pathogenesis of bile duct injury after NHB liver transplantation. 95

97 Bile salt toxicity and bile duct injury after NHB porcine liver transplantation Reference List 1. Recommendations for nonheartbeating organ donation. A position paper by the Ethics Committee, American College of Critical Care Medicine, Society of Critical Care Medicine. Crit Care Med 2001; 29: Neuberger J. Developments in liver transplantation. Gut 2004; 53: Reddy S, Zilvetti M, Brockmann J, McLaren A, Friend P. Liver transplantation from non-heart-beating donors: current status and future prospects. Liver Transpl 2004; 10: Zawistowski CA, Devita MA. Non-heartbeating organ donation: a review. J Intensive Care Med 2003; 18: Abt P, Crawford M, Desai N, Markmann J, Olthoff K, Shaked A. Liver transplantation from controlled non-heart- beating donors: an increased incidence of biliary complications. Transplantation 2003; 75: Abt PL, Desai NM, Crawford MD et al. Survival following liver transplantation from non-heart-beating donors. Ann Surg 2004; 239: Garcia-Valdecasas JC, Tabet J, Valero R et al. Evaluation of ischemic injury during liver procurement from non- heart-beating donors. Eur Surg Res 1999; 31: Lewis WD, Jenkins RL. Biliary strictures after liver transplantation. Surg Clin North Am 1994; 74: Verdonk RC, Buis CI, Porte RJ, Haagsma EB. Biliary complications after liver transplantation: a review. Scand J Gastroenterol 2006; Suppl: Strazzabosco M, Fabris L, Spirli C. Pathophysiology of cholangiopathies. J Clin Gastroenterol 2005; 39: S90-S Cameron AM, Busuttil RW. Ischemic cholangiopathy after liver transplantation. Hepatobiliary Pancreat Dis Int 2005; 4: Noack K, Bronk SF, Kato A, Gores GJ. The greater vulnerability of bile duct cells to reoxygenation injury than to anoxia. Implications for the pathogenesis of biliary strictures after liver transplantation. Transplantation 1993; 56: Lazaridis KN, Strazzabosco M, Larusso NF. The cholangiopathies: disorders of biliary epithelia. Gastroenterology 2004; 127: Geuken E, Visser D, Kuipers F et al. Rapid increase of bile salt secretion is associated with bile duct injury after human liver transplantation. J Hepatol 2004; 41: Hertl M, Harvey PR, Swanson PE et al. Evidence of preservation injury to bile ducts by bile salts in the pig and its prevention by infusions of hydrophilic bile salts. Hepatology 1995; 21: Hoekstra H, Porte RJ, Tian Y et al. Bile salt toxicity aggravates cold ischemic injury of bile ducts after liver 96

98 Chapter 5 transplantation in Mdr2+/- mice. Hepatology 2006; 43: Trauner M, Meier PJ, Boyer JL. Molecular pathogenesis of cholestasis. N Engl J Med 1998; 339: Monbaliu D, Crabbe T, Roskams T, Fevery J, Verwaest C, Pirenne J. Livers from non-heart-beating donors tolerate short periods of warm ischemia. Transplantation 2005; 79: Monbaliu D, van PJ, De VR et al. Primary graft nonfunction and Kupffer cell activation after liver transplantation from non-heart-beating donors in pigs. Liver Transpl 2007; 13: Oike F, Uryuhara K, Otsuka M et al. Simplified technique of orthotopic liver transplantation in pigs. Transplantation 2001; 71: Turley SD, Dietschy JM. Re-evaluation of the 3 alpha-hydroxysteroid dehydrogenase assay for total bile acids in bile. J Lipid Res 1978; 19: Banff schema for grading liver allograft rejection: an international consensus document. Hepatology 1997; 25: Schmucker DL, Ohta M, Kanai S, Sato Y, Kitani K. Hepatic injury induced by bile salts: correlation between biochemical and morphological events. Hepatology 1990; 12: Buis CI, Hoekstra H, Verdonk RC, Porte RJ. Causes and consequences of ischemic-type biliary lesions after liver transplantation. J Hepatobiliary Pancreat Surg 2006; 13: Arrese M, Trauner M. Molecular aspects of bile formation and cholestasis. Trends Mol Med 2003; 9: de Vree JM, Jacquemin E, Sturm E et al. Mutations in the MDR3 gene cause progressive familial intrahepatic cholestasis. Proc Natl Acad Sci U S A 1998; 95: Deleuze JF, Jacquemin E, Dubuisson C et al. Defect of multidrug-resistance 3 gene expression in a subtype of progressive familial intrahepatic cholestasis. Hepatology 1996; 23: Jacquemin E, de Vree JM, Cresteil D et al. The wide spectrum of multidrug resistance 3 deficiency: from neonatal cholestasis to cirrhosis of adulthood. Gastroenterology 2001; 120: Suchy FJ, Ananthanarayanan M. Bile salt excretory pump: biology and pathobiology. J Pediatr Gastroenterol Nutr 2006; 43 Suppl 1: S10-S Trauner M, Boyer JL. Bile salt transporters: molecular characterization, function, and regulation. Physiol Rev 2003; 83: Fickert P, Zollner G, Fuchsbichler A et al. Effects of ursodeoxycholic and cholic acid feeding on hepatocellular transporter expression in mouse liver. Gastroenterology 2001; 121: Trauner M, Fickert P, Wagner M. MDR3 (ABCB4) defects: a paradigm for the genetics of adult cholestatic syndromes. Semin Liver Dis 2007; 27: Miranda S, Vollrath V, Wielandt AM, Loyola G, Bronfman M, Chianale J. Overexpression of mdr2 gene by peroxisome proliferators in the mouse liver. J Hepatol 1997; 26:

99 Bile salt toxicity and bile duct injury after NHB porcine liver transplantation 34. Hooiveld GJ, Vos TA, Scheffer GL et al. 3-Hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors (statins) induce hepatic expression of the phospholipid translocase mdr2 in rats. Gastroenterology 1999; 117: Chianale J, Vollrath V, Wielandt AM et al. Fibrates induce mdr2 gene expression and biliary phospholipid secretion in the mouse. Biochem J 1996; 314:

100 6 Altered bile composition after liver transplantation is associated with the development of Nonanastomotic biliary strictures J of Hepatol. In Press Carlijn I Buis Erwin Geuken Dorien S Visser Folkert Kuipers Elizabeth B Haagsma Henkjan J Verkade Robert J Porte

101 Bile composition after liver transplantation and NAS Abstract Nonanastomotic biliary strictures are troublesome complications after liver transplantation. The pathogenesis of NAS is not completely clear, but experimental studies suggest that bile salt toxicity is involved. In 111 adult liver transplant bile samples were collected daily posttransplantation for determination of bile composition. Expression of bile transporters was studied perioperativly. Nonanastomotic biliary strictures were detected in 14 patients (13%) within one year after transplantation. Patient- and donor characteristics and postoperative serum liver enzymes were similar between patients who developed nonanastomotic biliary strictures and those who did not. Secretions of bile salts, phospholipids and cholesterol were significantly lower in patients who developed strictures. In parallel, biliary phospholipids/bile salt ratio was lower in patients developing strictures, suggestive for increased bile cytotoxicity. There were no differences in bile salt pool composition or in hepatobiliary transporter expression. Conclusion. Although patients who develop nonanastomotic biliary strictures are initially clinically indiscernible from patients who do not develop nonanastomotic biliary strictures, the biliary bile salts and phospholipids secretion, as well as biliary phospholipids/bile salt ratio in the first week after transplantation, was significantly lower in the former group. This supports the concept that bile cytotoxicity is involved in the pathogenesis of nonanastomotic biliary strictures. 100

102 Chapter 6 Introduction Biliary complications are a major cause of morbidity and graft failure in patients after liver transplantation (1-3). Nonanastomotic strictures (NAS) of the larger bile ducts are considered to represent the most troublesome biliary complication as they are frequently resistant to therapy (4). The reported incidence of NAS is 5-15% (5-11). The occurrence of NAS can be partly attributed to thrombosis of the hepatic artery. The pathogenesis of NAS that develop in the absence of hepatic artery thrombosis is less clear (1,12). In general, three mechanisms contributing to bile duct injury after liver transplantation have been postulated: preservation or ischemia-related injury (7,13-18), immunological processes (7,19,20) and injury induced by cytotoxicity of biliary bile salts (21-24). Bile salts have potent detergent properties and may damage cells by affecting the integrity of cellular membranes (22,25). In the biliary tree, the toxic effects of bile salts are usually reduced by the formation of mixed micelles with phospholipids (26,27). Studies in mice and pigs, as well as clinical studies in humans, have indicated that bile formation early after liver transplantation may be disturbed, resulting in more cytotoxic bile with a relatively low phospholipids / bile salt ratio (1,21-24,28,29). We previously showed a strong relationship between this ratio early after liver transplantation and injury of the small bile ducts in the liver (21,24,29). The small bile ducts, however, are lined by distinct cholangiocytes, that have different characteristics compared with cholangiocytes in larger bile ducts, i.e. the location of NAS (30-33). It is unknown whether bile toxicity is also involved in the pathogenesis of transplantation-related injury of the large bile ducts, which may lead to the development of NAS. In contrast to the cytotoxic properties of bile salts, evidence has accumulated that bile salts may also influence cholangiocyte proliferation and survival, especially in the larger bile ducts (31,34-36). Some bile salts, including taurocholate and taurolithocholate stimulate cholangiocyte proliferation in vitro and in vivo, and bile salts are considered a survival factor for cholangiocytes in the larger bile ducts (34,35). Cholangiocytes of the large bile ducts are able to take up bile salts from bile via the apical Na+-dependent bile acid transporter (ASBT, gene symbol SLC10A2). After basolateral secretion, bile salts are transported back to hepatocytes and resecreted into bile, thereby contributing to bile flow via the cholehepatic shunt pathway (30,37). Bile production and composition, is therefore not exclusively determined by hepatocytes. 101

103 Bile composition after liver transplantation and NAS It has remained unclear whether bile salts are detrimental or beneficial for cholangiocyte function in large bile ducts after human liver transplantation, and whether or not bile production and composition are involved in the pathogenesis of NAS. In contrast to the small bile ducts, bile salts may not only have toxic effects but could also exert a proliferative restoration or preservation of the biliary epithelial lining of large bile ducts after transplantation. If bile composition is involved in the pathogenesis of NAS, one would expect that the bile composition in the first week after liver transplantation is different in those patients who will develop NAS as compared to patients who will not develop NAS. We tested this hypothesis by prospectively assessing bile production and composition within one week after liver transplantation and the subsequent development of NAS in a large cohort of adult liver transplant recipients. Patients and Methods Patients Between August 2000 and December 2004 a total of 222 liver transplants were performed at the University Medical Center Groningen. After excluding children (<18 years; n=70) and non heart-beating donor liver transplants (n=5), 147 patients were potential candidates for the study. Thirty six cases were excluded, because of graft loss within 90 days (n=22), initial poor graft function (defined as in (38,39); n=12), or hepatic artery thrombosis (confirmed by either Doppler ultrasound or angiography; n=2). This resulted in a study population of 111 liver transplant procedures. Surgical technique and perioperative management were as previously described by our group (5,40,41). Clinical variables and laboratory data were prospectively collected in a computerized database. Tissue and data collection was performed according to the guidelines of the medical ethical committee of our institution and the Dutch Federation of Scientific Societies. Diagnosis of NAS NAS was defined as any stricture, dilatation, or irregularity of the intra- or extrahepatic bile ducts of the liver graft, occurring within the first year after transplantation (Figure 1). The diagnosis NAS was based on at least one adequate imaging study of the biliary tree, after exclusion of hepatic artery thrombosis by either Doppler ultrasound or conventional angiography. Imaging studies of the arterial vasculature were repeated over time if no other explanation for the 102

104 Chapter 6 NAS was found and to confirm patency of the hepatic artery (5). Severity of NAS was graded according to a semi-quantitative scale, as described previously (5). Isolated strictures at the bile duct anastomosis were not included in this analysis. The time of first presentation of NAS was recorded for all patients. A B Figure 1. Postoperative cholangiography in liver transplant recipients. (A) Example of normal cholangiogram, with smooth lining and equal filling of the biliary tree. (B) Example of non anastomotic biliary strictures (NAS), characterized by diffuse strictures and irregularities of both the extra- and intrahepatic bile ducts on both sides of the liver with intrahepatic dilatations. Collection of Liver Biopsies Specimens of liver tissue were obtained during routine diagnostic biopsies of the liver grafts. According to our protocol, three consecutive needle biopsies were collected: at the end of cold preservation, approximately 3 hours after reperfusion, and 1 week after transplantation. An aliquot of the biopsy specimen was immediately snap-frozen for isolation of total RNA, the remaining material was used for routine histological analysis. Pieces of normal liver tissue from hepatic resections for colorectal metastasis were collected after obtaining informed consent and served as controls (n=9). All liver biopsies were snap-frozen and stored at -80 C until further processing. 103

105 Bile composition after liver transplantation and NAS Collection and Analysis of Bile Samples Before transplantation, the gallbladder was removed and the bile ducts were flushed with preservation fluid on the backtable during preparation for implantation. During the transplantation an open tip silicon catheter was inserted in the recipient common bile duct and placed retrograde through the anastomosis. Via this open biliary tube, bile flow was entirely diverted outside the patient into a collection bag that was placed below the horizontal bed level (42). Interruption of the enterohepatic circulation in the patient was prevented by re-administration of bile via a percutaneous feeding jejunostomy catheter. Samples of bile were collected daily in the first postoperative week between 8:00 and 9:00 am. Bile samples were frozen and stored at -80 C until further processing. None of the patients received a statin or ursodeoxycholic acid during the first week after transplantation. Bile samples were analyzed for total bile salts, phospholipids, and cholesterol contents. Total bile salt concentrations were measured with fluorescent method using 3α-hydroxysteroid dehydrogenase (43). Phospholipid and cholesterol concentrations in bile were assayed spectrophotometrically, using commercially available enzymatic methods (Wako Chemicals GmbH, Neuss, Germany; and Roche Diagnostics GmbH, Mannheim, Germany; respectively). Postoperative secretion of bile components was defined as concentration multiplied by daily bile production per kilogram body weight of the donor. Bile salt composition of bile samples was determined by capillary gas chromatography in a 50μL bile sample on a Hewlett-Packard gas chromatograph (HP 5880A) equipped with a 50 m x 0.32 mm CP-Sil-19 fused silica column (Chrompack B.V., Middelburg, The Netherlands) (44). Subsequently the hydrophobicity of the bile salt pool was determined by the Heuman index (45). RNA Extraction and Reverse Transcription Polymerase Chain Reaction Isolation and reverse transcription of RNA was performed as described previously (21). Messenger RNA levels of following hepatobiliary transporters were analyzed: the most prominent bile salt uptake system (NTCP, Na+-dependent taurocholate cotransporting polypeptide: gene symbol SLC10A1) and secretion system (BSEP, bile salt export pump: gene symbol ABCB11), the phospholipid translocator (MDR3, multidrug resistance protein 3: gene symbol ABCB4) and the main canalicular organic anion transporter and driving force of the bile salt independent bile flow (MRP2, multidrug resistant associated protein-2: gene symbol ABCC2). Additionally, cholesterol 7α-hydroxylase (gene symbol CYP7A1) was analyzed by real-time polymerase chain reaction 104

106 Chapter 6 (PCR), using the ABI PRISM 7900 HT Sequence detector (Applied Biosystems, Foster City, CA, USA). Nucleotide sequences of Primers (Invitrogen, Paisly, Scotland) and Probes (Eurogentec, Herstal, Belgium) were designed using Primes Express software (Applied Biosystems, Foster City, CA, USA). Probes were 5 labeled by a 6-carboxy-fluoresceine (FAM) reporter and 3 labeled with a 6-carboxy-tetra-methyl-rhodamin (TAMRA) quencher and are listed in Table 1. Messenger RNA copy numbers of genes were normalized to those of 18S rrna. Real time PCR data were analyzed using the comparative cycle threshold (CT) method (46). Table 1. Sequences of Primers and Probes Used for Real-Time PCR Analysis Gene Alternative Name Primers and Probes PCR Product (bp) SLC10A1 NTCP sense 5 -TGA TAT CAC TGG TCC TGG TTC TCA antisense 5 -GCA TGT ATT GTG GCC GTT TG -3 probe 5 FAM-TCC TTG CAC CAT AGG GAT CGT CCT CA - TAMRA 3 ABCB11 BSEP sense 5 -ACA TGC TTG CGA GGA CCT TTA antisense 5 -GGA GGT TCG TGC ACC AGG TA -3 probe 5 FAM-CCA TCC GGC AAC GCT CCA AGT CT - TAMRA 3 ABCB4 MDR3 sense 5 -CTA TGG AAT TAC TTT TAG TAT CTC ACA AGC ATT antisense 5 -AGC GCA TAT GTC CAT TCA CAA T -3 probe 5 FAM-TTT TTC CTA TGC CGG TTG TTT - TAMRA 3 ABCC2 MRP-2 sense 5 -TGC AGC CTC CAT AAC CAT GAG antisense 5 -CTT CGT CTT CCT TCA GGC TAT TCA -3 5 FAM-CAG CTT TCG TCG AAC ACT TAG CCG CA - probe TAMRA 3 CYP7A1 sense 5 -GAG AAG GCA AAC GGG TGA AC antisense 5 -GGT ATG ACA AGG GAT TTG TGA TGA-3 5 FAM-TGG ATT AAT TCC ATA CCT GGG CTG TGC probe TCT-TAMRA 3 18S sense 5 -CGG CTA CCA CAT CCA AGG A antisense 5 -CCA ATT ACA GGG CCT CGA AA -3 probe 5 FAM-CGC GCA AAT TAC CCA CTC CCG A - TAMRA 3 105

107 Bile composition after liver transplantation and NAS Statistical Analysis Collection of laboratory values from the central laboratory database was conducted as described previously (46). Continuous variables were presented as medians with interquartile range (IQR) or means with standard error of the mean (SEM) when appropriate. Categorical variables were presented as numbers with percentages and compared using Pearson s chisquare test. Comparison of continuous variables was performed using the Mann-Whitney U test. Area under the curve (AUC) was analyzed by the trapezium method. The level of significance was set at Statistical analysis was performed using SPSS 14.0 (SPSS, Chicago, IL, USA). Results Development of NAS NAS was diagnosed in 14 of the 111 liver transplant recipients (13%) at a median time interval of 2.4 months (IQR months) after transplantation. Signs of NAS were mild/moderate in 12 patients and severe in 2 patients. There were no significant differences in donor and recipient characteristics or surgical variables in patients who developed NAS compared to patients who did not develop NAS (Table 2). Serum Markers of Hepatocellular Injury and Cholestasis Serum levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in the first week after transplantation, as markers of ischemia reperfusion injury, were similar in patients who did or did not develop NAS (Figure 2). Similarly, gamma glutamyltransferase (γgt) and alkaline phosphatase (ALP), markers of cholestasis, were not different between the two groups in the first postoperative week (Figure 2). 106

108 Chapter Serum AST (U/L) AUC p=0.73 Serum ALT (U/L) AUC p= A Days after transplantation 0 B Days after transplantation Serum ALP (U/L) AUC p=0.37 Serum GT (U/L) AUC p= C Days after transplantation 0 D Days after transplantation Figure 2. Comparison of median serum levels (IQR) of aspartate aminotransferase (AST; panel A), alanine aminotransferase (ALT; panel B), alkaline phosphatase (ALP; panel C), and gamma glutamyltransferase (γgt; panel D) during the first 8 days after liver transplantation in patients who later developed non anastomotic biliary strictures (NAS, closed squares) and patients who did not develop NAS (open triangles). Biliary Secretion of Bile Salts, Phospholipids and Cholesterol Bile production increased 7-fold during the first week after transplantation in both groups (Figure 3). Biliary bile salt secretion increased after transplantation in both groups. Bile flow increased in linear fashion with the higher bile salt secretion rate in both groups. The bile salt independent bile flow (Y-intercept) and the bile salt dependent bile flow (slope) were similar in both groups (NAS group: flow = x BS-secretion , r 2 =0.47; Controls: flow = x BS-secretion , r 2 = 0.56). However, in patients who did not develop NAS, the increase in bile salt secretion was over 1.5 fold higher compared to patients who did develop NAS (99 ± 23 μmol/day/kg versus 166 ± 27 μmol/day/kg at day 8) (Figure 3). In parallel with the relatively reduced bile salt secretion, secretion of phospholipids and cholesterol was also significantly lower in patients developing NAS (Figure 3). In patients who developed NAS, the secretion of 107

109 Bile composition after liver transplantation and NAS biliary phospholipids during the first week after transplantation was even more compromised than the secretion of bile salts. This resulted in a significantly lower biliary phospholipid / bile salt ratio in the patients developing NAS, compared to patients who did not develop NAS (Figure 4). Bile production (ml/day) A AUC p= Days after transplantation BS secretion (umol/day/kg) AUC p= B Days after transplantation PL secretion (umol/day/kg) C AUC p= Days after transplantation CH secretion (umol/day/kg) D AUC p= Days after transplantation Figure 3. Comparison of median (IQR) daily bile production (panel A), bile salt (BS; panel B) secretion, phospholipid (PL; panel C) secretion, and cholesterol (CH; panel D) secretion during the first 8 days after liver transplantation in patients who later developed non anastomotic biliary strictures (NAS, closed squares) and patients who did not develop NAS (open triangles). Overall BS, PL and CH secretion, as determined by the area under the curve (AUC), was significantly lower in the patients who developed NAS. 108

110 Chapter AUC p=0.02 PL/BS ratio Days after transplantation Figure 4. Comparison of the mean biliary phospholipid / bile salt (PL/BS) ratio in the first 8 days after liver transplantation in patients who developed non anastomotic biliary strictures (NAS, closed squares) and patients who did not develop NAS (open triangles). The PL/BS ratio was significantly lower in patients who developed NAS. Absolute amount (nmol) DC C CDC UDC Figure 5. Composition of bile salts in bile at day 3 after transplantation in patients who later developed non anastomotic biliary strictures (NAS, dark bars) and patients who did not develop NAS (open bars). There were no significant differences in the absolute amounts of the various bile salts between the two groups of patients. Abbreviations: DC: deoxycholate, C: cholate, CDC: chenodeoxycholate, UDC: ursodeoxycholate. 109

111 Bile composition after liver transplantation and NAS Table 2. Comparison of Donor, Recipient, Surgical and Postoperative Variables of Liver Grafts With or Without Non Anastomotic Strictures (NAS). NAS Control OLT (n = 14) (n = 97) P-value Donor variables Age (years) 47 (39-57) 48 (37-58) 0.98 Gender (male/female) 6 / 8 (43% / 57%) 44/53 (45% / 55%) Gender match (donor/recipient) 0.42 M/M 2 (14%) 28 (29%) F/F 5 (35%) 24 (25%) M/F 4 (29%) 16 (17%) F/M 3 (21%) 29 (30%) Body weight donor 72.5 ( ) 70 (65-80) 0.54 Laboratory variables* Hemoglobulin (mmol/l) 7.1 ( ) 7.0 ( ) 0.90 Total bilirubin (umol/l) 8.4 ( ) 10.1 ( ) 0.31 Alanine Amino transferase (U/L) 20 (13-25) 23 (15-45) 0.17 gamma Glutamyl transferase (U/L) 25 (20-63) 22 (14-38) 0.63 Alkaline phosphatase (U/L) 55 (50-86) 53 (39-66) 0.31 Cause of death 0.89 Cerebral Vascular Accident 11 (79%) 72 (74%) Trauma 2 (14%) 19 (20%) Miscellaneous 1 (7%) 6 (6%) Recipient variables Age (years) 54 (44-58) 50 (40-55)

112 Chapter 6 Gender (male/female) 5 / 9 (36% / 64%) 57 / 40 (59% / 41%) 0.10 Disease 0.42 Primary Sclerosing Cholangitis 4 (30%) 20 (21%) Primary and Secondary Biliary Cirrhosis 2 (14%) 9 (9%) Viral hepatitis 0 20 (21%) Auto immune hapatitis 2 (14%) 10 (10%) Alcoholic cirrhosis 2 (14%) 10 (10%) Cryptogenic cirrhosis 2 (14%) 5 (5%) Other 2 (14%) 23 (24%) Child Pugh Classification (A/ B/ C) 1 / 7 / 6 (7% / 50% / 43%) 19 / 38 / 37 (20% / 40% / 40%) 0.49 Re-transplantation 3 (21%) 15 (16%) 0.57 Surgical variables Preservation Solution 0.23 High viscosity (UW) 14 (0%) 88 (91%) Low viscosity (HTK) 0 (100%) 9 (9%) Cold ischemia time (minutes) 500 ( ) 489 ( ) 0.86 Warm ischemia time (minutes) 48 (42-54) 45 (40-51) 0.26 Revascularization time (minutes) 78 (64-98) 93 (80-109) 0.21 Bile duct reconstruction (duct to duct / Roux-Y) 11 / 3 (79% / 21%) 76 / 21 (78% / 22%) 0.99 Postoperative variables ICU-length of stay (days) 2.5 ( ) 2 ( ) 0.70 Acute rejection 5 (36%) 35 (36%)

113 Bile composition after liver transplantation and NAS ABCB11 Fold Induction A ** ** * Before After 1 week SLC10A1 Fold induction B ** ** Before After 1 week ABCB4 Fold Induction C Before After 1 week ABCC2 Fold Induction D Before After 1 week Figure 6. Relative gene expression of the bile transporters ABCB11 (panel A), SLC10A1 (panel B), ABCB4 (panel C) and ABCC2 (panel D) in human liver grafts. A comparison was made between patients who developed non anastomotic biliary strictures (NAS, dark bars) and patients who did not develop NAS (open bars). Genes of interest were standardized for 18S rrna. In livers that later developed NAS, a significant decrease in ABCB11 mrna expression was found immediately after transplantation, compared to pretransplant values. This decrease was not observed in livers that did not develop NAS. In both groups mrna expression of the bile salt transporters ABCB11 and SLC10A1 increased significantly during the first week after transplantation. However, there were no significant differences between the two groups. Before: before reperfusion. After: 3 hours after reperfusion. One week: one week after liver transplantation. *) p<0.05, when compared to values before transplantation. **) p<0.05, when compared to values after reperfusion. 112

114 Chapter 6 15 CYP7A1 Fold Induction NAS Control p=0.07 Figure 7. Relative CYP7A1 gene expression one week after transplantation in livers of patients who developed non anastomotic biliary strictures (NAS) and patients who did not develop NAS. CYP7A1 catalyzes the conversion of cholesterol into 7α-hydroxycholesterol and is considered to be the rate-controlling step in bile salt synthesis. Bile Salt Pool Analysis In a subset of 22 patients (9 NAS and 13 controls) bile salt pool composition at postoperative day 1, 2, 3 and 7 was analyzed using gaschromatography. This analysis did not reveal any significant differences between the two groups. Amounts of the various bile salts at postoperative day 3, when the difference in phospholipids / bile salt ratio between the two groups was most pronounced, are shown in Figure 5. In addition, no differences in biliary hydrophibicity, as reflected by the Heuman index, were found at any time point between the two groups. Hepatic Expression of Bile Transporters and CYP7A1 Perioperative changes in the hepatic expression of hepatobiliary transporters are presented in Figure 6. Compared to preoperative values, mrna levels of the bile salt transporter ABCB11 were significantly decreased at 3 hrs after reperfusion in livers that developed NAS, whereas this change was not observed livers that did not develop NAS. In both groups, mrna levels of the bile salt transporters ABCB11 and SLC10A1 increased significantly during the first week after transplantation. In contrast, no significant changes were observed in the hepatic expression of ABCB4, the phospholipid translocator, and ABCC2. There were no significant differences in transporter expression between the two groups at any time point. 113

115 Bile composition after liver transplantation and NAS In parallel with the low bile salt secretion, expression of CYP7A1 (the rate-controlling enzyme in de novo bile salt synthesis) at one week after transplantation was substantially lower in patients who developed NAS, compared to those who did not (Figure 7). Discussion In a prospective clinical study, we evaluated the potential role of bile composition and especially the relative contribution of bile salts and phospholipids in the development strictures of the large bile ducts, or NAS, after otherwise successful liver transplantation. Interestingly, the overall biliary secretion of bile salts, phospholipids and cholesterol during the first week after transplantation was significantly lower in patients who later developed NAS, compared to patients who did not develop NAS. The secretion of phospholipids was relatively more affected than bile salt secretion, resulting in a lower biliary phospholipids / bile salt ratio in patients who developed NAS. These findings indicate that the development of strictures of the large bile ducts is preceded by abnormal bile composition early after transplantation, several weeks before clinical symptoms of bile duct injury appear. This study supports the hypothesis that early changes in bile composition contribute to the relatively late stricturing of the large bile ducts, leading to the radiological diagnosis of NAS after transplantation. In the current study the incidence of NAS up to one year after transplantation was 13%. This rate is similar to data reported in most previous studies (6,7,10,46) but higher than reported in some others (15,18,47). Variations in the reported incidence of NAS among different studies can be explained by differences in study design (retrospective versus prospective) and differences in the diagnostic criteria used. Bile salts possess potent detergent properties and as such, are potentially cytotoxic (48,49). In case of relative excess of bile salts, either due to increased bile salt secretion or reduced secretion of phospholipids, micellar bile salts may cause cholangiocyte injury, pericholangitis and periductal fibrosis (50,51). In previous studies we have shown that toxic bile composition early after transplantation, characterized by a low biliary phospholipid / bile salt ratio, is associated with histological signs of injury of the small bile ducts in the liver (21,24,29). The role of bile salt toxicity in the pathogenesis of injury of the small intrahepatic bile ducts was also demonstrated in an experimental study using a liver transplant model in mice (24). Livers transplanted from Abcb4-/+ mice, which have only 50% expression of 114

116 Chapter 6 the phospholipids translocator Abcb4, into wild-type recipients developed signs of severe injury of the small intrahepatic bile ducts within two weeks after transplantation (24). In the current study we focused on the development of NAS, which is a disease of the large bile ducts (5,52). Our results suggest for the first time that bile salt toxicity is also involved in the development of large bile duct injury, leading to the clinical and radiological diagnosis of NAS. Despite the observed low phospholipid / bile salt ratio in patients developing NAS, reflecting bile toxicity, the overall biliary secretion of bile salts in these patients was lower than in patients who did not develop NAS. This observation was not expected and introduces the intriguing possibility that, apart from relative bile salt toxicity, relative bile salt deprivation could (also) contribute to cholangiocyte injury and the development of NAS. There is substantial evidence that bile salts are potent inducers of cholangiocyte proliferation and thus bile duct repair (31,34-36). Uptake of bile salts by cholangiocytes is mediated by the transporter ASBT (SLC10A2) at the ductular membrane of these cells (30,37). In contrast to cholangiocytes of the small bile ducts, cholangiocytes in larger bile ducts do express ASBT and, therefore, these cells can re-absorb bile salts from bile (30,37). This important difference between cholangiocytes from small and large bile ducts may explain why a previous clinical study focusing on posttransplant injury of the small bile ducts did not reveal a relationship between small bile duct injury and reduced bile salt secretion. Collectively, these observations raise the possibility that the pathogenesis of biliary injury after liver transplantation is different for small and large bile ducts. In this respect it would have been interesting to study the expression of ASBT (SLC10A2) in the large bile ducts in the current study. However, it is difficult to take serial biopsies of the large bile ducts in patients and we were unable to detect ASBT (SLC10A2) mrna expression in liver biopsies, which mainly contain small bile ducts (data not shown). Some bile salts have a more pronounced effect on cholangiocyte proliferation than others. Taurocholate, for example, may enhance proliferation, while ursodeoxycholate may reduce the proliferative effects of other bile salts (36,53). In the current study we found no differences in the bile salt pool composition in patients who developed NAS, compared to those who did not. Therefore, we have no evidence to suggest that differences in composition of the bile salt pool are involved in the altered overall bile salt secretion or in the pathogenesis of NAS after liver transplantation. A key question that emerges from this study is: what determines the low bile salt secretion in livers that are developing NAS? Theoretically, reduced biliary bile salt secretion can result 115

117 Bile composition after liver transplantation and NAS from a) decreased de novo synthesis, b) impaired hepatobiliary transport at the level of the canalicular membrane (ABCB11), and/or c) impaired intestinal bile salt re-absorption and fecal loss of bile salts leading to reduced bile salt pool size. In the classical pathway of de novo bile salt biosynthesis, CYP7A1 catalyzes the conversion of cholesterol into 7α-hydroxycholesterol, which is considered to be the rate-controlling step. In humans, the classical pathway accounts for approximately 80% of total bile salt synthesis (54,55). We observed a lower hepatic expression of CYP7A1 in patients who later developed NAS, compared to those who did not. It is tempting to ascribe the reduced bile salt secretion in patients who developed NAS to the lower expression of CYP7A1. Yet, three issues should be considered in this respect: a) the difference in CYP7A1 expression was striking, but it did not reach statistical significance, in contrast to the difference in bile secretion; b) no information is available on the correlation between CYP7A1 mrna levels and actual cholate synthesis in the early post-transplant period; and c) it can be anticipated that the amount of bile salts secreted after liver transplantation is increasingly derived from re-absorbed ( conserved ) bile salts from the intestine. To demonstrate or refute increased intestinal loss of bile salts as an explanation for the differences in biliary bile salt secretion we would have needed the collection of faeces. Although this was not performed, we have other arguments to assume that the observed differences in bile salt secretion are not caused by differences in intestinal bile salt loss. Reduced bile salt pool size due to impaired intestinal reabsorption would be expected to lead to an increased rather than a decreased hepatic CYP7A1 expression. In addition, a previous study from our centre has shown that serum bile salt concentrations increase during the first week after transplantation, which is not compatible with increased intestinal losses (56). Hepatobiliary secretion of bile salts is an active process which, under normal circumstances, is mainly influenced by the sinusoidal transporter SLC10A1 and the canalicular transporter ABCB11. Theoretically, impaired hepatobiliary transport could have resulted form a reduced expression of these transporter proteins. Compared to pretransplant values, ABCB11 mrna expression was decreased immediately after transplantation in livers that later developed NAS. Although this decrease was not observed in livers that did not develop NAS, there were no significant differences between the two groups either before or immediately after transplantation. In accordance with previous observations by Geuken et al. (21), we observed an increased mrna expression of the bile salt transporters SLC10A1 and ABCB11 in both groups after transplantation, while mrna levels of the phospholipid translocator ABCB4 did 116

118 Chapter 6 not change. These findings are compatible with the relatively low biliary phospholipid / bile salt ratio observed early after transplantation. However, there were no significant differences in the expression of the bile transporters between the two groups, suggesting that the observed differences in bile salt secretion cannot be explained by differences in gene transcription. Based on the current study, we cannot exclude that posttranscriptional processes or changes in transporter activity are involved. Unfortunately, we were unable to perform Western blot analyses for quantification of transporter protein levels due to the small amount of liver tissue obtained from needle biopsies. We also examined whether differences in bile composition between patients who developed NAS and those who did not could be explained by differences in phospholipids secreted per bile salt. Therefore we additionally analyzed the biliary hydrophobicity index and the bile salt independent bile flow. There were no significant differences in the hydrophobicity index or in the bile salt independent bile flow, indicating that these factors cannot explain the observed differences between the two groups (57,58). Several other factors have been shown to contribute to the development of NAS after liver transplantation, including long cold or warm ischemia times (7,9), inadequate washout and perfusion of the peribiliary capillary plexus (16,17), and immunological injury (19,59). In the current study we found no differences in the duration of cold and warm ischemia in livers with or without NAS. These data support previous suggestions that the pathogenesis of NAS is not only related to a direct ischemic injury of the biliary epithelium (1,12). In summary, the results of this prospective clinical study strongly support the hypothesis that bile composition is involved in the pathogenesis of NAS after liver transplantation. Patients who developed NAS within one year after liver transplantation were initially clinically indiscernible from patients who did not develop NAS. However, bile composition in this early postoperative period was different in these two groups. Patients who developed NAS were characterized by a reduced biliary secretion of bile salts and phospholipids and a decreased biliary phospholipid / bile salt ratio. We speculate that those early defects in bile formation, possibly genetically based, play a role in the injury of the biliary epithelium of large bile ducts early after transplantation, subsequently leading to the formation of biliary strictures. 117

119 Bile composition after liver transplantation and NAS Reference List 1. Buis CI, Hoekstra H, Verdonk RC, Porte RJ. Causes and consequences of ischemic-type biliary lesions after liver transplantation. J Hepatobiliary Pancreat Surg 2006;13: Calne RY. A new technique for biliary drainage in orthotopic liver transplantation utilizing the gall bladder as a pedicle graft conduit between the donor and recipient common bile ducts. Ann Surg 1976;184: Starzl TE, Marchioro TL, Vonkualla KN, Hermann G, Brittain RS, Waddell WR. Homotransplantation of the liver in humans. Surg Gynecol Obstet 1963 ;117: Verdonk RC, Buis CI, Porte RJ, Van der Jagt EJ, Limburg AJ, van den Berg AP, et al. Anastomotic biliary strictures after liver transplantation: Causes and consequences. Liver Transpl 2006;12: Buis CI, Verdonk RC, Van der Jagt EJ, van der Hilst CS, Slooff MJ, Haagsma EB, et al. Nonanastomotic biliary strictures after liver transplantation, part 1: Radiological features and risk factors for early vs. late presentation. Liver Transpl 2007;13: Campbell WL, Sheng R, Zajko AB, Abu-Elmagd K, Demetris AJ. Intrahepatic biliary strictures after liver transplantation. Radiology 1994;191: Guichelaar MM, Benson JT, Malinchoc M, Krom RA, Wiesner RH, Charlton MR. Risk factors for and clinical course of non-anastomotic biliary strictures after liver transplantation. Am J Transplant 2003;3: Pascher A, Neuhaus P. Bile duct complications after liver transplantation. Transpl Int 2005;18: Sanchez-Urdazpal L, Gores GJ, Ward EM, Maus TP, Wahlstrom HE, Moore SB, et al. Ischemic-type biliary complications after orthotopic liver transplantation. Hepatology 1992;16: Sawyer RG, Punch JD. Incidence and management of biliary complications after 291 liver transplants following the introduction of transcystic stenting. Transplantation ;66: Turrion VS, Alvira LG, Jimenez M, Lucena JL, Nuno J, Pereira F, et al. Management of the biliary complications associated with liver transplantation: 13 years of experience. Transplant Proc 1999;31: Verdonk RC, Buis CI, Porte RJ, Haagsma EB. Biliary complications after liver transplantation: a review. Scand J Gastroenterol Suppl 2006; 243: Abt P, Crawford M, Desai N, Markmann J, Olthoff K, Shaked A. Liver transplantation from controlled non-heart- beating donors: an increased incidence of biliary complications. Transplantation 2003;75: Foley DP, Fernandez L, Leverson G, Chin LT, Kreiger N, Cooper JT, et al. Donation After Cardiac Death: The University of Wisconsin Experience With Liver Transplantation. Ann Surg 2005; 242: Li S, Stratta RJ, Langnas AN, Wood RP, Marujo W, Shaw BW, Jr. Diffuse biliary tract injury after orthotopic liver transplantation. Am J Surg 1992;164:

120 Chapter Moench C, Moench K, Lohse AW, Thies J, Otto G. Prevention of ischemic-type biliary lesions by arterial back- table pressure perfusion. Liver Transpl 2003;9: Pirenne J, Van Gelder F, Coosemans W, Aerts R, Gunson B, Koshiba T, et al. Type of donor aortic preservation solution and not cold ischemia time is a major determinant of biliary strictures after liver transplantation. Liver Transpl 2001;7: Sankary HN, McChesney L, Frye E, Cohn S, Foster P, Williams J. A simple modification in operative technique can reduce the incidence of nonanastomotic biliary strictures after orthotopic liver transplantation. Hepatology 1995;21: Moench C, Uhrig A, Lohse AW, Otto G. CC chemokine receptor 5delta32 polymorphism-a risk factor for ischemic-type biliary lesions following orthotopic liver transplantation. Liver Transpl 2004;10: Sanchez-Urdazpal L, Sterioff S, Janes C, Schwerman L, Rosen C, Krom RA. Increased bile duct complications in ABO incompatible liver transplant recipients. Transplant Proc 1991;23: Geuken E, Visser D, Kuipers F, Blokzijl H, Leuvenink HG, de Jong KP, et al. Rapid increase of bile salt secretion is associated with bile duct injury after human liver transplantation. J Hepatol 2004;41: Hertl M, Harvey PR, Swanson PE, West DD, Howard TK, Shenoy S, et al. Evidence of preservation injury to bile ducts by bile salts in the pig and its prevention by infusions of hydrophilic bile salts. Hepatology 1995;21: Hertl M, Hertl MC, Kluth D, Broelsch CE. Hydrophilic bile salts protect bile duct epithelium during cold preservation: a scanning electron microscopy study. Liver transplantation 2000;6: Hoekstra H, Porte RJ, Tian Y, Jochum W, Stieger B, Moritz W, et al. Bile salt toxicity aggravates cold ischemic injury of bile ducts after liver transplantation in Mdr2+/- mice. Hepatology ;43: Palmeira CM, Rolo AP. Mitochondrially-mediated toxicity of bile acids. Toxicology ;203:1-15. De Vree JM, Jacquemin E, Sturm E, Cresteil D, Bosma PJ, Aten J, et al. Mutations in the MDR3 gene cause progressive familial intrahepatic cholestasis. Proc Natl Acad Sci U S A ;95: Smit J, Schinkel A, Oude Elferink R, Groen A, Wagenaar E, van Deemter. Homozygous disruption of the murine mdr2 P-glycoprotein gene leads to a. Cell 1993;75: Wagner M, Zollner G, Trauner M. Ischemia and cholestasis: more than (just) the bile ducts! Transplantation ;85: Yska MJ, Buis CI, Monbaliu D, Schuurs TA, Gouw ASH, Kahmann ONH, Visser DS, Pirenne J, Porte RJ. The role of bile salt toxicity in the pathogenesis of bile duct injury after non heart-beating porcine liver transplantation. Transplantation 2008;85: Alpini G, Glaser SS, Rodgers R, Phinizy JL, Robertson WE, Lasater J, et al. Functional expression of the apical Na+- dependent bile acid transporter in large but not small rat cholangiocytes. Gastroenterology 1997;113:

121 Bile composition after liver transplantation and NAS 31. Alpini G, Glaser S, Robertson W, Phinizy JL, Rodgers RE, Caligiuri A, et al. Bile acids stimulate proliferative and secretory events in large but not small cholangiocytes. Am J Physiol 1997;273:G518-G Glaser S, Francis H, Demorrow S, Lesage G, Fava G, Marzioni M, et al. Heterogeneity of the intrahepatic biliary epithelium. World J Gastroenterol ;12: Ueno Y, Alpini G, Yahagi K, Kanno N, Moritoki Y, Fukushima K, et al. Evaluation of differential gene expression by microarray analysis in small and large cholangiocytes isolated from normal mice. Liver Int 2003;23: Alpini G, Glaser SS, Ueno Y, Rodgers R, Phinizy JL, Francis H, et al. Bile acid feeding induces cholangiocyte proliferation and secretion: evidence for bile acid-regulated ductal secretion. Gastroenterology 1999;116: Alpini G, Glaser S, Alvaro D, Ueno Y, Marzioni M, Francis H, et al. Bile acid depletion and repletion regulate cholangiocyte growth and secretion by a phosphatidylinositol 3-kinase-dependent pathway in rats. Gastroenterology 2002;123: Xia X, Francis H, Glaser S, Alpini G, Lesage G. Bile acid interactions with cholangiocytes. World J Gastroenterol ;12: Lazaridis KN, Tietz P, Wu T, Kip S, Dawson PA, Larusso NF. Alternative splicing of the rat sodium/bile acid transporter changes its cellular localization and transport properties. Proc Natl Acad Sci U S A ;97: Ploeg RJ, D alessandro AM, Knechtle SJ, Stegall MD, Pirsch JD, Hoffmann RM, et al. Risk factors for primary dysfunction after liver transplantation--a multivariate analysis. Transplantation 1993;55: Buis CI, van der SG, Visser DS, Nolte IM, Hepkema BG, Nijsten M, et al. Heme oxygenase-1 genotype of the donor is associated with graft survival after liver transplantation. Am J Transplant 2008;8: Miyamoto S, Polak WG, Geuken E, Peeters PM, de Jong KP, Porte RJ, et al. Liver transplantation with preservation of the inferior vena cava. A comparison of conventional and piggyback techniques in adults. Clin Transplant 2004;18: Polak WG, Miyamoto S, Nemes BA, Peeters PM, de Jong KP, Porte RJ, et al. Sequential and simultaneous revascularization in adult orthotopic piggyback liver transplantation. Liver Transpl 2005;11: Lenzen R. In liver transplantation, T tube bile represents total bile flow. Liver transplantation and surgery 1999;5: Turley SD, Dietschy JM. Re-evaluation of the 3 alpha-hydroxysteroid dehydrogenase assay for total bile acids in bile. J Lipid Res 1978;19: Kok T, Hulzebos CV, Wolters H, Havinga R, Agellon LB, Stellaard F, et al. Enterohepatic circulation of bile salts in farnesoid X receptor-deficient mice: efficient intestinal bile salt absorption in the absence of ileal bile acidbinding protein. J Biol Chem 2003;278:

122 Chapter Heuman DM. Quantitative estimation of the hydrophilic-hydrophobic balance of mixed bile salt solutions. J Lipid Res 1989;30: Feller RB, Waugh RC, Selby WS, Dolan PM, Sheil AG, McCaughan GW. Biliary strictures after liver transplantation: clinical picture, correlates and outcomes. J Gastroenterol Hepatol 1996;11: Rull R, Garcia Valdecasas JC, Grande L, Fuster J, Lacy AM, Gonzalez FX, et al. Intrahepatic biliary lesions after orthotopic liver transplantation. Transpl Int 2001;14: Galle PR, Theilmann L, Raedsch R, Otto G, Stiehl A. Ursodeoxycholate reduces hepatotoxicity of bile salts in primary human hepatocytes. Hepatology 1990;12: Schmucker DL, Ohta M, Kanai S, Sato Y, Kitani K. Hepatic injury induced by bile salts: correlation between biochemical and morphological events. Hepatology 1990;12: Trauner M, Meier PJ, Boyer JL. Molecular pathogenesis of cholestasis. N Engl J Med ;339: Arrese M, Trauner M. Molecular aspects of bile formation and cholestasis. Trends Mol Med 2003;9: Verdonk RC, Buis CI, Van der Jagt EJ, Gouw AS, Limburg AJ, Slooff MJ, et al. Nonanastomotic biliary strictures after liver transplantation, part 2: Management, outcome, and risk factors for disease progression. Liver Transpl 2007;13: Alvaro D, Gigliozzi A, Attili AF. Regulation and deregulation of cholangiocyte proliferation. J Hepatol 2000;33: Vlahcevic ZR, Stravitz RT, Heuman DM, Hylemon PB, Pandak WM. Quantitative estimations of the contribution of different bile acid pathways to total bile acid synthesis in the rat. Gastroenterology 1997;113: Duane WC, Javitt NB. 27-hydroxycholesterol: production rates in normal human subjects. J Lipid Res 1999;40: Haagsma EB, Huizenga JR, Vonk RJ, Albers CJ, Grond J, Krom RA, et al. Composition of bile after orthotopic liver transplantation. Scand J Gastroenterol 1987;22: Verkade HJ. Inhibition of biliary phospholipid and cholesterol secretion by organic anions affects bile canalicular membrane composition and fluidity. J Gastroenterol 2000;35: Verkade HJ, Vonk RJ, Kuipers F. New insights into the mechanism of bile acid-induced biliary lipid secretion. Hepatology 1995;21: Carrasco L. Effects of cold ischemia time on the graft after orthotopic liver. Transplantation 1996;61:

123

124 7 Polymorphisms of hepatobiliary phospholipid transporter MDR-3 associated with non anastomotic strictures after human liver transplantation Submitted Carlijn I Buis Gerrit van der Steege Ilja M Nolte Dorien S Visser Robert J Porte

125 ABCB4 gene polymorphism and NAS Abstract Non-anastomotic biliary strictures (NAS) are an important and troublesome biliary complication after liver transplantation. The pathogenesis of NAS is not completely clear, but studies have suggested that bile salt toxicity, due to an imbalance between biliary bile salts and phospholipids is involved. Hepatobiliary transporter proteins are responsible for bile secretion and composition. Aim of this study was to assess whether genetic variations in these transporters are associated with the development of NAS. In 461 liver transplant procedures, we studied donor genotype of three of the most relevant hepatobiliary transporters: the bile salt export pump (ABCB11), the transporter of phospholipids (ABCB4) and the transporter of glutathione and bilirubin (ABCC2). Four to five tagging single nucleotide polymorphisms (SNPs) with an equal physical distribution per gene were selected using HapMap data. Haplotypes were constructed using an Expectation-Maximization algorithm to estimate haplotype frequencies. The occurrence of NAS was determined for livers with the various transporter genotypes. NAS were detected in 77 patients (16.7%) after transplantation. Patients who received a donor liver with ABCB4 haplotype AGGTA developed NAS almost twice as often as donor livers with other haplotypes (28% versus 15%; p = 0.007). In a multivariate Cox regression analysis, the AGGTA haplotype of the ABCB4 gene in the donor, was confirmed as an independent risk factor for NAS (HR=2.23, 95% CI= ; p = 0.004). Various haplotypes of the ABCB11 and the ABCC2 gene, or individual SNPs, were not associated with NAS. Conclusion: A common haplotype in the transporter of phospholipids (ABCB4) in donor livers is independently associated with a two-fold increased risk for NAS after liver transplantation. Transport of phospholipids into the bile in livers with this risk haplotype might be altered after liver transplantation, contributing to the development of NAS. 124

126 Chapter 7 Introduction Biliary complications are reported in 10 to 30% of the patients after liver transplantation representing a major cause of morbidity and mortality (1). Non-anastomotic biliary strictures (NAS) are considered to be the most troublesome biliary complication, because they may occur at multiple locations in the biliary tree and are frequently resistant to therapy (2,3). Graft survival is markedly reduced in patients developing NAS; 16% of all patients with NAS need a re-transplantation and 35% will require an interventional treatment (3). The occurrence of NAS can partly be attributed to thrombosis of the hepatic artery. The pathogenesis of NAS developing in the absence of hepatic artery thrombosis is less clear (1,4). In general, three mechanisms contributing to bile duct injury after liver transplantation have been postulated: preservation or ischemia-related injury (5-11), immunological processes (7,12,13) and injury induced by cytotoxicity of biliary bile salts (14-17). Bile salts have potent detergent properties and may damage cells in the absence of phospholipids by affecting the integrity of cellular membranes (15,18). Damage to the canalicular membrane of the biliary epithelial cells could result in progressive destruction of bile ducts (19). Normally, these toxic effects of bile salts are prevented through neutralization by phospholipids. We have recently shown that changes in bile formation, leading to cytotoxic bile with a relative low phospholipidto-bile salt ratio, are associated with bile duct injury and the development of NAS after liver transplantation (14,17,20,21). Bile production depends on an active process involving the transport of bile acids, phospholipids and other osmotic compounds across a concentration gradient into the bile canaliculus. Hepatobiliary transporter proteins play a rate-limiting role in this process. Genetic variations in the phospholipid translocator, multiple drug resistance protein 3 (MDR3, official name ATP binding cassette, subfamily B, member 4 or ABCB4) have been associated with abnormal phenotypes, characterized by the production of bile with a low biliary phospholipid content, leading to bile duct injury and intrahepatic cholestasis. A genetic variation inevitably leading to disease is the mutation in the ABCB4 gene associated with progressive familial intrahepatic cholestasis type III (PFIC III) (22). Characteristic clinical features of these patients are jaundice, recurrent cholangitis and elevated serum γ-glutamyltransferase levels, reflecting the destruction of cell membranes of the biliary epithelium. Other genetic variations of the ABCB4 gene have a less stable phenotype and may lead to symptoms only under specific 125

127 ABCB4 gene polymorphism and NAS circumstances. An example of this is intrahepatic cholestasis of pregnancy (ICP), which may occur in women who were previously without symptoms, but develop jaundice during pregnancy. (19,23-28). SNPs in the bile salt exporter pump (BSEP, official name ABCB11) have been related with a spectrum of clinical phenotypes such as the syndrome of benign recurrent intrahepatic cholestasis (BRIC) (29), PFIC-2 (30), and ICP (28,31). Multidrug resistance related protein 2 (MRP-2, official name ABCC2), which is a transporter of bilirubin and glutathion (GSH) into bile, is known from the benign human disorder Dubin Johnson, which is characterized by an increase of conjugated bilirubin without elevation of liver enzymes (32). It has remained unclear whether variations in these genes encoding for hepatobiliary transporters might contribute to the pathogenesis of NAS after liver transplantation. We hypothesized that there variations in these genes that do not result in an abnormal phenotype under normal, physiological conditions, but that are associated with to the development of bile duct injury under stressful conditions, such as liver transplantation. Such a mechanism would be similar to the development of ICP in women with certain ABCB4 polymorphisms. The aim of the present study was to determine whether genetic variations in the genes encoding for hepatobiliary transporters are associated with the development of NAS in a large prospective cohort of 461 adult liver transplant recipients. Patients and Methods Patients Between January 1990 and January 2005, 720 liver transplantations were performed in 621 patients in our center. After exclusion of pediatric patients (n=160), 461 adult liver transplant recipients remained. Cryopreserved splenocyts from the donors were used for the genotyping. Recipient follow-up was until December 2007, resulting in a median follow-up of 8.2 years (interquartile range years). Surgical procedure and postoperative management have been described previously (2). In short, ABO blood group-identical or compatible grafts from brain-death donors with normal or near normal liver function tests were used for all patients. Immunosuppressive protocols were based on a calcineurin inhibitor (tacrolimus or cyclosporine A) either with or without azathioprine and a rapid taper of steroids. Biopsy-proven acute rejection was treated when clinically indicated with a bolus of methylprednisolone on 126

128 Chapter 7 three consecutive days. Doppler ultrasound was performed routinely at postoperative days 1, 3, and 7 and later on demand to rule out vascular or biliary complications or parenchymal lesions. Cholangiography via a biliary drain was routinely performed between postoperative day and later on demand (i.e. for rising cholestatic laboratory parameters or dilatation of bile ducts on ultrasound). Donor data was collected from the donor forms and checked and completed with information from the archives of the Eurotransplant Organization, Leiden, The Netherlands. Recipient data were obtained from a prospectively collected computer database. If necessary the original patient notes were reviewed for missing information. Tissue and data collection was performed according to the guidelines of the medical ethical committee of our institution and the Dutch Federation of Scientific Societies. Diagnosis of NAS Primary outcome parameter in this study was the development of NAS. For this study NAS were defined as any stricture, dilatation, or irregularity of the intra- or extrahepatic bile ducts of the liver graft, either with or without biliary sludge formation, after exclusion of hepatic artery thrombosis by either Doppler ultrasound or conventional angiography. Isolated strictures at the bile duct anastomosis were, by definition, excluded from this analysis and have been described elsewhere (33). Selection of Hepatobiliary Transporter SNPs and Genotyping The following genes were studied: the phospholipid translocator (ABCB4), the most prominent bile salt transporter (ABCB11), and the main canalicular organic anion transporter and driving force of the bile salt independent bile flow (ABCC2). Genomic DNA was isolated from donor splenocytes using a commercial kit (Gentra Systems, Minneapolis, MN, USA). SNPs were selected based on data from the HapMap database (release #16, using the Haploview tagging tool. Per locus only those SNPs were selected that tagged haplotypes with a frequency in the HapMap Caucasian dataset of more than 10% and with a minor allele frequency around 20%. In addition, we tried to combine this selection criterion with an equal physical distribution across the genes, preferably with exonic location. TaqMan assays were used to genotype the ABC transporter SNPs. These were obtained from Applied Biosystems (Foster City, CA, USA) by the assay-on-demand or the assay-by-design services. Details of the various SNPs are given in Table

129 ABCB4 gene polymorphism and NAS Table 1. Marker data Gene Applied assay ID SNP Position Nucleotide change Amino Acid change MAF * ABCB11 (BSEP) hcv rs UTR G --> A A (30%) chromosome 2 hcv rs intron 8 G --> A A (30%) hcv rs intron 10 G --> A A (48%) hcv rs UTR G --> A A (31%) ABCB4 (MDR3) hcv rs exon 3 G --> A synonymous Leu --> Leu A (11%) chromosome 7 hcv rs exon 5 A --> G synonymous Asn --> Asn G (33%) design rs intron 9 T --> G G (17%) design rs31674 intron 13 C --> T T (18%) hcv rs intron 23 A --> C C (11%) ABCC2 (MRP2) hcv rs intron 3 A --> T T (40%) chromosome 10 hcv rs intron 7 T --> G G (45%) design rs exon 10 G --> A nonsynonymous Ile --> Val A (16%) design rs intron 19 C --> T T (30%) hcv rs UTR G --> A A (23%) * MAF: Minor allele frequency 128

130 Chapter 7 Statistical Analysis Descriptive, continuous variables are reported as median and interquartile ranges (IQR) and categorical variables are reported as numbers with percentage. The power of this study was greater than 80% to detect an odds ratio (HR) of 1.71 or larger for SNPs with a minor allele frequency of at least 20% at the statistical significant level of 5%. Prior to all analyses, Hardy Weinberg equilibrium was confirmed for all genotypes using the chi-squared test. Single SNP analysis was performed with chi-squared test. Haplotype analysis was performed using an Expectation- Maximization algorithm to estimate haplotype frequencies (in-house software). The frequencies were estimated on the total group of patients and these frequencies were used to estimate the haplotype frequencies among patients with and patients without NAS. In addition for each specific haplotype a log-likelihood ratio test was performed to test whether the frequencies of this haplotype differed between patients with and without NAS. Incidence rate of NAS was estimated with the Kaplan Meier actuarial method and compared with the log-rank test. Furthermore, if the probability of a specific haplotype combination (using frequencies of the total group) was higher than 90%, that combination was assumed to the true combination. In this way, being carrier of specific haplotypes could be determined for use as a covariate in a multivariate Cox regression model. Statistical analyses were performed using SPSS Version 14.0 for Windows (SPSS Inc., Chicago, IL, USA). All p-values were two-tailed and considered as statistically significant at levels <

131 ABCB4 gene polymorphism and NAS Results Incidence NAS After Liver Transplantation Clinical characteristics of donors and recipients, as well as perioperative variables of the entire series are presented in Table 2. NAS were detected in 77 of the 461 (16.7%) liver grafts studied. Among the liver grafts that developed NAS, 67 were first transplants and 10 were re-transplants Cumulative ris sk of NAS Log Rank p=0.007 Carrier AGGTA yes no Years after transplantation 20 Figure1. Cumulative risk of NAS during the first 10 years after liver transplantation in carriers of the risk haplotype versus non-carriers of the risk haplotype AGGTA. The incidence of NAS was almost doubled in the group of patients carrying the risk haplotype AGGTA (log-rank test p < 0.01) Association between bile transporter haplotypes and NAS In a univariate analysis, we found no relationship between any of the selected individual SNPs in the ABCB11, ABCB4, or ABCC2 gene and the occurrence of NAS (data not shown). We next constructed different haplotypes of these genes, as is presented in Table 3. In 33 patients haplotypes could not be assigned, because the probability of a specific haplotype combination was below 90%. Haplotype analysis of the phospholipid transporter ABCB4 showed a strong correlation with NAS. The haplotype variant AGGTA was 2-times more frequent in patients who developed NAS, compared to patients who did not develop NAS (13.2% versus 6.7%, p=0.01). 130

132 Chapter 7 Analyses of haplotype variations of the ABCB11 and ABCC2 genes were not associated with the occurrence of NAS (data not shown). As shown in figure 1, the cumulative incidence of NAS in livers with the AGGTA risk haplotype was almost two-times higher than in livers that did not carry the AGGTA risk haplotype (28% versus 15%; p < 0.01). To determine whether the AGGTA haplotype of the ABCB4 gene is an independent risk factor for NAS, we next performed a multivariate Cox regression analysis. In this analysis we included all accepted clinical risk factors for NAS that have been described previously, including type of perfusion solution, cold ischemia time, warm ischemia time, indication for transplantation, gender match, as well as the risk haplotype AGGTA. In this multivariate Cox regression model, the donor AGGTA haplotype of the ABCB4 gene was independently associated with NAS (HR=2.23, 95% CI= ; p=0.004). 131

133 ABCB4 gene polymorphism and NAS Table 2. Donor, Recipient, Surgical and Postoperative Variables of Liver Grafts (n=461)* Donor variables Age (years) 43 (30-51) Gender (male/female) 231 / 230 (50% / 50%) Recipient variables Age (years) 46 (35-54) Gender (male/female) 238 / 223 (52% / 48%) Disease Cholestatic disease 142 (31)% Parenchymal disease 205 (44)% Metabolic liver disease 54 (11)% Vascular liver disease 12 (3)% Acute liver failure 32 (7)% Liver tumor 5 (1)% Other 11 (2)% Child Pugh Classification (A / B / C) 72 / 189 / 200 (15% / 41% / 43%) Retransplantation 62 (13%) Surgical variables Preservation solution Low viscosity / High viscosity 23 / 438 (5% / 95%) Cold ischemia time (minutes)** 564 ( ) Warm ischemia time (minutes)# 54 (45-63) Revascularization time (minutes)## 96 (79-115) Bile duct reconstruction (duct-to-duct / Roux-Y) 381 / 73 (82% / 16%) Type of graft (whole / reduced size) 447 / 14 (97% / 3%) Postoperative variables ICU-length of stay (days) 4 (2-8) Acute rejection 150 (33%) * Continuous variables are presented as median and interquartile range, categorical variables as numbers with percentage. ** Cold ischemia time; between start cold perfusion in the donor and end of cold preservation of the liver graft # Warm ischemia time; between the end of cold ischemic preservation of the liver and portal vein reperfusion ## Revascularization time; between the end of cold ischemic preservation of the liver and arterial reperfusion 132

134 Chapter 7 Table 3. Haplotype frequencies among donor livers with and without NAS. ABCB11 NAS no-nas overall SNP1 SNP2 SNP3 SNP4 n=77 n=384 LR p-value A G A G 25,7% 23,9% 24,2% 0,20 0,66 G G A G 30,4% 31,1% 31,0% 0,01 0,91 A A A G 6,8% 8,8% 8,4% 0,65 0,42 G A A G 5,1% 6,7% 6,4% 0,52 0,47 A G G A 2,6% 1,2% 1,4% 1,39 0,24 A A G A 9,4% 11,9% 11,5% 0,71 0,40 G A G A 16,9% 13,7% 14,2% 0,86 0,35 Total * 97% 97% 97% ABCB4 NAS no-nas overall SNP1 SNP2 SNP3 SNP4 SNP5 n=77 n=384 LR p-value G A T C A 48,7% 48,8% 48,8% 0,14 0,71 G G T C A 19,3% 19,4% 19,4% 0,00 1,00 G A G C A 2,6% 3,2% 3,1% 0,09 0,76 A G T C A 1,5% 1,5% 1,5% -0,02 1,00 A G G T A 13,2% 6,7% 7,7% 6,46 0,01 A G G T C 4,6% 3,3% 3,5% 0,65 0,42 G A T C C 2,5% 5,0% 4,6% 1,83 0,18 G G G T A 3,3% 4,3% 4,1% 0,21 0,65 G G T T A 1,5% 1,9% 1,9% 0,04 0,84 Total * 97% 94% 95% ABCC2 NAS no-nas overall SNP1 SNP2 SNP3 SNP4 SNP5 n=77 n=384 LR p-value A T G C G 8,0% 13,7% 12,8% 3,58 0,06 A T G T G 30,0% 25,4% 26,2% 0,92 0,34 A T A C A 27,0% 19,1% 20,4% 3,49 0,06 T T G C G 5,0% 4,3% 4,4% 0,16 0,69 T T A C G 13,7% 15,3% 15,1% 0,24 0,63 T G A C G 16,0% 20,8% 20,0% 1,58 0,21 Total * 100% 99% 99% * Only haplotypes with a frequency > 1% are shown 133

135 ABCB4 gene polymorphism and NAS Discussion We have tested the hypothesis that genetic variability in hepatobiliary transporters in donor livers is associated with the development of NAS after transplantation. We have evaluated this in a large cohort of 461 liver transplant recipients. The most important finding in this study was a strong association between the phospholipid translocator ABCB4 genotype and the development of NAS after liver transplantation. A common haplotype in the ABCB4 gene was significantly more present in livers that developed NAS, compared to those that did not develop NAS. A multivariate Cox regression analysis confirmed that the risk haplotype of the ABCB4 gene is an independent risk factor for the development of NAS. We found no association between haplotypes of the ABCB11 and ABCC2 gene and the occurrence of NAS after liver transplantation. There is accumulating evidence from both clinical and experimental studies that altered bile composition with decreased phospholipid secretion may contribute to biliary injury and subsequent intrahepatic biliary strictures after liver transplantation (14,17,20). Although previous studies have suggested that changes in the expression of the phospholipids translocator ABCB4 may play a role in the reduced biliary excretion of biliary phospholipids after liver transplantation, the impact of a genetic predisposition has not been reported before (14,21). Although we did not perform bile analysis in this large cohort of patients, the observed association between the AGGTA ABCB4 haplotype strongly suggests that biliary phospholipid secretion in livers with this haplotype is reduced after liver transplantation. Reduced biliary secretion of phospholipids results in a lower biliary phospholipids-to-bile salt ratio, which has been associated with increased bile duct damage. Normally, the toxic effects of bile salts are prevented by the neutralization of bile salts by phospholipids through the formation of mixed micelles in bile. In case of relative excess of bile salts, either due to increased bile salt secretion or reduced secretion of phospholipids, free non-micellar bile salts may cause cholangiocyte injury, pericholangitis and periductal fibrosis (34,35). The role of diminished biliary phospholipids secretion and increased bile salt toxicity in the pathogenesis of bile duct injury has previously been demonstrated in an experimental study using a murine liver transplant model (17). In this study livers from Abcb4-/+ mice, expressing only 50% of the phospholipids translocator Abcb4, were transplanted into wild-type recipients. Although livers and bile ducts from Abcb4-/+ mice are phenotypically normal under normal circumstances, 134

136 Chapter 7 these livers developed severe injury of the intrahepatic bile ducts after transplantation. This finding indicates that, although a reduction of biliary phospholipid secretion of up to 50% alone does not result in bile duct injury, this may result in overt bile duct injury when a second insult is present, such as ischemia / reperfusion injury (17). This animal study suggest that the impact of cold ischemia and reperfusion on ABCB4 function specifically in genetically susceptible individuals with an ABCB4 genetic variation could contribute to bile duct injury following liver transplantation (36). Several genetic variations in hepatobiliary transporters have been linked to various types of cholestatic disorders and cholangiopathies (19,36). A large number of clinicall relevant SNPs of the ABCB4 gene have been reported in literature (22,24,26-28,37-39). For two reasons we found it not rational to analyse these individual SNPs in the current study. First of all, some of the reported gene variations have been linked to a known and permanent cholestatic phenotype, such as PFIC-3, and it is unlikely that patients with such a phenotype were selected as organ donor. Secondly, the prevelance of most of the individual SNPs reported in the literature is low, and it is unlikely that very rare SNPs could accountable for a complication such as NAS with an incidence of around 16%. In conclusion, in this large series of 461 liver transplant recipients, we established a strong association between donor ABCB4 haplotype and the development of NAS after liver transplantation. Livers with the AGGTA haplotype of the ABCB4 gene were found to have a two-times higher risk of developing NAS, compared to livers without this haplotype. These data contribute to the accumulating evidence that a (relative) reduction in biliary phospholipid secretion, resulting in the increased toxicity of bile salts, play an important role in the development of bile duct injury and NAS after liver transplantation, and that alterations in bile composition after transplantation may have part of its origin in the donor. 135

137 ABCB4 gene polymorphism and NAS Reference List 1. Buis CI, Hoekstra H, Verdonk RC, Porte RJ. Causes and consequences of ischemic-type biliary lesions after liver transplantation. J Hepatobiliary Pancreat Surg 2006;13: Buis CI, Verdonk RC, Van der Jagt EJ, van der Hilst CS, Slooff MJ, Haagsma EB, et al. Nonanastomotic biliary strictures after liver transplantation, part 1: Radiological features and risk factors for early vs. late presentation. Liver Transpl 2007;13: Verdonk RC, Buis CI, Van der Jagt EJ, Gouw AS, Limburg AJ, Slooff MJ, et al. Nonanastomotic biliary strictures after liver transplantation, part 2: Management, outcome, and risk factors for disease progression. Liver Transpl 2007;13: Verdonk RC, Buis CI, Porte RJ, Haagsma EB. Biliary complications after liver transplantation: a review. Scand J Gastroenterol Suppl 2006; Abt P, Crawford M, Desai N, Markmann J, Olthoff K, Shaked A. Liver transplantation from controlled non-heart- beating donors: an increased incidence of biliary complications. Transplantation 2003;75: Foley DP, Fernandez L, Leverson G, Chin LT, Kreiger N, Cooper JT, et al. Donation After Cardiac Death: The University of Wisconsin Experience With Liver Transplantation. Ann.Surg. 242, Guichelaar MM, Benson JT, Malinchoc M, Krom RA, Wiesner RH, Charlton MR. Risk factors for and clinical course of non-anastomotic biliary strictures after liver transplantation. Am J Transplant 2003;3: Li S, Stratta RJ, Langnas AN, Wood RP, Marujo W, Shaw BW, Jr. Diffuse biliary tract injury after orthotopic liver transplantation. Am J Surg 1992;164: Moench C, Moench K, Lohse AW, Thies J, Otto G. Prevention of ischemic-type biliary lesions by arterial back- table pressure perfusion. Liver Transpl 2003;9: Pirenne J, Van Gelder F, Coosemans W, Aerts R, Gunson B, Koshiba T, et al. Type of donor aortic preservation solution and not cold ischemia time is a major determinant of biliary strictures after liver transplantation. Liver Transpl 2001;7: Sankary HN, McChesney L, Frye E, Cohn S, Foster P, Williams J. A simple modification in operative technique can reduce the incidence of nonanastomotic biliary strictures after orthotopic liver transplantation. Hepatology 1995;21: Moench C, Uhrig A, Lohse AW, Otto G. CC chemokine receptor 5delta32 polymorphism-a risk factor for ischemic-type biliary lesions following orthotopic liver transplantation. Liver Transpl 2004;10: Sanchez-Urdazpal L, Sterioff S, Janes C, Schwerman L, Rosen C, Krom RA. Increased bile duct complications in ABO incompatible liver transplant recipients. Transplant Proc 1991;23(1 Pt 2):

138 Chapter Geuken E, Visser D, Kuipers F, Blokzijl H, Leuvenink HG, de Jong KP, et al. Rapid increase of bile salt secretion is associated with bile duct injury after human liver transplantation. J Hepatol 2004;41: Hertl M, Harvey PR, Swanson PE, West DD, Howard TK, Shenoy S, et al. Evidence of preservation injury to bile ducts by bile salts in the pig and its prevention by infusions of hydrophilic bile salts. Hepatology 1995;21: Hertl M, Hertl MC, Kluth D, Broelsch CE. Hydrophilic bile salts protect bile duct epithelium during cold preservation: a scanning electron microscopy study. Liver transplantation 2000;6: Hoekstra H, Porte RJ, Tian Y, Jochum W, Stieger B, Moritz W, et al. Bile salt toxicity aggravates cold ischemic injury of bile ducts after liver transplantation in Mdr2+/- mice. Hepatology ;43: Palmeira CM, Rolo AP. Mitochondrially-mediated toxicity of bile acids. Toxicology 2004 ;203:1-15. De Vree JM, Jacquemin E, Sturm E, Cresteil D, Bosma PJ, Aten J, et al. Mutations in the MDR3 gene cause progressive familial intrahepatic cholestasis. Proc Natl Acad Sci U S A ;95: Buis CI, Geuken E, Visser D, Kuipers F, Haagsma EB, Verkade HJ, et al. Altered bile composition after liver transplantation is associated with the development of non-anastomotic biliary strictures. J Hepatol. In press Yska MJ, Buis CI, Monbaliu D, Schuurs TA, Gouw AS, Kahmann ON, et al. The role of bile salt toxicity in the pathogenesis of bile duct injury after non-heart-beating porcine liver transplantation. Transplantation ;85: Jacquemin E, De Vree JM, Cresteil D, Sokal EM, Sturm E, Dumont M, et al. The wide spectrum of multidrug resistance 3 deficiency: from neonatal cholestasis to cirrhosis of adulthood. Gastroenterology 2001;120: Deleuze JF, Jacquemin E, Dubuisson C, Cresteil D, Dumont M, Erlinger S, et al. Defect of multidrug-resistance 3 gene expression in a subtype of progressive familial intrahepatic cholestasis. Hepatology 1996;23: Jacquemin E. Heterozygous non-sense mutation of the MDR3 gene in familial intrahepatic cholestasis of pregnancy. The lancet 1999;353: Dixon PH, Weerasekera N, Linton KJ, Donaldson O, Chambers J, Egginton E, et al. Heterozygous MDR3 missense mutation associated with intrahepatic cholestasis of pregnancy: evidence for a defect in protein trafficking. Hum Mol Genet ;9: Gendrot C, Bacq Y, Brechot MC, Lansac J, Andres C. A second heterozygous MDR3 nonsense mutation associated with intrahepatic cholestasis of pregnancy. J Med Genet 2003;40:e Lucena JF, Herrero JI, Quiroga J, Sangro B, Garcia-Foncillas J, Zabalegui N, et al. A multidrug resistance 3 gene mutation causing cholelithiasis, cholestasis of pregnancy, and adulthood biliary cirrhosis. Gastroenterology 2003;124:

139 ABCB4 gene polymorphism and NAS 28. Pauli-Magnus C, Lang T, Meier Y, Zodan-Marin T, Jung D, Breymann C, et al. Sequence analysis of bile salt export pump (ABCB11) and multidrug resistance p-glycoprotein 3 (ABCB4, MDR3) in patients with intrahepatic cholestasis of pregnancy. Pharmacogenetics 2004;14: van Mil SW, van der Woerd WL, van der Brugge G, Sturm E, Jansen PL, Bull LN, et al. Benign recurrent intrahepatic cholestasis type 2 is caused by mutations in ABCB11. Gastroenterology 2004;127: Strautnieks SS, Bull LN, Knisely AS, Kocoshis SA, Dahl N, Arnell H, et al. A gene encoding a liver-specific ABC transporter is mutated in progressive familial intrahepatic cholestasis. Nat Genet 1998;20: Noe J, Kullak-Ublick GA, Jochum W, Stieger B, Kerb R, Haberl M, et al. Impaired expression and function of the bile salt export pump due to three novel ABCB11 mutations in intrahepatic cholestasis. J Hepatol 2005;43: Toh S. Genomic structure of the canalicular multispecific organic anion-transporter gene (MRP2/cMOAT) and mutations in the ATP-binding-cassette region in Dubin-Johnson syndrome. American journal of human genetics 1999;64: Verdonk RC, Buis CI, Porte RJ, Van der Jagt EJ, Limburg AJ, van den Berg AP, et al. Anastomotic biliary strictures after liver transplantation: Causes and consequences. Liver Transpl 2006;12: Trauner M, Meier PJ, Boyer JL. Molecular pathogenesis of cholestasis. N Engl J Med ;339: Arrese M, Trauner M. Molecular aspects of bile formation and cholestasis. Trends Mol Med 2003;9: Trauner M, Fickert P, Wagner M. MDR3 (ABCB4) defects: a paradigm for the genetics of adult cholestatic syndromes. Semin Liver Dis 2007;27: Rosmorduc O, Hermelin B, Poupon R. MDR3 gene defect in adults with symptomatic intrahepatic and gallbladder cholesterol cholelithiasis. Gastroenterology 2001;120: Eloranta ML. Association of single nucleotide polymorphisms of the bile salt export pump gene with intrahepatic cholestasis of pregnancy. Scandinavian journal of gastroenterology 2003;38: Pauli-Magnus C, Kerb R, Fattinger K, Lang T, Anwald B, Kullak-Ublick GA, et al. BSEP and MDR3 haplotype structure in healthy Caucasians, primary biliary cirrhosis and primary sclerosing cholangitis. Hepatology 2004;39:

140

141

142 Part III HO-1 and hepatobiliary injury after liver transplantation

143

144 8 Expression of Heme oxygenase -1 in human livers before transplantation correlates with graft injury and function after transplantation Am J Transplant. 2005; 5: Erwin Geuken Carlijn I Buis Dorien S Visser Hans Blokzijl Han Moshage Balazs Nemes Henri GD Leuvenink Koert P de Jong Paul MJG Peeters Maarten JH Slooff Robert J Porte

145 HO-1 before transplantation and graft injury and function after transplantation Abstract Upregulation of heme oxygenase-1 (HO-1) has been proposed as an adaptive mechanism protecting against ischemia/reperfusion (I/R) injury. We investigated HO-1 expression in 38 human liver transplants and correlated this with I/R injury and graft function. Before transplantation, median HO-1 mrna levels were 3.4-fold higher (range ) than in normal controls. Based on the median value, livers were divided into two groups: low and high HO-1 expression. These groups had similar donor characteristics, donor serum transaminases, cold ischemia time, HSP-70 expression, and distribution of HO-1 promoter polymorphism. After reperfusion, HO-1 expression increased significantly further in the initial low HO-1 expression group, but not in the high HO-1 group. Postoperatively, serum transaminases were significantly lower and bile salt secretion was higher in the initial low HO-1 group, compared to high expression group. Immunofluorescence staining identified Kupffer cells as the main localization of HO-1. In conclusion, human livers with initial low HO-1 expression (< 3.4 times controls) are able to induce HO-1 further during reperfusion and this is associated with less injury and better function than initial high HO-1 expression (> 3.4 times controls). These data suggest that increase in HO-1 during transplantation is more protective than a high HO-1 expression before transplantation. 144

146 Chapter 8 Introduction Orthotopic liver transplantation (OLT) is an effective treatment for end-stage liver diseases (1). However, ischemia and subsequent reperfusion of the liver remain a major cause of graft injury, causing liver dysfunction and even failure after transplantation (2). This is particularly true for livers from older donors and steatotic livers, which have a higher susceptibility to ischemia/ reperfusion (I/R) injury (3,4). During organ procurement and transplantation, the liver is exposed to oxidative stress. Besides the ischemia during cold storage, hypoxia may occur before or during procurement due to hypotension or cardiac arrest in the donor. After graft reperfusion, several cascades are triggered leading to the formation of reactive oxygen species (ROS), which are well-known sources of oxidative stress. Methods to protect liver grafts against I/R injury have considerable clinical consequences and are therefore of great interest. It is increasingly recognized that cells respond to stressful events, such as ischemia, hypoxia and ROS, by the activation of various cytoprotective genes and pathways. Heme oxygenase-1 (HO-1) has recently been proposed as a graft survival gene (5,6). Up-regulation of HO-1 is considered to be one of the most critical cellular protection mechanisms (7,8). It is rapidly induced under various conditions of oxidative stress, including hypoxia, hyperoxia and ROS (9). HO-1 catalyzes the rate-limiting step in the oxidative detoxification of excess heme, by cleaving the a-methene bridge into equimolar amounts of free iron, biliverdin and carbon monoxide (CO) (9). Free iron, catalyzing oxidative reactions, is bound by iron regulatory proteins that stimulate synthesis of ferritin, thereby preventing iron-dependent oxidative stress (10,11). Biliverdin is subsequently converted into bilirubin and both have the ability to scavenge ROS (12-15). CO has been shown to serve as an endogenous regulator for maintaining microvascular blood flow of the liver (16,17). Two- to three-fold induction of HO-1 by pharmacologic agents or genetic engineering has been shown to reduce I/R injury in rat liver grafts after extended cold ischemia time (6). Moreover, steatotic livers from genetically obese Zucker rats are markedly protected against I/R injury after exogenous upregulation of HO-1 (5). Based on these observations, exogenous induction of HO-1 prior to transplantation has been proposed as a potentially powerful therapeutic option to protect liver grafts against I/R injury (5,6). Molecules such as HO-1, however, are probably not exclusively cytoprotective and each of the products generated by the action of heme oxygenase (Fe 2+, bilirubin and CO) can cause injury under certain circumstances (18). Indeed, 145

147 HO-1 before transplantation and graft injury and function after transplantation several experimental studies have shown that excessive overexpression of HO-1 is directly related with increased injury (19-21). Recently, also a (GT) n dinucleotide repeat polymorphism that modulates the level of HO-1 inducibility was identified in the promoter region of the human HO-1 gene. Short GT repeats (<25) are associated with highly significant upregulation of HO-1 in response to inflammatory stimuli (22,23). Therefore, it is critically important to understand the endogenous changes in HO-1 expression under clinical conditions, such as transplantation, before the exogenous induction of HO-1 can be safely attempted as a possible therapeutic or prophylactic measure to reduce I/R injury. We have therefore studied the changes in endogenous HO-1 expression in human liver grafts before and after transplantation and correlated these with biochemical markers of graft injury and hepatobiliary function. This study provides important new information on the role of endogenous HO-1 expression during human liver transplantation Patients and Methods Patient and Donor Data Thirty-eight patients undergoing OLT were included. All patients received livers from brain death, multi-organ donors. In the control group (n=5), biopsies were collected in patients undergoing partial hepatectomy for metastatic tumors. Tissue and data collection was performed according to the guidelines of the medical ethical committee of our institution and the Dutch Federation of Scientific Societies. Collection of Liver Biopsies and Bile Samples from Recipients Three sequential needle biopsies were taken from each liver graft: at the end of cold storage, 3 hours after reperfusion and 1 week after transplantation. Biopsies were immediately divided: one part was snap-frozen in liquid nitrogen for RNA and protein isolation and one part was frozen in isopentane at -80 C for histology studies. During transplantation a bile drain was routinely placed into the common bile duct, allowing collection of bile (24). To avoid interruption of the entero-hepatic circulation bile was daily readministered via a jejunostomy catheter. After the transplantation, bile samples were collected daily between 8 and 9 am. Liver and bile specimens were stored at -80 C. 146

148 Chapter 8 RNA Isolation and Reverse-Transcriptase Polymerase Chain Reaction Total RNA was isolated from liver biopsies using TRIzol (Invitrogen Life Technologies, Breda, the Netherlands) and quantified using Ribogreen (Molecular Probes, Inc., Eugene, OR). Reverse transcription was performed on 3.36 μg RNA using random primers in a final volume of 75 μl (Reverse Transcription System, Promega, Madison, WI). For quantitative real-time detection RT-PCR (25,26), sense and antisense primers (Invitrogen, Paisley, Scotland) and fluorogenic probes (Eurogentec, Herstal, Belgium) for HO-1, HSP-70 and 18S were designed using Primer Express software (PE Applied Biosystems, Foster City, CA). For HO-1, the primers and probe used were 5 -GACTGCGTTCCTGCTCAACAT-3 (sense), 5 -GCTCTGGTCCTTGGTGTCATG-3 (antisense), and 5 -TCAGCAGCTCCTGCAACTCCTCAAAGAG-3 (probe), generating a 75 base pair PCR product. For heat shock protein-70 (HSP-70), used as a molecular stress marker, the following primers and probe were used: 5 -TCTTCTCGCGGATCCAGTCT-3 (sense), 5 -GGTTCCCTGCTCTCTGTCG-3 (antisense), and 5 -CCGTTTCCAGCCCCCAATCTCAG-3 (probe), generating a 70 base pair PCR product. For 18S, the primers and probe used were 5 -CGGCTACCACATCCAAGG-3 (sense), 5 -CCAATTACAGGGCCTCGAAA-3 (antisense), and 5 -CGCGCAAATTACCCACTCCCGA-3 (probe), generating a 109-base pair PCR fragment. The ABI PRISM 7700 (Applied Biosystems, Foster City, CA) was used for PCR. Protein Isolation and Western Blot Analysis Frozen liver tissue was homogenized in buffer containing protease inhibitors. Protein concentrations were measured using a standard Lowry assay. Fifteen μg of protein was fractioned on a 5% SDS-PAGE gel and transferred to PVDF membranes (Pall Life Sciences, Ann Arbor, MI). The membranes were blocked with 1% SKIM milk (Fluka BioChemica, Buchs, Switzerland) and labeled with the anti HO-1 polyclonal antibody (dilution, 1:5000, StressGen, Victoria, British Columbia, Canada). After washing in PBS/0.05% Tween-20 (Sigma, Malden, The Netherlands), blots were incubated with a horseradish peroxidase-labled goat anti-rabbit IgG (dilution, 1:2000, DAKO, Glostrup, Denmark). Finally membranes were developed with ECL (Amersham, Chalfont St Giles, UK). Five separate cases were examined in each group. 147

149 HO-1 before transplantation and graft injury and function after transplantation HO-1 Genotype assessment Genomic DNA was isolated from donor splenocytes using a commercial kit (Gentra Systems, Minneapolis, MN). PCR and genotyping procedures were similar as described by de Jong et al. (27). The 5 -flanking region of the HO-1 gene containing the poly (GT) n repeat was amplified by PCR using as forward primer 5 -CAGCTTTCTGGAACCTTCTGG-3, carrying a 6-FAM flourescent label (Sigma, Malden, the Netherlands), and as reversed primer 5 -GAAACAAAGTCTGGCCATAGGAC-3. Sequence analysis of the amplification products of individuals homozygous for the 222 and 229 basepairs alleles showed correspondence with GT numbers 26 and 29, respectively (results not shown). We divided allelic repeats into two subclasses using a classification as previously described in transfection studies (28). Short repeats, with less than 25 GT repeats (amplicons of 220 basepairs and less), were designated as allele class S (short), and long repeats with 25 or more GT repeats as allele class L (long). Recipients of class S allele liver transplants (homozygous S/S and heterozygous S/L) were compared with recipients of non-class S allele transplants (L/L). Immunofluorescence Microscopy Frozen liver sections were stained for HO-1 and the Kupffer cell marker CD68, using an anti-ho-1 polyclonal antibody (dilution, 1:100, StressGen) and an anti-human CD68 monoclonal antibody KP-1 (dilution, 1:2000, DAKO). After washing, sections were subsequently incubated with a goat anti-rabbit IgG with a red fluorescent label (Alexa Fluor 568, Molecular Probes, Leiden, the Netherlands), and with a goat anti-mouse IgG with a green fluorescent label (Alexa Fluor 488, Molecular Probes). Double-positive cells were identified as those stained yellow. Percentages of HO-1-positive Kupffer cells were calculated by dividing the number of cells stained yellow by the number of cells stained green (29). Five different high power fields (x400) were analyzed in an individual liver sample, and five separate cases were examined in each group. Images were taken with a Leica DM LB fluorescence microscope (Leica, Wetzlar, Germany). Total Bile Salt Secretion and Serum Biochemistry Postoperatively, bile flow was expressed as daily bile production in ml per kg body weight of the donor. Total bile salt concentration was measured spectrophotometrically with 3a-hydroxysteroid dehydrogenase (30). Serum samples were analyzed for aspartate- and alanine aminotransferase (AST and ALT) and gamma glutamyltransferase (GGT), by routine clinical chemistry testing. 148

150 Chapter 8 Statistics Statistical analyses were performed using SPSS Version 11.5 for Windows (SPSS Inc., Chicago, IL). All data are reported as median and interquartile ranges (IQR). Groups were compared with the Mann-Whitney U-tests, Wilcoxon Signed Ranks-tests, Pearson X2-tests and the Fisher s Exact Test where appropriate. Postoperative biochemical variables were compared using the daily values, but also the total course during the first week was compared by calculating the area under the curve (AUC), using the trapezium rule. All P values were 2-tailed and considered as statistically significant at levels < Results Effects of OLT on HO-1 Gene and Protein Expression. Before transplantation, the median HO-1 mrna level was 3.4-times higher in donor livers than in normal control livers (P = 0.001; Figure 1), suggesting that HO-1 is already induced in brain-death donors or during organ procurement. P = P = P = Relative HO O-1 mrna levels control livers Before OLT 3 hours after reperfusion 1 week after OLT liver grafts Figure 1. HO-1 mrna levels in human liver grafts (n=38) and normal control livers (n=5). HO-1 mrna was standardized for 18S rrna. HO-1 expression in control livers was set to 1.0. Values represent medians and interquartile ranges. 149

151 HO-1 before transplantation and graft injury and function after transplantation At 3 hours after reperfusion, there was no significant overall change in HO-1 expression. One week after transplantation, HO-1 gene expression decreased by 38% compared to the values after reperfusion (P = 0.002; Figure 1). However, HO-1 expression remained strongly elevated during the first postoperative week compared to normal control livers (Figure 1). A P = P = Initial low HO-1 expression Initial high HO-1 expression P = P = mrna levels Relative HO P = P = 0.03 control livers before OLT 3 hours after reperfusion before OLT 3 hours after reperfusion liver grafts B HO-1 protein (32 kd) Figure 2. A) Course of HO-1 mrna levels in human liver grafts with low or high HO-1 expression before transplantation; initial low HO-1 expression group (n=19) and initial high HO-1 expression group (n=19), respectively. HO-1 mrna was standardized for 18S rrna. HO-1 mrna levels in normal control livers was set to 1.0. Values represent medians and interquartile ranges. B) Western blot analysis of HO-1 protein expression in the initial low and initial high HO-1 group. 150

152 Chapter 8 A wide variation in HO-1 gene expression was detected in liver biopsies that were collected before transplantation, ranging from 0.7- to 9.3-times the levels in normal control livers. To be able to identify donor variables that are associated with HO-1 induction, and to study the possible impact of HO-1 on I/R injury and graft viability after transplantation, we decided to divide liver grafts into two groups based on the level of HO-1 expression before transplantation. A low HO-1 expression group (n=19) was formed by livers with an initial HO-1 mrna level below the median value (< 3.4-times control levels) and a high HO-1 expression group (n=19) was formed by livers with an initial HO-1 gene expression above the median value (> 3.4-times control levels). Median HO-1 expression in the low and high expression group was 2.0- and 5.0-times higher than in control livers (Figure 2A). Interestingly, HO-1 mrna level increased significantly by 43% after reperfusion in the initial low expression group, whereas HO-1 expression decreased by 23% after reperfusion in the inital high expression group (Figure 2A). In both groups, HO-1 gene expression remained significantly elevated during the first postoperative week, compared to controls (data not shown). Changes in HO-1 protein concentrations, as detected by Western blot analysis, were similar to the changes in HO-1 mrna expression. HO-1 protein concentration was low in normal control livers, compared to the donor livers. After reperfusion, HO-1 protein expression increased further in the initial low HO-1 expression group, but not in the initial high HO-1 group (Figure 2B). Comparison of Donor Data for Livers with Low and High HO-1 Expression A large number of donor characteristics and laboratory values were investigated in an attempt to explain the differences in HO-1 gene expression before transplantation. Several events that are known to induce HO-1 expression in animal models, such as hypotension, cardiac arrest, blood transfusions and ischemia, may also occur in brain-death donors or during organ procurement. In addition to this, some drugs (i.e. dopamine) have been shown to induce HO-1 expression (31). We have compared all these donor-related events and variables in the two groups, but were unable to find statistically significant differences (Table 1). 151

153 HO-1 before transplantation and graft injury and function after transplantation Table 1. Comparison of donor, recipient and surgical variables in initial low HO-1 expression group 152 and initial high HO-1 expression group. Low HO-1 Expression High HO-1 Expression Donor variables Age (years; median [IQR]) 39 (25-60) 48 (41-58) Gender (M/F) 7/12 8/11 ICU stay (days; median [IQR]) 2.5 ( ) 1.2 ( ) Duration of liver procurement 150 (51-177) 150 (67-195) (minutes; median [IQR]) Hypotension (no. of donors)a 7/19 11/19 Cardiac arrest (no. of donors)b 2/19 3/19 Dopamine (no. of donors)c 8/19 11/19 Bloodtransfusion (no. of donors)c 5/19 7/19 Temperature (oc; median [IQR]) 36.1 ( ) 36.5 ( ) Diuresis last hour (ml; median [IQR]) 220 ( ) 200 ( ) Bloodpressure (mmhg; median [IQR]) 120/60 (110/60-124/73) 120/67 (110/65-137/78) po2 (kpa; median [IQR]) 16.5 ( ) 13.6 ( ) FiO2 (%; median [IQR]) 40 (36-47) 40 (40-57) AST (U.L-1; median [IQR]) 27 (15-93) 42 (19-67) ALT (U.L-1; median [IQR]) 24 (18-61) 25 (14-45) GGT (U.L-1; median [IQR]) 20 (15-29) 20 (13-63) Total Bilirubin (U.L-1; median [IQR]) 4.0 ( ) 10.0 ( ) Hemoglobin (mmol.l-1; median [IQR]) 7.6 ( ) 7.0 ( ) Recipient and Surgical variables Age (years; median [IQR]) 45 (28-58) 47 (35-54) Gender (M/F) 9/10 13/6 ICU stay (days; median [IQR]) 3 (2-6) 2 (2-7) Acute rejection of the graft (no. of recipients)d 11/19 4/19 1st Warm Ischemia Time, WIT 43 (36-57) 42 (28-49) (minutes; median [IQR])e Cold Ischemia Time, CIT 465 ( ) 574 ( ) (minutes; median [IQR]) 2nd WIT (minutes; median [IQR])f 43 (37-47) 48 (43-56) a) Donors who suffered at least one episode of hypotension or b) cardiac arrest within 24 hrs prior to procurement of the liver. c) Number of donors who were administered dopamine or blood within 24 hrs before donor hepatectomy. d) Number of recipients who suffered from rejection of the graft within the first week after transplantation. e) 1st WIT: time between start cold perfusion in the donor and procurement of the liver graft. f) 2nd WIT: time between the end of cold ischemic preservation of the liver and start of reperfusion in the recipient. There were no statistical significant differences for any variables between the two groups (Mann Whitney U-test or Pearson Chi-Square-test).

154 Chapter 8 There were also no significant differences in the time between start of in situ cold perfusion in the donor and actual hepatectomy (1st relatively warm ischemia) or in the duration of cold storage (Table 1). Interestingly, there were also no differences in donor serum markers of liver injury (AST, ALT and GGT) or liver function (bilirubin) between the two groups (Table 1). Moreover, there was no significant difference in pretransplant mrna expression of the stress protein HSP-70 in the low and high HO-1 group (1.18 [ IQR ] versus 0.57 [IQR ]; p = 0.44). These data suggest that differences in HO-1 expression in liver grafts before transplantation cannot simply be explained by a higher number of compromised donors in the high HO-1 expression group. The Effect of HO-1 Donor Genotype To examine whether the differences in initial HO-1 expression could be explained by the the number of (GT) n repeats in the HO-1 promoter region, HO-1 donor genotypes were analyzed. Allele class S/S was present in 8% of the donors, 35% of the donors were heterozygous for class S alleles (S/L), and 57% of the donors were non-carriers of the class S allele (L/L). Distribution of the numbers of (GT) n repeats was not different for donor livers in the initial low and high HO-1 expression group (Figure 3). There were also no significant differences in the distribution of class S allele donor livers (S/S and S/L) and non-class S donor livers (L/L) in the two groups (Table 2). Table 2. Distribution of HO-1 genotype in the livers with initial low or high HO-1 mrna expression. Initial HO-1 Expression Genotype* Low High Short Allele (SS or SL) 8 (42%) 8 (44%) Long Allele (LL) 11 (58%) 10 (56%) p-value = 1 19 (100%) 18** (100%) a) Short allele (S) status defined as < 25 (GT) repeats in the HO-1 promoter region; Long allele (L) status defined as > 25 (GT) repeats in the HO-1 promter region. b) Genomic DNA for gene sequencing was not available in one donor. 153

155 HO-1 before transplantation and graft injury and function after transplantation Allele Freque ency (%) Initial low HO-1 expression Initial high HO-1 expression Number of (GT) n Repeats Figure 3. Allele frequencies of the HO-1 (GT) n repeat promoter polymorphism in liver grafts with initial low or high HO-1 mrna expression. Post-transplant Outcome in Relation to HO-1 Expression To examine whether the magnitude of HO-1 induction was associated with differences in outcome after transplantation, laboratory values and recipient characteristics were analyzed. Posttransplant serum levels of AST and ALT were used as well-accepted markers of I/R injury. Although there were no differences in serum AST levels in the donors, we found a significant positive correlation between serum AST levels in the recipient on postoperative day 1 and HO-1 expression in the donor liver before transplantation (Figure 4). When comparing the two groups, serum AST levels on postoperative days 1 through 3 were significantly higher in recipients of livers with high HO-1 expression (Figure 5A). Also serum ALT levels were significantly higher on postoperative day 1 in recipients of livers with high HO-1 expression (Figure 5B). Hepatobiliary function, as reflected by biliary bile salt secretion, was significantly worse in the group with high HO-1 expression, compared to the group with low expression (Figure 5C). When groups were categorized based on the ability of increasing HO-1 expression during reperfusion of the liver graft, serum AST levels in the induction group (n=15) were significantly lower on postoperative days 2 and 3 than in the HO-1 reduction group (n=23). Serum ALT levels and biliary bile salt secretion however, did nof differ between the groups in the latter classification (data not shown). 154

156 Chapter 8 Serum AST level on POD R 2 = 0.15 P = mrna HO-1 before transplantation Figure 4. Correlation between hepatic HO-1 mrna expression before transplantation and serum AST level on postoperative day 1 (POD 1) in all liver transplant recipients (n=38). These findings indicate that liver grafts with an initial high (> 3.4-fold) HO-1 expression before transplantation exhibited more I/R injury and have poorer hepatobiliary function after transplantation than grafts with an initial low (< 3.4-fold) HO-1 expression, despite the fact that there were no differences in biochemical or molecular markers of graft injury in the donor before organ procurement. 155

157 HO-1 before transplantation and graft injury and function after transplantation A AST (U/L) * * * AUC P < B C ALT (U/L) Postoperative days * AUC P < Postoperative days Biliary Bile Salt Output (µmol day -1 kg -1 ) AUC P < Postoperative days Figure 5. Serum AST (panel A) and ALT (panel B) levels and biliary bile salt secretion (panel C) in the first week after OLT in the initial low (open bars; n=19) and high HO-1 (closed bars; n=19) expression groups. Values represent medians and interquartile ranges. The asterisks indicate significant differences between the groups (p<0.05). Total course during the first week was calculated as the area under the curve (AUC), using the trapezium rule. 156

158 Chapter 8 Immunofluorescence Microscopy Specific immunostaining showed that HO-1 was predominantly localized in irregular and star-shaped cells. These characteristics suggested that HO-1 protein is mainly expressed in Kupffer cells, which was confirmed by double-color immunofluorescence labeling, using the anti-ho-1 and anti-human CD68 MoAb KP-1, a marker of Kupffer cells. As shown in Figure 6, the distribution of anti-ho-1 positive (red) cells overlapped with that of KP-1-positive (green) cells, resulting in a yellow staining. In control livers, a considerable proportion of Kupffer cells did not express HO-1-associated immunoreactivity and displayed mainly a green staining (Figure 6A). In contrast with this, almost all Kupffer cells in liver grafts demonstrated positive staining for HO-1 (Figure 6B-E). Indeed, morphometrical analysis showed significantly higher percentages of HO-1-positive Kupffer cells in liver grafts before transplantation, compared to normal control livers (low and high HO-1 expression group 88% and 95%, respectively, compared to 50% in normal control livers, P < 0.02 for both groups; Table 3). After reperfusion, HO-1 expression in Kupffer cells increased further, resulting in a positive staining of all Kupffer cells in both groups (Table 3). Although, after reperfusion, all Kupffer cells in both groups stained positive for HO-1, the red staining (HO-1) per cell was far more intense in the group with high HO-1 expression than in the low expression group (Figure 6C and E). This indicates that not only the percentage of Kupffer cells expressing HO-1 is increased in liver grafts, but that also the HO-1 protein expression per Kupffer cell is enhanced, where the latter seems to discriminate the group with high HO-1 expression from the livers with low HO-1 expression. This is in line with the higher HO-1 mrna and protein levels after reperfusion in the group with high HO-1 expression, compared to the low expression group. 157

159 HO-1 before transplantation and graft injury and function after transplantation A. Normal control liver B. Initial low HO-1 expression: before OLT C. Initial low HO-1 expression: after reperfusion D. Initial high HO-1 expression: before OLT E. Initial high HO-1 expression: after reperfusion Figure 6. Immunofluorescence double-staining of liver biopsies. Sections are stained for HO-1 (red) and the Kupffer cell marker CD68 (green). Colocalization of these two colours can be recognized by the yellow colour. Panel A; normal control liver. Panel B; pretransplant biopsy of a liver with low initial HO-1 mrna expression. Panel C; postreperfusion (3hrs) biopsy of a liver with low initial HO-1 mrna expression. Panel D; pretransplant biopsy of a liver with high initial HO-1 mrna expression. Panel E; postreperfusion biopsy (3hrs) of a liver with high initial HO-1 mrna expression. 158

160 Chapter 8 Table 3. Morphometrical analysis of cell type specific expression of HO-1 in human liver transplants with low or high HO-1 expression and control livers. Control Initial Low HO-1 Expression Initial High HO-1 Expression Before OLT After Reperfusion Before OLT After Reperfusion Single immunostaining HO-1(+) (no. of cells; median [IQR]) a 20 [17-23] 31 [27-47] e 27 [17-36] e 40 [37-44] e,f 37 [27-44] e CD68(+) (no. of cells; median [IQR]) b 37 [35-43] 31 [25-40] 30 [23-36] 42 [40-44] f 36 [33-43] g Double immunostaining HO-1(+) Kupffer cells (no. of cells; median [IQR]) c 20 [17-23] 28 [23-35] 23 [13-30] 40 [37-43] f 35 [30-43] g % HO-1(+) Kupffer cells (%; median [IQR]) d 50 [45-63] 88 [78-99] e 100 [40-100] e 95 [93-100] e 100 [88-100] e a) Number of HO-1 and b) CD68 positive cells. c) Number and d) percentage of HO-1 positive Kupffer cells. Analyses based on observations in five different high power fields within one liver biopsy at 400X e) P < 0.02, compared with the control group f) P < 0.03, compared with the values before OLT of the initial low expression group g) P < 0.01, compared with the values after reperfusion of the initial low expression group 159

161 HO-1 before transplantation and graft injury and function after transplantation Discussion We have investigated HO-1 expression in human liver allografts during transplantation and correlated this with clinical signs of graft injury and hepatobiliary function. There are three novel findings in this study. First, we have shown that, compared to normal control livers, HO-1 gene and protein expression in human liver grafts from brain-death donors is induced already prior to transplantation. After reperfusion, HO-1 expression increased further in livers with relatively low initial HO-1 expression (< 3.4 times controls), but not in livers with initial high HO-1 expression (> 3.4 times controls). Second, allografts with initial high expression of HO-1 demonstrated significantly more I/R injury and had worse hepatobiliary function than grafts with a low upregulation of HO-1. Finally, we were able to identify Kupffer cells as the main site of HO-1 protein expression in human liver grafts. While about 50% of the Kupffer cells in normal control liver expressed HO-1, positive staining for HO-1 was found in 100% of the Kupffer cells of transplanted livers. These findings provide important new information on the endogenous regulation of HO-1 during human liver transplantation. There is accumulating evidence that the HO-1 system has important vasoregulatory properties and actively maintains hepatic microperfusion and tissue oxygenation via the production of CO (16). In addition to this, the HO-1 system has been shown to have anti-oxidant, antiinflammatory, anti-apoptotic and platelet aggregation-inhibiting properties and, therefore, it has been proposed a graft survival gene. Animal studies have suggested that exogenous induction of HO-1 before transplantation may confer cytoprotective and immune regulatory functions (6,32-34) and could become a novel and potentially powerful strategy to protect (marginal) liver grafts from I/R injury (5,8). Induction of HO-1 can be obtained by a variety of methods, such as administration of HO-1 inducers (i.e. cobalt protoporphyrin) or adenoviral HO-1 genetransfer (5,8). These methods generally lead to a 2 to 3-fold upregulation of HO-1 activity (5). There is increasing evidence that overexpression of HO-1 higher than this is not exclusively cytoprotective (19,21). In fibroblast cell cultures, low induction of HO-1 (less than 5-fold) was shown to be cytoprotective against hyperoxia, but excessive HO-1 activation resulted in the accumulation of free divalent iron and increased oxidative injury (19). Moreover, it has been shown that highly increased (about eight- to nine-fold) activity of HO-1 contributes to endotoxin-induced shock in rats, due to the increased production of CO, a potent vasorelaxant (21). Therefore, it is of paramount importance that the endogenous changes in 160

162 Chapter 8 HO-1 expression during transplantation, as well as the therapeutic window of protection, are well defined before clinical application of HO-1 inducing protocols are attempted. All donor livers in our study were obtained from brain-death multi-organ donors. The increased HO-1 mrna and protein expression observed in these livers before transplantation suggests that HO-1 is induced in brain-death donors. This observation is in line with studies in kidney allografts from brain-death donors (35). We have tried to identify variables which could have contributed to the increased expression of HO-1 in the donor livers before transplantation. Several factors have been shown to induce HO-1 gene expression in vivo, including hypotension (36), hypoxia (37-39), hyperoxia (9,40), blood transfusions (41,42), and inotropic drugs like dopamine (31). All of these factors may also occur in postmortem organ donors. Comparison of these known inducers of HO-1 gene expression, as well as several other donor and procurement related variables, however, did not show any statistically significant differences between the two groups. Variations in initial HO-1 expression could also not be explained by differences in the distribution of the (GT) n repeat polymorphism of the HO-1 promoter. The functionally relevant short allele status (<25 repeats) was not found more frequently in livers with initial low HO-1 expression. Further studies will be necessary to elucidate the mechanisms of endogenous HO-1 induction in organs from brain death donors. Although we did not find differences in biochemical (liver enzymes) or molecular (HSP-70) markers of liver injury before transplantation between the liver grafts with low or high HO-1 expression, we did observe a significant correlation between postoperative serum AST in the recipients and initial HO-1 expression. In parallel with this, serum AST levels were significantly higher and biliary bile salt output significantly lower after transplantation in recipients of livers with high HO-1 expression, compared to grafts with low HO-1 expression. Liberation of divalent iron is one of the effects resulting from increased HO-1 activity (9). Iron is a mediator of the generation of ROS and it has been shown to play an important role in I/R injury (43,44). We, therefore, speculate that exaggerated HO-1 activity in liver grafts may cause increased injury due to the liberation of iron, resulting in a pro-oxidant condition and higher susceptibility to I/R injury. The apparent paradox of one molecule or pathway causing both cytoprotection and cytotoxicicty has also been found in other systems, like the nitric oxide system (45). More studies will be needed to clarify this issue. Interestingly, a significant further increase in HO-1 expression was found after reperfusion of livers with an initially low expression, whereas a small, but significant decrease in HO-1 expression was 161

163 HO-1 before transplantation and graft injury and function after transplantation observed in livers with initially high HO-1 expression. This data could imply that HO-1 mrna expression cannot be further upregulated upon reperfusion when levels are already high to start with, whereas further upregulation can occur in livers with moderately elevated HO-1 expression before reperfusion. Although we observed a better postoperative outcome in the initial low HO-1 expression group, it remains indefinite whether it is the initial low HO-1 expression or the ability to increase HO-1 expression upon reperfusion that confers cytoprotection. We identified Kupffer cells as the main site of HO-1 expression in human livers. Makino et al. (29) have recently reported similar findings in human cirrhotic livers. These studies in human liver are in contrast with data from rat livers, where considerable expression of HO-1 has also been found in hepatocytes (46). While in our study about 50% of the Kupffer cells in the control livers expressed HO-1, this was more than 80% in the liver grafts before transplantation and even 100% after transplantation. These findings suggest that a subpopulation of Kupffer cells, which does not express HO-1 under normal circumstances may induce HO-1 expression. It has been suggested that Kupffer cells may serve as sensor cells detecting local hemodynamic changes and mechanical forces in sinusoids (29,47). By increasing HO-1 activity and the generation of the vasorelaxing gaseous CO, Kupffer cells are able to maintain microvascular blood flow in the liver (29). On the other hand, it is well-known that Kuppfer cells play a critical role in the pathogenesis of I/R injury of the cold preserved liver through the production of ROS and cytokines, like tumor necrosis factor-α (48,49). Our data suggests that high overexpression of HO-1 in Kupffer cells prior to transplantation contributes to the deleterious effects of these cells in I/R injury. Although there is a large body of evidence suggesting that exogenous up-regulation of HO-1 in transplant models in animals confers cytoprotective effects (5,32-34), our findings caution against an uncontrolled application of non-cell specific methods to induce HO-1 expression in human organ donors. Exogenous induction of HO-1 in postmortem organ donors could further increase an already elevated HO-1 expression, resulting in potentially detrimental effects instead of cytoprotection. The main difference between our study in patients undergoing liver transplantation and studies in animal models of liver transplantation is that in the clinical situation liver grafts are usually obtained from brain death organ donors, whereas healthy animals are used as donors in experimental models. Moreover, cellular localization of HO-1 expression in human liver transplantation is predominantly restricted to the Kupffer cells, whereas in stress-exposed rat livers, HO-1 is also upregulated in hepatocytes (46). 162

164 Chapter 8 Our data suggest a dual role for HO-1 in human liver transplants, with either cytoportection or increased cytotoxicity, depending on the initial level of overexpression. New pharmacological interventions should probably not focus on the induction of HO-1 prior to transplantation, but rather aim for induction during transplantation. 163

165 HO-1 before transplantation and graft injury and function after transplantation Reference List 1. Starzl TE, Demetris AJ. Liver transplantation: a 31-year perspective. Part III. Curr Probl Surg 1990; 27: Clavien PA, Harvey PR, Strasberg SM. Preservation and reperfusion injuries in liver allografts. An overview and synthesis of current studies. Transplantation 1992; 53: D Alessandro AM, Kalayoglu M, Sollinger HW, Hoffmann RM, Reed A, Knechtle SJ et al. The predictive value of donor liver biopsies for the development of primary nonfunction after orthotopic liver transplantation. Transplantation 1991; 51: Strasberg SM, Howard TK, Molmenti EP, Hertl M. Selecting the donor liver: risk factors for poor function after orthotopic liver transplantation. Hepatology 1994; 20: Amersi F, Buelow R, Kato H, Ke B, Coito AJ, Shen XD et al. Upregulation of heme oxygenase-1 protects genetically fat Zucker rat livers from ischemia/reperfusion injury. J Clin Invest 1999; 104: Redaelli CA, Tian YH, Schaffner T, Ledermann M, Baer HU, Dufour JF. Extended preservation of rat liver graft by induction of heme oxygenase-1. Hepatology 2002; 35: Fujita T, Toda K, Karimova A, Yan SF, Naka Y, Yet SF et al. Paradoxical rescue from ischemic lung injury by inhaled carbon monoxide driven by derepression of fibrinolysis. Nat Med 2001; 7: Coito AJ, Buelow R, Shen XD, Amersi F, Moore C, Volk HD et al. Heme oxygenase-1 gene transfer inhibits inducible nitric oxide synthase expression and protects genetically fat Zucker rat livers from ischemia-reperfusion injury. Transplantation 2002; 74: Maines MD. The heme oxygenase system: a regulator of second messenger gases. Annu Rev Pharmacol Toxicol 1997; 37: Vile GF, Tyrrell RM. Oxidative stress resulting from ultraviolet A irradiation of human skin fibroblasts leads to a heme oxygenase-dependent increase in ferritin. J Biol Chem 1993; 268: DeRusso PA, Philpott CC, Iwai K, et al. Expression of a constitutive mutant of iron regulatory protein 1 abolishes iron homeostasis in mammalian cells. J Biol Chem 1995; 270: Kutty RK, Maines MD. Purification and characterization of biliverdin reductase from rat liver. J Biol Chem 1981; 256: McCoubrey WK, Jr., Cooklis MA, Maines MD. The structure, organization and differential expression of the rat gene encoding biliverdin reductase. Gene 1995; 160: Stocker R, Yamamoto Y, McDonagh AF, Glazer AN, Ames BN. Bilirubin is an antioxidant of possible physiological importance. Science 1987; 235:

166 Chapter Stocker R, Glazer AN, Ames BN. Antioxidant activity of albumin-bound bilirubin. Proc Natl Acad Sci U S A 1987; 84: Suematsu M, Kashiwagi S, Sano T, Goda N, Shinoda Y, Ishimura Y. Carbon monoxide as an endogenous modulator of hepatic vascular perfusion. Biochem Biophys Res Commun 1994; 205: Suematsu M, Goda N, Sano T, Kashiwagi S, Egawa T, Shinoda Y et al. Carbon monoxide: an endogenous modulator of sinusoidal tone in the perfused rat liver. J Clin Invest 1995; 96: Platt JL, Nath KA. Heme oxygenase: protective gene or Trojan horse. Nat Med 1998; 4: Suttner DM, Dennery PA. Reversal of HO-1 related cytoprotection with increased expression is due to reactive iron. FASEB J 1999; 13: Dennery PA, Sridhar KJ, Lee CS, Wong HE, Shokoohi V, Rodgers PA et al. Heme oxygenase-mediated resistance to oxygen toxicity in hamster fibroblasts. J Biol Chem 1997; 272: Yet SF, Pellacani A, Patterson C, Tan L, Folta SC, Foster L et al. Induction of heme oxygenase-1 expression in vascular smooth muscle cells. A link to endotoxic shock. J Biol Chem 1997; 272: Exner M, Schillinger M, Minar E, Mlekusch W, Schlerka G, Haumer M et al. Heme oxygenase-1 gene promoter microsatellite polymorphism is associated with restenosis after percutaneous transluminal angioplasty. J Endovasc Ther 2001; 8: Schillinger M, Exner M, Mlekusch W, Ahmadi R, Rumpold H, Mannhalter C et al. Heme oxygenase-1 genotype is a vascular anti-inflammatory factor following balloon angioplasty. J Endovasc Ther 2002;9: Lenzen R, Bahr A, Eichstadt H, Marschall U, Bechstein WO, Neuhaus P. In liver transplantation, T tube bile represents total bile flow: physiological and scintigraphic studies on biliary secretion of organic anions. Liver Transpl Surg 1999; 5: Gibson UE, Heid CA, Williams PM. A novel method for real time quantitative RT-PCR. Genome Res 1996; 6: Heid CA, Stevens J, Livak KJ, Williams PM. Real time quantitative PCR. Genome Res 1996; 6: De Jong MM, Nolte IM, De Vries EG, Schaapveld M, Kleibeuker JH, Oosterom E et al. The HLA class III subregion is responsible for an increased breast cancer risk. Hum Mol Gen 2003; 12: Funk M, Endler G, Schillinger M, Mustafa S, Hsieh K, Exner M et al. The effect of a promoter polymorphism in the heme oxygenase-1 gene on the risk of ischemic cerbrovascular events: The influence of other vascular risk factors. Thromb Res 2004; 113: Makino N, Suematsu M, Sugiura Y, Morikawa H, Shiomi S, Goda N et al. Altered expression of heme oxygenase-1 in the livers of patients with portal hypertensive diseases. Hepatology 2001; 33: Turley SD, Dietschy JM. Re-evaluation of the 3 alpha-hydroxysteroid dehydrogenase assay for total bile acids in bile. J Lipid Res 1978; 19:

167 HO-1 before transplantation and graft injury and function after transplantation 31. Berger SP, Hunger M, Yard BA, Schnuelle P, Van der Woude FJ. Dopamine induces the expression of heme oxygenase-1 by human endothelial cells in vitro. Kidney Int 2000; 58: Maines MD, Raju VS, Panahian N. Spin trap (N-t-butyl-alpha-phenylnitrone)-mediated suprainduction of heme oxygenase-1 in kidney ischemia/reperfusion model: role of the oxygenase in protection against oxidative injury. J Pharmacol Exp Ther 1999; 291: Squiers EC, Bruch D, Buelow R, Tice DG. Pretreatment of small bowel isograft donors with cobalt-protoporphyrin decreases preservation injury. Transplant Proc 1999; 31: Katori M, Buelow R, Ke B, Ma J, Coito AJ, Iyer S et al. Heme oxygenase-1 overexpression protects rat hearts from cold ischemia/reperfusion injury via an antiapoptotic pathway. Transplantation 2002; 73: Nijboer WN, Schuurs TA, Van der Hoeven JA, Fekken S, Wiersema-Buist J, Leuvenink HG et al. Effect of brain death on gene expression and tissue activation in human donor kidneys. Transplantation 2004; 78: Rensing H, Jaeschke H, Bauer I, Patau C, Datene V, Pannen BH et al. Differential activation pattern of redox- sensitive transcription factors and stress-inducible dilator systems heme oxygenase-1 and inducible nitric oxide synthase in hemorrhagic and endotoxic shock. Crit Care Med 2001; 29: Motterlini R, Foresti R, Bassi R, Calabrese V, Clark JE, Green CJ. Endothelial heme oxygenase-1 induction by hypoxia. Modulation by inducible nitric-oxide synthase and S-nitrosothiols. J Biol Chem 2000; 275: Morita T, Perrella MA, Lee ME, Kourembanas S. Smooth muscle cell-derived carbon monoxide is a regulator of vascular cgmp. Proc Natl Acad Sci U S A 1995; 92: Borger DR, Essig DA. Induction of HSP 32 gene in hypoxic cardiomyocytes is attenuated by treatment with N-acetyl-L-cysteine. Am J Physiol 1998; 274(3Pt2): H Otterbein LE, Kolls JK, Mantell LL, Cook JL, Alam J, Choi AM. Exogenous administration of heme oxygenase-1 by gene transfer provides protection against hyperoxia-induced lung injury. J Clin Invest 1999; 103: Abraham NG, Lavrovsky Y, Schwartzman ML, Stoltz RA, Levere RD, Gerritsen ME et al. Transfection of the human heme oxygenase gene into rabbit coronary microvessel endothelial cells: protective effect against heme and hemoglobin toxicity. Proc Natl Acad Sci U S A 1995; 92: Jeney V, Balla J, Yachie A, Varga Z, Vercellotti GM, Eaton JW et al. Pro-oxidant and cytotoxic effects of circulating heme. Blood 2002; 100: Arora AS, Gores GJ. The role of metals in ischemia/reperfusion injury of the liver. Semin Liver Dis 1996; 16:

168 Chapter Wyllie S, Seu P, Gao FQ, Goss JA. Deregulation of iron homeostasis and cold-preservation injury to rat liver stored in University of Wisconsin solution. Liver Transpl 2003; 9: Clemens MG. Nitric oxide in liver injury. Hepatology 1999; 30: 1-5. Bauer I, Wanner GA, Rensing H, Alte C, Miescher EA, Wolf B et al. Expression pattern of heme oxygenase isoenzymes 1 and 2 in normal and stress-exposed rat liver. Hepatology 1998; 27: Schemmer P, Connor HD, Arteel GE, Raleigh JA, Bunzendahl H, Mason RR et al. Reperfusion injury in livers due to gentle in situ organ manipulation during harvest involves hypoxia and free radicals. J Pharmacol Exp Ther 1999; 290: Sindram D, Porte RJ, Hoffman MR, Bentley RC, Clavien PA. Synergism between platelets and leukocytes in inducing endothelial cell apoptosis in the cold ischemic rat liver: a Kupffer cell-mediated injury. FASEB J 2001; 15: Cutrin JC, Perrelli MG, Cavalieri B, Peralta C, Rosell Catafau J, Poli G. Microvascular dysfunction induced by reperfusion injury and protective effect of ischemic preconditioning. Free Radic Biol Med 2002; 33:

169

170 9 Heme oxygenase-1 genotype of the donor is associated with graft survival after liver transplantation Am J Transplant. 2008; 8: Carlijn I Buis Gerrit van der Steege Dorien S Visser Ilja M Nolte Bouke G Hepkema Maarten Nijsten Maarten JH Slooff Robert J Porte

171 HO-1 genotype of the donor and graft survival Abstract Heme oxygenase 1 (HO-1) has been suggested as a cytoprotective gene during liver transplantation. Inducibility of HO-1 is modulated by a (GT) n polymorphism and a single nucleotide polymorphism (SNP) A(-413)T in the promoter. Both a short (GT) n allele and the A-allele have been associated with increased HO-1 promoter activity. In 308 liver transplantations, we assessed donor HO-1 genotype and correlated this with outcome variables. For (GT) n genotype, livers were divided into two classes: short alleles (<25 repeats; class-s) and long alleles ( 25 repeats; class-l). In a subset, hepatic mrna expression was correlated with genotypes. Graft survival at 1 year was significantly better for A-allele genotype compared to TT-genotype (84% versus 63%, p=0.004). Graft loss due to primary dysfunction occurred more frequently in TT-genotype compared to A-receivers (p=0.03). Recipients of a liver with TT-genotype had significantly higher serum transaminases after transplantation and hepatic HO-1 mrna levels were significantly lower compared to the A-allele livers (p=0.03). No differences were found for any outcome variable between class S and LL-variant of the (GT) n polymorphism. Haplotype analysis confirmed dominance of the A(-413)T SNP over the (GT) n polymorphism. In conclusion, HO-1 genotype is associated with outcome after liver transplantation. These findings suggest that HO-1 mediates graft survival after liver transplantation. 170

172 Chapter 9 Introduction Orthotopic liver transplantation (OLT) is the best available treatment for patients with endstage liver failure. It is well recognized that, during the transplant procedure, livers are exposed to various stressful stimuli such as ischemia and reperfusion injury. Heme oxygenase 1 (HO-1) has been shown to provide cytoprotection during liver ischemia and reperfusion. Moreover, it has been suggested to have an immune modulating effect (1). In various experimental OLT models, upregulation of HO-1 has been shown to protect livers from I/R injury and to improve graft survival (2,3). HO-1 catalyzes the oxidative detoxification of excess heme resulting in equimolar amounts of free iron (Fe 2+ ), biliverdin and carbon monoxide. All products formed in this process possess potential beneficial effects in the transplant setting. CO has vasodilatating effects, thereby maintaining microvascular hepatic blood flow (4,5). Biliverdin and the subsequently formed bilirubin possess potent anti-oxidant effects (6-9). Free iron is highly reactive by itself, however cellular Fe 2+ released via heme degradation up-regulates the expression of the Fe 2+ sequestrating protein ferritin as well as that of an Fe 2+ pump, thereby limiting the amount of free iron and preventing the generation of reactive oxygen species (10-12). We previously studied the endogenous regulation of HO-1 during human liver transplantation and showed a dual role for HO-1, with either cytoprotection or increased cytotoxicity, depending on the initial level of overexpression (13). However, none of the clinical variables analyzed in this study could explain the variation in initial expression of HO-1 in the donor livers. We therefore decided to study the impact of genetic differences on HO-1 expression and outcome after OLT. Expression of the HO-1 gene is modulated by two functional polymorphisms in the promoter: a (GT) n polymorphism and a single nucleotide polymorphism (SNP) (14-19). (GT) n is the most frequent of the simple repeats scattered throughout the human genome and many of these exhibit a length polymorphism (20). Most of these variable sites are not expected to have any functional effect, since they are located in intragenic regions and introns. However, the HO-1 (GT) n repeat resides in a regulatory sequence and a short (GT) n allele has been associated with enhanced transcriptional activity of the gene (14,17-19). In kidney transplantation the influence of HO-1 (GT) n polymorphism has recently been studied by Baan et. al. (21) and Exner et. al. (22), who found a positive correlation between a short GT repeat and graft function and 171

173 HO-1 genotype of the donor and graft survival survival after transplantation. In addition to the (GT) n polymorphism, the A(-413)T SNP has been identified as a functionally relevant variation of the HO-1 gene (15,16). Using a transient transfection assay of HO-1 promoter luciferase genes in bovine aortic endothelial cells Ono et al have shown that the A-allele of this SNP is associated with a higher promoter activity than the T-allele. Interestingly, the A(-413)T SNP appeared in vitro to be more important for HO-1 promoter activity than the (GT) n polymorphism (15,16). Only limited work has been conducted evaluating the A(-413)T SNP in clinical research (15). Based on the accumulating evidence that HO-1 is an important enzyme influencing graft survival after transplantation, we hypothesized that these two functionally relevant polymorphisms in the HO-1 promoter are associated with outcome after OLT. Therefore, we analyzed the two functional HO-1 promoter polymorphisms in donor genomic DNA in relation to outcome after human liver transplantation. Furthermore we studied the functional relevance of these polymorphisms by measuring hepatic mrna expression. Patients and methods Patients Between January 1996 and January 2005, a total number of 465 consecutive OLT s were performed at the University Medical Center Groningen. After exclusion of children (<18 years), 320 transplants in 282 adult patients were included in this study. Of 308 donors (96%) cryopreserved splenocytes were available for the HO-1 genotyping. Median follow up time for this cohort was 4 years and 6 months (range months). ABO blood group-identical or compatible grafts from brain-death donors with normal or near normal liver function tests were used for all patients. A standardized technique was used for implantation, as has been described previously (23,24). During the study period, immunosuppressive protocols were based on tacrolimus or cyclosporine A, either with or without azathioprine and a rapid taper of steroids. Biopsy-proven acute rejection was treated when clinically indicated, with a bolus of methylprednisolone on three consecutive days. Steroid-resistant rejections were treated either by conversion to tacrolimus in patients on cyclosporine A, or by giving 5 doses of antithymocyte globulin (4 mg/kg i.v.) on alternating days. Doppler ultrasound was performed routinely at postoperative days 1, 3, and 7 and on demand to rule out vascular or biliary complications or parenchymal lesions. Cholangiography via a 172

174 Chapter 9 biliary drain was routinely performed between postoperative day and later on demand (i.e. for rising cholestatic parameters or dilatation of bile ducts on ultrasound). Tissue and data collection was performed according to the guidelines of the medical ethical committee of our institution and the Dutch Federation of Scientific Societies. HO-1 genotype assessment Genomic DNA was isolated from donor splenocytes using a commercial kit (Gentra Systems, Minneapolis, MN, USA). The 5 -flanking region of the HO-1 gene containing the (GT) n polymorphism was amplified by polymerase chain reaction (PCR) using as forward primer 5 -CAG CTT TCT GGA ACC TTC TGG-3 (sense), carrying a 6-FAM fluorescent label (Sigma, Malden, the Netherlands), and as reversed primer 5 -GAA ACA AAG TCT GGC CAT AG GAC-3 (antisense). PCR and genotyping procedures were similar as described earlier (25). Sequence analysis of the amplification products of individuals homozygous for the 222 and 229 base-pairs alleles showed correspondence with GT numbers 26 and 29, respectively (results not shown). Allelic repeats were divided into two subclasses using a similar classification based on transfection studies as described previously (26). A short allele, with less than 25 GT repeats, were designated as class S, and long allele with 25 or more GT repeats (amplicons of 220 base-pairs and more), as class L (26). Recipients of class S allele liver transplants (homozygous SS and heterozygous SL) were compared with recipients of non-class S allele transplants (LL). The single nucleotide polymorphism A(-413)T (rs ) was analyzed using the ABI7900HT TaqMan system (Applied Biosystems, Foster City, CA, USA) with a probe/primer assay hcv , developed by and purchased from Applied Biosytems (Assay-on-Demand). Recipients of at least one A-allele liver transplants (homozygous AA and heterozygous AT) were compared with recipients of heterozygous T-allele recipients (TT). Collection of liver biopsies, RNA isolation and reverse-transcriptase polymerase chain reaction In a subset of 38 patients we collected liver biopsies at the end of cold storage to compare HO-1 mrna expression in the various genotype groups. RNA isolation and cdna synthesis were performed as described before (13). cdna levels of HO-1 and 18S were measured by Real Time Polymerase Chain Reaction (PCR) using the ABI PRISM 7900 HT Sequence 173

175 HO-1 genotype of the donor and graft survival detector (Applied Biosystems). Nucleotide sequences of Primers (Invitrogen) and Probes (Eurogentec) were designed using Primes Express software (PE Applied Biosystems). Probes were 5 labeled by a 6-carboxy-fluoresceine (FAM) reporter and 3 labeled with a 6-carboxytetra-methyl-rhodamin (TAMRA) quencher. Real time RT-PCR data were analyzed using the comparative cycle threshold (CT) method. Briefly, the difference in cycle times, CT, was determined as the difference between the tested gene and the reference RNA, 18S. We then obtained CT by finding the difference compared to a control group of liver biopsies from patients undergoing an hemihepatectomy for colorectal metastasis. The fold induction (FI) was calculated as 2 - CT. Clinical Outcome parameters Outcome parameters included serum concentrations of aspartate aminotransferase (AST) and alanine aminotransferase (ALT), as marker for ischemia / reperfusion injury after OLT, graft survival, incidence of acute rejection, and causes of graft loss. Recipient data were obtained from a prospectively collected database. Donor data were extracted from the national and hospital s donor databases. Acute rejection was suspected on the basis of daily liver function tests, fever and deterioration of the clinical condition and proven by needle biopsy of the liver. The degree of acute rejection was histologically graded according to the Banff classification (27). Only rejections within the first three months with grade II and III, or grade I with a clinical indication for treatment, were considered in this study. As individual causes of graft loss, five different etiologies were identified: 1) Primary dysfunction (PDF), defined as either primary non function (PNF) or graft loss due to initial poor function (IPF). PNF was defined as non life sustaining function of the liver requiring retransplantation or leading to death within seven days after OLT. IPF was defined as early graft dysfunction characterized by serum AST levels > 2000 U/l on any day between postoperative day 2-7, and a prothrombine time (PT) >16 sec (modified according to Ploeg et. al. (28)), which was not explained by biliary or vascular complications; 2) Hepatic artery thrombosis, which was always confirmed by doppler ultrasonography and or angiography; 3) Non-anastomotic biliary strictures, as detected on imaging studies of the biliary tree and in the absence of arterial complications (29); 4) Recurrent disease, and 5) non-graft related graft loss, including extrahepatic conditions that contributed to the loss of the donor liver, such as postoperative sepsis and multi-organ failure. 174

176 Chapter 9 Statistics All data are reported as median and interquartile ranges (IQR) or number with percentage. Collection of laboratory values from the central hospital database was conducted as follows. For every postoperative biochemical variable of each patient a time curve was constructed before further analysis. In case multiple measurements of a parameter were performed on one day, these values were averaged to a single value before further analysis. Likewise, in case laboratory values were missing on certain days, these values were interpolated. Extrapolations were not performed. Groups were compared with the chi-square test or Mann Whitney U test, where appropriate. Biochemical variables were compared using the daily values, but also the total course during the first two post operative weeks was compared by calculating the area under the curve (AUC), using the trapezium rule. Graft survival curves were calculated according to the Kaplan-Meier method and compared using the log-rank test. A two-tailed p-value of < 0.05 was considered statistically significant. Statistical analyses were performed using SPSS version (SPSS Inc., Chicago, IL, USA). To study linkage disequilibrium between the two polymorphisms in the promoter of the HO-1 gene, the frequencies of the combined genotypes of the (GT) n polymorphism and A(-413)T were counted. Linkage disequilibrium is the occurrence of two or more polymorphism variants together on the chromosome more often than could be expected based on recombination possibilities, most likely due to their close locations, but it may also arise when the combination confers a selective advantage. A haplotype is a vector of polymorphisms. Haplotype frequencies were estimated from the genotype counts using the expectation-maximization algorithm (own software). Linkage disequilibrium is then determined from these haplotype frequencies by means of D and the correlation coefficient R 2, which both range from 0 to 1 with 1 implying the strongest possible linkage disequilibrium and 0 as no linkage disequilibrium. 175

177 HO-1 genotype of the donor and graft survival e frequency (%) Allele Number of GT repeats of the HO-1 promoter in 308 liver donors Figure 1. Allele distribution of the (GT) n polymorphism in 308 liver donors. Results Distribution of HO-1 genotypes in the donor population. The allelic distribution of the (GT) n polymorphism in the HO-1 promoter of liver donors is given in figure 1. The distribution of (GT) n is bimodal, with a peak at 22 repeats (22%) and the other at 29 repeats (45%). Forty two (14%) patients received a liver from a donor homozygous for class S allele, 130 (42.2%) from a heterozygote (SL) and 136 (44.2%) from a donor homozygous for the class L allele. With respect to the T(-413)A SNP, the distribution of the genotypes was as follows: 92 (30%) patients received a liver from an AA genotype donor, 153 (50%) from an AT genotype donor, and 61 (20%) from a TT genotype donor, (in two samples genotyping of A(-413)T failed). There were no significant differences in donor- and recipient characteristics of patients receiving a liver from a donor with a class S allele (SS or SL) or from a donor without a class S allele (LL). Also no significant differences were found between the group of patients who received a liver from a donor with an A-allele (AA or AT) and the group of patients who received a liver without an A-allele (TT genotype) (table 2). 176

178 Chapter 9 Are the two HO-1 polymorphisms in linkage disequlibrium? Haplotype frequencies were estimated from data in table 1A and are presented in table 1B. The two most prevalent haplotypes were A(-413)_(GT)29 and (-413) T_(GT)22 indicating that the favorable A-allele is in linkage disequilibrium with the unfavorable class L genotype. The linkage disequilibrium measures D and R 2 were 0.87 and 0.50, respectively indicating strong linkage disequilibrium between the two promoter polymorphisms. The class L genotype cosegregates at -413 more often than expected with the A-allele, the same holds for the combination of the class S genotype and T-allele at Table 1A. Number of A(-413)T genotypes in each genotype of the (GT) n polymorphism (GT) n repeat length polymorphism TT AT AA (21, 29) (22, 22) (22, 23) (22, 24) (22, 29) (22, 36) (23, 29) (23, 30) (24, 29) (25, 29) (26, 29) (28, 29) (29, 29) (29, 30) (29, 36) Combinations occurring less then 4 times are not shown in this table 177

179 HO-1 genotype of the donor and graft survival Table 1B. Estimated haplotype frequencies from table 1A with the EM algorithm. Haplotypes with a frequency of less than 1% are not shown. (GT) n number of repeats repeats -413 Estimated haplotype frequency (%) 21 T A T T T T A T A A T A T 5.4 Is there an association between HO-1 genotypes and mrna expression? In a subgroup of 38 livers, material was available to measure hepatic HO-1 mrna expression. The fold induction of the HO-1 mrna in liver biopsies, retrieved at the end of the cold storage period, was significantly higher in A-receiver genotype livers compared to the TT-genotype (p=0.03) (figure 2). No difference in mrna expression was found for the recipients of class S or LL- livers. Although these findings provide support for a functional relevance of the A(-413)T SNP and not for the (GT) n polymorphism, they do not demonstrate the dominance of one of these polymorphisms. Therefore we next studied HO-1 mrna expression in the various haplotype combinations (figure3). Within the group of non class S allele transplants (LL), HO-1 mrna was higher in livers with an A allele (haplotype A-L, A-L) compared to livers with a T-allele (haplotype T-L, T-L). A similar comparison of different haplotypes within the group of class S livers was not possible due to the low frequency of the S-A, S-A haplotype (table 1B). 178

180 Chapter 9 P=0.03 HO-1 Fold Inductio on in liver biopsy TT genotype A-allele genotype (n=38) Figure 2. Fold induction of the HO-1 gene in biopsies taken at the end of cold storage in a group of 38 patients. Liver grafts with at least one A-allele had a significantly higher expression of HO-1 mrna compared to TT genotype liver grafts (p=0.03). Are HO-1 polymorphisms associated with outcome after liver transplantation? Survival. In the entire cohort of 308 transplants, overall actuarial graft survival rate at 1 and 5 years was 80% and 71%, respectively. Graft survival rates were significantly better in recipients of livers with at least one A-allele, compared to recipients of a TT genotype liver; log rank p=0.004 (Figure 4). In addition, within the group of livers with at least one A-allele, there were no differences between AA and AT genotypes. No differences were found between recipients of class S or LL- livers. Ischemia / reperfusion injury. Postoperative serum levels of AST and ALT as a marker of ischemia / reperfusion injury, are presented in Figure 5 A and B. Recipients of a liver with TT genotype had significantly higher serum transaminase levels, as expressed by the AUC for the first two weeks (AST (p=0.01, ALT p=0.009)). There were no significant differences in serum AST or ALT in recipients of a class S liver, compared to recipients of a non class S liver (LL). 179

181 HO-1 genotype of the donor and graft survival Table 2. Comparison of donor and recipient characteristics in relation to donor HO-1 genotype. Donor (GT) n polymorphism S-Receiver LL (n = 172) (n = 136) P value Donor age (years) 46 (35-55) 46 (38-53) 0.67 Gender (M/F) 76 / 96 (44% / 56%) 70 /64 (53% / 47%) 0.11 Laboratory variables** Hemoglobulin (mmol/l) 7.1 ( ) 7.3 ( ) 0.46 Total bilirubin (umol/l) 10 (7-16) 11 (7-16) 0.90 AST (U/L) 28 (18-48) 27 (18-44) 0.60 ALT (U/L) 21 (14-34) 22 (14-42) 0.57 γ-gt (U/L) 20 (11-37) 24 (14-40) 0.13 AP (U/L) 53 (40-72) 55 (42-79) 0.33 Dopamine use (n=177) 100 (58%) 77 (57%) 0.92 Blood transfusion 76 (44%) 54 (40%) 0.43 Cause of death 0.07 Cerebral Vascular Accident 124 (73%) 98 (73%) Trauma 44 (21%) 28 (26%) Miscellaneous 4 (7%) 10 (2%) Recipient Recipient age (years) 49 (37-55) 46 (35-53) 0.08 Gender (M/F) 95 / 77 (55 % / 45%) 71 / 65 (52% / 48%) 0.60 Diagnosis 0.04 Cirrhosis 147 (85.5%) 102 (75%) Acute Failure 12 (7%) 11 (8%) Tumors 1 (.5%) 6 (4.5%) Non-cirrhotic 12 (7%) 17 (12.5%) MELD Score 15 (10-22) 14 (11-19) 0.52 Preservation Solution 0.80 High viscosity 157 (92%) 125 (93%) Low viscosity 14 (8%) 10 (7%) CIT (minutes) 515 ( ) 526 ( ) 0.13 WIT (minutes) 50 (43-62) 45 (45-60) 0.40 LOS at ICU 3 (2-10) 3 (2-7) 0.10 *) SNP analysis failed for two donors. **) At time of donor procedure Continuous variables are presented as median and interquartile range, categorical variables as numbers with percentage. 180

182 Chapter 9 A(-413)T SNP * A-Receiver TT (n=245) (n=61) P value 45 (37-54) 47 (37-55) / 125 (49% / 51%) 34 / 27 (43% / 57%) ( ) 7.2 ( ) (7-16) 12 (7-14) (18-44) 32 (20-54) (13-36) 27 (18-46) (12-39) 24 (15-37) (41-76) 49 (36-68) (56%) 40 (66%) (44%) 22 (36%) (73%) 44 (73%) 57 (23%) 15 (25%) 11 (10%) 2 (3%) 46 (35-55) 49 (38-54) / 114 (53% / 47%) 33 / 28 (54% / 46%) (80%) 51 (84%) 19 (8.5%) 4 (6.5%) 5 (1.5%) 2 (3%) 25 (10%) 4 (6.5%) 14 (11-20) 16 (11-24) (92%) 58 (95%) 20 (8%) 3 (5%) 519 ( ) 531 ( ) (44-60) 50 (42-63) (2-7) 3 (2-12)

183 HO-1 genotype of the donor and graft survival Acute rejection. The overall incidence of clinically relevant acute rejection within the first three months after OLT was 34%. There were no statistically significant differences in the incidence of acute rejection between any of the genotypes. Moreover, no differences were found in the severity of rejection among the different genotype groups (Table 3). 3 HO-1 Fold Indu uction in liver biopsy 2,5 2 1,5 1 0,5 0 (T-L,T-L) (A-L,A-L) Haplotype combinations Figure 3. HO-1 m RNA expression in relation to HO-1 haplotypes. Within the group of LL livers, HO-1 mrna expression was higher when the LL allele variant was combined with two A-alleles, compared to LL allele carriers combined with two T-alleles. 182

184 Chapter 9 Table 3. Incidence of acute rejection within the first three months after OLT in relation to donor HO-1 genotype. (GT) n polymorphism A(-413)T SNP * S-Receiver LL A-Receiver TT (n = 172) (n = 136) P value (n=245) (n=61) P value Acute Rejection 60 (35%) 44 (32%) (34%) 22 (36%) 0.70 Grade** I 15 (25%) 15 (35%) 24 (30%) 6 (29%) II 32 (55%) 20 (47%) 44 (55%) 8 (38%) III 11 (19%) 8 (19%) 12 (15%) 7 (33%) *) SNP analysis failed for two donors. **) Grades of rejection according to the BANFF classification. For three patients no histological data were available. Categorical variables as numbers with percentage. Table 4. Causes of graft loss grouped by donor HO-1 genotype (GT) n polymorphism A(-413)T SNP * S-Receiver LL A-Receiver TT (n = 172) (n = 136) P value (n=245) (n=61) P value Primary dysfunction 6 (3%) 5 (4%) (2%) 6 (10%) 0.03 Hepatic artery thrombosis 7 (4%) 5 (4%) (3%) 4 (7%) 0.66 Non anastomotic bilary strictures 4 (2%) 5 (4%) (3%) 2 (3%) 0.68 Recurrent disease 4 (2%) 5 (4%) (3%) 1 (2%) 0.23 Not graft related 25 (15%) 18 (13%) (13%) 10 (16%) 0.33 Miscellaneous 5 (3%) (3%) 2 (3%) 0.54 *) SNP analysis failed for two donors. 183

185 HO-1 genotype of the donor and graft survival Causes of graft loss. The number of grafts lost in patients receiving a liver with a S-allele was 50 (29%), the number of grafts lost in patients receiving a liver with LL genotype was 37 (27%). The number of grafts lost in patients receiving a liver with an A-allele was 62 (25%), the number of grafts lost in patients receiving a liver with TT genotype was 25 (41%) (p=0.004). To find an explanation for the observed differences in overall graft survival in relation to the A(-413)T SNP, we next examined the individual causes of graft loss (Table 4). Primary graft dysfunction was a significantly more frequent cause of graft loss in the group of TT-genotype livers (10%) compared to livers with an A-allele (2%); odds ratio 3.73 (95% Confidence interval 1.02 to 13.60; p=0.03). For the other most common causes of graft loss, including hepatic artery thrombosis, non anastomotic biliary strictures, recurrent disease and non graft related causes, no significant differences were found in the distribution among the different genotypes (Table 4). 100 Actuarial gr raft survival (%) A-allele genotype TT genotype 0 Log-rank p = Days post OLT Figure 4. Kaplan Meier 1-year survival curve for liver grafts in relation to donor HO-1 A(-413)T SNP. Log-rank test for livers with an A-allele (AA or AT genotype) versus no A-allele (TT genotype): p-value =

186 Chapter 9 A AUC p = 0.01 TT genotype A-allele genotype Serum AST level (U/l) ** ** * ** ** * * P < 0.05 ** P < 0.01 B Postoperative day AUC p = Serum ALT level (U/l) * * * * * * Postoperative day Figure 5. A. Serum levels of AST in the first two weeks after OLT. On day 8,9,11-14, recipients of a liver with at least one A-allele had significant lower AST levels. Total course during the first two weeks, calculated by the area under the curve, was significantly lower in liver grafts with at least one A-allele (p=0.01). B. Serum levels ALT in the first two weeks after OLT. On day 9 14, recipients of a liver with at least one A-allele had significant lower ALT levels. Total course during the first two weeks, as calculated by the area under the curve, was significantly lower in liver grafts with at least one A-allele (p<0.01). 185

187 HO-1 genotype of the donor and graft survival Discussion In this study we have examined the relationship between two functionally relevant polymorphisms in the promoter of the HO-1 gene in the donor and postoperative outcome in a large cohort of 308 liver transplant recipients. There are three novel findings in this study. Firstly we observed significantly worse outcome in patients receiving a liver from a TT genotype donor, compared to recipients from donors with at least one A allele. Secondly, we have shown that the A(-413)T SNP and the (GT) n polymorphism are in linkage disequilibrium with each other in this predominantly Caucasian population. Finally, we have shown, for the fist time in a human population, the differences in functional relevance of these two HO-1 promoter polymorphisms. Our association study of the various haplotypes and actual HO-1 mrna expression suggests that the A(-413)T SNP is of greater functional relevance than the (GT) n polymorphism. No differences were found in any outcome parameter between the class S and the LL-receivers of the (GT) n polymorphism. The power of this study with an overall sample size of 308 subjects was greater than 80% to detect a difference of 13% in graft survival at the statistical significant lever of 5%. An association with functional polymorphisms of the HO-1 gene and clinical outcome parameters has also been found in other pathological conditions, such as pulmonary emphysema, certain cardiovascular diseases and malignancies (14,19,30-35). With respect to transplantation, two groups have previously reported an association between HO-1 polymorphism in the donor and outcome after kidney transplantation (21,22). Baan et al and Exner et al have shown a positive correlation between the presence of a short (GT) n allele in the HO-1 promoter and a favorable outcome after kidney transplantation (21,22). Although our data and the two studies in kidney transplant recipients all point towards a critical role for the HO-1 / CO pathway in maintaining graft function after solid organ transplantation, in detail the studies are different. The two studies in kidney transplantation revealed an association between the (GT) n polymorphism and outcome after transplantation, whereas we found an association with the A(-413)T SNP. Unfortunately, this SNP was not tested in the two previous studies in kidney transplant recipients. Moreover, a third large genetic association study between the (GT) n polymorphism and outcome after kidney transplantation did not provide evidence for a protective effect of class S alleles on kidney graft survival (36). The linkage disequilibrium between the short (GT) n variant and the T-allele at -413 in the current study, in combination 186

188 Chapter 9 with the known dominant effect of the A(-413)T SNP in relation to the (GT) n polymorphism (16), could possibly explain the inconsistent results of studies in kidney transplant recipients focusing on the (GT) n polymorphism only. It could well be that HO-1 expression has actually been lower in kidney grafts with a short (GT) n allele. Unfortunately, tissue levels of HO-1 mrna, as a marker of actual HO-1 gene expression, were not measured in the three studies in kidney transplantation. In our study population, which mainly consisted of Caucasians, we found the A(-413)T SNP and the (GT) n polymorphism within the promoter of the HO-1 gene to be in linkage disequilibrium. The two most frequent haplotypes were the A-allele at -413 in combination with a long (29) (GT) n allele (43.2%) and the T-allele at -413 in combination with a short (22) (GT) n allele (20.3%). This finding is in accordance with results from previous studies in Japanese populations (15,16). Theoretically, these combinations are counterproductive, as the A-allele at -413 and a short (GT) n allele are both associated with enhanced expression of HO-1, whereas the T-allele and a long (GT) n allele are associated with reduced HO-1 expression. Our data, however, consistently point towards a dominant effect of the A(-413) T SNP over the (GT) n polymorphism. Not only clinical outcome parameters but also hepatic HO-1 mrna correlated with the A(-413)T SNP, but not with the (GT) n polymorphism. To our knowledge this is the first study in humans suggesting an association between mrna expression and the various HO-1 haplotypes. Similar observations have been made by Ono et al. who have studied the functional role of the A(-413)T SNP and the (GT) n polymorphism in an in vitro system of bovine aortic endothelial cells, using a luciferase reporter assay (15). These investigators suggested that, with respect to HO-1 promoter activity, the A(-413)T SNP is dominant over the (GT) n polymorphism. In a previous study we have shown the HO-1 mrna expression correlates well with protein expression in human livers (13). The exact mechanisms explaining the clinical observations in this study are incompletely understood. Experimental studies have previously shown that upregulation of HO-1 protects liver grafts against ischemia / reperfusion injury and improves graft survival (2,3,37-39) Especially, steatotic livers, which are highly sensitive to ischemic injury, seem effectively protected against this type of injury by induction of HO-1 (37,40). The inferior outcomes of livers with the unfavorable TT genotype (associated with a reduced HO-1 promoter activity) in the current study supports these previous findings. The effect of HO-1 genotype on graft survival could, at least partly, be explained by a higher incidence of PDF in livers with a TT-genotype, 187

189 HO-1 genotype of the donor and graft survival compared to livers with an A-allele (p=0.03). However, when the Kaplan Meier curves are carefully observed, the lines started to separate from day 25 and were further divergent later after transplantation. In accordance with this, the differences in serum transaminases became more pronounced in the second week after transplantation. These observations suggest that the observed differences in graft survival are not only explained by differences in ischemia / reperfusion injury, but also result from other mechanisms. In fact, the absolute number of grafts lost due to PDF is relatively small, again suggesting that other factors have also contributed to the observed differences in graft survival. Apparently, the impact HO-1 is not limited to the early postoperative period. We speculate that other, possibly immune-mediated processes, could explain the more late effects of HO-1 on graft survival. Several studies have shown that HO-1 is a key enzyme in certain immune processes. Nevertheless, we observed no differences in the incidence or severity of acute rejection between the various genotypes. However, it would be of interest to study HO-1 mrna expression in donor livers more longterm after transplantation and to see if differences in HO-1 expression persist. Unfortunately, we had no access to repeated biopsies during long term follow-up after OLT. More studies on the mechanisms underlying the more long-term effects of HO-1 on graft survival will be needed. In conclusion, in this large series of 308 liver transplant recipients, we found an association between donor HO-1 genotype and outcome after liver transplantation. Livers with at least one A-allele of the A(-413)T SNP had significantly better graft survival rate and a lower rate of PDF than livers with the TT genotype. In addition, our data indicate a functional dominance of the A(-413)T SNP over the (GT) n polymorphism. These data suggest that HO-1 is critically involved in maintaining graft function during and after liver transplantation. 188

190 Chapter 9 Reference List 1. Choi AM, Alam J. Heme oxygenase-1: function, regulation, and implication of a novel stress-inducible protein in oxidant-induced lung injury. Am J Respir Cell Mol Biol 1996;15: Amersi F, Buelow R, Kato H et al. Upregulation of heme oxygenase-1 protects genetically fat Zucker rat livers from ischemia/reperfusion injury. J Clin Invest 1999;104: Kato H, Amersi F, Buelow R et al. Heme oxygenase-1 overexpression protects rat livers from ischemia/ reperfusion injury with extended cold preservation. Am J Transplant 2001;1: Suematsu M, Goda N, Sano T et al. Carbon monoxide: an endogenous modulator of sinusoidal tone in the perfused rat liver. J Clin Invest 1995;96: Suematsu M, Ishimura Y. The heme oxygenase-carbon monoxide system: a regulator of hepatobiliary function. Hepatology 2000;31: Kutty RK, Maines MD. Purification and characterization of biliverdin reductase from rat liver. J Biol Chem 1981;256: McCoubrey WK, Jr., Cooklis MA, Maines MD. The structure, organization and differential expression of the rat gene encoding biliverdin reductase. Gene 1995;160: Stocker R, Yamamoto Y, McDonagh AF, Glazer AN, Ames BN. Bilirubin is an antioxidant of possible physiological importance. Science 1987;235: Stocker R, Glazer AN, Ames BN. Antioxidant activity of albumin-bound bilirubin. Proc Natl Acad Sci U S A 1987;84: Vile GF, Tyrrell RM. Oxidative stress resulting from ultraviolet A irradiation of human skin fibroblasts leads to a heme oxygenase-dependent increase in ferritin. J Biol Chem 1993;268: Ferris CD, Jaffrey SR, Sawa A et al. Haem oxygenase-1 prevents cell death by regulating cellular iron. Nat Cell Biol 1999;1: DeRusso PA, Philpott CC, Iwai K, Mostowski HS, Klausner RD, Rouault TA. Expression of a constitutive mutant of iron regulatory protein 1 abolishes iron homeostasis in mammalian cells. J Biol Chem 1995;270: Geuken E, Buis CI, Visser DS et al. Expression of heme oxygenase-1 in human livers before transplantation correlates with graft injury and function after transplantation. Am J Transplant 2005;5: Yamada N, Yamaya M, Okinaga S et al. Microsatellite polymorphism in the heme oxygenase-1 gene promoter is associated with susceptibility to emphysema. Am J Hum Genet 2000;66:

191 HO-1 genotype of the donor and graft survival 15. Ono K, Goto Y, Takagi S et al. A promoter variant of the heme oxygenase-1 gene may reduce the incidence of ischemic heart disease in Japanese. Atherosclerosis 2004;173: Ono K, Mannami T, Iwai N. Association of a promoter variant of the haeme oxygenase-1 gene with hypertension in women. J Hypertens 2003;21: Okinaga S, Takahashi K, Takeda K et al. Regulation of human heme oxygenase-1 gene expression under thermal stress. Blood 1996;87: Hirai H, Kubo H, Yamaya M et al. Microsatellite polymorphism in heme oxygenase-1 gene promoter is associated with susceptibility to oxidant-induced apoptosis in lymphoblastoid cell lines. Blood 2003;102: Chen YH, Lin SJ, Lin MW et al. Microsatellite polymorphism in promoter of heme oxygenase-1 gene is associated with susceptibility to coronary artery disease in type 2 diabetic patients. Hum Genet 2002;111: Naylor LH, Clark EM. d(tg).d(ca) sequences upstream of the rat prolactin gene form Z-DNA and inhibit gene n n transcription. Nucleic Acids Res 1990;18: Baan C. Fundamental role for HO-1 in the self-protection of renal allografts. American journal of transplantation 2004;4: Exner M. Donor heme oxygenase-1 genotype is associated with renal allograft function. Transplantation 2004;77: Miyamoto S, Polak WG, Geuken E et al. Liver transplantation with preservation of the inferior vena cava. A comparison of conventional and piggyback techniques in adults. Clin Transplant 2004;18: Polak WG, Miyamoto S, Nemes BA et al. Sequential and simultaneous revascularization in adult orthotopic piggyback liver transplantation. Liver Transpl 2005;11: de Jong MM, Nolte IM, de Vries EG et al. The HLA class III subregion is responsible for an increased breast cancer risk. Hum Mol Genet 2003;12: Funk M. The effect of a promoter polymorphism in the heme oxygenase-1 gene on the risk of ischaemic cerebrovascular events: the influence of other vascular risk factors. Thrombosis research 2004;113: Banff schema for grading liver allograft rejection: an international consensus document. Hepatology 1997;25: Ploeg RJ, D Alessandro AM, Knechtle SJ et al. Risk factors for primary dysfunction after liver transplantation--a multivariate analysis. Transplantation 1993;55: Buis CI, Verdonk RC, Van der Jagt EJ et al. Nonanastomotic biliary strictures after liver transplantation, part 1: Radiological features and risk factors for early vs. late presentation. Liver Transpl 2007;13: Chen YH, Chau LY, Lin MW et al. Heme oxygenase-1 gene promotor microsatellite polymorphism is associated with angiographic restenosis after coronary stenting. Eur Heart J 2004;25:

192 Chapter Exner M, Schillinger M, Minar E et al. Heme oxygenase-1 gene promoter microsatellite polymorphism is associated with restenosis after percutaneous transluminal angioplasty. J Endovasc Ther 2001;8: Exner M. The role of heme oxygenase-1 promoter polymorphisms in human disease. Free radical biology 2004;37: Kaneda H, Ohno M, Taguchi J et al. Heme oxygenase-1 gene promoter polymorphism is associated with coronary artery disease in Japanese patients with coronary risk factors. Arterioscler Thromb Vasc Biol 2002;22: Schillinger M, Exner M, Mlekusch W et al. Heme oxygenase-1 genotype is a vascular anti-inflammatory factor following balloon angioplasty. J Endovasc Ther 2002;9: Lo SS, Lin SC, Wu CW et al. Heme Oxygenase-1 Gene Promoter Polymorphism is Associated with Risk of Gastric Adenocarcinoma and Lymphovascular Tumor Invasion. Ann Surg Oncol 2007;14: Courtney AE, McNamee PT, Middleton D, Heggarty S, Patterson CC, Maxwell AP. Association of functional heme oxygenase-1 gene promoter polymorphism with renal transplantation outcomes. Am J Transplant 2007;7: Coito AJ, Buelow R, Shen XD et al. Heme oxygenase-1 gene transfer inhibits inducible nitric oxide synthase expression and protects genetically fat Zucker rat livers from ischemia-reperfusion injury. Transplantation 2002;74: Lai IR, Ma MC, Chen CF, Chang KJ. The protective role of heme oxygenase-1 on the liver after hypoxic preconditioning in rats. Transplantation 2004;77: Yang Z, Tsui T, Ho D, Tang T, Fan S. Heme oxygenase-1 potentiates the survival of small-for-size liver graft. Liver transplantation 2004;10: Lehmann TG, Wheeler MD, Froh M et al. Effects of three superoxide dismutase genes delivered with an adenovirus on graft function after transplantation of fatty livers in the rat. Transplantation 2003;76:

193

194 10 Summary, general discussion and future perspectives

195 Summary, discussion and future perspectives Summary Chapter 1 provides a short introduction of the facts and figures in liver transplantation. Furthermore, the aims of this thesis are discussed. These aims were to evaluate the molecular and biochemical mechanisms of bile duct injury after liver transplantation. In Chapter 2 the literature regarding the causes and consequences of non-anastomotic strictures (NAS) is reviewed. The aim of this chapter was to describe the current knowledge about the pathophysiological mechanisms, the clinical presentation, and the treatment of NAS. NAS is a radiological diagnosis, characterized by intrahepatic strictures and dilatations on a cholangiogram. NAS were first described after liver transplantation in association with hepatic artery thrombosis (HAT). In case of early HAT after liver transplantation the biliary tree becomes ischemic and eventually necrotic, resulting in a typical cholangiographic picture of biliary strictures, dilatations and intraductal cast formation. However, these cholangiographic abnormalities of strictures and dilatations can also be seen in patients who do not have a hepatic artery thrombosis, so the term ischemic-type biliary lesions emerged. In this thesis the term NAS was used to describe intrahepatic biliary strictures and dilatations in the confirmed absence of HAT. The incidence of NAS varies around 15% in different series. Several risk factors for NAS have been identified, strongly suggesting a multifactorial origin. Main categories include ischemia related injury, immunological induced injury and cytotoxic injury by bile salts. However, in many cases no specific risk factor can be identified. The clinical presentation of patients with NAS is often not specific. Symptoms may include fever, abdominal complaints and increased cholestatic liver function tests. The diagnosis is made by imaging studies of the bile ducts. Treatment starts with relieving symptoms of cholestasis and dilatation of the stenosed bile ducts by endoscopic retrograde cholangiopancreaticography (ERCP) or percutaneous transhepatic cholangiodrainage (PTCD), if possible followed by stenting. Eventually up to 50% of the patients with NAS will require a re-transplantation or may die. In selected cases, a re-transplantation can be avoided or delayed by surgical intervention. In the clinical study described in Chapter 3 we aimed to identify clinical risk factors for the development of NAS after liver transplantation. A total of 487 adult liver transplants with a median follow-up of 7.9 years were studied. All imaging studies of the biliary tree were reviewed. 194

196 Chapter 10 Localization of NAS at first presentation was categorized into 4 anatomical zones of the biliary tree. Severity of NAS was semiquantified as mild, moderate, or severe. NAS developed in 81 livers (16.6%). In 85% of the cases, anatomical localization of NAS was around or below the bifurcation of the common bile duct. The severity of biliary strictures was classified as mild in 43 (55%) and as moderate to severe in 35 (45%) of the cases. The cumulative incidence of moderate to severe NAS in the entire population of liver transplant recipients was 7.3%. A large variation was observed in the time interval between liver transplantation and first presentation of NAS (median 4.1 months; range months). NAS presenting early (<1 year) after liver transplantation were associated with preservation related risk factors. Cold and warm ischemia times were significantly longer in patients with early NAS compared with NAS presenting late (>1 year) after transplantation, and early NAS were more frequently located in the central bile ducts. NAS presenting late after transplantation were more frequently found in the periphery of the liver and were more frequently associated with immunological factors, such as primary sclerosing cholangitis as the indication for liver transplantation. By separating cases of NAS on the basis of the time of presentation after transplantation, we were able to identify differences in risk factors, indicating different pathogenic mechanisms depending on the time of initial presentation. The population of patients suffering from NAS as described in Chapter 3 is further studied in Chapter 4. The aim of this particular study was to describe the treatment, and identify risk factors for radiological progression of bile duct abnormalities, recurrent cholangitis, biliary cirrhosis and retransplantation in patients with NAS. Progression of disease was noted in 68% of cases in whom follow-up radiology was available. Radiological progression was more common in patients with early NAS ( 1 year) and with one or more episodes of bacterial cholangitis, and less prevalent in patients with extrahepatic biliary abnormalities. Recurrent bacterial cholangitis (3 or more episodes) was more frequently seen in patients with a Rouxen-Y anastomosis. Severe fibrosis or cirrhosis developed in 23 cases, especially in cases with peripheral biliary abnormalities. Graft survival, but not patient survival, was influenced by the presence of NAS. Thirteen patients (16%) were retransplanted for NAS. The conclusion of the study is that especially patients with a hepatico-jejunostomy, those with an early diagnosis of NAS, and those with NAS presenting at the level of the peripheral branches of the biliary tree, are at risk for progressive disease with severe outcome. 195

197 Summary, discussion and future perspectives In Chapter 5 we took a closer look at the role of bile composition in the development of bile duct injury after liver transplantation, in a porcine model of non heart-beating liver transplantation. After non-heart-beating (NHB) liver transplantation, the occurrence of NAS is a serious and often encountered complication. Bile salt toxicity has been identified as an important factor in the pathogenesis of bile duct injury and cholangiopathies in general. The role of bile salt toxicity in the development of biliary strictures after NHB liver transplantation was, however, unclear. In a porcine model of NHB liver transplantation, we studied the effect of different periods of warm ischemia in the donor on bile composition and subsequent bile duct injury after transplantation. After induction of cardiac arrest in the donor, liver procurement was delayed for 0 min (group A), 15 min (group B), and 30 min or more (group C). Subsequently livers were transplanted after 4 hr of cold preservation. In the recipients, bile flow was measured, and bile samples were collected daily to determine the phospholipids-to-bile salt ratio. Severity of bile duct injury was semi quantified by using a histological grading scale. Survival after transplantation was directly related to the duration of warm ischemia in the donor. The phospholipids-to-bile salt ratio in bile produced early after transplantation was significantly higher in group C, compared with group A and B. Histopathologic examination showed the highest degree of bile duct injury in group C. Based on these results, it was concluded that prolonged warm ischemia in NHB donors is associated with the formation of toxic bile after transplantation, characterized by a low biliary phospholipids-to-bile salt ratio. These data suggest that bile salt toxicity contributes to the pathogenesis of bile duct injury after NHB liver transplantation. The previous chapter, as well as other studies from our group, have indicated that bile formation early after liver transplantation may be disturbed, resulting in more cytotoxic bile with a relatively low phospholipids-to-bile salt ratio. It was unknown whether bile toxicity is also involved in the pathogenesis of NAS, a disease of the larger bile ducts. If bile composition is involved in the pathogenesis of NAS, one would expect that the bile composition in the first week after liver transplantation is different in those patients who will develop NAS than in patients who will not develop NAS. We tested this hypothesis in a prospective clinical study, described in Chapter 6. In this study, bile production and composition within one week after liver transplantation were correlated with the subsequent development of NAS in a large cohort of adult liver transplant recipients. In 111 adult liver transplants bile samples were 196

198 Chapter 10 collected daily after transplantation for determination of bile composition. Expression of bile transporters was studied perioperatively. NAS were detected in 14 patients (13%) within one year after transplantation. Patient and donor characteristics and postoperative serum liver enzymes were similar between patients who developed NAS and those who did not. Secretions of bile salts, phospholipids and cholesterol were significantly lower in patients who developed NAS. In parallel, biliary phospholipids-to-bile salt ratio was lower in patients developing NAS, suggestive for increased bile cytotoxicity. There were no differences in bile salt pool composition or in hepatobiliary transporter mrna expression. Although patients who develop NAS were initially clinically indiscernible from patients who did not develop NAS, the biliary bile salts and phospholipids secretion, as well as biliary phospholipids-to-bile salt ratio in the first week after transplantation, was significantly lower in the former group. This supports the concept that bile cytotoxicity is involved in the pathogenesis of NAS. In the previous chapter we have shown that altered bile composition, with a lower phospholipidsto-bile salt ratio is associated with NAS after liver transplantation. Hepatobiliary transporter proteins are responsible for the biliary secretion of phospholipids and bile salts. Aim of Chapter 7 was to assess whether variations in the genes in the donor encoding for these transporters are associated with the occurrence of NAS in the recipient. Without transplantation, genetic variations itself may not result in bile duct injury. However, early after transplantation, when the graft is still recovering from I/R injury, these variations might be a critical second factor in the sequence of events leading to bile duct injury. A similar phenomenon can be found in other diseases, such as intrahepatic cholestasis of pregnancy (ICP), where patients with a genetic variation in hepatobiliary transporters display an abnormal phenotype only during pregnancy. Of 458 procedures in adults, cryopreserved splenocytes were available form the donors and used for genotyping. The following genes were studied: bile salt export pump (ABCB11), transporter of phospholipids (ABCB4) and transporter of glutathione and bilirubin (ABCC2). Four to five tagging single nucleotide polymorphisms (SNPs) with an equal physical distribution per gene were selected using HapMap data. Haplotypes were constructed using an Expectation-Maximization algorithm to estimate haplotype frequencies. NAS was detected in 77 patients (16%) after transplantation. Patients who received a donor liver with ABCB4 haplotype AGGTA developed NAS almost twice as often (28%) as donor livers with other 197

199 Summary, discussion and future perspectives haplotypes (15%) (p=0.007). Analysis in a multivariate Cox regression model showed AGGTA haplotype of ABCB4 from the donor to be an independent risk factor for NAS (p=0.004, OR=2.23, 95% CI= ). ABCB11 and ABCC2 haplotypes or single SNPs, were not associated with NAS. These data indicate that a common haplotype in the transporter of phospholipids (ABCB4) in donor livers is independently associated with a two-fold increased risk for NAS after liver transplantation. Transport of phospholipids into the bile in livers which are carriers of this risk haplotype might be altered in the time period early after transplantation. Upregulation of heme oxygenase-1 (HO-1) has been considered an adaptive and protective mechanism against ischemia/reperfusion (I/R) injury. In Chapter 8 we studied the role of endogenous HO-1 expression in human liver transplants in relation to early postoperative hepatobiliary injury and dysfunction. Before transplantation, median HO-1 mrna levels were 3.4-fold higher (range: ) in donors than in normal controls. Based on the median value, livers were divided into two groups: low and high HO-1 expression. There were no differences in donor characteristics, donor serum transaminases or cold ischemia time between the two groups. Postoperatively, however, serum transaminases were significantly lower and the bile salt secretion was higher in the group with an initial low HO-1 expression, compared to the high expression group. Immunofluorescence staining identified Kupffer cells as the main localization of HO-1. To study possible effects of HO-1 induction upon reperfusion, we categorized groups based on the ability to increase HO-1 expression during reperfusion of the liver graft. In this analysis, serum AST levels immediately after liver transplantation were significantly lower in the group with an increase in HO-1 expression compared to livers without upregulation of HO-1 upon reperfusion. These findings suggest that the ability to induce HO-1 expression at the time of graft reperfusion may confer hepatobiliary protection. Further research will be necessary to determine which is more important: a low expression of HO-1 before liver transplantation, or the ability to induce HO-1 at the time of graft reperfusion. In the previous chapter, the endogenous regulation of HO-1 during human liver transplantation was studied. None of the clinical variables analyzed in this study could explain the variation in initial expression of HO-1 in the donor livers. We therefore hypothesized that genetic variations 198

200 Chapter 10 may be responsible for the differences in HO-1 expression and subsequent outcome after liver transplantation. The inducibility of HO-1 is modulated by a (GT) n polymorphism and a single nucleotide polymorphism (SNP) A(-413)T in the promoter. Both a short (GT) n allele and the A-allele have been associated with increased HO-1 promoter activity. In Chapter 9, a study is described in which HO-1 genotype in the donor was tested and correlated with outcome in 308 adult patients. For (GT) n genotype, livers were divided into two classes: short alleles (<25 repeats; class-s) and long alleles ( 25 repeats; class-l). For the A(-413)T SNP, livers were grouped as A-carriers (AT or AA) versus TT-genotype livers. In a subset of each group, hepatic mrna expression was correlated with genotypes. Graft survival at 1 year was significantly better for A-allele genotype compared to TT-genotype (84% versus 63%, p=0.004). Graft loss due to primary dysfunction occurred more frequently in TT-genotype compared to A-receivers (p=0.03). No differences were found for the occurrence of NAS in both groups. Recipients of a liver with TT-genotype had significantly higher serum transaminases after transplantation. Hepatic HO-1 mrna levels were significantly lower in TT genotype livers compared to the A-allele livers (p=0.03). No differences were found for any outcome variable between class S and LL-variant of the (GT) n polymorphism. Haplotype analysis indicated the dominance of the A(-413)T SNP over the (GT) n polymorphism. The main conclusion of this study was that the HO-1 promoter polymorphism A(-413)T is associated with outcome after liver transplantation. The TT variant is linked with worse graft survival, more primary dysfunction, increased I/R injury and reduced HO-1 mrna levels. Furthermore we provided evidence for a greater functional relevance of the A(-413)T SNP over the (GT) n polymorphism. 199

201 Summary, discussion and future perspectives General discussion and future perspectives Part I: Non-anastomotic biliary complications after liver transplantation The specific aims of this section were to describe the various forms of NAS and the accompanying clinical risk factors as well as to study the risk factors for progression of NAS. We found a difference in risk factors for NAS presenting early ( 1 year) and NAS presenting late (>1 year) after transplantation. NAS occurring early after transplantation were correlated with prolonged ischemia times. NAS occurring late after transplantation were more strongly associated with immunological risk factors. These data suggest that there are different subtypes of NAS that have different etiologies. This aspect should be considered in future studies. The following groups of patients were found to have an increased risk for disease progression: patients with a hepaticojejunostomy, those with an early diagnosis of NAS, and those with NAS presenting at the level of the peripheral branches of the biliary tree. In clinical practice it is important to identify these patients for a close follow up and early intervention. Our newly proposed classification system for NAS is a promising tool to better classify patients with NAS. However, to become useable and successful in the currently expanding international field of liver transplantation, our system should be validated. This would enable us to confirm our findings on the relevance of the localization of NAS and subsequent consequences for prognosis and management. Aligning many different international centres with different protocols, facilities and expertise for a prospective study into this classification system might be complex and time consuming. Therefore and second best, this validation could be achieved by retrospectively studying other cohorts of liver transplant patients by reviewing the images of the biliary tree and correlating the classification with risk factors and level of progression. It is likely that recurrent PSC may have been accountable for the occurrence of late NAS in a number of patients. On the basis of radiological evaluation, however, recurrent PSC cannot be distinguished from a late presentation of NAS. Although some of our patients fit well within the definition of recurrent PSC, more than half of our patients who presented with NAS late after transplantation were not transplanted for PSC. In an attempt to reduce the occurrence of early NAS, it remains important to focus on a further reduction of ischemic times, in particular 200

202 Chapter 10 the cold ischemia time. However, many centres have already put a lot of effort in this, and it is questionable whether a substantial further reduction of cold ischemic time is feasible. New perspectives in preservation of the liver graft might realize these assiduously soughtafter improvements of graft quality. Maintaining organ viability via (normothermic) machine perfusion during preservation might be effective in reducing postoperative bile duct injury. The central concept behind (normothermic) perfusion is to maintain normal function of the liver during the whole period of preservation and enable immediate graft function and protect the vulnerable biliary epithelial cells from I/R injury. Currently great efforts are being taken to better understand the concepts of machine perfusion, as well as to find the ideal preservation fluid and to create possibilities to implement machine perfusion in daily practice (1). Part II: Bile physiology after liver transplantation The specific aims of this second section were to evaluate the contribution of bile composition to the development of bile duct injury. We found supporting evidence that toxic bile, characterized by a low phospholipids-to-bile salt ratio, contributes to the development of bile duct injury, not only at a microscopic level but also at a macroscopic level, like NAS. The questions whether bile duct injury and toxic bile composition are not just two consequences of the same underlying factor, has been studied previously by our group. Using mice heterozygous for disruption of the Mdr2 gene (equivalent of human MDR3), Hoekstra et al. confirmed that there is indeed a cause-effect relationship between toxic bile formation and bile duct injury after liver transplantation and ruled out the possibility that toxic bile composition and bile duct injury both result from the same underlying factor (2). In this study it was demonstrated that endogenous bile salts act synergistically with I/R in the origin of bile duct injury in vivo. The question of which role toxic bile plays in bile duct injury is of great interest. What is exactly happening on a cellular level? What is happening on epithelial level? What is the sequence of events before the epithelial cells are damaged so severely that we can detect it by radiological examination? Fickert et al proposed a very appealing mechanism similar to the pathogenesis of primary sclerosing cholangitis in humans. They stated that due to a lack of phospholipids the nonmiccellar-bound, free bile acids might damage the tight junctions and basement membranes of the epithelial lining, leading to leakage of potentially toxic bile acids 201

203 Summary, discussion and future perspectives into the periductal area. As a result the inflammatory response is induced, ultimately resulting in fibrosis and narrowing of the biliary ducts (3). Therapeutic strategies to modify intrahepatic cholestasis and to prevent bile duct injury after OLT may include the administration of the hydrophilic bile salt ursodeoxycholic acid. Daily oral administration of ursodeoxycholic acid is a well-known therapy to reduce bile salt toxicity by replacement of the hydrophobic bile salts in the bile salt pool (4,5). Although the exact mechanisms underlying its cytoprotective effect are not fully understood, it may reduce bile salt induced injury by replacing the toxic hydrophobic biliary bile salts. In addition, it has been shown to stimulate cannalicular transport and biliary excretion, enhancing bile flow and reducing the exposure time of biliary epithelium to toxic bile salts (4). The potentially beneficial effects of ursodeoxycholic acid make this drug an interesting strategy to prevent NAS. Current experimental and clinical research provides strong support for a prospective clinical trial focussing on the abilities of ursodeoxycholic acid to prevent NAS early after liver transplantation. Another interesting therapeutic target could be the MDR3 gene, given the key role of biliary phospholipids in protecting bile duct epithelium from potentially toxic, aggressive biliary content (5). Administration of fibrates, statins, or peroxisome proliferators in mice, have been shown to stimulate biliary phospholipid secretion by the induction of MDR3 making bile less toxic (7-9). Further research in this direction seems justified. Part III: HO-1 and hepatobiliary injury after liver transplantation The specific aim of the third section was to study the role of HO-1 in relation to postoperative hepatobiliary injury and graft function. We showed that upregulation of HO-1 during liver transplantation correlates with better hepatobiliary function after transplantation. Furthermore we demonstrated that patients possessing a polymorphism that is associated with reduced HO-1 expression on mrna level have a worse hepatobiliary function after transplantation and an increased risk of graft loss on the long run. The role of HO-1 as a cytoprotective protein was confirmed by these studies. It was noted that HO-1 is already upregulated in many livers form brain death donors. The variations in the observed upregulation of HO-1 mrna levels could not be explained by a larger number of marginal donors in the group with high HO-1 expression. Moreover, factors associated with major hemodynamic alterations in the donor and several surgical variables 202

204 Chapter 10 were similarly distributed amongst the donor groups with initial low expression of HO-1, compared to the donor group with initial high expression of HO-1. To find an explanation for these differences we studied two functional polymorphisms in the promoter region of the gene: a (GT) n polymorphism and the single nucleotide polymorphism A(-413)T SNP. The finding that the A(-413)T SNP exerts its effect not only in the immediate moments after the transplant procedure, but has also consequences in the longer term (figure 4 in chapter 9), is interesting. This could indicate that not only attenuation of I/R injury by a favourable HO-1 phenotype is beneficial but that HO-1 mediated processes may also play a role in later phases after the transplantation. Clinical application of interventions in the HO-1 system should be considered. However, we should bear in mind that the beneficial effects of HO-1 may have a narrow therapeutic window as shown in chapter 8. Highly overexpressed HO-1 displays pro-oxidant properties secondary to iron accumulation, and may therefore be harmful instead of cytoprotective. It would be very interesting to focus this research on the specific effect of HO-1 on biliary epithelial cells which are especially vulnerable for I/R injury. We know from the study described in chapter 8 that human HO-1 in the liver is mainly located in the Kupffer cells, and not abundantly present in biliary epithelial cells. Strategies to enter HO-1 in these cells might be of great interest to study whether HO-1 over expression could protect the bile ducts from injury resulting from I/R injury or bile toxicity. In summary, new insights are provided into the molecular and biochemical mechanisms of bile duct injury after liver transplantation. We have proposed a classification system of NAS based on the localization and severity of the biliary abnormalities. This classification system appeared valuable in identifying different etiologies of NAS and also allowed the identification of patients with NAS who are more at risk for complications or disease progression. Toxic bile composition, characterized by a low phospholipids-to-bile salt ratio was discovered as a contributing mechanism in the development of bile duct injury and NAS after liver transplantation. Further interventional studies aimed at prevention of NAS based on the principle of this altered bile composition are warranted. Finally, we have demonstrated a cytoprotective role of HO-1 in liver transplantation, opening new avenues for the development of novel preventive strategies or therapies. 203

205 Summary, discussion and future perspectives References 1. Maathuis MH, Leuvenink HG, Ploeg RJ. Perspectives in organ preservation. Transplantation ;83: Hoekstra H, Porte RJ, Tian Y, Jochum W, Stieger B, Moritz W, et al. Bile salt toxicity aggravates cold ischemic injury of bile ducts after liver transplantation in Mdr2+/- mice. Hepatology ;43: Fickert P, Fuchsbichler A, Wagner M, Zollner G, Kaser A, Tilg H et al. Regurgitation of bile acids from leaky bile ducts causes sclerosing cholangitis in Mdr2 (Abcb4) knockout mice. Gastroenterology 2004;127: Fickert P, Zollner G, Fuchsbichler A, Stumptner C, Pojer C, Zenz R, et al. Effects of ursodeoxycholic and cholic acid feeding on hepatocellular transporter expression in mouse liver. Gastroenterology 2001;121: Trauner M,Fickert P,Wagner M. MDR3(ABCB4) defects: Aparadigm for the genetics of adult cholestatic syndromes. Semin Liver Dis 2007; 27: Trauner M, Boyer JL. Bile salt transporters: Molecular characterization, function, and regulation. Physiol Rev 2003; 83: Miranda S, Vollrath V, Wielandt AM, et al. Overexpression of mdr2 gene by peroxisome proliferators in the mouse liver. J Hepatol 1997; 26: Hooiveld GJ, Vos TA, Scheffer GL, et al. 3-Hydroxy-3-methylglutarylcoenzymeAreductase inhibitors (statins) induce hepatic expression of the phospholipid translocase mdr2 in rats. Gastroenterology 1999; 117: Chianale J, Vollrath V, Wielandt AM, et al. Fibrates induce mdr2 gene expression and biliary phospholipid secretion in the mouse. Biochem J 1996; 314:

206 Nederlandse samenvatting

207 Nederlandse samenvatting Samenvatting Levertransplantatie is de aangewezen behandeling voor patiënten met eindstadium leverfalen. Het succespercentage van een transplantatie is groot, na 5 jaar is meer dan 75% van de patiënten nog in leven. Er kunnen echter complicaties optreden, onder meer van de galwegen. Complicaties van de galwegen betreffen lekkage, stricturen van de anastomose (AS) en stricturen en verwijdingen van de galwegen in de lever, de non-anastomotische stricturen (NAS). In dit proefschrift zijn NAS nader onderzocht, we hebben gekeken naar moleculaire en biochemische mechanismen van deze complicatie. NAS is een diagnose die gesteld wordt door de radioloog op basis van afbeelding van de galwegen, een cholangiogram. Het beeld wordt gekarakteriseerd door vernauwingen en verwijdingen van de galwegen in de lever (intrahepatisch). NAS na levertransplantatie zijn initieel veel beschreven in combinatie met een trombose van de arteria hepatica (HAT). In het geval van een vroege HAT na levertransplantatie worden de galwegen ischemisch en uiteindelijk necrotisch, hetgeen resulteert in een typisch beeld op het cholangiogram met vernauwingen en verwijdingen. Deze typische cholangiografische afwijkingen van vernauwingen en verwijdingen worden echter soms ook gezien in de afwezigheid van HAT, vandaar dat de term ischemic-type biliary lesions (ITBL) is ontstaan. In dit proefschrift is de term NAS gebruikt om intrahepatische vernauwingen en verwijdingen te beschrijven in de bevestigde afwezigheid van HAT. De incidentie van NAS varieert rond 15% in verschillende onderzoeken. Er zijn meerdere risico factoren voor NAS geïdentificeerd wat sterk suggereert dat er een multifactoriele origine is. De belangrijkste categorieën zijn ischemisch gerelateerde schade, immunologisch geïnduceerde schade en schadelijke effecten door galzouten. In sommige gevallen kan echter geen specifieke risicofactor worden aangewezen. De klinische presentatie van patiënten met NAS is vaak niet specifiek. Symptomen als koorts, buikklachten en afwijkende lever waarden in het bloed duidend op cholestase kunnen voorkomen. De diagnose wordt gesteld met behulp van beeldvormende studies van de galwegen. De behandeling begint met het verlichten van de klachten veroorzaakt door de cholestase en verwijding van de eventueel gestenoseerde galwegen met behulp van endoscopisch retrograde 206

208 cholangiopancreaticography (ERCP) of percutanes transhepatische cholangiodrainage (PTCD) zo mogelijk gevolgd door het achterlaten van een stent in de galwegen. Uiteindelijk moet er bij een deel van de patiënten een re-transplantatie plaatsvinden of komen ze te overlijden. In geselecteerde gevallen kan een re-transplantatie worden vermeden of in ieder geval uitgesteld door chirurgische interventie. Part I: Non-anastomotische galwegstricturen na levertransplantatie In dit eerste gedeelte beschrijven we een tweetal studies met als doel a) de verschillende vormen van NAS en bijbehorende klinische risico factoren te beschrijven, b) te achterhalen welke patiënten met NAS een risico lopen op het ontwikkelen van ernstige problemen en de behandeling daarvan. In hoofdstuk 3 presenteren we een nieuwe classificatie van NAS. Met deze methode hebben we alle beeldvorming van de patiënten met NAS van de afgelopen jaren in het UMCG opnieuw beoordeeld. Verder hebben we gekeken naar risicofactoren voor het ontstaan van NAS, het moment na de transplantatie waarop NAS zich presenteren en de progressie van de ziekte in de loop van de jaren na de transplantatie. In een groep van 487 volwassen transplantatie patiënten met een mediane follow-up van bijna 8 jaar waren er 81 (16.6%) patiënten die NAS ontwikkelden. We hebben aanwijzingen gevonden dat er 2 vormen van NAS bestaan. NAS welke zich vroeg, binnen 1 jaar na de transplantatie, presenteren en NAS welke zich na meer dan 1 jaar presenteren. Vroege NAS zijn geassocieerd met preservatie en ischemisch gerelateerde risocofactoren, zoals een langere koude en warme ischemie tijd. Tevens presenteren de vroege NAS zich vaker centraal in de lever. Late NAS daarentegen zijn meer geassocieerd met immunologische risico factoren, zoals primair scleroserende cholangitis als indicatie voor de transplantatie. Deze late vorm van NAS werd vaker gezien in de periferie van de galwegen. We kunnen dus zeggen dat er verschillende pathogenetische processen een rol spelen bij het ontstaan van NAS. Als we verder kijken naar de groep patiënten met diagnose NAS, in hoofdstuk 4, konden we de volgende risicofactoren identificeren voor radiologische progressie, wat bij bijna 70% van de patiënten optrad: vroege NAS en één of meerdere episodes van bacteriële cholangitis. Bij patiënten met NAS bleek een galwegreconstructie met een Roux-Y hepaticojejunostormie een risicofactor voor het ontstaan van bacteriële cholangitis. Ernstige fibrose 207

209 Nederlandse samenvatting of cirrose ontstond in 23 gevallen, vooral in gevallen waarbij de NAS perifeer in de lever gelokaliseerd was. Transplantaatoverleving, maar niet patiëntenoverleving, werd beïnvloed door de aanwezigheid van NAS. Dertien patiënten (16%) onderging een re-transplantatie vanwege de NAS. De conclusie van deze studie was dat vooral patiënten met een Roux-Y reconstructie, patiënten met vroege NAS en patiënten met NAS in de periferie van de lever risico lopen op voortschrijdende ziekte met ernstige uitkomsten. Part II: Gal fysiologie na levertransplantatie De specifieke doelen van dit tweede gedeelte waren te evalueren welke bijdrage de gal samenstelling heeft op het ontstaan van galwegschade. Van galzouten is het bekend dat ze een detergente, vetoplossende werking hebben (zie de titelpagina verklaring). Galzouten, zonder beschermende fosfolipiden, hebben een schadelijke werking. Het is bekend dat ze een rol spelen in het ontstaan van galwegschade en cholangiopathie bij vele andere ziektebeelden. Galformatie direct na de transplantatie kan verstoord zijn hetgeen resulteert in een dergelijke schadelijke samenstelling met een lage fosfolipiden galzouten ratio. De rol van schadelijke galsamenstelling in de ontwikkeling van galwegstricturen na levertransplantatie is echter onduidelijk. Na non heart-beating (NHB) levertransplantatie zijn NAS een vaak voorkomende complicatie. In hoofdstuk 5 wordt in een varkensmodel van NHB levertransplantatie het effect van verschillende periodes van warme ischemie in de donor op galsamenstelling en daaropvolgende schade aan de galwegen na transplantatie bestudeerd. In drie groepen werd een oplopende vertraging voor uitname van de lever toegepast, waarna het orgaan wordt getransplanteerd na 4h koude bewaartijd. De galflow werd gemeten en galmonsters werden dagelijks verzameld om de fosfolipiden galzout ratio te bepalen. De mate van schade aan de galwegen werd gescoord door middel van een histologische scoringsschaal. De resultaten toonden dat de fosfolipiden galzouten ratio in de gal die vlak na de transplantatie werd geproduceerd significant lager is in groep met de langste warme ischemietijd in de donor. In deze groep was ook de grootste mate van galwegschade te zien. Op basis van de resultaten van deze studie werd geconcludeerd dat langere warme ischemie in NHB donoren is geassocieerd met de vorming van schadelijke gal na transplantatie, deze bevindingen suggereren dat schade door galzouten bijdraagt aan het ontstaan van galwegschade na NHB levertransplantatie. 208

210 In het voorgaande hoofdstuk en ook andere studies uit onze groep hebben aangetoond dat galformatie direct na de transplantatie verstoord kan zijn wat resulteert in een schadelijke samenstelling van de gal met een lage fosfolipiden - galzouten ratio. Het was onbekend of deze veranderde samenstelling ook en rol speelt bij de ontwikkeling van NAS, een aandoening van de grotere galwegen. Indien veranderingen in de galsamenstelling een rol spelen bij het ontstaan van NAS na transplantatie dan is te verwachten dat de galsamenstelling na transplantatie anders is bij patiënten die NAS ontwikkelen ten opzichte van patiënten die geen NAS ontwikkelen. Deze hypothese werd getest in een prospectieve klinische studie welke beschreven is in hoofdstuk 6. In deze grote cohort studie bij volwassen levertransplantatiepatiënten werd galproductie en samenstelling in de eerste week na transplantatie gecorreleerd aan het ontstaan van NAS in het verdere beloop na transplantatie. In 111 levertransplantatiepatiënten werden dagelijks galmonsters verzameld om de samenstelling te analyseren. NAS werden gediagnosticeerd in 14 patiënten (13%) binnen 1 jaar na transplantatie. Patiënten die uiteindelijke NAS ontwikkelden bleken minder galzouten, fosfolipiden en cholesterol uit te scheiden in de gal. Tegelijkertijd was de biliare fosfolipiden - galzouten ratio lager in patiënten die NAS ontwikkelden, wat duidt op mogelijk meer schadelijke samenstelling van de gal. Deze bevindingen passen in het concept dat galzouten betrokken is bij het ontstaan van NAS. In het voorgaande hoofdstuk hebben we laten zien dat schadelijke gal samenstelling met een verlaagde fosfolipiden - galzouten ratio geassocieerd is met NAS na levertransplantatie. Hepatobiliare transporteiwitten zijn verantwoordelijk voor de secretie van fosfolipiden en galzouten vanuit de hepatocyten naar de gal. Omdat de verschillen in de galsamenstelling niet te verklaren waren door klinische variaties bij de donor of ontvanger was het doel van hoofdstuk 7 om te analyseren of variaties in de genen die coderen voor deze transporters in de donor geassocieerd zijn met het ontstaan van NAS in de ontvanger. Zonder transplantatie leiden deze variaties op zichzelf niet tot galwegschade. Echter, direct na transplantatie, op het moment dat de lever nog herstellende is van de schade van de ischemie en reperfusie (I/R), kunnen deze variaties juist een belangrijke factor zijn in de serie van gebeurtenissen die leidt tot het ontstaan van galweschade. Een vergelijkbare situatie wordt gezien bij patiënten met intrahepatische cholestase tijdens de zwangerschap. Wanneer deze patiënten niet zwanger zijn hebben zij geen klachten, echter op het moment dat er iets bijzonders gebeurt, een zwangerschap vertonen ze ziekte verschijnselen. Bij 458 levertransplantatie procedures 209

211 Nederlandse samenvatting konden we de verschillende hepatobiliare transport eiwitten genotyperen. Zevenenzeventig patiënten (16%) ontwikkelden NAS na transplantatie. Patiënten die een donorlever ontvingen met een genetische variatie in de fosfolipidentransporter ontwikkelde bijna 2 keer zo vaak NAS (28%) als patiënten die een donorlever ontvingen zonder deze variatie (15%). Ook in een multivariate analyse was deze variatie een onafhankelijke risicofactor voor het ontstaan van NAS. Hoewel we dat in deze studie niet hebben onderzocht, zou het transport van fosfolipiden naar de gal in levers welke drager zijn van het risico haplotype veranderd kunnen zijn in de direct postoperatieve periode en op deze wijze een bijdrage kunnen leveren aan het ontstaan van NAS. Part III: HO-1 en hepatobiliaire schade na lever transplantatie Het specifieke doel van het derde deel was om de rol van heme oxygenase-1 (HO-1) te bestuderen in relatie tot hepatobiliaire schade en leverfunctie. Opregulatie van HO-1 wordt beschouwd als een belangrijk beschermingsmechanisme tegen I/R schade bij levertransplantatie. In hoofdstuk 8 hebben we in 38 volwassen levertransplantatiepatiënten de rol van endogene HO-1 expressie, voor tijdens en na transplantatie, bestudeerd in relatie tot postoperatieve hepatobiliaire schade en functie direct na transplantatie. Voorafgaand aan de operatie was de mediane HO-1 expressie reeds 3,4-keer verhoogd (spreiding 0,7 tot 9,3-keer verhoogd). Deze spreiding was niet te verklaren door de klinische condities of behandelingen van de donoren. We vonden dat in de groep van levers die het vermogen hadden om het HO-1 tijdens de transplantatie verder op te reguleren, de schade aan de lever minder was dan bij de patiënten waarbij de HO-1 expressie in de lever niet toenam. Dit suggereert dat levers die tijdens reperfusie het HO-1 kunnen induceren beter beschermd zijn tegen I/R-schade dan donorlevers die dit niet kunnen. Verder onderzoek zal nodig zijn om te achterhalen wat belangrijker is: een lage HO-1 expressie voor aanvang van de transplantatie, of het vermogen om HO-1 tijdens de reperfusie te induceren. In het voorgaande hoofdstuk is de endogene regulatie van HO-1 tijdens levertransplantatie onderzocht. Omdat de variatie die werd gevonden in de initiële HO-1 expressie in de donor levers kon niet worden verklaard door klinische variabelen, werd de hypothese opgevat dat genetische verschillen verantwoordelijke zouden kunnen zijn voor de variatie in HO- 1. De expressie van HO-1 wordt in belangrijke mate bepaald door 2 variaties in het gen, zogenaamde polymorfismen. Eén daarvan is het single nucleotide polymorfisme (SNP) 210

212 A(-413)T. Aangezien ieder mens 2 allelen heeft kunnen de volgende variaties ontstaan: AA, AT en TT. De A-variant is geassocieerd met een verhoogde HO-1 activiteit. In hoofdstuk 9 beschrijven we een studie waarin de genetische variatie van de donor werd geanalyseerd en gecorreleerd aan uitkomsten na transplantatie in een groep van 308 volwassen patiënten die een levertransplantatie ondergingen. In een subgroep werd de HO-1 genexpressie in de lever gecorreleerd aan de genotypen. Overleving van het transplantaat na 1 jaar was beter voor A-varianten in vergelijking met de TT-genotypes. Verlies van het transplantaat als gevolg van primaire disfunctie werd vaker waargenomen bij levers met het TT-genotype. Er werd geen verschil gezien in de incidentie van NAS in beide groepen. Ontvangers van een TT-genotype lever hadden meer schade aan de lever direct na transplantatie. HO-1 genexpressie in de lever was lager in de levers met het TT-genotype, in vergelijking met levers met een A-allel. De belangrijkste conclusie van deze studie was dat het A(-413)T polymorphisme in de HO-1 promoter geassocieerd is met uitkomsten na levertransplantatie. Tot besluit kunnen we stellen dat onderzoek naar de moleculaire en biochemische mechanismen van het ontstaan van galwegschade belangrijke nieuwe gezichtspunten hebben opgeleverd. We hebben een nieuw classificatie systeem voor NAS voorgesteld, dat is gebaseerd op de lokalisatie en ernst van de galwegafwijkingen. Dit classificatiesysteem bleek waardevol in het identificeren van verschillende ontstaansmechanismen van NAS, tevens was het mogelijk patiënten te identificeren die een groter risico liepen op complicaties en progressie van de ziekte. Schadelijke samenstelling van de gal, gekarakteriseerd door een lage fosfolipiden - galzouten ratio, werd geïdentificeerd als een belangrijk bijdragende factor aan het ontstaan van galwegschade en NAS na levertransplantatie. Verder interventie onderzoeken gericht op het voorkómen van NAS gebaseerd op de bevindingen van deze veranderde gal samenstelling zijn nu het aangewezen vervolg. Tot slot hebben we een beschermende rol aangetoond voor HO-1 in levertransplantatie, dit opent nieuwe wegen voor het ontwikkelen van preventieve strategieën en therapieën. 211

213

214 List of contributing authors

215 List of Contributing Authors List of Contributing Authors Dr. H. Blokzijl Department of Gastroenterology and Hepatology University Medical Centre Groningen Groningen, the Netherlands Dr. W. Geuken Surgical Research Laboratory, Department of Surgery. Currently Department of Pathology University Medical Centre Groningen Groningen, the Netherlands Dr. A.S.H. Gouw Department of Pathology University Medical Centre Groningen Groningen, the Netherlands Dr. E.B. Haagsma Department of Gastroenterology and Hepatology University Medical Centre Groningen Groningen, the Netherlands Dr. B.G. Hepkema Department of Laboratory medicine, Transplantation Immnology University Medical Centre Groningen Groningen, the Netherlands Drs. C.S. van der Hilst Section Hepatobiliary Surgery and Liver Transplantation, Department of Surgery University Medical Centre Groningen Groningen, the Netherlands 214

216 Drs. H.H. Hoekstra Section Hepatobiliary Surgery and Liver Transplantation, Department of Surgery University Medical Centre Groningen Groningen, the Netherlands Dr. E.J. Van der Jagt Department of Radiology University Medical Centre Groningen Groningen, the Netherlands Dr. K.P. de Jong Section Hepatobiliary Surgery and Liver Transplantation, Department of Surgery University Medical Centre Groningen Groningen, the Netherlands O.N.H. Kahmann Surgical Research Laboratory University Medical Centre Groningen Groningen, the Netherlands Prof. dr. H. Kleibeuker Department of Gastroenterology and Hepatology University Medical Centre Groningen Groningen, the Netherlands Prof. dr. F. Kuipers Pediatric Gastroenterology, Department of Pediatrics University Medical Centre Groningen Groningen, the Netherlands 215

217 List of Contributing Authors Dr. H.G.D. Leuvenink Surgical Research Laboratory University Medical Centre Groningen Groningen, the Netherlands Dr. A.J. Limburg Department of Gastroenterology and Hepatology University Medical Centre Groningen Groningen, the Netherlands Dr. D. Monbaliu Department of Abdominal Transplant Surgery and coordination University Hospitals Leuven Leuven, Belgium Prof. H. Moshage Department of Gastroenterology and Hepatology University Medical Centre Groningen Groningen, the Netherlands Dr. B.A. Nemes Section Hepatobiliary Surgery and Liver Transplantation, Department of Surgery University Medical Centre Groningen Groningen, the Netherlands Dr. M. Nijsten Surgical Intensive Care Unit University Medical Centre Groningen Groningen, the Netherlands 216

218 Dr. I.M. Nolte Department of Epidemiology University Medical Centre Groningen Groningen, the Netherlands Dr. P. M.J.G. Peeters Section Hepatobiliary Surgery and Liver Transplantation, Department of Surgery University Medical Centre Groningen Groningen, the Netherlands Prof. dr. J. Pirenne Department of Abdominal Transplant Surgery and coordination University Hospitals Leuven Leuven, Belgium Prof. dr. R.J. Porte Section Hepatobiliary Surgery and Liver Transplantation, Department of Surgery University Medical Centre Groningen Groningen, the Netherlands Dr. T.A. Schuurs Surgical Research Laboratory University Medical Centre Groningen Groningen, the Netherlands Prof. dr. M.J.H. Slooff Section Hepatobiliary Surgery and Liver Transplantation, Department of Surgery University Medical Centre Groningen Groningen, the Netherlands 217

219 List of Contributing Authors Dr. G. van der Steege Department of Genetics University Medical Centre Groningen Groningen, the Netherlands Dr. R.C. Verdonk Department of Gastroenterology and Hepatology University Medical Centre Groningen Groningen, the Netherlands Prof. dr. H.J. Verkade Pediatric Gastroenterology, Department of Pediatrics University Medical Centre Groningen Groningen, the Netherlands Ing. D.S. Visser Surgical Research Laboratory, Department of Surgery University Medical Centre Groningen Groningen, the Netherlands Drs. M.J. Yska Section Hepatobiliary Surgery and Liver Transplantation, Department of Surgery Surgical Research Laboratory University Medical Centre Groningen Groningen, the Netherlands 218

220 List of publications

221 List of Publications List of Publications Buis CI, Bakker SJL. ACE- inhibitie niet effectief bij voorkómen van restenose na coronaire stentimplantatie. Ned Tijdschr Geneeskd 2001; 145:2051. (short review paper). Buis CI, Wijdicks EFM. Serial MR imaging in central pontine myelinolysis. Liver Transpl 2002; 8: Buis CI, Wiesner RH, Krom RAF, Kremers WK, Wijdicks EFM. Acute confusional state following liver transplantation for alcoholic liver disease. Neurology 2002; 59: Geuken E, Buis CI, Visser DS, Blokzijl J, Moshage H, Nemes B, Leuvenink HGD, Jong de KP,Peeters PMJG, Slooff MJH, Porte RJ. Expression of Heme oxygenese-1 in human livers before transplantation correlates with graft injury and function after transplantation. Am J Transplant 2005; 5: Buis CI, Porte RJ, Slooff MJH. Levertransplantatie. In: H.G. Smeenk, N.W.L. Schep, W.M.U. van Grevenstein ed. Leidraad chirurgie. Houten: Bohn Stafleu van Loghum, 2005; 211. Buis CI, Porte RJ, Slooff MJH. Niertransplantatie. In: H.G. Smeenk, N.W.L. Schep, W.M.U. van Grevenstein ed. Leidraad chirurgie. Houten: Bohn Stafleu van Loghum, 2005; 219. Verdonk RC, Buis CI, Porte RJ, Haagsma EB. Biliary complications after liver transplantation, A review. Scand J Gastroenterol 2006; 243: Buis CI, Hoekstra H, Verdonk RC, Porte RJ. Causes and Consequences of Ischemic Type Biliary Lesions After Liver Transplantation. J Hepatobiliary Pancreat Surg 2006; 13: Su Huawei, van Dam GM, Buis CI, Visser DS, Hesselink JW, Schuurs TA, Leuvenink HGD, Contag CH, Porte RJ. Spatiotemporal Expression of heme oxygenase-1 Detected by in vivo bioluminescence after hepatic ischemia in HO-1/Luc mice. Liver Transpl 2006; 12:

222 Verdonk RC, Buis CI, Porte RJ, van der Jagt EJ, Limburg AJ, vd Berg AP, Slooff MJH, Peeters PMJG, de Jong KP, Kleibeuker JH, Haagsma EB. Anastomotic biliary strictures after liver transplantation: prevalence, presentation, management and outcome. Liver Transpl 2006; 12: Mantel HTJ, Buis CI, Homan van der Heide JJ, van den Berg AP, Verkade HJ, Haagsma EB, Peeters PMGJ, de Jong KP, Slooff MJH, Porte RJ. Gecombineerde lever-niertransplantaties: Indicaties en resultaten in het UMC Groningen. Ned Tijdschr Geneeskd 2006; 150: Buis CI, Verdonk RC, van der Jagt EJ, van der Hilst CS, Slooff MJH, Haagsma EB, Porte RJ. Non-anastomotic biliary strictures after adult liver transplantation part one: Radiological features and risk factors for early versus late presentation. Liver Transpl 2007; 13: Verdonk RC, Buis CI, van der Jagt EJ, Gouw ASH, Limburg AJ, Slooff MJH, Kleibeuker JH, Porte RJ, Haagsma EB. Non-anastomotic biliary strictures after adult liver transplantation part two: Management, outcome and risk factors for disease progression. Liver Transpl 2007; 13: Buis CI, Steege vd G, Visser DS, Nolte IM, Hepkema BG, Nijsten M, Slooff MJH, Porte RJ. Heme oxygenase-1 genotype of the donor is associated with graft survival after liver transplantation. Am J Transplant 2008; 8: Buis CI, Hofker HS, Nieuwenhuijs VB. Diverticulitis of the Jejunum, an uncommon diagnosis. Dig Surg. 2008; 25:83-4. Yska MJ, Buis CI, Monbaliu D, Schuurs TA, Gouw ASH, Kahmann ONH, Visser DS, Pirenne J, Porte RJ. The role of bile salt toxicity in the pathogenesis of bile duct injury after non heartbeating porcine liver transplantation. Transplantation 2008; 85: Buis CI, Geuken E, Visser DS, Kuipers F, Haagsma EB, Verkade HJ, Porte RJ. Altered bile composition is associated with the development of nonanastomotic biliary strictures. J of Hepatol, in press. 221

223 List of Publications Buis CI, Steege vd G, Visser DS, Nolte IM, Porte RJ. Polymorphism of hepatobiliary phospholipid transporter ABCB4 associated with nonanastomotic biliary strictures after human liver transplantation. Submitted. Hoekstra H, Buis CI, Verdonk RC, van der Hilst CS, van der Jagt EJ, Haagsma EB, Porte RJ. Is Roux-Y choledochojejeunostomy an indipendant risk factor for non-anastomotic biliary strictures after liver transplantation? Submitted. 222

224 Dankwoord

225 Dankwoord Dankwoord Het állerleukste van promoveren is dat je met zoveel verschillende mensen mag samenwerken en van hen kan leren. Graag w il ik velen van hen in dit meest gelezen hoofdstuk noemen. Professor dr. R.J. Porte, beste Robert, vanzelfsprekend ben jij de eerste, de allerbelangrijkste. Jij hebt voor mij de mogelijkheid gecreëerd om AGIKO te worden. Jij hebt mij in staat gesteld dit mooie (al zeg ik het zelf) proefschrift af te leveren. Het is fantastisch om met je te werken! Je bezieling voor wetenschappelijk onderzoek is ongelooflijk groot en voortdurend aanstekelijk omdat het is gebaseerd op een zeer scherpe en heldere analyse van de onderwerpen. Iedere keer na overleg met jou had ik nóg meer inspiratie om aan de slag te gaan, jammer dat de promotie nu klaar is. Gelukkig is er in ieder geval nog één artikel dat we samen verder mogen polijsten, zodat de samenwerking op deze manier nog even door mag gaan. Tot slot, je hebt een uitermate groot talent om een hele goede, leuke en bovenal gezellige groep van onderzoekers om je heen te verzamelen, het is genieten om daar onderdeel van uit te mogen maken. Professor dr. M.J.H. Slooff, beste professor, u bent het boegbeeld van de hepatobiliare chirurgie en levertransplantatie in Groningen. Op de achtergrond bent u voor mij heel belangrijk geweest voor het welslagen van deze promotie. Uw wijsheid en warmte die ik heb leren kennen voor het vak, maar zeker ook voor de andere mooie dingen van het leven zal ik niet vergeten. Dank voor het beoordelen van dit proefschrift. Professor dr. H.J. Verkade, beste Henkjan, ik heb heel veel van je geleerd. Voor mij ben je hét voorbeeld van combinatie van klinische top zorg met top wetenschappelijk onderzoek. Je analyseert, denkt (en praat) zó snel dat het voor een gewone sterveling zoals ik vaak moeilijk is bij te houden. Maar ik heb je altijd alle vragen mogen stellen totdat ik het begreep, dank voor alle heldere uitleg. Dank voor het beoordelen van dit proefschrift. Professor dr. H.J. Metselaar, beste Herold, ik heb je (en de onderzoeksgroep uit Rotterdam) mogen ontmoeten op vele mooie plaatsten in de wereld. Dank voor het beoordelen van dit proefschrift. 224

226 Professor dr. H.J. ten Duis, beste opleider, zonder uw steun was dit proefschrift nooit in deze vorm tot stand gekomen. Graag wil ik u danken voor het vertrouwen dat u al heel vroeg in mij heeft gesteld. Dr. M. Eeftinck Schattenkerk, beste dr. Schattenkerk, graag wil ik ook u danken voor het vertrouwen dat u reeds vroeg in mij gesteld heeft, dat ik mijn opleiding juist in Deventer mag vervolgen. Nu dit proefschrift succesvol is afgerond kijk ik er erg naar uit onder uw leiding ook mijn chirurgische vaardigheden verder te ontwikkelen. Drs. M.T. de Boer, lieve Marieke, samen kunnen wij de wereld aan! Rio de Janiero, Milaan, San Fransisco, Los Angeles, Amsterdam, Mumbai, (New York...?). Een voor een hoogtepunten. Kijk uit naar (het feest van) jouw promotie. De manier waarop jij je vak bedrijft is een groot voorbeeld voor me, ik ben apetrots dat jij mijn paranimf bent. Dr. M.H.J. Maathuis, lieve Hugo, wat een feest was het om tegelijk met jou onderzoek te doen. Het is fantastisch om met jou samen te werken, je bent ongelooflijk positief, organisatorisch de beste, en een echte teamspeler, superlatieven te kort. Van het organiseren van het SEOHS heb ik dan ook intens genoten. Ik weet zeker dat je succesvol en gelukkig zal worden in je nieuwe functie. Ik ben waanzinnig vereerd dat je mijn paranimf bent. Dr. W. Geuken, beste Erwin, dank voor het mede opzetten van de lijn galwegcomplicaties na levertransplantatie in het lab. Dr. R.C. Verdonk, beste Robert, nu is het mijn beurt jou te bedanken voor het samenwerken. Dank ook voor alle biopten waar ik een stukje van mocht hebben. Ik kom graag op je oratie over een paar jaar! Drs. M.J. Yska, beste Marit, jij hebt als student in het lab een uitzonderlijke prestatie geleverd! Dank voor het trekken van het Leuven project, en veel succes in je verdere carrière. Ing D.S. Visser, Beste Dorien, dank voor je grote hulp bij alle labbepalingen! We hebben fantastisch samengewerkt, hetgeen ook blijkt uit het feit dat jullie zoon pas werd geboren toen al het werk voor dit proefschrift klaar was! Veel geluk met het leven op Ameland! Dr. G. van der Steege, beste Gerrit, jij nam alle tijd om mij in te wijden in de wereld van de polymorfismen en haplotypes. Het was genieten om samen te puzzelen achter jouw computer. 225

227 Dankwoord Hoofdstuk 9 is inmiddels heel mooi gepubliceerd, en ik weet zeker dat dit ook met Hoofdstuk 7 gaat lukken! Dr. E.J. van der Jagt, beste dr. van der Jagt, dank voor de vele uren dat u samen met Robert Verdonk en mij alle beeldmaterieel van mogelijke NAS patiënten heeft gereviseerd en gescoord. De uitkomsten van dit werk waren essentieel voor dit proefschrift. Professor dr. F. Kuipers, beste Folkert, hoe fantastisch is het ergens te mogen werken waar je grootheden als jij gewoon tegen het lijf loopt bij het koffieapparaat. Dank voor je warme betrokkenheid bij mijn promotie onderzoek en in het bijzonder hoofdstuk 6. Dr. I.M. Nolte, beste Ilja, jouw inzicht in statistiek is onnavolgbaar, toch slaagde je erin mij te laten begrijpen wat je deed. Dank voor je hulp bij Hoofdstuk 7 en 9. Dr. E.B. Haagsma, beste dr. Haagsma, als begeleider van Robert Verdonk bent u betrokken geweest bij het welslagen van Part I van mijn proefschrift, dank daarvoor. Professor dr. J. Pirenne en dr. D. Monbaliu, beste professor, beste Diethard, dank voor de succesvolle samenwerking en de warme ontvangst in Leuven, ook voor Marit. De medeauteurs van de artikelen en nog niet eerder genoemd; Dr. A.S.H. Gouw, beste Annette, dank voor uw hulp bij alle pathologische vraagstukken. Dr. B.G. Hepkema, beste Bouke, dank voor de hulp vanuit de transplantatie immunologie bij hoofdstuk 7 en 9. Dr. M. Nijsten, beste Maarten, dank voor de hulp bij het verkrijgen van alle labwaarden van de levertransplantatiepatiënten. Dr. H. Blokzijl, beste Hans, alweer lang geleden heb jij mijn eerste schreden in het lab begeleid, dank daarvoor en voor al het advies along the way. Drs. C.S. van der Hilst, beste Christian, al mijn statistische kennis heb ik van jou! Dank voor je voortdurende uitleg. Drs. H.H. Hoekstra, beste Harm, ik heb respect voor jouw eigen manier waarop je je onderzoek bedrijft. Je muizen studie is mechanistisch gezien voor dit proefschrift van groot belang geweest. HPB en Levertransplantatie chirurgen en fellows uit Groningen dank ik voor alle uitleg en het verzamelen van materiaal zodat er onderzoek gedaan kon worden; Dr. P.M.J.G. Peeters, beste Paul, de precisie en toewijding waarmee jij opereert en klinische zorg verleent heb ik als keuzeco leren kennen en zal voor altijd een voorbeeld blijven. Daarnaast heb ik de gesprekken over zeilen, schilderen en het leven bijzonder gewaardeerd. Dr. K.P. de Jong, beste Koert, na ons gestrande KOROCA project durfde ik bijna niet opnieuw bij de HPB club in Groningen aan te kloppen, ik ben blij dat ik toch de stap heb genomen. Dr. I.Q. Molenaar, beste Q, hoe kan ik jou bedanken? Het feestje had al ruim 2 jaar geleden kunnen zijn ;-). Je bent een waanzinnig 226

228 groot voorbeeld van een ambitieuze chirurg met een prachtgezin! Dr. W.G. Polak, beste Wojtek, dr. B.A. Nemes, dear Balasz, dr. S. Eguchi, dear Susumu, dr. A. Soyama, dear Aki en dr. E. Sieders, beste Ger, thank you so much for all your explanations during the past years. Professor dr. H. Kleibeuker, dr. A.J. Limburg, dank voor uw bijdrage aan hoofdstuk 4 van dit proefschrift. Dr. A.P. van den Berg, beste Aad, dank voor al je uitleg en warme belangstelling tijdens en na mijn keuzecoschap tijd. Graag wil ik de volgende mensen danken voor hun bijdrage aan mijn proefschrift. Tina Crabbé uit Leuven is onmisbaar geweest voor het uitvoeren van de experimenten uit Hoofdstuk 5, zodat wij met de verkregen monsters onderzoek konden doen. Jan Bottema, dank voor het verzamelen van gal voor Hoofdstuk 6, zodat ik ook eens een weekendje weg kon! Renze Boverhof, dank voor het uitvoeren van de gaschromatografie van de galmonsters in hoofdstuk 6. Mariska Geuken, Fjodor van der Sluijs en Petra Suichies-Ottens, voor jullie technische ondersteuning, en niet alleen voor hoofdstuk 8! Marcel Mulder, dank voor het uitvoeren van de genotypering van Hoofdstuk 7 en 9. Tot slot Daniëlle Nijkamp, dank voor vele zaken, maar ook voor de gezellige tijd in het oude archief van Eurotransplant in Leiden alwaar we alle oude donordata hebben nagezocht! De omgeving waar ik mijn onderzoek heb uitgevoerd is het Chirurgisch Onderzoekslaboratorium. Vanuit de kliniek uiteraard professor dr. R.J. Ploeg, dank voor uw vele scherpe vragen tijdens de labbesprekingen, en voor het meenemen van de jonge dokter op donor (vlucht (!) met whisky). Dr. G.M. van Dam, je bent een waanzinnig enthousiaste wetenschapper en een super begeleider van een jonge assistent. Organisatorisch waren het soms roerige tijden, maar het chirurgisch lab is een fantastische plek om te werken. Veel dank, dr. T. Lisman, beste Ton, voor je komst om te beginnen, je wetenschappelijke inzicht en gezelligheid! Dr. H.G.D. Leuvenink, beste Henri, dank voor je opvang van de jonge en onervaren onderzoeker in het lab die ik in het begin was! Dr. T.A Schuurs, beste Theo, dank voor je wetenschappelijk inzicht en je begeleiding van Marit en het totstandkomen van Hoofdstuk 5. Ing J.J Zwaagstra, beste Jacco, je weet dat ik groot respect heb voor hoe jij het hoofdanalist-schap invult! Veel dank ook aan Ing A.van Dijk, beste Anthony, dank voor het dierexperimentele werk, ik heb veel van je geleerd op microchirurgisch gebied, 227

229 Dankwoord Ing J. Wiersema-Buist, beste Janneke, dank voor je algemene ondersteuning. Het laboratorium kindergeneeskunde en MDL wil ik graag danken voor hun gastvrijheid en voortdurende hulp tijdens mijn onderzoek. Beter een goede buur dan een verre vriend! Met name dr. K.N. Faber en professor dr. A.J. Moshage. Drs. J. Mulder, beste Jaap, jammer dat we (nog) geen artikel samen hebben geschreven, wel veel dank voor al je advies (op PCR gebied). Huawei Su, MD, dear Su, it was great to get to know and work with you! You published a wonderful paper in the American Journal and I am pleased with my first citation in a Chinese journal! Collega onderzoekers en kamergenoten, Mijntje Nijboer (collega, we blijven elkaar zeker nog tegenkomen!), Jayant Janandunsing (aan de andere kant van het doek), Cyril Moers (collega, de lat is gelegd), Lyan koudstaal (succes met afronden van jouw proefschrift), Anne Margot Roskott (dank voor de overheerlijke cappu s!), Micheal Sutton (lang leve de galwegen), Tan Hongtao (thank you so much for your contribution in the rapamycin project), Hugo Maathuis (paranimf!) en Ilona Peereboom (dank voor het zijn van een hele hele leuke collega, dat dat nog maar lang mag duren!), dank voor alle gezellige momenten en (wetenschappelijke) reflectie. Graag wil ik ook op deze plaats de mensen bedanken die mijn eerste schreden op het wetenschappelijke pad hebben begeleid en gestimuleerd. Professor dr. T.H. The, door mij te selecteren voor de JSM en goede adviezen nadien. Professor dr. C.H. Gips, zoals gezegd, de eerste basis is gelegd bij de GISH-T! Dankzij mijn succesvolle afstuderen in the Mayo Clinics ben ik gestimuleerd door te gaan met wetenschap. Professor dr. R.A.F. Krom, icoon op het gebied van levertransplantatie, dank voor uw begeleiding op en naast het wetenschappelijke gebied en uw voortdurende belangstelling in mijn (helaas chirurgische) carrière. Professor dr. E.F.M. Wijdicks, uw kunde om in een razend tempo een goed artikel te schrijven zal me altijd bijblijven! Het was fantastisch met u samen te werken! Dr. C.B. Rosen, dear Chuck, our small project together resulted in the first international oral presentation in my career, thanks for the opportunities you created for me as a medical student to come to the OR and join in liver transplant and donor procedures. 228

230 Het leverteam is een prachtige Groningse traditie! Dank aan alle studenten die in de loop van de jaren hebben geholpen met het verzamelen van biopten tijdens de transplantaties! Verder wil ik ook graag de andere studenten waar ik kort of lang mee heb mogen samenwerken bedanken: Danka, Olivier (medeauteur H5!), Mohammed, Bakhtawar, Mickey, Henk-Jan (Mayo-collega en auteur van CLKTx stuk), Maurits (weer terug!) en natuurlijk Fraukje en Heleen. It has been a great honour to work with professor dr. G.J. Barrit, head of the department of Medical Biochemistry, Flinders Medical School, Adelaide, Australia. Dear Greg and dear Yabin Zhou, thank you for your hospitality in and outside the lab. Dr. V.B. Nieuwenhuijs, beste Vincent, dankzij jouw contacten was het voor mij mogelijk naar Greg s lab te gaan en een nieuw project, dat we hier ook samen met Robert hebben geschreven, daar op te zetten. Dutchies! Marije, Heleen, Fraukje, Claire, Meike, Claire en Judith, dank voor de ongelooflijke gezelligheid, op het lab en daarbuiten en voor alle hele mooie tripjes die we hebben gemaakt! Onderzoekers van het eerste uur uit het TRIADE gebouw Anne Brecht (dank voor het precedent van een uitgebreid dankwoord), Tjeerd, Lucas, Annemarie, Kirsten, Marten, Eric, Esther en Martin. In mijn eerste maanden werd ik meteen opgenomen in de club. Het was als jonge onderzoeker fantastisch om collegae te hebben! SEOHS bestuur 2006, Hugo, Marcel, Justine (OH s 4ever), Coralien, Hilke, Anton, Marinus en Martin, het was een topcongres! Dank voor de heerlijk afwisseling in het wetenschappelijke werk in de vorm van onze vele vergaderingen. Onderzoek en congresbezoek-collegae; Quintus, Marieke, Nienke, Sander, Daantje, Christian, Harm, Ilona, Margijske het is fantastisch met jullie de halve wereld over te vliegen!! Collegae assistenten en chirurgen van het UMCG. Het voelde fantastisch om als jonkie meteen opgenomen te worden in de club. Veel dank voor alle gezelligheid tijdens mijn tijd als onderzoeker. Veel dank voor het wegwijs maken van datzelfde jonkie 2 jaar later in de kliniek toen ik eindelijk mocht. En alvast voor de toekomst: veel dank voor alle gezelligheid en het wegwijs blijven maken in de chirurgie! 229

231 Dankwoord Ongelooflijk veel dank aan Linda, de regassen van C4 en alle andere secretaresses, voor het bestellen van vele statussen en andere regeldingen! Veel dank ook aan de medewerkers van C4 waar ik niet alleen begon als keuzeco, maar ook nog heel vaak kwam om gal te verzamelen. Veel mensen die niet direct bij het onderzoek betrokken zijn geweest, maar wel in mijn leven een belangrijke rol spelen; Kickers (vanaf nu meer tijd om die band voor het leven weer wat meer aandacht te geven), GGGG (lieve oud-huisgenootjes: wat wij hebben, heeft niemand! ), Boswandeling (weekend van 22 januari 2009 staat geblokkeerd!), en alle verschillende hockeyteams die mijn tijd in Groningen van de nodige inspanning en ontspanning hebben voorzien. Een klein stukje gedicht voor jullie allen (naar Eric Brey): Ik denk terug aan duizendtallen hapjes, nipjes, slokken Maar t was vooral m n Ziel die daar zo gulzig zat te schrokken Dat wij zo weer eens met elkaar langdurig haute-cuisine den: Kom, laat er altijd Eten zijn, en Drinken zijn, met Vrienden... Lieve Pappa & Mamma en Hanne & Rikkert, Oom Wouter, dank voor alle 1001 leuke momenten die wij met elkaar hebben! Mamma, dank voor alle wijze adviezen die ik gevraagd en ongevraagd van je krijg op zoveel terreinen. Pappa, dank voor het inzicht dat ik van je heb mogen leren (afkijken eigenlijk) terwijl jij alles maakte wat je bedacht (boot!). Wel spannend dat ik straks als chirurg sta te knutselen zonder jou! Lieve Hanne, dank voor het zijn van mijn steun en toeverlaat! Het is zo heerlijk om je te realiseren dat jullie voor altijd mijn Pappa, Mamma en Zussie zijn!! Mijn allerliefste Luitzen, Je zoenen zijn zoeter dan zoeter dan honing en ik vind je mooier en liever, liever en aardiger nog dan de koning Naar Judith Herzberg en Herman van Veen 230

232 Curriculum Vitae