Lessons from the toxic bile concept for the pathogenesis and treatment of cholestatic liver diseases

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1 Wien Med Wochenschr (2008) 158/19 20: DOI /s Ó Springer-Verlag 2008 Printed in Austria Lessons from the toxic bile concept for the pathogenesis and treatment of cholestatic liver diseases Michael Trauner, Peter Fickert, Emina Halilbasic and Tarek Moustafa Laboratory of Experimental and Molecular Hepatology, Division of Gastroenterology and Hepatology, Department of Internal Medicine, Medical University of Graz, Graz, Austria Received December 12, 2007, accepted January 7, 2008 Bedeutung des Galle-Toxizit at Konzeptes f ur die Pathogenese und Therapie von cholestatischen Lebererkrankungen Zusammenfassung. St orungen der Gallensekretion k onnen auf hepatozellul arer und cholangiozellul arer Ebene eine Cholestase verursachen. Die Bildung einer,,toxischen Galle als Folge einer abnormen Gallezusammensetzung kann zu einer Sch adigung der Hepatozyten und vor allem der Galleng ange f uhren. Die kanalikul are Phospholipid Flippase (Mdr2/MDR3) bewerkstelligt normalerweise die bili are Exkretion von Phospholipiden welche in weiterer Folge gemischte Mizellen mit Gallens auren und Cholesterin bilden, und dadurch das Gallengangsepithel vor der Detergenzienwirkung der potentiell toxischen Gallens auren sch utzen. Mdr2 Knockout M ause sind nicht in der Lage Phospholipide in die Galle zu sezernieren und entwickeln eine Gallengangssch adigung mit den makroskopischen und mikroskopischen Zeichen einer sklerosierenden Cholangitis. MDR3 Mutationen k onnen beim Menschen ein breites Spektrum hepatobili arer Erkrankungen verursachen, welche von der progressiven famili aren intrahepatischen Cholestase beim Neugeborenen uber die intrahepatische Schwangerschaftscholestase, medikament os-induzierte Cholestasen, intrahepatische Cholelithiasis bis hin zu sklerosierenden Cholangitis und bili aren Zirrhose beim Erwachsenen reichen. Andere Beispiele f ur eine Gallengangssch adigung als Folge einer toxischen Galle sind die Cholangiopathie im Rahmen einer zystischen Fibrose, nach Litochols auref utterung im Mausmodell, sowie Vanishing Bile Duct Syndrome durch Medikamente und Xenobiotika. Der therapeutische Ansatz f ur Cholangiopathien kann auf eine Modulation der Gallezusammensetzung im Sinne einer Reduktion der Toxizit at bzw. eine Protektion des Gallengangsepithels abzielen. Die Ursodeoxychols aure (UDCA) weist einige dieser Eigenschaften auf, zeigte jedoch in der Therapie von Cholangiopathien beim Menschen nur eine limitierte klinische Effektivit at. Im Gegensatz Correspondence: Michael Trauner, M.D., Division of Gastroenterology and Hepatology, Department of Internal Medicine, Medical University of Graz, Auenbruggerplatz 15, 8036 Graz, Austria. Fax: þþ , michael.trauner@meduni-graz.at,, zu UDCA, unterliegt die Seitenketten verk urzte norudca einem cholehepatischen Shunting und induziert eine Bikarbonat-reiche Hypercholerese. Weiters hat norudca anti-inflammatorische, anti-fibrotische und anti-proliferative Effekte und stimuliert die Gallens aurendetoxifikation. Geplante klinische Studien werden erst zeigen m ussen ob norudca oder andere Seitenketten-modifizierte Gallens auren auch beim Menschen klinisch effektiv sind. Neue Therapieans atze beinhalten auch die M oglichkeit uber Kernrezeptoren wie FXR und PPARa die Gallezusammensetzung und Galletoxizit at zu beeinflussen. Schl usselw orter: Cholestase, sklerosierende Cholangitis, Gallens auren, Phospholipide Summary. Alterations in bile secretion at the hepatocellular and cholangiocellular levels may cause cholestasis. Formation of toxic bile may be the consequence of abnormal bile composition and can result in hepatocellular and/or bile duct injury. The canalicular phospholipid flippase (Mdr2/MDR3) normally mediates biliary excretion of phospholipids, which normally form mixed micelles with bile acids and cholesterol to protect the bile duct epithelium from the detergent properties of bile acids. Mdr2 knockout mice are not capable of excreting phospholipids into bile and spontaneously develop bile duct injury with macroscopic and microscopic features closely resembling human sclerosing cholangitis. MDR3 mutations have been linked to a broad spectrum of hepatobiliary disorders in humans ranging from progressive familial intrahepatic cholestasis in neonates to intrahepatic cholestasis of pregnancy, drug-induced cholestasis, intrahepatic cholelithiasis, sclerosing cholangitis and biliary cirrhosis in adults. Other examples for bile injury due to the formation of toxic bile include the cholangiopathy seen in cystic fibrosis, after lithocholate feeding (in mice) and vanishing bile duct syndromes induced by drugs and xenobiotics. Therapeutic strategies for cholangiopathies may target bile composition/toxicity and the affected bile duct epithelium itself, and ideally should also have anti-cholestatic, anti-fibrotic and anti-neoplastic properties. Ursodeoxycholic acid (UDCA) shows some of these properties, but is of limited efficacy in the treatment of human cholangiopathies. By contrast to UDCA, its side chain-shortened homologue norudca undergoes cholehepatic shunting leading to a bicarbonate-rich /2008 wmw

2 hypercholeresis. Moreover, norudca has anti-inflammatory, anti-fibrotic and anti-proliferative effects, and stimulates bile acid detoxification. Upcoming clinical trials will have to demonstrate whether norudca or other side chain-modified bile acids are also clinically effective in humans. Finally, drugs for the treatment of cholangiopathies may target bile toxicity via nuclear receptors (FXR, PPARa) regulating biliary phospholipid and bile acid excretion. Key words: Cholestasis, sclerosing cholangitis, bile acids, phospholipids Introduction Bile is a complex solution consisting of inorganic and organic compounds including bile acids, bilirubin, cholesterol, phospholipids, and proteins such as albumin and immunoglobins [1]. Bile acids are actively excreted into bile where they form mixed micelles with phospholipids and cholesterol, thereby promoting their biliary elimination [1]. Formation of mixed micelles is also crucial for reducing the detergent activity of monomeric bile acids in the bile duct lumen, thus preventing toxicity of high (mm) biliary bile acid concentrations to cholangiocytes [1 3]. Alterations in bile composition and flow may give rise to cholestatic syndromes at the hepatocellular and cholangiocellular levels [2 4]. This article aims to give an overview on toxic bile as a pathogenetic factor and potential therapeutic target for cholestatic liver diseases with a special focus on cholangiopathies. The toxic bile concept Under normal conditions bile is already a considerably toxic fluid. Bile toxicity to hepatocytes and bile duct epithelial cells is prevented by high apical membrane cholesterol, and sphingomyelin content, micellar binding of bile acids, bile hydration and alkalinization, mucin formation (in the gallbladder and larger ducts) and, last but not least, bile flow, that is the movement of bile limiting the interaction time with cell membrane domains [5, 6]. Disturbances of normal hepatobiliary transport and bile composition may result in the formation of toxic bile (Fig. 1) and subsequent hepatocellular and/or bile duct injury (Table 1 for examples) [4]. Whether biliary enzymes contribute to bile toxicity remains to be investigated. Apart from changes in bile composition, stasis of bile flow, formation of precipitates and biliary casts, as well as increases in biliary pressure may also contribute to bile toxicity (Table 1). Fig. 1: Transporter alterations contributing to bile toxicity. Under normal conditions bile salts (BS) are excreted into bile via the canalicular bile salt export pump (BSEP). Phosphatidylcholine (PC) is exported via the phospholipid Flippase (MDR3). Following their excretion into bile, BA normally form mixed biliary micelles with PC and cholesterol, which prevents BA toxicity. Reduced biliary excretion of PC (in relation to BS) results in toxic bile and bile duct injury. Reduced biliary excretion of glutathione (GSH; via MRP2) and cholesterol (chol; via ABCG5/8) may also contribute to bile toxicity. Defects of CFTR and/or AE2 result in impaired bicarbonate excretion, alkalinization and hydration of bile. Toxification of bile may cause bile duct injury per se or aggravate any other (e.g., immune mediated) bile duct injury Tab. 1: Potential causes of toxic bile formation 1) Abnormal bile composition a) Increased bile acid/phospholipid ratio i) Hereditary MDR3/Mdr2 defects ii) Imbalance in BSEP/MDR3 expression (e.g. following liver transplantation) iii) Impaired MDR3 function (e.g., lack of enteral nutrition/ total parenteral nutrition) b) Decreased bicarbonate excretion and hydration of bile i) Hereditary CFTR defects ii) AE2 defects (e.g. PBC) c) Primarily toxic bile acids (e.g., lithocholic acid feeding in mice) d) Xenobiotics/drugs and their metabolites (e.g. 3,5- diethoxycarbonyl-1,4-dihydrocollidine, alphanaphthylisothiocyanate) 2) Biliary enzymes? 3) Stasis, increased biliary pressure and shear stress 4) Biliary casts (e.g. rapidly progressive sclerosing cholangitis after sepsis/shock) [For review see refs. 4, 21] The Mdr2 knockout mouse model proof of the toxic bile concept The canalicular phospholipid floppase (Mdr2 in rodents/mdr3 in humans; gene name Abcb4/ABCB4) mediates translocation of phosphatidylcholine (PC) into the outer leaflet of the canalicular cell membrane, thus facilitating their subsequent extraction by bile acids and excretion into bile [1 4, 7]. Mdr2 (Abcb4) knockout mice (Mdr2/Abcb4 = ) are not capable of excreting PC into bile, which renders bile toxic because of an increased concentration of free non-micellar bile acids which damage cell membranes and cell junctions [7, 8]. In addition, Mdr2 = mice have reduced wmw 19 20/2008 Trauner et al. Lessons from the toxic bile concept for the pathogenesis 543

3 cholesterol excretion, indicating that bile acid/pc micelles are important in facilitating biliary excretion of cholesterol [7]. Since PC in bile is required for the formation of mixed micelles with bile acids and cholesterol to protect the bile duct epithelium from the detergent properties of bile acids, Mdr2 = mice spontaneously develop bile duct injury with macroscopic and microscopic features closely resembling those observed in human (primary) sclerosing cholangitis [8 10]. Bile duct injury in Mdr2 = mice can be explained by a sequence of events including an increased concentration of free non-micellar bound bile acids, increased tight junction permeability of bile duct epithelium, leakage of bile, pericholangitis, periductal fibrosis with ductular proliferation and finally sclerosing cholangitis [8, 9]. In addition, Mdr2 = mice spontaneously develop cholesterol cholecysto- and hepatolithiasis as a result of impaired cholesterol solubility in PC-deficient bile and hepatocellular carcinomas due to chronic inflammation [10 14]. Other conditions with formation of toxic bile Another example for a transport defect associated with bile duct injury is cystic fibrosis [15 21]. Since the cystic fibrosis transmembrane conductance regulator (CFTR/ ABCC7) drives biliary bicarbonate secretion via a chloride/bicarbonate anion exchanger (AE2/ SLC4A2), impaired function in CFTR and/or AE2 will result in impaired hydration and alkalinization of bile, inspissated bile and bile duct injury [21] (Table 1, Fig. 1). Cftr = mice show progressive liver disease, with hepatosteatosis, focal cholangitis, inspissated secretions, bile duct proliferation and the progression to focal biliary cirrhosis after 1 year of age [22]. Interestingly, the induction of colitis in Cftr = mice aggravates bile duct injury [23, 24]. Xenobiotics and drugs may also lead to bile duct injury and biliary fibrosis [21]. Toxic drug metabolites excreted into bile may cause a drug-induced vanishing bile duct syndrome and eventually biliary cirrhosis (Table 1). Feeding of 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) is a valuable model to investigate the mechanisms of xenobiotic-induced chronic cholangiopathies and its sequels including biliary fibrosis [25]. In this model, DDC feeding leads to increased biliary porphyrin secretion, induction of VCAM, osteopontin, and TNF-a expression in bile duct epithelial cells. This is associated with a pronounced pericholangitis with significantly increased number of CD11b-positive cells, ductular reaction and activation of periductal myofibroblasts leading to a biliary type of liver fibrosis. Another example includesbileduct injury induced by alpha-napthyl isocyanate [26]. Lithocholic acid (LCA) causes bile duct injury without the reduction of biliary PC excretion, since LCA per se is already highly hydrophobic and toxic. Thus, LCA feeding in mice leads to segmental bile duct obstruction, destructive cholangitis, periductal fibrosis, [27]. At the ultrastructural level, small bile ducts are frequently obstructed by crystals in this model, suggesting that mechanical injury/obstruction to small ducts may play a crucial role. Relevance for pathogenesis of cholestatic liver diseases in humans Interfering with normal bile composition may affect membrane fluidity and bile secretory function at the hepatocellular and cholangiocellular levels. Therefore, the clinical scenarios associated with the formation of toxic bile comprise a range of cholestatic syndromes presenting as hepatocellular forms of cholestasis (e.g. drug-induced cholestasis, intrahepatic cholestasis of pregnancy) or cholangiopathies (e.g. sclerosing cholangitis) [4]. Role of canalicular phospholipid export pump (MDR3) In analogy to the Mdr2 = mouse model of sclerosing cholangitis and the toxic bile concept, MDR3 defects resulting in the formation of PC-deficient toxic bile could play an important role in the pathogenesis of various cholangiopathies in humans [2 4]. MDR3 mutations have so far been linked to a broad spectrum of hepatobiliary disorders ranging from progressive familial intrahepatic cholestasis (PFIC3) in neonates to intrahepatic cholestasis of pregnancy, drug-induced cholestasis, intrahepatic cholelithiasis, sclerosing cholangitis and adult biliary cirrhosis [2 4, 28 30]. Moreover, MDR3 mutations and polymorphisms may (pre) determine individual susceptibility to acquired cholestatic liver injury caused by drugs and inflammatory cytokines [4]. In addition to PFIC3, impaired biliary PC excretion (in relation to bile acid excretion) as the hardest functional evidence for MDR3 deficiency has so far only been demonstrated in patients lacking enteral nutrition and patients with oriental cholangiopathy or bile duct injury following liver transplantation [4, 31, 32]. Intrahepatic cholestasis of pregnancy and druginduced cholestasis may now be viewed as classic examples for decompensation of a heterozygous state of a transporter gene (MDR3) defect under hormonal or drug challenge in a subset of patients who under 544 Trauner et al. Lessons from the toxic bile concept for the pathogenesis 19 20/2008 wmw

4 basal conditions still have sufficient MDR3 activity [4]. Moreover, MDR3 (and other transporter) variants could play a role as disease modifier genes in the pathogenesis of various cholangiopathies such as primary sclersoing cholangitis (PSC), primary biliary cirrhosis and adulthood idiopathic ductopenia/biliary fibrosis [4]. A heterozygous MDR3 mutation was found to be specificto PSC and one MDR3 haplotype was encountered more frequently in PSC patients [29, 33]. More recently, MDR3 variants have been indentified together with variants of the organic solute transporter as disease modifier genes in patients with PSC [34]. However, it is important to emphasize that the current data, as expected, do not support a major, causative role for MDR3 genetic variations/mutations in the pathogenesis of PSC. Since PSC may represent a mixed bag of diseases with variable pathogenetic background in individual subgroups, a general role of MDR3 mutations in unselected PSC patients would be very surprising. Rather, MDR3 genetic variations should be further explored in selected PSC subgroups, for example, small duct PSC (making up only about 5% of PSC cases) and pediatric patients [4]. Role of cystic fibrosis transmembrane conductance regulator (CFTR) Increased life expectancy has led to an increasing recognition of hepatobiliary complications in cystic fibrosis patients including biliary tract complications such as stones and sclerosing cholangitis [4, 21]. Cholangiographic features of sclerosing cholangitis with strictures and beading of the larger intrahepatic bile ducts on endoscopic retrograde and magnetic resonance cholangiography have been reported in 4 to 18% of adult CF patients [4, 21]. Since sclerosing cholangitis is increasingly encountered in adult patients with CF, this raises the question for a role of CFTR variants in PSC [4, 21] (Fig. 1, Table 1). Patients with inflammatory bowel disease who are (heterozygous) the carriers of CFTR mutations may be at increased risk of developing PSC. As proof of concept, experimental colitis in exon 10 cftr = mice, a mouse model that does not develop liver disease spontaneously, results in bile duct injury characterized by portal tract and bile duct infiltration with mononuclear inflammatory cells and bile duct proliferates [16]. Colonic inflammation may impair cftr expression via impairment of PPARa expression [24], which has also been implicated in the transcriptional regulation of Mdr2 [1 3]. In a subset of patients with inflammatory bowel disease (IBD) and PSC, an increased prevalence of CFTR abnormalities was observed compared with IBD patients lacking liver disease [15]. A recent study also observed a high prevalence of CFTR mutations and decreased CFTR function in childhood PSC [17]. However, other studies could not confirm an association of disease-causing CFTR mutations with PSC [18 29] and the role of CFTR in PSC is still under debate [4]. Role of Anion Exchanger 2 (AE2) Several studies found reduced expression and function of anion exchanger AE2 (SLC4A2), a Cl /HCO 3 exchanger, in PBC, which may contribute to reduced bile flow and cholestasis in PBC [35 38]. Moreover, decreased AE2 expression in salivary and lacrimal glands could explain the frequently associated sicca syndrome in these patients [39] and pointed towards a more generalized glandular failure in PBC. Pro-inflammatory cytokines, which play a role in the pathogenesis of PBC, may also contribute to altered transporter expression [40]. However, reduced AE2 expression is not secondary to inflammation in PBC as suggested by unchanged Ae2 expression in cytokine-treated rat cholangiocytes [41]. Whether AE2 repression is a primary mechanism in the pathogenesis of PBC is not clear and the mutations of the AE2 gene in PBC have not yet been reported. Interestingly, Ae2 knockout mice develop features of human PBC (Jesus Prieto, personal communication, EASL Monothematic PBC Conference, Newcastle, December 2007). Moreover, AE2 variants may be associated with a biochemical response to ursodeoxycholic acid (UDCA) (R. Poupon, personal communication, Annual AASLD Meeting, Boston, November 2007). However, most changes in hepatocellular transporter gene expression levels encountered in PBC represent secondary adaptive changes [40]. Potential therapeutic implications Current pharmacological treatment for chronic cholangiopathies urgently needs improvement [42, 43]. Drugs for cholangiopathies may target bile toxicity/aggressiveness of bile, and should have bile duct-protective, anti-cholestatic, anti-fibrotic and anti-neoplastic properties. UDCA shows some of these properties, but is of limited efficacy in the treatment of cholangiopathies such as PSC and PBC [44, 45]. Sidechain shortening of bile acids could increase their therapeutic efficacy since this modification significantly alters the physiological properties and pharmacological profile of bile acids [46 48]. norudca is a side chain-shortened C 23 homologue of UDCA possessing one less methylene group in its wmw 19 20/2008 Trauner et al. Lessons from the toxic bile concept for the pathogenesis 545

5 Fig. 2: Therapeutic strategies to counteract toxic bile-mediated bile duct injury. Strategies counteracting toxic bile resulting from an imbalance between bile salt and phospholipid excretion, or impaired biliary bicarbonate excretion (left) include (i) reducing bile salt hydrophobicity (ii) promoting biliary phospholipid excretion via the phospholipid export pump Mdr2 (by FXR and PPARa agonists) and (iii) stimulating bicarbonate excretion (right). Biliary enrichment of hydrophilic norudca or UDCA increases the hydrophilicity of bile. Moreover, norudca undergoes cholehepatic shunting and thus flushes injured ducts with a hydrophilic alkalinized (bicarbonate-rich) bile. norudca also induces bile salt and bilirubin detoxification via phase I and II enzymes and alternative export pumps at the sinusoidal membrane of hepatocytes thus permitting their renal elimination (not shown). These mechanisms together with norudca's anti-inflammatory, anti-fibrotic and anti-proliferative effects result in healing of sclerosing cholangitis side chain [46]. As a result of this chemical modification norudca is more resistant to conjugation with taurine or glycine than UDCA, but instead is secreted into bile mostly in unchanged form [46 48]. The secreted nor- UDCA undergoes absorption by cholangiocytes, returns to the liver and is resecreted into bile [46 48]. Such cholehepatic shunting leads to a bicarbonate-rich hypercholeresis and may also result in improved targeting to the injured bile duct epithelium and liver (Fig. 2). These unique mechanisms make norudca an attractive candidate drug for the treatment of cholangiopathies [46 48]. norudca (but not conventional UDCA) reverses sclerosing cholangitis in the Mdr2 = cholangiopathy model within 4 weeks of treatment [48]. Its suggested therapeutic mechanisms include (i) amelioration of bile hydrophobicity by biliary enrichement with hydrophilic norudca and its metabolites, (ii) flushing of injured bile ducts by the stimulation of bile flow and bicarbonate-rich choleresis, which dilutes toxic biliary content, (iii) induction of alternative bile acid detoxification (phase I and II enzymes) and elimination routes for bile acids (multidrug-resistance associated proteins 3 and 4), and (iv) direct anti-inflammatory and antifibrotic properties [48]. Future studies will show whether norudca is capable of preventing the development of hepatocellular carcinomas in Mdr2 = mice. These effects make norudca an attractive candidate drug for the treatment of cholangiopathies, particularly PSC. Other modified bile acids and nonbile acid compounds which also undergo cholehepatic shunting (e.g. sulindac) may also be explored for the treatment of cholangiopathies in the future [49 50]. Another therapeutic strategy in the treatment of cholestasis may represent the use of agonists for nuclear receptors such as the peroxisome proliferatoractivated receptor-alpha (PPARa), which promote biliary phospholipid excretion [51, 52]. Cholesterollowering drugs such as fibrates and inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme. A reductase (statins) indirectly activate PPARa and administration of these substances to patients with primary biliary cirrhosis resulted in a beneficial effect on liver enzymes [51, 52]. This could at least in part be explained by the stimulation of the biliary phospholipid excretion pump Mdr2, since phospholipids protect the bile duct epithelium from detergent bile salts by the formation of mixed micelles. In addition, statins activate pregnane X receptor (PXR), which induces bile detoxification and alternative export [53]. Unfortunately, atorvatstatin did not improve cholestasis in patients with PBC who had an incomplete biochemical response to UDCA [54]. Ligands for the nuclear bile acid/farnesoid X receptor (FXR) target multiple genes involved in bile formation, inflammation and fibrosis and have already entered phase II clinical trials in North America and Europe [51, 52]. The future should bring more tissue and genespecific nuclear receptor ligands to more efficiently target bile toxicity. Acknowledgements This work was supported by grants P B05 and P19118-B05 from the Austrian Science Fund and a GEN-AU grant from the Austrian Ministry for Science (GZ /1-IV/1/2006, GaTib to M.T.). References [1] Trauner M, Boyer JL. Bile salt transporters: molecular characterization, function, and regulation. Physiol Rev, 83: , [2] Oude Elferink RP, Paulusma CC. Function and pathophysiological importance of ABCB4 (MDR3 P-glycoprotein). Pflugers Arch, 453: , [3] Oude Elferink RP, Paulusma CC, Groen AK. 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