The pathology of chronic allograft dysfunction

Transcription

1 & 2010 International Society of Nephrology review The pathology of chronic allograft dysfunction Lorraine C. Racusen 1 and Heinz Regele 2 1 Department of Pathology, The Johns Hopkins University School of Medicine, Baltimore, Maryland, USA and 2 Department of Internal Medicine, Clinical Institute of Pathology, University of Vienna, Vienna, Austria Chronic allograft dysfunction is associated with a variety of fibrosing/sclerosing changes in the allograft. Fibrosis is multifactorial, a final pathway following varying types of injury. Using a range of diagnostic criteria, the pathologist can and should define specific lesions enabling identification of pathogenic processes affecting the allograft. Although some cases remain interstitial fibrosis and tubular atrophy, no specific cause, specific diagnoses can be made in most cases. Drug toxicity, bacterial or viral infection, hypertension, obstruction, recurrent or de novo renal diseases, and acute and chronic cell- and/or antibody-mediated rejection can be diagnosed in this setting. Of particular concern is a combination of persistent inflammation and fibrosis, which has repeatedly been shown to be correlated with poor graft outcomes. Identification of ongoing activity, and the stage of evolution of fibrosis/sclerosis provides important diagnostic and therapeutic information for patient management. Histological, immunohistological, ultrastructural, and molecular studies may be needed to adequately assess the kidney in the setting of chronic allograft dysfunction. Protocol biopsies may provide diagnostic insights in early stages of late graft deterioration, or even before evident dysfunction develops. ; doi: /ki KEYWORDS: allograft; fibrosis; pathology; protocol biopsies; rejection TO CITE THIS ARTICLE: Racusen LC, Regele H. The pathology of chronic allograft dysfunction. Kidney Int 2010; 78 (Suppl 119): S27 S32. Correspondence: Lorraine C. Racusen, Department of Pathology, The Johns Hopkins University School of Medicine, 711 Pathology Building, 600 North Wolfe Street, Baltimore, Maryland 21287, USA. lracusen@jhmi.edu Chronic allograft dysfunction is associated with a variety of fibrosing/sclerosing changes in the allograft. Any anatomical component of the allograft kidney may undergo such changes. Glomeruli may have progressive increase in the matrix and/or may develop segmental or global sclerosis. There may be thickening in the arterial intima by fibrosis, and accumulation of hyaline material in small arteries and arterioles. Peritubular capillaries may develop multilayered basal lamina, and there may be thickening of glomerular capillary walls with reduplication of basement membrane and mesangial interposition.there may be progressive increase in the interstitial matrix downstream, eventuating in interstitial fibrosis (IF) and collagen scar, with progressive tubular atrophy (TA) an almost inevitable accompaniment. Loss of interstitial capillaries is associated with these tubulointerstitial changes. It is the challenge and responsibility of the pathologist to interpret these changes, and make differential diagnoses in this context. 1 Fibrosis in the renal allograft is multifactorial, representing a final common pathway after various types of injury. 2 5 Chronic allograft nephropathy/can had been coined as a general term for fibrosis in the allograft, in recognition that not all fibrosis is chronic rejection. 6,7 However, CAN became a diagnostic entity, distracting from the recognition of more specific etiologically relevant diagnoses. Thus, in 2005, the Banff consensus conference developed recommendations to do away with CAN, make specific diagnoses when possible, and denote IF/TA no cause identified as necessary. 1 To fully assess chronic features in the allograft, it is critical to have a baseline biopsy of the allograft In the era of extended criteria deceased donors, there are frequently preexisting donor-related changes in the graft, including glomerulosclerosis, 11 and glomerular disease (especially diabetic glomerulopathy), arteriosclerosis, and hyaline arteriolosclerosis, and IF and TA. In addition, there is a trend to use older live donors, 12 with the likelihood of significant attendant pathology in the allograft from these donors. Even younger live or deceased donors may have unsuspected pathology in the allograft, including glomerular disease, glomerular sclerosis, and chronic vascular disease. 13 As outlined in the Banff 2005 meeting report, 1 possible causes of IF/TA include drug toxicity (especially calcineurin inhibitor toxicity), bacterial or viral infection (for example, polyoma virus), hypertension, obstruction, recurrent and de novo glomerular and tubulointerstitial diseases, and of S27

2 review LC Racusen and H Regele: Multifactorial pathogenesis of allograft fibrosis course chronic rejection. Each has specific morphological features that may be identifiable. Calcineurin inhibitor toxicity is characterized by progressive arteriolar hyalinosis, especially with peripheral hyaline nodules, with progressive downstream fibrosis in an ischemic striped pattern. Often, there is isometric vacuolization in tubular cells as a sign of ongoing exposure. 14 Chronic pyelonephritis may be recognized by nodular lymphoid aggregates and focal intratubular neutrophils. Polyoma virus nephropathy is an important cause of allograft fibrosis. Early stages are characterized by intranuclear inclusions and inflammation, which may include plasma cells and neutrophils. However, in later stages, fibrosis with some inflammation and relatively few inclusions may be the major finding. 15,16 The characteristic pale purple inclusions may not be prominent, and immunostaining or in situ hybridization to detect viral antigens must be used to make a definitive diagnosis these staining procedures should be carried out on all late allograft biopsies. Hypertension is characterized by arterial fibrointimal thickening, with reduplication of elastic and small vessel hyaline change in the intima. Glomerulosclerosis, including focal segmental glomerulosclerosis with hyalinosis, often develops in the setting of hypertension. Obstruction in the allograft may be functional, without visible hydronephrosis on imaging studies. Tubular dilatation, large Tamm-Horsfall protein casts with extravasation into the interstitium and/or vascular channels are features of obstruction identifiable on renal biopsy. Recurrent disease is a major cause of fibrosis in the allograft, 17 becoming more significant with time after transplant. Recurrent focal segmental glomerulosclerosis is particularly likely to lead to fibrosis and graft dysfunction, 18 but other glomerular diseases, especially immune complex diseases and vascular and tubulointerstitial diseases may also recur. With more long-term graft survival, processes such as hypertensive nephropathy and diabetic glomerulopathy may appear, with risk potentially exacerbated by diabetogenic immunosuppressive agents. Identification of these processes potentially requires pathological evaluation not just by light microscopy but by immunofluorescence and electron microscopy as well. 19 A full panel of immunostaining procedures for immunoglobulins and early and late complement components should be performed on any allograft biopsy at 1-year posttransplant and beyond, and even earlier in patients with proteinuria, hematuria, or who are at risk. FIBROSIS/SCLEROSIS RELATED TO REJECTION Chronic rejection due to alloimmunity in the allograft may be cell- or antibody mediated. Once again, specific pathological features can be identified to make a definitive diagnosis of chronic rejection. 1 Active lymphocytic tubulitis or arterititis in the graft with fibrosis signals smoldering alloimmune injury. Chronic transplant glomerulopathy and arterial changes of accelerated graft arteriosclerosis, especially with lymphocytes in the fibrosing intima, splintering of the internal elastic, and formation of neo-media and neo-intima inside the vessel, are reliable features. Microvascular injury is typical of antibody-mediated processes, and significant glomerulitis and peritubular margination of cells are signs of ongoing antibody-mediated injury in the fibrosing graft. 20 Both chronic transplant glomerulopathy (CTG) and peritubular capillary basement membrane multilayering are strongly associated, not only with each other but also with positive staining for C4d in peritubular capillaries and with monocyte/macrophages in capillaries. 21,22 It is important to look for evidence of ongoing injury in allografts, even in the context of significant fibrosis. Mauiyyedi et al. 23 demonstrated that cases of chronic rejection with marginating cells and C4d in peritubular capillaries had superior outcome compared with those with bland fibrosis with treatment. A number of recent series have demonstrated that it is the combination of some fibrosis and evidence of active ongoing immune injury that is most deleterious to the graft. 24,25 The specification of diagnostic criteria for chronic rejection and other entities by the Banff conference was an important achievement. 1 The current definition, however, requires already established chronic lesions such as arterial intimal fibrosis or CTG. These lesions represent an advanced stage of the rejection process, are likely irreversible, and are markers of unfavorable outcome. It is crucial to identify chronic rejection at the earliest stage possible to prevent irreversible damage to the graft by timely application of specific treatment. Early and reliable detection of possibly minor, clinically unapparent, alloimmune reactions is, however, still a complex diagnostic challenge. Unfortunately, we are not yet able to fully translate our quite sophisticated immunologic concepts of rejection mechanisms into diagnostic criteria detectable by histology. Discrimination between cellular and humoral mechanisms of graft rejection is an important step toward a more pathogenesis-based classification of rejection processes. Persisting inflammation causes graft fibrosis and functional deterioration Protocol biopsies, performed at predefined times after transplantation, irrespective of graft function at the time of biopsy, have contributed crucially to our understanding of the mechanisms and the clinical course of chronic rejection. Rush and co-workers 26,27 were among the first to use protocol biopsies for specifically investigating the predictive value of histomorphological lesions in clinically stable renal allografts for long-term graft function. They observed morphological signs of rejection (at 1, 2, or 3 months after transplant) in 30% of grafts with normal function 26 and concluded that this might represent subclinical rejection (SCR). 27 Nankivell and colleagues 28,29 confirmed and extended the concept of SCR as being causative for chronic allograft injury. In a cohort of 119 patients monitored by protocol biopsies for up to 10 years after transplantation, they observed that SCR (including Banff borderline lesions) was a frequent finding early after transplantation (in 61 and 46% of biopsies at 1 and 3 months after transplant). If SCR persisted, it was not S28

3 LC Racusen and H Regele: Multifactorial pathogenesis of allograft fibrosis review only associated with a higher degree of IF and TA but also resulted in significantly decreased renal function at 2 years after transplantation. 29 Their proposed definition of true cellmediated chronic rejection was SCR persisting for at least 2 years. A more recent study from Hannover also seems to support the view that persistence of inflammation might be crucial for the subsequent development of graft dysfunction. Mengel et al. 30 calculated the cumulative inflammatory burden by summing up the number of inflammatory infiltrates in sequential biopsies (at 1.5, 3, and 6 months after transplant). Persistence of inflammation in sequential biopsy specimens regardless of its severity, localization, or cellular composition predicted creatinine clearance at 1 and 2 years. 30 The concept of persisting inflammation (even if low grade) causing progressive tissue injury that only becomes clinically apparent at advanced stages of tissue fibrosis is biologically plausible, and is also in line with clinical experience from other fields of medicine (that is, longstanding hypertension or diabetes mellitus causing chronic vascular injury). The role of alloantibodies and complement in chronic graft injury Can we apply the concept of subclinical immune injury causing cumulative organ damage to antibody-mediated rejection (AMR)? Many studies linking the presence of circulating anti-human lymphocyte antigen (HLA) antibodies to impaired long-term outcome indeed support this view In a recent review article, Terasaki and Cai 36 reviewed evidence for the causative role of alloantibodies in chronic rejection. Serological studies on the impact of alloantibodies on long-term graft outcome share some important limitations. They only provide circumstantial evidence for a causal role of antibodies for graft injury and loss. Methods for detecting circulating alloantibodies are quite variable, both in terms of specificity and sensitivity. Moreover, the definitions of positive results are not standardized and depend on the testing system. In addition, almost all of the studies cited above also demonstrate that a certain proportion of recipients with circulating antibodies (sometimes even the majority of patients) do not experience clinical problems that are likely due to alloantibodies. To specifically investigate the predictive value of circulating alloantibodies, we tested sequential serum samples from 164 patients with functioning grafts for circulating anti-hla-antibodies (by flow cytomeric crossmatch testing and highly sensitive solid-phase assays). We identified a subgroup of 34 patients with an uneventful 1-year posttransplant course and excellent graft function. Nine of these patients (27%) had circulating anti-hla antibodies (donor-specific antibodies in five cases). Frequencies of positive test results were not significantly lower than those documented for the other 130 patients. Remarkably, in patients with excellent 1-year graft function, anti-hla reactivity was not associated with reduced growth factor receptor or proteinuria during follow-up (median 65 months). 37 In patients without graft dysfunction, circulating antibodies (even if donor specific) are not necessarily associated with accelerated graft loss, and are thus of only limited predictive value and likely inadequate for individualized risk assessment. Other studies investigating allograft biopsies for the concurrent presence of alloantibody (detected by serology and/or C4d deposition within the graft) and CTG (the morphological hallmark of chronic rejection) also supported an association and even causal relationship between alloantibody and chronic rejection. 21,23,38 It thus seems likely that persisting antibody-mediated injury eventually leads to progressively accumulating chronic tissue damage (mainly affecting the vasculature) that results in clinically overt graft dysfunction only at advanced stages (similar to the suspected mechanisms in SCR). Diagnostic information from indication biopsies, performed for clinically overt dysfunction, might therefore come too late and be inadequate for guiding successful treatment. It is our diagnostic challenge (in both cell-mediated and antibody-mediated SCR) to detect clinically silent immune mechanisms as early as possible, before likely irreversible chronic tissue injury occurs. As mentioned above, it seems questionable whether serological testing alone is sufficient for immunological monitoring and accurate risk assessment in individual patients. A promising strategy for a refined risk assessment might be the staining of protocol biopsies for C4d, which is of proven utility as a marker of AMR in patients with graft dysfunction. 20 Does C4d staining in protocol biopsy samples predict chronic rejection? Mengel et al. 39 investigated 551 non-selected protocol biopsy samples for C4d deposition, which was detected in 4.4% of cases but was not associated with inferior outcome (a median follow-up of 43 months). A more recent study by Yoon et al. 40 detected C4d in 4 of 79 (5.1%) protocol biopsy specimens and also did not observe graft dysfunction during a median follow-up of 30 months. Even more remarkable, studies on protocol biopsy specimens from ABO-incompatible transplants revealed that C4d was present in 80% (ref. 41) to 94% (ref. 42) of biopsies but was not associated with morphological signs of AMR 41,42 or adverse outcome. 42 C4d, a highly reliable indicator of AMR in patients preselected for graft dysfunction, seems to be of more limited diagnostic value in grafts with stable function. One possible explanation for the apparent insensitivity of many ABO-incompatible and some ABO-compatible transplants to antibody- and complement deposition is accommodation, an acquired state of resistance to antibodymediated injury. 43 The mechanisms of accommodation are, however, not well understood, making it difficult to reliably confirm its presence in individual grafts and to assess its frequency and role in recipients with anti-hla antibodies. Another explanation for C4d positivity without clinical or morphological implications might be that humoral reactivity is only transient. The studies cited above did not S29

4 review LC Racusen and H Regele: Multifactorial pathogenesis of allograft fibrosis systematically analyze whether C4d deposition was continuously present in sequential biopsy specimens. However, Yoon et al. 40 reported a loss of C4d staining in the only two followup biopsies that were performed in four C4d-positive recipients. A study by Haas et al. 44 on the course of subclinical AMR detected in 10 of 83 highly sensitized crossmatch-positive patients successfully transplanted after desensitization showed that the presence of subclinical AMR (C4d positive and capillaritis/glomerulitis) in a first biopsy was predictive for more severe chronic lesions in follow-up biopsies. Interestingly, in this study, the persistence of at least focal C4d staining was observed in 9 of 10 follow-up biopsies. Persistence of C4d positivity in sequential protocol biopsies might thus be of practical diagnostic relevance for discriminating potentially harmful sustained alloantibody activity from transient, clinically innocous responses. Capillaritis/glomerulitis might be a diagnostically helpful feature of clinically relevant SCR. This lesion was only rarely (4 18%) found in ABO-incompatible protocol biopsies 42 but was almost always present (86 100%) in subclinical and clinical cases of AMR reported by Haas et al. 41 Lerut et al. 45 observed in a small series of protocol biopsies that peritubular capillaritis at 3 months was significantly associated with signs of chronic AMR at 1 year. Can chronic AMR occur without C4d deposition within the graft? C4d reliably indicates the presence of AMR in patients with acute allograft dysfunction. It seems likely that complement activation within the graft is not only a valuable diagnostic marker but also represents a crucial step in the pathogenesis of AMR. 46,47 Reports on the prevalence of C4d in biopsies with CTG are, however, somewhat controversial. Most studies show a statistical association of C4d with CTG 21 but C4d is far from being universally present in grafts with CTG, even in cases with serologically detectable anti-hla antibodies. Issa et al. 48 found that (c4d) was positively correlated with increasing levels of (pretransplant) anti-hla class II antibodies but could demonstrate C4d in only 24% of the biopsies investigated. Previous studies from the same group and others detected anti-hla in 82% (ref. 49) and 70% (ref. 22) of CTG cases, but C4d deposits were present in only 25 and 36%, respectively, of antibody-positive cases. Akalin et al. 50 reported a prevalence of only 36% for donorspecific antibodies and 14% for C4d staining in a cohort of 28 CTG cases. This could mean that previously active AMR might have resolved at the time of biopsy, leaving behind capillary lesions but no more complement deposits or that CTG is not at all specific for AMR and might also be caused by antibody-independent mechanisms. However, we also have to consider that low-level complement deposits might escape detection by immunohistochemistry, but still might be biologically active. Furthermore, it is conceivable that anti-hla antibodies without the ability to activate complement could still mediate injury to endothelial cells. The latter two options (low-level complement or complement-independent action of antibodies) might represent potential mechanisms of alloantibody-mediated tissue damage that are not recognized by current diagnostic definitions, requiring C4d deposition for the diagnosis of AMR. 51 However, recent experimental data from animal models and in vitro research support the concept of anti-hla antibodymediated, complement-independent injury to endothelial cells that still might result in progressively accumulating vascular damage. Uehara et al. 52 demonstrated that chronic transplant arteriopathy could be induced in hearts transplanted to RAG1 KO mice (devoid of T cells and B cells) by passive transfer of anti-major histocompatibility complex antibodies. Repeated administration of antibody over 4 weeks induced chronic transplant arteriopathy. Endothelial C4d deposits were present on antibody treatment but disappeared during further follow-up (56 days), whereas chronic transplant arteriopathy did not diminish. 52 A series of in vitro studies by the group of Elaine Reed 53 demonstrated that (monoclonal) antibodies against HLA class I antigens exert effects on human endothelial cells by complement-independent activation of signaling pathways involved in cell survival and proliferation. It is of particular interest that signaltransduction pathways are activated in a time- and dosedependent manner, with low doses of antibody inducing protective mechanisms (Bcl-2, Bcl-xL upregulation), whereas higher titers of the same antibody might induce expression of fibroblast growth factor receptor, thereby enhancing cell proliferation and possibly contributing to vascular damage. 54 Yamakuchi et al. 55 demonstrated that anti-hla antibodies triggered exocytosis of von Willebrand factor and P-selectin from endothelial cells in a complement-free in vitro system. This finding was confirmed in human skin transplants to nude mice perfused with the anti-hla antibody in vivo. Although none of the studies cited above were performed in kidneys and we are not aware of similar investigations in renal transplant models, the findings are likely of universal importance as they highlight the complexity of antibodymediated responses that go far beyond complementmediated cell activation/damage. Duration and dosage (titer) of antibody exposure are obviously crucial in shaping the molecular events triggered by antibody binding to cell surfaces. A recent study from the group in Edmonton strongly supported the concept of complement-independent (or at least C4d negative) chronic allograft rejection 56 in human kidney transplantation as well, by investigating the transcriptional response of endothelial cell allograft biopsies. They observed the typical transcriptional signature of AMR also in cases without C4d deposition and calculated a much higher specificity of increased endothelial cell transcriptional activity (71%) for graft loss as compared with C4d (31%). These findings from studies in transplanted hearts and endothelial cell culture and the emerging evidence from clinical investigations should stimulate the search for novel diagnostic markers of chronic AMR and/or accommodation. S30

5 LC Racusen and H Regele: Multifactorial pathogenesis of allograft fibrosis review EVOLUTION OF FIBROSIS/SCLEROSIS An additional important concept is that the development of fibrosis is a multiphase process. 57,58 The process begins with an early signaling phase involving cytokines and chemokines 59 and inflammatory cells, and activation of fibroblasts and myofibroblasts. Macrophages have a pivotal role in this process. 60 Deposition of an early relatively immature matrix is characterized by the presence of desmin, laminin, and immature collagen. Generation of immature collagen is then followed by polymerization of collagen into more mature scar. The pathologist should indicate whether the fibrosing process is in an early active phase, evolving (often in the context of inflammation), or advanced. In the context of this evolution, antifibrogenic therapy may have general efficacy. Epithelial mesenchymal transition is a potentially important pathogenic component that may be identifiable as well. 61 To make timely and meaningful diagnoses, biopsy should be performed as graft function begins to deteriorate. 62 These biopsies should be performed with the expectation that specific pathological processes can and will be identified, and that intervention can be and often is possible. In addition, identification of the evolving fibrotic process and its level of activity may be used to guide initiation of antifibrogenic therapies. 58 The hope is to identify, abrogate, and even reverse fibrosis and atrophy in the graft. Finally, there is considerable rationale for performing surveillance biopsy to detect early development of fibrosis in the graft. 10,62,63 Serum creatinine is not a sensitive indicator of graft damage, potentially rising only after significant chronic changes have evolved. Earlier surveillance may enable more precise definition of cause of evolving changes, as clues to etiology may be easier to detect. There may be clinical clues that a given allograft is at risk for these changes. As reviewed above, a major factor is development of anti-hla antibody, and especially donor-specific antibody. Endothelial/capillary deposition of C4d may precede development of capillary damage. 48 SCR in surveillance biopsies may predispose to the development of later fibrosis. 27,29,41 With a more proactive approach to diagnosis, the inevitable development of chronic allograft dysfunction may be obviated. The pathologist should have an important role in this effort. DISCLOSURE The authors declared no competing interests. ACKNOWLEDGMENTS LR has received consulting fees from BMS. HR has submitted a grant proposal to the Austrian Science Fund. REFERENCES 1. Solez K, Colvin RB, Rcusen LC et al. 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