- Email: [email protected]
Immune monitoring and biomarkers to predict chronic allograft dysfunction
review
http://www.kidney-international.org & 2010 International Society of Nephrology
Immune monitoring and biomarkers to predict chronic allograft dysfunction Roslyn B. Mannon1 1
Division of Nephrology, Department of Medicine, University of Alabama at Birmingham, Birmingham, Alabama, USA
Late failure of a kidney transplant continues to be a major problem after transplantation, in spite of more potent immunosuppressive strategies and the focus of clinical management shifting toward prolonging long-term graft survival. It is now recognized that graft failure occurs because of two major complications: death with a functioning graft and intrinsic allograft failure. Recent studies of late kidney graft loss have indicated a complexity of findings, including etiologies that are both immune and non-immune. These studies suggest that late graft failure is not an inevitable fact and that further investigation into the etiology of transplant graft failure may lead to a new understanding of the biology that will provide novel therapeutic strategies and biomarkers. In this review, we will focus on late allograft failure due to intrinsic injury to the transplant. The role of immune monitoring will be discussed in the context of monitoring for ongoing injury or for identifying late injury. A variety of methodologies have been used, including genomics, proteomics, and metabolomics, not only for monitoring allograft injury but also for identifying markers of graft failure that are more sensitive than serum creatinine. The available studies, as they relate to late or chronic graft injury, will also be reviewed. Kidney International (2010) 78 (Suppl 119), S59–S65; doi:10.1038/ki.2010.425 KEYWORDS: biomarker; biopsy; ELISpot; monitoring; proteomics; transplantation TO CITE THIS ARTICLE: Mannon RB. Immune monitoring and biomarkers to predict chronic allograft dysfunction. Kidney Int 2010; 78 (Suppl 119): S59–S65.
Correspondence: Roslyn B. Mannon, Division of Nephrology, Department of Medicine, University of Alabama at Birmingham, 1900 University Boulevard, THT 611G, Birmingham, Alabama 35294, USA. E-mail: [email protected] Kidney International (2010) 78 (Suppl 119), S59–S65
WHAT CAUSES LATE GRAFT FAILURE?
Over the last decade, graft failure more than 3 months after transplantation has been ascribed to an entity called chronic allograft nephropathy. This term came into use through the Banff classification schema, which organized biopsy specimens into those with acute and chronic injuries.1 This histological picture included fibrosing changes in the biopsy interstitium, as well as tubular atrophy (TA). Inflammation, when present, was only assessed in regions of biopsy viability and was often referred to as ‘chronic rejection’.2 These changes could also be accompanied by arterial thickening and, more recently, may have included the entity of transplant glomerulopathy.1 Thus, establishing criteria for allograft pathology could facilitate agreement and establish diagnostic standards to support clinical trials and the study of specific entities. However, it was recognized that many factors could contribute to allograft fibrosis, including hypertension, recurrent disease, ischemic injury, drug toxicity, infection such as BK nephropathy, and ongoing alloimmune injury; however, all these became synonymous with one term, ‘chronic allograft nephropathy’. As such, various entities with different etiologies were lumped together, and various animal models were studied to simulate different aspects of human disease, all seemingly used to describe one entity.3 The results were another revision of the Banff criteria, classifying in an isolated manner both interstitial fibrosis (IF) and TA in the absence of etiological factors in a biopsy as IF/TA.4 Other ‘chronic’ lesions included chronic T-cell-mediated rejection, chronic antibody-mediated rejection,5 as well as contributory entities to graft failure (‘other’). Although these changes supported the notion that not all chronic allograft nephropathy was rejection, it continued to de-emphasize the need to understand the causes of injury that could be reversed or ameliorated. Recent studies, however, have suggested that individual diseases can be identified as the causes of late graft failure. These insights are critical if we are to formulate strategies for prevention and disease identification. A large single-center study of over 1000 recipients reviewed allograft biopsies over a 10-year period.6 A total of 330 allografts were lost during this period, and of these, 46% were because of allograft failure. Nearly all of these individuals had an allograft biopsy in a median of 4.7 months before graft loss. In all, 95% of individuals with failing allografts had an identifiable S59
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diagnosis—this included 37% of such failures due to transplant glomerulopathy and recurrent disease, 12% with acute rejection, and 31% with a diagnosis of IF/TA. Of those with IF/TA, 80% of biopsy samples had an ascribable cause to their fibrosis, including such entities as BK polyomavirus nephropathy, ongoing cellular or antibody-mediated rejection, or recurrent pyelonephritis. Another surprising feature of these data was the lack of frequency of calcineurin inhibitor toxicity. Although calcineurin inhibitor use has been attributed to be commonly seen in kidney allograft recipients, and is a key factor to graft loss,7 only 0.6% of allografts failed because of calcineurin inhibitor toxicity. Thus, allograft failure, in the current generation of immunosuppression, can take on many etiologies, and immune-mediated injury is one of them. Strategies that can assist in diagnosis of these etiologies are important adjuncts to biopsy diagnosis and are discussed below. A recent consortium study of failing allografts and their biopsies (the Decline in Kidney Allograft Function (DeKAF) study) has suggested that, although IF/TA may be uniformly present on biopsy, histopathological subscoring by cluster analysis can identify groups of patients with varying outcomes.8 Indeed, the presence of tubulitis, interstitial inflammation, and vascular lesions was associated with worse prognosis. Similarly, a study of 234 biopsy samples for cause associated Banff ’s scoring patterns into patterns of disease, including microcirculation change, tubulointerstitial inflammation, and tubulointerstitial scarring, supporting the notion that grouping of related lesions into diagnoses can explain the stress on various compartments in the kidney and may guide the interpretation of the biology of the lesion by acknowledging their relationships.9 The development of donor-specific alloantibody has long been recognized as a negative prognostic factor in graft survival.10 With the detection of C4d deposition in the allograft as a marker of antibody deposition and complement activation, it is now recognized that alloantibody can be a critical cause of late graft failure (reviewed by Colvin and Smith11). Although controversy still exists about the association of C4d and transplant glomerulopathy,12 the use of molecular analysis of allograft biopsies suggests that endothelial cell activation detected by gene expression is strongly associated with graft failure, even in the absence of C4d staining.13 Thus, it is becoming apparent that screening for alloantibody is critical if we are to make any impact on antibody-mediated late injury. This will be discussed in more depth below. A number of studies have indicated that inflammation in the allograft is strongly associated with poorer graft outcome. In an analysis of nearly 300 protocol biopsy samples at 1-year after transplantation, Cosio et al.14 demonstrated that fibrosis and changes in inflammation, even in the absence of meeting criteria for acute rejection, together had a 8.5-fold higher risk of graft failure than did biopsies showing no histological abnormalities. Similarly, the presence of subclinical rejection, identified as Banff ’s acute cellular rejection or borderline inflammation, in 435 biopsies performed in S60
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recipients with normal function and without proteinuria at 6 months after transplantation, in association with IF/TA, was an independent risk factor for graft failure.15 These types of studies, as well as recent data implicating that inflammation in areas of scarring and atrophy may contribute to graft failure,16 emphasize the need to identify ongoing inflammation in the allograft even in the absence of functional impairment or abnormality. MONITORING STRATEGIES: OVERVIEW
Using allograft histology as the ‘gold standard’, a number of approaches have been suggested as non-invasive strategies for diagnosis and monitoring.17,18 A major issue in the literature is that many of the studies are single center or consist of small numbers of patients from multiples centers and have yet to be validated in large patient populations. The recently conducted Clinical Trials in Organ Transplantation is a series of consortium studies in immune monitoring sponsored by the National Institute of Allergy and Infectious Diseases. Insights from these studies have to date been published in abstract format only, but suggest that, rather than an individual analyte, analysis should take into account a number of methodologies simultaneously to identify those at risk of ongoing immune injury.19 The opportunities for monitoring immune responses are plenty in kidney transplant recipients. These include assaying the serum, peripheral blood mononuclear cells (PBMCs), urine, and of course the allograft through biopsy. A distinct advantage for an assay is the utilization of a non-invasive method that may ultimately be easy to use and with limited risk or side effects when performing such tests. Methodology may be further affected by the immunosuppressive strategy. For example, with the increasing frequency of use of depletional induction therapy, PBMC availability may be limited, at least in the first 6 months after induction. As already noted, with inflammation in the first 6–12 months correlating with poor outcome, the lack of ability to survey this cell population is a limitation of this approach. Urine markers seem ideal as urine is in close contact with the microenvironment of the kidney. This approach may be complicated by the presence of proteinuria, an obvious marker of renal injury, or if there is severe allograft dysfunction. Thus, defining an approach needs to take into account not only reproducibility, cost, and ease but also the limitations of a specific site of sampling. A critical feature of late graft failure, as noted above, is an ongoing alloimmune response. This may be cellular or antibody mediated and may be observed as proinflammatory markers such as chemokines and cytokines, and as the presence of donor-specific antibody. Other contributory factors in late allograft failure may include elevated growth factor expression supporting a profibrotic milieu.20 Thus, we will review approaches taken on the basis of sample location, recognizing that an approach that uses multiple methods and biological specimens may ultimately be the required approach. Kidney International (2010) 78 (Suppl 119), S59–S65
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SERUM AND PERIPHERAL BLOOD MARKERS OF ALLOGRAFT INJURY Serum antibodies
The detection and monitoring of antibody-mediated injury has become a primary focus of postrenal transplant management, as evidence of the contribution of donor-specific antibody to injury continues to mount. In addition to the association of transplant glomerulopathy with endothelial cell injury and complement activation,21 graft failure has been associated strongly with alloantibody development.22–26 A recent prospective multicenter study of over 1100 recipients of deceased donor kidney transplants demonstrated that human leukocyte antigen (HLA) class I antibodies present before transplantation were associated with a higher rate of delayed graft function and acute rejection episodes during the first 3 months after transplantation, and ultimately in an increased risk of graft loss by 3 years after transplantation.27 Although the majority of these studies support donor-specific HLA antibodies, preclinical studies in rodents suggest that autoantibodies to kidney antigens may also be of functional significance.28,29 As yet, the role of alloantibody detection in allograft monitoring has not been fully defined. Key factors include detecting the onset of development and specificity of donorreactive antibody, as well as correlating their presence with specific tissue damage. Using luminex technology, the ability to detect HLA antibodies has occurred in about a third of recipients at 5 years after transplantation, and of these, about 30% have donor-specific antibody.30 Serial monitoring indicates that the occurrence of repeated negative results over 5 years is associated with a high rate of graft survival (95%) compared with those recipients with HLA antibody (79%). With the increased sensitivity to detect alloantibodies, there is also a significant rise in cost. A monitoring protocol following transplantation that incorporated such assays would potentially identify the development of donor-specific antibody; however, in non-sensitized first-graft recipients, the frequency may be so low that the assay may not be costeffective. Another critical concern is the limited effect in therapeutics following a positive study. Studies in sensitized patients suggest that B-lymphocyte depletion31 or plasma cell inhibition32 may reduce the production of anti-HLA antibodies and might be therefore effective in limiting chronic antibody-mediated injuries. However, additional studies are needed in a broad clinical population to identify their cost-effectiveness and clinical utility. Serum proteins
The analysis of serum proteins has been another focus of blood sample monitoring. The upregulation of transforming growth factor-b (TGFb)33,34 remains an inconsistent finding in recipients with biopsy-proven IF/TA.35 Indeed, our evaluation of connective tissue growth factor, a downstream effector of TGFb, indicates that, although serum levels in a cohort of recipients were elevated compared with normal, healthy controls, these elevations did not distinguish acute Kidney International (2010) 78 (Suppl 119), S59–S65
from chronic allograft injury, whereas urinary levels had an excellent correlation to renal pathology.36 Other serum proteins upregulated in recipients with IF/TA include advanced glycation end products and oxidative stress proteins,37 C-reactive protein,38 sCD30 and neopterin,39 metzincins and related proteins,40 and tribbles-1 protein, which is expressed by activated endothelial cells and antigenpresenting cells;41 however, these targets have not been validated in larger studies. Finally, proteomic approaches have largely focused on urine (see below) rather than serum in identifying, on a more global scale, proteins associated with chronic allograft injury. PBMCs
One approach to monitor and detect the extent of alloreactivity is the enzyme-linked immunosorbent spot, a variation of the standard enzyme-linked immunosorbent assay to detect cytokine produced by individual antigenspecific T cells. This assay has been studied over the past decade, providing a sensitive quantitation of antigen-specific T cells, and assessing immune responses in both animal models and in human solid organ transplant recipients (reviewed by Dinavahi and Heeger42). Enzyme-linked immunosorbent spot provides information on both cell frequency and cytokine function. Before transplantation, high frequencies of donor-reactive interferon-g-producing cells have correlated with posttransplant acute rejection episodes.43 Serial monitoring after transplantation has also identified recipients at risk for acute rejection, with worse renal function in those with higher posttransplant frequencies of both donor and third-party responses.44,45 The ability to predict those more at risk for immune injury may provide an opportunity to intervene in the peritransplant period, as well as encourage more intense clinical follow-up. Further testing is underway to establish its role in immune therapy management. As an alternative approach, Kurian et al.46 have reported some fascinating results using combined gene microarray with tandem mass spectroscopy of RNA and proteins extracted from PBMCs from a small set of recipients with either mild IF/TA changes on allograft biopsy or more severe IF/TA. In mild disease, 1066 genes falling into 27 networks were identified, including, most commonly, those related to toll-like receptor signaling, stress-activated protein kinase/cJun NH2-terminal kinase, apoptosis, notch, and death receptor and interferon signaling. Finding 1066 significantly differentially expressed genes is a first indication that peripheral blood leukocyte transcript profiling is capable of classifying subjects defined by chronic allograft nephropathy biopsy histology. In moderate-to-severe IF/TA, 62 differentially expressed genes were identified. Proteomic analysis by shotgun tandem mass spectroscopy of PBMCs identified 206 differentially expressed proteins between Banff 0 and Banff grade I changes, including those linked to program cell death, cell signaling, and post-translational protein modification. In more severe 2–3 IF/TA, 282 proteins were differentially S61
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identified compared with Banff 0 biopsies and 95 proteins with grade 1 IF/TA, including proteins linked to cellular morphology, growth, proliferation, and signaling through extracellular signal-regulated protein kinase/mitogen-activated protein kinase, acute-phase reactants, peroxisome proliferatoractivated receptor-a/retinoid X receptor alpha, and insulin-like growth factor. Importantly, using only differentially expressed proteins, moderate-to-severe IF/TA was correctly classified 83% of the time. Thus, a series of potential candidate biomarkers has been proposed to provide near-perfect identification of biopsy pathology. These transcripts and proteins are undergoing further validation in a comprehensive multicenter prospective clinical trial and may be a source of potential therapeutic targets for chronic allograft failure. URINE: THE MICROENVIRONMENT OF THE ALLOGRAFT
The use of proteomic technology has changed the focus from a specific factor related to outcome to the identification of multiple factors that may be interrelated in allograft failure. Initial studies have primarily studied acute cellular rejection, distinguishing this injury from stable functioning allografts.47–49 Although not directly detecting chronic injury, one could suggest that the detection of ongoing acute injury may be beneficial in identifying allografts at risk for later graft failure. One approach has been through the use of liquid chromatography and tandem mass spectroscopy of urine specimens. In their initial report, Quintana et al.51 identified 6000 protein ions from 32 recipients with IF/TA and chronic antibody-mediated injury and from 18 stable recipients. Preliminary studies demonstrated that 14 proteins differed between those with IF/TA and those with antibody-mediated processes.50 A further analysis of these patient samples identified uromodulin at 638.03 m/z and a high expression of 642.61 m/z as diagnostic of chronic allograft dysfunction in nearly all cases. By two-dimensional difference gel electrophoresis, this group has also demonstrated 19 different proteins related to the histological diagnosis of IF/TA.52 In an alternative strategy, proteomic analysis of urine specimens from recipients enrolled in the DeKAF study8 undergoing allograft biopsy for late allograft dysfunction demonstrated qualitatively different magnitude spectra, and maximal discriminatory spectral subregions were identified in IF/TA biopsies without inflammation versus those with ongoing inflammation, suggesting the opportunity to recognize ongoing inflammatory processes that may lead to allograft failure.53 Further identification of the specific peptides is underway, as well as further validation to assess the diagnostic/prognostic capabilities of all classifiers. A number of investigators have begun to analyze urine samples of recipients with documented IF/TA changes on their allograft biopsy using a more focused analytical approach. On the basis of a microarray analysis of allograft biopsy samples with IF/TA (n ¼ 11) compared with normal kidney tissue (n ¼ 7), Mas et al.35 identified hundreds of probe sets that were differentially regulated, with S62
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upregulation of genes associated with matrix deposition and fibrosis, such as TGFb and matrix metalloproteinase-9, as well as immunoglobulin genes and B- and T-cell markers, and downregulation of epidermal growth factor receptor and fibroblast growth factor-2 receptor. Using these derived markers, urine samples from IF/TA recipients similarly demonstrated elevations in TGFb but downregulation of epidermal growth factor receptor, consistent with the biopsy analysis. Recent investigation into calcineurin inhibitor toxicity effects on renal tubular epithelial cells has demonstrated an altered expression of 38 proteins after incubation with cyclosporine in vitro.54 These include proteins involved in protein metabolism, as well as in response to cell damage, cell organization and cytoskeleton, energy metabolism, cell cycle, and nucleotide metabolism, and will provide a potential opportunity to non-invasively measure the toxic effects of these agents beyond allograft biopsy. In clinical settings, drug-related tubular injury may also be identified by measuring urine N-acetyl-b-D-gluosaminidase relative to total urine protein.55 Distinguishing drug-related injury before clinical disease would be a useful adjunct of immune monitoring tools beyond simply following trough drug levels. Urine mRNA
Suthanthiran and colleagues56 have advocated the use of mRNA isolation and analysis from urine specimens to noninvasively diagnose ongoing allograft pathology. Again, most of this work has focused on acute cellular rejection rather than later developing processes in the course of allograft failure.19 However, on the basis of microarray analysis of a limited number of allograft biopsies already discussed above,35 Mas et al.57 recently demonstrated, albeit in a small sample number, differential expression of angiotensin, epidermal growth factor receptor, and TGFb mRNA in urine samples from recipients with IF/TA compared with those with stable function. The detection of interferon producing protein-10 and CXCR3 was also associated with acute inflammation allograft biopsies.58 This type of approach may be a useful adjunct to monitoring ongoing inflammation that may contribute to late allograft injury. Further investigations using this technique should be considered and focused targets may be developed from genomics study discussed below. ALLOGRAFT BIOPSY: INVASIVE BUT INFORMATIVE Genomics
At present, allograft biopsy is the best technique to identify the cause of renal dysfunction, although there are circumstances in which it may provide limited information regarding the clinical impact and prognosis of a process, such as the presence of inflammatory cell infiltrates in biopsy samples unassociated with tubulitis, distinguishing BK polyomavirus disease from acute cellular rejection, identifying functionally significant rejection from SCR, and distinguishing the etiologies of IF/TA. The histological Kidney International (2010) 78 (Suppl 119), S59–S65
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classification of antibody-mediated injuries has also been recently revised to accept the notion that a number of different histological lesions, in the proper context, indicate alloantibody participation,5 although the pathobiology of this injury is not well defined. With the focus on the human genome, there has been an explosion of transcriptional information that is being applied to solid organ transplantation, again with the potential to identify markers and targets for therapeutic management. Microarray analysis allows for the interrogation of thousands of genes simultaneously, and is an increasingly robust and reproducible technique. The focus of these studies has been the identification of acute cellular rejection, identifying the molecular heterogeneity of allograft rejection, with differences detected by transcription that are not evident by light microscopy alone. Using an approach incorporating pathogenesis-based transcript sets identified in mouse kidney biopsy samples and correlated to response of human immune cells,59–62 Mueller et al.63demonstrated that these transcript sets correlate with biopsy diagnosis but have limited ability in the context of distinguishing so-called borderline rejection and acute cellular rejection, identifying some of the limitations of Banff ’s classification. In recipients with transplant glomerulopathy, the presence of endothelial activation and stress transcripts identifies a subset of recipients with worsened allograft outcome, even in the absence of C4d staining.13 These studies support the notion that histology has limited ability to explain a clinical situation and suggests an adjunct role of molecular analysis. Several groups have now incorporated the speed, technical ease, and quantitative capabilities of real-time PCR with the increasing availability of candidate transcripts, resulting in what may be the future—a rapid turnaround platform for analysis of fixed sets of 20–100 genes. These low-density arrays specifically limit the assessment of expression to a set of gene transcripts as opposed to a more global interrogation. Arrays may be customized or purchased commercially, depending on the study circumstances. Such expression data have demonstrated a number of unique findings in such clinical settings as ischemia–reperfusion injury,64,65 stable function graft function, acute rejection, and subclinical rejection.66 For example, use of low-density arrays has identified the development of allograft fibrosis and tubular injury in the setting of BK polyomavirus nephropathy.67 Transcripts associated with the induction of graft fibrosis and epithelial–mesenchymal transformation are markedly upregulated in BK polyomavirus nephropathy compared with biopsy samples with acute cellular rejection. Thus, the inflammatory immune response to BK has functional differences seen at the level of transcription that are not appreciable using standard histological techniques. Such studies demonstrate that specific transcriptional patterns may be identified that could be produced into a strategy with clinically applicable turnaround similar to that for standard histology. As such, it is now feasible to supplement biopsy information with contemporaneously available transcriptional analysis on a routine basis. Kidney International (2010) 78 (Suppl 119), S59–S65
Surveillance histology
Allograft biopsy remains the gold standard to clarify the etiology of allograft dysfunction. Protocol or surveillance biopsies have been recently used as a monitoring tool, albeit invasive, to recognize whether there is ongoing immune injury. Although the use of these biopsies has been debated over the years, SCR, findings of cellular rejection in the absence of any functional impediment, has been detected anywhere from 4 to 51% of biopsies (reviewed by Nankivell and Chapman68). The coincident appearance of SCR with IF/ TA in 6-month posttransplant biopsy samples is similarly a poor marker of outcome.15 Indeed, when found in repeated protocol biopsies, SCR is associated with more rapid allograft failure.69–71 The impact has also been debated showing benefit in non-randomized studies.72,71 However, in a recent randomized study, no effect of treatment was seen,73 perhaps in part because of the relatively low frequency of the event or because intervention was so early after transplantation. Further investigation into this area at later time points might provide additional clues to its utility. SUMMARY
Long-term graft failure continues to plague kidney allografts, in spite of potent immunosuppressive therapies. Both immune-dependent and -independent factors continue to contribute to failure. A number of promising observations made in human kidney recipients suggest unique protein and genetic signatures that may identify biomarkers of injury, as well as potential targets of therapy. Some of these may be obtained through noninvasive methods and may thus be extremely useful in the clinical realm. As discussed, these markers are undergoing further identification and validation to document their applicability and usefulness. In spite of the complexity of the immune response to a kidney allograft, these studies continue to move us forward and provide progress to the dismal area of late graft injury. DISCLOSURE
The author declared no competing interests. ACKNOWLEDGMENTS
This work was supported in part by funding from the University of Alabama at Birmingham Dean’s IMPACT and Division of Nephrology funds, as well as by U01-AI58013 and U19-AI070119. I thank Paolo Cravedi for his thoughtful discussion of this paper and Wendy Bailey for administrative assistance. REFERENCES 1. 2.
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Campistol JM, Inigo P, Larios S et al. Role of transforming growth factorbeta1 in the progression of chronic allograft nephropathy. Nephrol Dial Transplant 2001; 16(Suppl 1): 114–116. Sharma VK, Bologa RM, Xu GP et al. Intragraft TGF-beta 1 mRNA: a correlate of interstitial fibrosis and chronic allograft nephropathy. Kidney Int 1996; 49: 1297–1303. Mas V, Maluf D, Archer K et al. Establishing the molecular pathways involved in chronic allograft nephropathy for testing new noninvasive diagnostic markers. Transplantation 2007; 83: 448–457. Cheng O, Thuillier R, Sampson E et al. Connective tissue growth factor is a biomarker and mediator of kidney allograft fibrosis. Am J Transplant 2006; 6: 2292–2306. Raj DS, Lim G, Levi M et al. Advanced glycation end products and oxidative stress are increased in chronic allograft nephropathy. Am J Kidney Dis 2004; 43: 154–160. Sezer S, Akcay A, Ozdemir FN et al. Post-transplant C-reactive protein monitoring can predict chronic allograft nephropathy. Clin Transplant 2004; 18: 722–725. Weimer R, Susal C, Yildiz S et al. Post-transplant sCD30 and neopterin as predictors of chronic allograft nephropathy: impact of different immunosuppressive regimens. Am J Transplant 2006; 6: 1865–1874. Rodder S, Scherer A, Raulf F et al. Renal allografts with IF/TA display distinct expression profiles of metzincins and related genes. Am J Transplant 2009; 9: 517–526. Ashton-Chess J, Giral M, Mengel M et al. Tribbles-1 as a novel biomarker of chronic antibody-mediated rejection. J Am Soc Nephrol 2008; 19: 1116–1127. Dinavahi R, Heeger PS. T-cell immune monitoring in organ transplantation. Transplantation 2009; 88: 1157–1158. Heeger PS, Greenspan NS, Kuhlenschmidt S et al. Pretransplant frequency of donor-specific, IFN-gamma-producing lymphocytes is a manifestation of immunologic memory and correlates with the risk of posttransplant rejection episodes. J Immunol 1999; 163: 2267–2275. Augustine JJ, Siu DS, Clemente MJ et al. Pre-transplant IFN-gamma ELISPOTs are associated with post-transplant renal function in African American renal transplant recipients. Am J Transplant 2005; 5: 1971–1975. Hricik DE, Rodriguez V, Riley J et al. Enzyme linked immunosorbent spot (ELISPOT) assay for interferon-gamma independently predicts renal function in kidney transplant recipients. Am J Transplant 2003; 3: 878–884. Kurian SM, Heilman R, Mondala TS et al. Biomarkers for early and late stage chronic allograft nephropathy by proteogenomic profiling of peripheral blood. PLoS One 2009; 4: e6212. Clarke W, Silverman BC, Zhang Z et al. Characterization of renal allograft rejection by urinary proteomic analysis. Ann Surg 2003; 237: 660–664. O’riordan E, Orlova TN, Mei JJ et al. Bioinformatic analysis of the urine proteome of acute allograft rejection. J Am Soc Nephrol 2004; 15: 3240–3248. Wittke S, Haubitz M, Walden M et al. Detection of acute tubulointerstitial rejection by proteomic analysis of urinary samples in renal transplant recipients. Am J Transplant 2005; 5: 2479–2488. Quintana LF, Sole-Gonzalez A, Kalko SG et al. Urine proteomics to detect biomarkers for chronic allograft dysfunction. J Am Soc Nephrol 2009; 20: 428–435. Quintana LF, Campistol JM, Alcolea MP et al. Application of label-free quantitative peptidomics for the identification of urinary biomarkers of kidney chronic allograft dysfunction. Mol Cell Proteomics 2009; 8: 1658–1673. Banon-Maneus E, Diekmann F, Carrascal M et al. Two-dimensional difference gel electrophoresis urinary proteomic profile in the search of nonimmune chronic allograft dysfunction biomarkers. Transplantation 2010; 89: 548–558. Rush D, Somorjai R et al. Urine magnetic resonance spectroscopy (UMRS): results from the DeKAF study. (abstract) Am J Transplant 2009; 9 (Suppl 2): 350. Puigmule M, Lopez-Hellin J, Sune G et al. Differential proteomic analysis of cyclosporine A-induced toxicity in renal proximal tubule cells. Nephrol Dial Transplant 2009; 24: 2672–2686. Bone JM, Amara AB, Shenkin A et al. Calcineurin inhibitors and proximal renal tubular injury in renal transplant patients with proteinuria and chronic allograft nephropathy. Transplantation 2005; 79: 119–122. Li B, Hartono C, Ding R et al. Noninvasive diagnosis of renal-allograft rejection by measurement of messenger RNA for perforin and granzyme B in urine. N Engl J Med 2001; 344: 947–954. Mas VR, Mas LA, Archer KJ et al. Evaluation of gene panel mRNAs in urine samples of kidney transplant recipients as a non-invasive tool of graft function. Mol Med 2007; 13: 315–324.
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Tatapudi RR, Muthukumar T, Dadhania D et al. Noninvasive detection of renal allograft inflammation by measurements of mRNA for IP-10 and CXCR3 in urine. Kidney Int 2004; 65: 2390–2397. Einecke G, Broderick G, Sis B et al. Early loss of renal transcripts in kidney allografts: relationship to the development of histologic lesions and alloimmune effector mechanisms. Am J Transplant 2007; 7: 1121–1130. Einecke G, Melk A, Ramassar V et al. Expression of CTL associated transcripts precedes the development of tubulitis in T-cell mediated kidney graft rejection. Am J Transplant 2005; 5: 1827–1836. Famulski KS, Einecke G, Reeve J et al. Changes in the transcriptome in allograft rejection: IFN-gamma-induced transcripts in mouse kidney allografts. Am J Transplant 2006; 6: 1342–1354. Famulski KS, Kayser D, Einecke G et al. Alternative macrophage activationassociated transcripts in T-Cell-mediated rejection of mouse kidney allografts. Am J Transplant 2010; 10: 490–497. Mueller TF, Einecke G, Reeve J et al. Microarray analysis of rejection in human kidney transplants using pathogenesis-based transcript sets. Am J Transplant 2007; 7: 2712–2722. Avihingsanon Y, Ma N, Pavlakis M et al. On the intraoperative molecular status of renal allografts after vascular reperfusion and clinical outcomes. J Am Soc Nephrol 2005; 16: 1542–1548. Hoffmann SC, Kampen RL, Amur S et al. Molecular and immunohistochemical characterization of the onset and resolution of
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human renal allograft ischemia-reperfusion injury. Transplantation 2002; 74: 916–923. Hoffmann SC, Hale DA, Kleiner DE et al. Functionally significant renal allograft rejection is defined by transcriptional criteria. Am J Transplant 2005; 5: 573–581. Mannon RB, Hoffmann SC, Kampen RL et al. Molecular evaluation of BK polyomavirus nephropathy. Am J Transplant 2005; 5: 2883–2893. Nankivell BJ, Chapman JR. The significance of subclinical rejection and the value of protocol biopsies. Am J Transplant 2006; 6: 2006–2012. Choi BS, Shin MJ, Shin SJ et al. Clinical significance of an early protocol biopsy in living-donor renal transplantation: ten-year experience at a single center. Am J Transplant 2005; 5: 1354–1360. Rush DN, Jeffery JR, Gough J. Sequential protocol biopsies in renal transplant patients. Clinico-pathological correlations using the Banff schema. Transplantation 1995; 59: 511–514. Shishido S, Asanuma H, Nakai H et al. The impact of repeated subclinical acute rejection on the progression of chronic allograft nephropathy. J Am Soc Nephrol 2003; 14: 1046–1052. Rush D, Nickerson P, Gough J et al. Beneficial effects of treatment of early subclinical rejection: a randomized study. J Am Soc Nephrol 1998; 9: 2129–2134. Rush D, Arlen D, Boucher A et al. Lack of benefit of early protocol biopsies in renal transplant patients receiving TAC and MMF: a randomized study. Am J Transplant 2007; 7: 2538–2545.
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Copyright of Kidney International is the property of Nature Publishing Group and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use.
Copyright of Kidney International Supplement is the property of Nature Publishing Group and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use.
http://www.kidney-international.org & 2010 International Society of Nephrology
Immune monitoring and biomarkers to predict chronic allograft dysfunction Roslyn B. Mannon1 1
Division of Nephrology, Department of Medicine, University of Alabama at Birmingham, Birmingham, Alabama, USA
Late failure of a kidney transplant continues to be a major problem after transplantation, in spite of more potent immunosuppressive strategies and the focus of clinical management shifting toward prolonging long-term graft survival. It is now recognized that graft failure occurs because of two major complications: death with a functioning graft and intrinsic allograft failure. Recent studies of late kidney graft loss have indicated a complexity of findings, including etiologies that are both immune and non-immune. These studies suggest that late graft failure is not an inevitable fact and that further investigation into the etiology of transplant graft failure may lead to a new understanding of the biology that will provide novel therapeutic strategies and biomarkers. In this review, we will focus on late allograft failure due to intrinsic injury to the transplant. The role of immune monitoring will be discussed in the context of monitoring for ongoing injury or for identifying late injury. A variety of methodologies have been used, including genomics, proteomics, and metabolomics, not only for monitoring allograft injury but also for identifying markers of graft failure that are more sensitive than serum creatinine. The available studies, as they relate to late or chronic graft injury, will also be reviewed. Kidney International (2010) 78 (Suppl 119), S59–S65; doi:10.1038/ki.2010.425 KEYWORDS: biomarker; biopsy; ELISpot; monitoring; proteomics; transplantation TO CITE THIS ARTICLE: Mannon RB. Immune monitoring and biomarkers to predict chronic allograft dysfunction. Kidney Int 2010; 78 (Suppl 119): S59–S65.
Correspondence: Roslyn B. Mannon, Division of Nephrology, Department of Medicine, University of Alabama at Birmingham, 1900 University Boulevard, THT 611G, Birmingham, Alabama 35294, USA. E-mail: [email protected] Kidney International (2010) 78 (Suppl 119), S59–S65
WHAT CAUSES LATE GRAFT FAILURE?
Over the last decade, graft failure more than 3 months after transplantation has been ascribed to an entity called chronic allograft nephropathy. This term came into use through the Banff classification schema, which organized biopsy specimens into those with acute and chronic injuries.1 This histological picture included fibrosing changes in the biopsy interstitium, as well as tubular atrophy (TA). Inflammation, when present, was only assessed in regions of biopsy viability and was often referred to as ‘chronic rejection’.2 These changes could also be accompanied by arterial thickening and, more recently, may have included the entity of transplant glomerulopathy.1 Thus, establishing criteria for allograft pathology could facilitate agreement and establish diagnostic standards to support clinical trials and the study of specific entities. However, it was recognized that many factors could contribute to allograft fibrosis, including hypertension, recurrent disease, ischemic injury, drug toxicity, infection such as BK nephropathy, and ongoing alloimmune injury; however, all these became synonymous with one term, ‘chronic allograft nephropathy’. As such, various entities with different etiologies were lumped together, and various animal models were studied to simulate different aspects of human disease, all seemingly used to describe one entity.3 The results were another revision of the Banff criteria, classifying in an isolated manner both interstitial fibrosis (IF) and TA in the absence of etiological factors in a biopsy as IF/TA.4 Other ‘chronic’ lesions included chronic T-cell-mediated rejection, chronic antibody-mediated rejection,5 as well as contributory entities to graft failure (‘other’). Although these changes supported the notion that not all chronic allograft nephropathy was rejection, it continued to de-emphasize the need to understand the causes of injury that could be reversed or ameliorated. Recent studies, however, have suggested that individual diseases can be identified as the causes of late graft failure. These insights are critical if we are to formulate strategies for prevention and disease identification. A large single-center study of over 1000 recipients reviewed allograft biopsies over a 10-year period.6 A total of 330 allografts were lost during this period, and of these, 46% were because of allograft failure. Nearly all of these individuals had an allograft biopsy in a median of 4.7 months before graft loss. In all, 95% of individuals with failing allografts had an identifiable S59
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diagnosis—this included 37% of such failures due to transplant glomerulopathy and recurrent disease, 12% with acute rejection, and 31% with a diagnosis of IF/TA. Of those with IF/TA, 80% of biopsy samples had an ascribable cause to their fibrosis, including such entities as BK polyomavirus nephropathy, ongoing cellular or antibody-mediated rejection, or recurrent pyelonephritis. Another surprising feature of these data was the lack of frequency of calcineurin inhibitor toxicity. Although calcineurin inhibitor use has been attributed to be commonly seen in kidney allograft recipients, and is a key factor to graft loss,7 only 0.6% of allografts failed because of calcineurin inhibitor toxicity. Thus, allograft failure, in the current generation of immunosuppression, can take on many etiologies, and immune-mediated injury is one of them. Strategies that can assist in diagnosis of these etiologies are important adjuncts to biopsy diagnosis and are discussed below. A recent consortium study of failing allografts and their biopsies (the Decline in Kidney Allograft Function (DeKAF) study) has suggested that, although IF/TA may be uniformly present on biopsy, histopathological subscoring by cluster analysis can identify groups of patients with varying outcomes.8 Indeed, the presence of tubulitis, interstitial inflammation, and vascular lesions was associated with worse prognosis. Similarly, a study of 234 biopsy samples for cause associated Banff ’s scoring patterns into patterns of disease, including microcirculation change, tubulointerstitial inflammation, and tubulointerstitial scarring, supporting the notion that grouping of related lesions into diagnoses can explain the stress on various compartments in the kidney and may guide the interpretation of the biology of the lesion by acknowledging their relationships.9 The development of donor-specific alloantibody has long been recognized as a negative prognostic factor in graft survival.10 With the detection of C4d deposition in the allograft as a marker of antibody deposition and complement activation, it is now recognized that alloantibody can be a critical cause of late graft failure (reviewed by Colvin and Smith11). Although controversy still exists about the association of C4d and transplant glomerulopathy,12 the use of molecular analysis of allograft biopsies suggests that endothelial cell activation detected by gene expression is strongly associated with graft failure, even in the absence of C4d staining.13 Thus, it is becoming apparent that screening for alloantibody is critical if we are to make any impact on antibody-mediated late injury. This will be discussed in more depth below. A number of studies have indicated that inflammation in the allograft is strongly associated with poorer graft outcome. In an analysis of nearly 300 protocol biopsy samples at 1-year after transplantation, Cosio et al.14 demonstrated that fibrosis and changes in inflammation, even in the absence of meeting criteria for acute rejection, together had a 8.5-fold higher risk of graft failure than did biopsies showing no histological abnormalities. Similarly, the presence of subclinical rejection, identified as Banff ’s acute cellular rejection or borderline inflammation, in 435 biopsies performed in S60
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recipients with normal function and without proteinuria at 6 months after transplantation, in association with IF/TA, was an independent risk factor for graft failure.15 These types of studies, as well as recent data implicating that inflammation in areas of scarring and atrophy may contribute to graft failure,16 emphasize the need to identify ongoing inflammation in the allograft even in the absence of functional impairment or abnormality. MONITORING STRATEGIES: OVERVIEW
Using allograft histology as the ‘gold standard’, a number of approaches have been suggested as non-invasive strategies for diagnosis and monitoring.17,18 A major issue in the literature is that many of the studies are single center or consist of small numbers of patients from multiples centers and have yet to be validated in large patient populations. The recently conducted Clinical Trials in Organ Transplantation is a series of consortium studies in immune monitoring sponsored by the National Institute of Allergy and Infectious Diseases. Insights from these studies have to date been published in abstract format only, but suggest that, rather than an individual analyte, analysis should take into account a number of methodologies simultaneously to identify those at risk of ongoing immune injury.19 The opportunities for monitoring immune responses are plenty in kidney transplant recipients. These include assaying the serum, peripheral blood mononuclear cells (PBMCs), urine, and of course the allograft through biopsy. A distinct advantage for an assay is the utilization of a non-invasive method that may ultimately be easy to use and with limited risk or side effects when performing such tests. Methodology may be further affected by the immunosuppressive strategy. For example, with the increasing frequency of use of depletional induction therapy, PBMC availability may be limited, at least in the first 6 months after induction. As already noted, with inflammation in the first 6–12 months correlating with poor outcome, the lack of ability to survey this cell population is a limitation of this approach. Urine markers seem ideal as urine is in close contact with the microenvironment of the kidney. This approach may be complicated by the presence of proteinuria, an obvious marker of renal injury, or if there is severe allograft dysfunction. Thus, defining an approach needs to take into account not only reproducibility, cost, and ease but also the limitations of a specific site of sampling. A critical feature of late graft failure, as noted above, is an ongoing alloimmune response. This may be cellular or antibody mediated and may be observed as proinflammatory markers such as chemokines and cytokines, and as the presence of donor-specific antibody. Other contributory factors in late allograft failure may include elevated growth factor expression supporting a profibrotic milieu.20 Thus, we will review approaches taken on the basis of sample location, recognizing that an approach that uses multiple methods and biological specimens may ultimately be the required approach. Kidney International (2010) 78 (Suppl 119), S59–S65
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SERUM AND PERIPHERAL BLOOD MARKERS OF ALLOGRAFT INJURY Serum antibodies
The detection and monitoring of antibody-mediated injury has become a primary focus of postrenal transplant management, as evidence of the contribution of donor-specific antibody to injury continues to mount. In addition to the association of transplant glomerulopathy with endothelial cell injury and complement activation,21 graft failure has been associated strongly with alloantibody development.22–26 A recent prospective multicenter study of over 1100 recipients of deceased donor kidney transplants demonstrated that human leukocyte antigen (HLA) class I antibodies present before transplantation were associated with a higher rate of delayed graft function and acute rejection episodes during the first 3 months after transplantation, and ultimately in an increased risk of graft loss by 3 years after transplantation.27 Although the majority of these studies support donor-specific HLA antibodies, preclinical studies in rodents suggest that autoantibodies to kidney antigens may also be of functional significance.28,29 As yet, the role of alloantibody detection in allograft monitoring has not been fully defined. Key factors include detecting the onset of development and specificity of donorreactive antibody, as well as correlating their presence with specific tissue damage. Using luminex technology, the ability to detect HLA antibodies has occurred in about a third of recipients at 5 years after transplantation, and of these, about 30% have donor-specific antibody.30 Serial monitoring indicates that the occurrence of repeated negative results over 5 years is associated with a high rate of graft survival (95%) compared with those recipients with HLA antibody (79%). With the increased sensitivity to detect alloantibodies, there is also a significant rise in cost. A monitoring protocol following transplantation that incorporated such assays would potentially identify the development of donor-specific antibody; however, in non-sensitized first-graft recipients, the frequency may be so low that the assay may not be costeffective. Another critical concern is the limited effect in therapeutics following a positive study. Studies in sensitized patients suggest that B-lymphocyte depletion31 or plasma cell inhibition32 may reduce the production of anti-HLA antibodies and might be therefore effective in limiting chronic antibody-mediated injuries. However, additional studies are needed in a broad clinical population to identify their cost-effectiveness and clinical utility. Serum proteins
The analysis of serum proteins has been another focus of blood sample monitoring. The upregulation of transforming growth factor-b (TGFb)33,34 remains an inconsistent finding in recipients with biopsy-proven IF/TA.35 Indeed, our evaluation of connective tissue growth factor, a downstream effector of TGFb, indicates that, although serum levels in a cohort of recipients were elevated compared with normal, healthy controls, these elevations did not distinguish acute Kidney International (2010) 78 (Suppl 119), S59–S65
from chronic allograft injury, whereas urinary levels had an excellent correlation to renal pathology.36 Other serum proteins upregulated in recipients with IF/TA include advanced glycation end products and oxidative stress proteins,37 C-reactive protein,38 sCD30 and neopterin,39 metzincins and related proteins,40 and tribbles-1 protein, which is expressed by activated endothelial cells and antigenpresenting cells;41 however, these targets have not been validated in larger studies. Finally, proteomic approaches have largely focused on urine (see below) rather than serum in identifying, on a more global scale, proteins associated with chronic allograft injury. PBMCs
One approach to monitor and detect the extent of alloreactivity is the enzyme-linked immunosorbent spot, a variation of the standard enzyme-linked immunosorbent assay to detect cytokine produced by individual antigenspecific T cells. This assay has been studied over the past decade, providing a sensitive quantitation of antigen-specific T cells, and assessing immune responses in both animal models and in human solid organ transplant recipients (reviewed by Dinavahi and Heeger42). Enzyme-linked immunosorbent spot provides information on both cell frequency and cytokine function. Before transplantation, high frequencies of donor-reactive interferon-g-producing cells have correlated with posttransplant acute rejection episodes.43 Serial monitoring after transplantation has also identified recipients at risk for acute rejection, with worse renal function in those with higher posttransplant frequencies of both donor and third-party responses.44,45 The ability to predict those more at risk for immune injury may provide an opportunity to intervene in the peritransplant period, as well as encourage more intense clinical follow-up. Further testing is underway to establish its role in immune therapy management. As an alternative approach, Kurian et al.46 have reported some fascinating results using combined gene microarray with tandem mass spectroscopy of RNA and proteins extracted from PBMCs from a small set of recipients with either mild IF/TA changes on allograft biopsy or more severe IF/TA. In mild disease, 1066 genes falling into 27 networks were identified, including, most commonly, those related to toll-like receptor signaling, stress-activated protein kinase/cJun NH2-terminal kinase, apoptosis, notch, and death receptor and interferon signaling. Finding 1066 significantly differentially expressed genes is a first indication that peripheral blood leukocyte transcript profiling is capable of classifying subjects defined by chronic allograft nephropathy biopsy histology. In moderate-to-severe IF/TA, 62 differentially expressed genes were identified. Proteomic analysis by shotgun tandem mass spectroscopy of PBMCs identified 206 differentially expressed proteins between Banff 0 and Banff grade I changes, including those linked to program cell death, cell signaling, and post-translational protein modification. In more severe 2–3 IF/TA, 282 proteins were differentially S61
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identified compared with Banff 0 biopsies and 95 proteins with grade 1 IF/TA, including proteins linked to cellular morphology, growth, proliferation, and signaling through extracellular signal-regulated protein kinase/mitogen-activated protein kinase, acute-phase reactants, peroxisome proliferatoractivated receptor-a/retinoid X receptor alpha, and insulin-like growth factor. Importantly, using only differentially expressed proteins, moderate-to-severe IF/TA was correctly classified 83% of the time. Thus, a series of potential candidate biomarkers has been proposed to provide near-perfect identification of biopsy pathology. These transcripts and proteins are undergoing further validation in a comprehensive multicenter prospective clinical trial and may be a source of potential therapeutic targets for chronic allograft failure. URINE: THE MICROENVIRONMENT OF THE ALLOGRAFT
The use of proteomic technology has changed the focus from a specific factor related to outcome to the identification of multiple factors that may be interrelated in allograft failure. Initial studies have primarily studied acute cellular rejection, distinguishing this injury from stable functioning allografts.47–49 Although not directly detecting chronic injury, one could suggest that the detection of ongoing acute injury may be beneficial in identifying allografts at risk for later graft failure. One approach has been through the use of liquid chromatography and tandem mass spectroscopy of urine specimens. In their initial report, Quintana et al.51 identified 6000 protein ions from 32 recipients with IF/TA and chronic antibody-mediated injury and from 18 stable recipients. Preliminary studies demonstrated that 14 proteins differed between those with IF/TA and those with antibody-mediated processes.50 A further analysis of these patient samples identified uromodulin at 638.03 m/z and a high expression of 642.61 m/z as diagnostic of chronic allograft dysfunction in nearly all cases. By two-dimensional difference gel electrophoresis, this group has also demonstrated 19 different proteins related to the histological diagnosis of IF/TA.52 In an alternative strategy, proteomic analysis of urine specimens from recipients enrolled in the DeKAF study8 undergoing allograft biopsy for late allograft dysfunction demonstrated qualitatively different magnitude spectra, and maximal discriminatory spectral subregions were identified in IF/TA biopsies without inflammation versus those with ongoing inflammation, suggesting the opportunity to recognize ongoing inflammatory processes that may lead to allograft failure.53 Further identification of the specific peptides is underway, as well as further validation to assess the diagnostic/prognostic capabilities of all classifiers. A number of investigators have begun to analyze urine samples of recipients with documented IF/TA changes on their allograft biopsy using a more focused analytical approach. On the basis of a microarray analysis of allograft biopsy samples with IF/TA (n ¼ 11) compared with normal kidney tissue (n ¼ 7), Mas et al.35 identified hundreds of probe sets that were differentially regulated, with S62
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upregulation of genes associated with matrix deposition and fibrosis, such as TGFb and matrix metalloproteinase-9, as well as immunoglobulin genes and B- and T-cell markers, and downregulation of epidermal growth factor receptor and fibroblast growth factor-2 receptor. Using these derived markers, urine samples from IF/TA recipients similarly demonstrated elevations in TGFb but downregulation of epidermal growth factor receptor, consistent with the biopsy analysis. Recent investigation into calcineurin inhibitor toxicity effects on renal tubular epithelial cells has demonstrated an altered expression of 38 proteins after incubation with cyclosporine in vitro.54 These include proteins involved in protein metabolism, as well as in response to cell damage, cell organization and cytoskeleton, energy metabolism, cell cycle, and nucleotide metabolism, and will provide a potential opportunity to non-invasively measure the toxic effects of these agents beyond allograft biopsy. In clinical settings, drug-related tubular injury may also be identified by measuring urine N-acetyl-b-D-gluosaminidase relative to total urine protein.55 Distinguishing drug-related injury before clinical disease would be a useful adjunct of immune monitoring tools beyond simply following trough drug levels. Urine mRNA
Suthanthiran and colleagues56 have advocated the use of mRNA isolation and analysis from urine specimens to noninvasively diagnose ongoing allograft pathology. Again, most of this work has focused on acute cellular rejection rather than later developing processes in the course of allograft failure.19 However, on the basis of microarray analysis of a limited number of allograft biopsies already discussed above,35 Mas et al.57 recently demonstrated, albeit in a small sample number, differential expression of angiotensin, epidermal growth factor receptor, and TGFb mRNA in urine samples from recipients with IF/TA compared with those with stable function. The detection of interferon producing protein-10 and CXCR3 was also associated with acute inflammation allograft biopsies.58 This type of approach may be a useful adjunct to monitoring ongoing inflammation that may contribute to late allograft injury. Further investigations using this technique should be considered and focused targets may be developed from genomics study discussed below. ALLOGRAFT BIOPSY: INVASIVE BUT INFORMATIVE Genomics
At present, allograft biopsy is the best technique to identify the cause of renal dysfunction, although there are circumstances in which it may provide limited information regarding the clinical impact and prognosis of a process, such as the presence of inflammatory cell infiltrates in biopsy samples unassociated with tubulitis, distinguishing BK polyomavirus disease from acute cellular rejection, identifying functionally significant rejection from SCR, and distinguishing the etiologies of IF/TA. The histological Kidney International (2010) 78 (Suppl 119), S59–S65
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classification of antibody-mediated injuries has also been recently revised to accept the notion that a number of different histological lesions, in the proper context, indicate alloantibody participation,5 although the pathobiology of this injury is not well defined. With the focus on the human genome, there has been an explosion of transcriptional information that is being applied to solid organ transplantation, again with the potential to identify markers and targets for therapeutic management. Microarray analysis allows for the interrogation of thousands of genes simultaneously, and is an increasingly robust and reproducible technique. The focus of these studies has been the identification of acute cellular rejection, identifying the molecular heterogeneity of allograft rejection, with differences detected by transcription that are not evident by light microscopy alone. Using an approach incorporating pathogenesis-based transcript sets identified in mouse kidney biopsy samples and correlated to response of human immune cells,59–62 Mueller et al.63demonstrated that these transcript sets correlate with biopsy diagnosis but have limited ability in the context of distinguishing so-called borderline rejection and acute cellular rejection, identifying some of the limitations of Banff ’s classification. In recipients with transplant glomerulopathy, the presence of endothelial activation and stress transcripts identifies a subset of recipients with worsened allograft outcome, even in the absence of C4d staining.13 These studies support the notion that histology has limited ability to explain a clinical situation and suggests an adjunct role of molecular analysis. Several groups have now incorporated the speed, technical ease, and quantitative capabilities of real-time PCR with the increasing availability of candidate transcripts, resulting in what may be the future—a rapid turnaround platform for analysis of fixed sets of 20–100 genes. These low-density arrays specifically limit the assessment of expression to a set of gene transcripts as opposed to a more global interrogation. Arrays may be customized or purchased commercially, depending on the study circumstances. Such expression data have demonstrated a number of unique findings in such clinical settings as ischemia–reperfusion injury,64,65 stable function graft function, acute rejection, and subclinical rejection.66 For example, use of low-density arrays has identified the development of allograft fibrosis and tubular injury in the setting of BK polyomavirus nephropathy.67 Transcripts associated with the induction of graft fibrosis and epithelial–mesenchymal transformation are markedly upregulated in BK polyomavirus nephropathy compared with biopsy samples with acute cellular rejection. Thus, the inflammatory immune response to BK has functional differences seen at the level of transcription that are not appreciable using standard histological techniques. Such studies demonstrate that specific transcriptional patterns may be identified that could be produced into a strategy with clinically applicable turnaround similar to that for standard histology. As such, it is now feasible to supplement biopsy information with contemporaneously available transcriptional analysis on a routine basis. Kidney International (2010) 78 (Suppl 119), S59–S65
Surveillance histology
Allograft biopsy remains the gold standard to clarify the etiology of allograft dysfunction. Protocol or surveillance biopsies have been recently used as a monitoring tool, albeit invasive, to recognize whether there is ongoing immune injury. Although the use of these biopsies has been debated over the years, SCR, findings of cellular rejection in the absence of any functional impediment, has been detected anywhere from 4 to 51% of biopsies (reviewed by Nankivell and Chapman68). The coincident appearance of SCR with IF/ TA in 6-month posttransplant biopsy samples is similarly a poor marker of outcome.15 Indeed, when found in repeated protocol biopsies, SCR is associated with more rapid allograft failure.69–71 The impact has also been debated showing benefit in non-randomized studies.72,71 However, in a recent randomized study, no effect of treatment was seen,73 perhaps in part because of the relatively low frequency of the event or because intervention was so early after transplantation. Further investigation into this area at later time points might provide additional clues to its utility. SUMMARY
Long-term graft failure continues to plague kidney allografts, in spite of potent immunosuppressive therapies. Both immune-dependent and -independent factors continue to contribute to failure. A number of promising observations made in human kidney recipients suggest unique protein and genetic signatures that may identify biomarkers of injury, as well as potential targets of therapy. Some of these may be obtained through noninvasive methods and may thus be extremely useful in the clinical realm. As discussed, these markers are undergoing further identification and validation to document their applicability and usefulness. In spite of the complexity of the immune response to a kidney allograft, these studies continue to move us forward and provide progress to the dismal area of late graft injury. DISCLOSURE
The author declared no competing interests. ACKNOWLEDGMENTS
This work was supported in part by funding from the University of Alabama at Birmingham Dean’s IMPACT and Division of Nephrology funds, as well as by U01-AI58013 and U19-AI070119. I thank Paolo Cravedi for his thoughtful discussion of this paper and Wendy Bailey for administrative assistance. REFERENCES 1. 2.
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Copyright of Kidney International Supplement is the property of Nature Publishing Group and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use.