REVIEW URRENT C OPINION
Chronic allograft nephropathy or interstitial fibrosis and tubular atrophy: what is in a name? Mark Haas
Purpose of review Chronic allograft nephropathy has fallen into disfavor as a morphologic term to describe parenchymal scarring in the renal allograft, with a recommendation that this be replaced by the more descriptive term ‘interstitial fibrosis and tubular atrophy’. However, neither term addresses the underlying cause of the scarring. This review focuses on whether all interstitial fibrosis and tubular atrophy in the renal allograft has the same implications for long-term graft survival, and whether there are specific features of interstitial fibrosis and tubular atrophy that can be used to identify its underlying cause. Recent findings Results from a number of studies indicate that interstitial fibrosis and tubular atrophy, when associated with interstitial inflammation, is a strong predictor of graft loss, much more so than interstitial fibrosis and tubular atrophy alone. Most notably, findings from the multicenter Long-Term Deterioration of Kidney Allograft Function study, designed to identify the causes of late allograft dysfunction, showed interstitial inflammation in the areas of interstitial fibrosis and tubular atrophy (i-IF/TA) was predictive of reduced time to graft failure, even after adjustment for serum creatinine. In addition, the presence of i-IF/TA correlates with increased acute kidney injury gene transcripts. However, neither interstitial fibrosis and tubular atrophy nor i-IF/TA is associated with any specific cause of chronic graft injury. Summary Although (i-IF/TA), especially when widespread, is clearly associated with reduced renal allograft survival and molecular markers of active graft injury and repair, there is presently no reliable way, using either morphology alone, immunohistochemistry, or molecular techniques, to differentiate i-IF/TA (or interstitial fibrosis and tubular atrophy alone) resulting from different causes. Keywords chronic rejection, interstitial inflammation, kidney injury, renal transplantation
INTRODUCTION Despite improved immunosuppressive regimens that have significantly reduced the acute rejection rates during the first year after renal transplantation, there has been little or no improvement in the longterm graft outcomes resulting from the continuing problem of late graft loss [1,2,3 ,4]. The treatment of late graft dysfunction has been limited in large part because there are many potential causes of such dysfunction that, unlike the case with early graft injury, often cannot be fully distinguished on a renal allograft biopsy. The term ‘chronic allograft nephropathy’, or CAN, was introduced with the original Banff working classification for renal allograft disease (Banff ’93) and defined as mild (grade I), moderate (grade II), or severe (grade III) tubular atrophy and interstitial fibrosis, often seen in the presence of &&
mononuclear cell inflammation [5]. CAN was distinguished from chronic rejection, the latter being suggested by arterial intimal fibrosis of new onset and transplant glomerulopathy [5]. Still, in the 12 years that followed the publication of the original Banff classification, there were over 550 PubMed citations using the term ‘CAN’, creating the misconception that CAN is an allograft-specific disease, rather than just a term for parenchymal Department of Pathology and Laboratory Medicine, Cedars-Sinai Medical Center, Los Angeles, California, USA Correspondence to Mark Haas, MD, PhD, Department of Pathology and Laboratory Medicine, Cedars-Sinai Medical Center, 8700 Beverly Boulevard, Los Angeles, CA 90048, USA. Tel: +1 310 248 6695; fax: +1 310 423 5881; e-mail: [email protected] Curr Opin Nephrol Hypertens 2014, 23:245–250 DOI:10.1097/01.mnh.0000444811.26884.2d
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Renal immunology and pathology
KEY POINTS The use of the term ‘interstitial fibrosis and tubular atrophy’ is preferable to ‘CAN’, although neither term is indicative of specific underlying disease. Interstitial inflammation in the areas of i-IF/TA represents a lesion that is clearly more deleterious to the graft than is bland interstitial fibrosis and tubular atrophy. At present, there is no reliable way, using either immunohistochemical or molecular techniques, to differentiate interstitial fibrosis and tubular atrophy (with or without associated inflammation) resulting from different causes. One must rely on the clinical history, serologic markers, and established morphologic findings to most accurately determine the cause of interstitial fibrosis and tubular atrophy.
scarring resulting from one or more different causes, some of which may be seen in native kidneys as well as allografts [6]. Furthermore, the use of CAN as a quasi-specific diagnosis may inhibit efforts to identify specific causes of graft function that may still be amenable to treatment. Finally, it has been shown that a primary or secondary diagnosis of CAN on renal allograft biopsies taken for new onset of late dysfunction does not correlate with graft survival [7]. It is for these reasons that the Banff group, at their 2005 biennial conference, recommended discontinuation of the term ‘CAN’, and replacement by the more descriptive (but morphologically equivalent, according to the original Banff schema) term tubular atrophy and interstitial fibrosis [5]. It should be recognized, however, that interstitial fibrosis and tubular atrophy is no more or less disease-specific than CAN, only less apt to be misinterpreted by some clinicians.
DOES ALL INTERSTITIAL FIBROSIS AND TUBULAR ATROPHY HAVE THE SAME PROGNOSTIC SIGNIFICANCE? Clearly, some morphologic lesions seen on renal allograft biopsies performed for late dysfunction are strongly associated with, although not entirely specific for, individual causes of such dysfunction. Examples include the association of transplant glomerulopathy [8–10] and of chronic arteriopathy not present at the time of transplantation [10,11] with chronic antibody-mediated rejection (AMR), although there are multiple other such examples that are beyond the scope of this article. 246
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However, beyond that associated with a specific diagnosis, two important questions remain regarding interstitial fibrosis and tubular atrophy. First, are qualitative differences in the types of interstitial fibrosis and tubular atrophy, such as that with and without associated interstitial inflammation (Fig. 1), associated with differences in prognosis for long-term graft function? Second, is it possible by examining interstitial fibrosis and tubular atrophy using morphology alone, or in combination with immunohistochemical and molecular methods, to accurately discern the underlying cause of the scarring? The widely used Banff criteria for the diagnosis of acute cell-mediated rejection do not consider interstitial inflammation in scarred areas and tubulitis involving atrophic tubules [12]. However, when Mengel et al. [13] compared the predictive value for graft survival of the Banff i score, which grades the extent of interstitial inflammation in nonscarred areas of the renal cortex, with that of a total-i (or ti) score grading inflammation in both scarred and nonscarred cortex with a similar semi-quantitative scale as the i score, they found that the ti score was a far better predictor of graft survival in a series of 104 renal allografts biopsied for cause. Furthermore, when only biopsies with at least mild interstitial fibrosis and tubular atrophy were considered, the i score was not correlated with graft survival, whereas the ti score was [13]. Finally, the ti score, but not the i score, correlated significantly with the level of expression of a set of cytotoxic T-cell-associated transcripts [13]. The above findings were confirmed and extended by the multicenter Long-Term Deterioration of Kidney Allograft Function (DeKAF) study [14]. Mannon et al. [14] examined the effects of both interstitial inflammation and tubulitis in the areas of interstitial fibrosis and tubular atrophy in 337 renal allograft biopsies performed for new onset of late (median 5.7 years after transplantation) graft dysfunction. These investigators found a strong association between the extent of i-IF/TA (termed ‘iatr’ in this study) and death-censored graft survival (Fig. 2). This effect remained significant when adjusted for tubular atrophy (Banff ct score), interstitial fibrosis (i score), serum creatinine (SCr), C4d, and donor-specific antibodies (DSAs). Tubulitis in the areas of interstitial fibrosis and tubular atrophy was also significantly associated with reduced graft survival, although less strongly than i-IF/TA [14]. Molecular studies of 171 renal allograft biopsies with a wide variety of lesions performed for late (>1 year after transplantation) graft dysfunction showed that high levels of expression of tissue injury Volume 23 Number 3 May 2014
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Fibrosis in the renal allograft Haas
(a)
(b)
FIGURE 1. (Panel a) Bland interstitial fibrosis and tubular atrophy (IF and TA) and (panel b) IF and TA associated with inflammation (i-IF/TA). The interstitial inflammation is composed mainly of lymphocytes with some admixed plasma cells, as is quite typical for i-IF/TA. Periodic acid-Schiff (PAS) stain, original magnification 200 (both panels).
and repair response-associated transcripts (IRRATs) independently predicted graft loss in a multivariate analysis, including estimated glomerular filtration rate and proteinuria at the time of biopsy and renal scarring (Banff ci score) [15 ]. In this same study, among 188 grafts with biopsies showing no rejection (even borderline), 31 were lost; 22 of these had IRRAT gene expression in the top tertile and 20 of the latter also had i-IF/TA. Finally, in these latter 188 grafts, i-IF/TA, but not interstitial fibrosis and tubular atrophy alone, was associated with a mean &&
IRRAT signal that was significantly higher than that in biopsies without interstitial fibrosis and tubular atrophy [15 ]. These data confirm the morphologic findings [13,14] that i-IF/TA is associated with active parenchymal injury. However, it was noted that multiple disease processes, including those related to rejection, infection, and others that could not be specifically identified morphologically, all were capable of triggering the injury–repair response (IRRAT) signal that was associated with an increased risk of graft loss [15 ]. &&
&&
100 90
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80 70 60 50 40 30
Iatr = 0 Iatr = 1 Iatr = 2 Iatr = 3
20 10
Logrank = 30.39
p = 0.0000
0 0
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FIGURE 2. Death-censored renal allograft survival as a function of increasing severity of i-IF/TA (iatr), semiquantitatively scored as absent (0), mild (1), moderate (2), and severe (3). i-IF/TA, inflammation in the areas of interstitial fibrosis and tubular atrophy. Data from Mannon et al. [14], with permission from John Wiley and Sons. 1062-4821 ß 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins
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CAN INTERSTITIAL FIBROSIS AND TUBULAR ATROPHY OF DIFFERENT CAUSES BE DISTINGUISHED BY MORPHOLOGIC OR MOLECULAR METHODS? The earliest attempts to associate histologic and immunohistologic patterns of interstitial fibrosis and tubular atrophy with a specific cause involved the diagnosis of chronic calcineurin inhibitor (CNI), particularly cyclosporine (CSA) nephrotoxicity. A ‘striped’ pattern of interstitial fibrosis and tubular atrophy in the renal cortex was observed in the native kidneys of human recipients of cardiac allografts treated with CSA and in an animal model of CSA nephrotoxity [16–18], suggesting this lesion to be relatively specific for CSA nephrotoxicity as opposed to chronic rejection. The cause of this pattern of interstitial fibrosis and tubular atrophy is not clear, although animal studies indicate that it may occur independently from structurally apparent arteriolopathy [19,20]. Localized hypoxia resulting from vasoconstriction induced by CSA (or tacrolimus) may lead to the formation of free radicals and reactive oxygen species that cause tubular cell injury [21–23]. In animal models, the interstitial fibrosis and tubular atrophy is preceded by an influx of macrophages and increased TGF-beta expression [18,20]. In a study of 120 diabetic recipients of renal allografts (kidney–pancreas transplants in all but one) who underwent protocol biopsies at regular intervals for 10 years after transplantation, Nankivell et al. [24] found that 90 and 100% of the 5-year and 10-year biopsies, respectively, showed arteriolar hyalinosis, and 68 and 87%, respectively, showed striped fibrosis. This led the authors to conclude that chronic CNI nephrotoxicity was almost universal by 10 years after transplantation and is a major cause of late renal allograft dysfunction. However, data from the DeKAF study [7] found evidence of CNI toxicity in only 30% of renal allograft biopsies taken a median of 5.7 years (mean 7.5 years) after transplantation. Furthermore, in a retrospective study comparing 10-year protocol biopsy results in patients who did and did not receive a CNI (CSA), it was found that although arteriolar and tubulointerstitial lesions commonly attributed to CNI nephrotoxicity were seen significantly more often (and with greater severity) in CNItreated patients, these lesions were not sufficiently specific to diagnose CNI nephrotoxicity [25]. In a recent review, Naesens et al. [23] list potential alternative causes of striped interstitial fibrosis and tubular atrophy to include ischemia–reperfusion injury, chronic bacterial and viral infections, chronic obstructive nephropathy, and chronic 248
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ischemia because of renal arterial lesions and size discrepancy in pediatric renal transplantation. Abrass et al. [26] used an immunohistologic approach to differentiate interstitial fibrosis related to rejection from that related to CNI nephrotoxicity. They found that whereas rejection led to new expression of the alpha-3 chain of type IV collagen (COL4A3) and laminin beta-2 along cortical tubular basement membranes, CSA nephrotoxicity was associated with generalized interstitial staining for collagen types I and III. However, in a later study [27] analyzing renal cortical collagen types I and III, COL4A3, and laminin beta-2 mRNA levels in biopsies showing chronic rejection and chronic CSA toxicity, no significant differences were seen between the latter two groups, although the levels of COL4A3 and laminin beta-2 mRNAs in both groups were higher than those in 6-month protocol biopsies from grafts with stable function. The findings for laminin beta-2 were also confirmed by immunohistochemistry [27]. Anglicheau et al. [28 ] reported that a model composed of levels of urinary cell mRNAs for vimentin, the Na–K–Cl cotransporter NKCC2, E-cadherin, and 18S ribosomal RNA predicted renal allograft fibrosis with a sensitivity of 77–94% and a specificity of 84–88%. The mean composite score was significantly higher in urine samples from patients with interstitial fibrosis and tubular atrophy with interstitial inflammation compared with samples from patients with interstitial fibrosis and tubular atrophy without inflammation; however, the model did not differentiate between mild, moderate, or severe fibrosis [28 ]. The authors did not test whether composite scores varied with different causes of interstitial fibrosis and tubular atrophy, and excluded biopsies with evidence of active rejection, BK virus nephropathy, and CNI nephrotoxicity from the control (no interstitial fibrosis and tubular atrophy) groups. Other potential noninvasive diagnostic markers for interstitial fibrosis and tubular atrophy within renal allograft biopsies are metzincins, including matrix metalloproteases (MMPs), and metzincinrelated proteins that are involved in remodeling of extracellular matrix [29]. Rodder et al. [29] found from microarray analysis of renal allograft biopsies that gene sets for both metzincins and metzincinrelated proteins showed progressively increased expression with increasing severity of interstitial fibrosis and tubular atrophy. Furthermore, serum levels of MMP-2 and MMP-7 were significantly higher in patients with renal allografts showing interstitial fibrosis and tubular atrophy than in those having normal allograft function and biopsies without interstitial fibrosis and tubular atrophy. &
&
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Fibrosis in the renal allograft Haas
However, it was not examined whether expression of these proteins differed with different causes of interstitial fibrosis and tubular atrophy. A number of investigators have examined microRNA (miRNA) profiles of urine and renal biopsy tissue from renal allograft recipients with and without interstitial fibrosis and tubular atrophy. Scian et al. [30] found 56 miRNAs that were differentially expressed in renal allograft biopsies with or without interstitial fibrosis and tubular atrophy. They further examined a set of five miRNAs with a large degree of differential expression in these two groups of biopsies in a second (validation) set of patients with and without interstitial fibrosis and tubular atrophy. In addition to confirming differential expression of these miRNAs in biopsies with and without interstitial fibrosis and tubular atrophy, three of the miRNAs were differentially expressed in urine samples from patients with and without interstitial fibrosis and tubular atrophy in their allograft biopsies [29]. Ben Dov et al. [31 ] identified several miRNAs, miRNA clusters, and miRNA sequence families having two-fold to ten-fold higher expression in biopsies with interstitial fibrosis and tubular atrophy compared with histologically unremarkable protocol biopsies. These miRNAs were initially identified in a discovery set of 4 biopsies with interstitial fibrosis and tubular atrophy and 4 with normal histology, and validated on a second set of 18 biopsies (10 interstitial fibrosis and tubular atrophy, and 8 controls). However, expression of these miRNAs was not different in biopsies showing interstitial fibrosis and tubular atrophy with and without chronic AMR, although a separate miRNA that was not significantly overexpressed in interstitial fibrosis and tubular atrophy had a two-fold higher expression in interstitial fibrosis and tubular atrophy alone versus interstitial fibrosis and tubular atrophy with chronic AMR [30]. It thus remains to be determined whether molecular analysis of renal allograft biopsies or urine will prove to be of use in differentiating interstitial fibrosis and tubular atrophy of different causes. &
CONCLUSION Use of the term ‘chronic allograft nephropathy’ (CAN) is best avoided, mainly because it implies a type of allograft-specific injury, when in fact this is not (and has never been) the case. Interstitial fibrosis and tubular atrophy better portrays the nonspecific nature of this lesion, although this term provides no information regarding the mechanism and in itself is not a useful diagnosis. i-IF/TA, although not contributory toward a diagnosis of acute cell-mediated rejection by the current Banff criteria, represents a lesion that is clearly more deleterious to the graft
than bland interstitial fibrosis and tubular atrophy (without associated inflammation). Compared with interstitial fibrosis and tubular atrophy alone, i-IF/ TA is associated with decreased graft survival, especially when widespread, and with greater expression of molecular markers of active tissue injury and repair. However, as is the case in interstitial fibrosis and tubular atrophy alone, i-IF/TA is not specific for any given cause (e.g., chronic cell-mediated or antibody-mediated rejection, CNI nephrotoxicity, BK virus nephropathy, glomerulonephritis, etc.). At present, there is no reliable way, using either immunohistochemistry or molecular techniques, to differentiate interstitial fibrosis and tubular atrophy (with or without associated inflammation) resulting from different causes. One must rely on the clinical history, serologic markers (e.g., DSA), and established morphologic findings (e.g., transplant glomerulopathy, vascular lesions, viral inclusions, C4d, immunofluorescence and electron microscopic studies of glomeruli, etc.) to most accurately establish the cause of the interstitial fibrosis and tubular atrophy. Just as in native kidney diseases, interstitial fibrosis and tubular atrophy, particularly when moderate to severe, appears to potentiate the rate of graft loss resulting from a number of types of graft injury, including cell-mediated and antibodymediated rejection, BK virus nephropathy, and recurrent or de novo glomerular diseases. The lack of a significant effect of interstitial fibrosis and tubular atrophy on graft loss from multivariate analysis in some studies that include molecular parameters is likely related to overlap between interstitial fibrosis and tubular atrophy and one or more of the molecular signals also included in the analysis. Acknowledgements None. Conflicts of interest There are no conflicts of interest.
REFERENCES AND RECOMMENDED READING Papers of particular interest, published within the annual period of review, have been highlighted as: & of special interest && of outstanding interest 1. Hariharan S, Johnson CP, Bresnahan BA, et al. Improved graft survival after renal transplantation in the United States. N Engl J Med 2000; 342:605–612. 2. Pascual M, Theruvath T, Kawai T, et al. Strategies to improve long-term outcomes after renal transplantation. N Engl J Med 2002; 346:580–590. 3. Sellares J, de Freitas DG, Mengel M, et al. Understanding the causes of kidney && transplant failure: the dominant role of antibody-mediated rejection and nonadherence. Am J Transplant 2012; 12:388–399. A study of 315 renal allografts, 60 of which failed. The two most common individual causes of graft failure were chronic or chronic and active rejection (64%; mostly antibody mediated) and recurrent or de novo glomerulonephritis (18%). Nearly half of graft losses resulting from rejection were associated with nonadherence with immunosuppressive medications.
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Renal immunology and pathology 4. Lamb KE, Lodhi S, Meier-Kriesche H-U. Long-term renal allograft survival in the United States: a critical reappraisal. Am J Transplant 2011; 11:450–462. 5. Solez K, Axelsen RA, Benediktsson H, et al. International standardization of criteria for the histologic diagnosis of renal allograft rejection: The Banff working classification of kidney transplant pathology. Kidney Int 1993; 44:411–422. 6. Solez K, Colvin RB, Racusen LC, et al. Banff ’05 meeting report: differential diagnosis of chronic allograft injury and elimination of chronic allograft nephropathy (‘CAN’). Am J Transplant 2007; 7:518–526. 7. Gourishankar S, Leduc R, Connett J, et al. Pathological and clinical characterization of the ‘troubled transplant’: data from the DeKAF study. Am J Transplant 2010; 10:324–330. 8. Gloor JM, Sethi S, Stegall MD, et al. Transplant glomerulopathy: subclinical incidence and association with alloantibody. Am J Transplant 2007; 7:2124– 2132. 9. Sis B, Campbell PM, Muller T, et al. Transplant glomerulopathy, late antibodymediated rejection and the ABCD tetrad in kidney allograft biopsies for cause. Am J Transplant 2007; 7:1743–1752. 10. Mauiyyedi S, Pelle PD, Saidman S, et al. Chronic humoral rejection: identification of antibody-mediated chronic allograft rejection by C4d deposits in peritubular capillaries. J Am Soc Nephrol 2001; 12:574–582. 11. Hill GS, Nochy D, Bruneval P, et al. Donor-specific antibodies accelerate arteriosclerosis after kidney transplantation. J Am Soc Nephrol 2011; 22:975–983. 12. Racusen LC, Solez K, Colvin RB, et al. The Banff 97 working classification of renal allograft pathology. Kidney Int 1999; 55:713–723. 13. Mengel M, Reeve J, Bunnag S, et al. Scoring total inflammation is superior to the current Banff inflammation score in predicting outcome and the degree of molecular disturbance in renal allografts. Am J Transplant 2009; 9:1859–1867. 14. Mannon RB, Matas AJ, Grande J, et al. Inflammation in areas of tubular atrophy in kidney allograft biopsies: a potent predictor of allograft failure. Am J Transplant 2010; 10:2066–2073. 15. Famulski KS, Reeve J, de Freitas DG, et al. Kidney transplants with progres&& sing chronic disease express high levels of acute kidney injury transcripts. Am J Transplant 2013; 13:634–644. A study of expression of genes associated with tissue injury and repair (IRRATs) in a large series of indication renal allograft biopsies, including biopsies showing Tcell-mediated and antibody-mediated rejection and a number of nonrejection lesions. IRRAT gene expression was a strong predictor of future graft loss, and in biopsies without rejection were strongly correlated with inflammation in the areas of fibrosis. 16. Nizze H, Mihatsch MJ, Zollinger HU, et al. Cyclosporine-associated nephropathy in patients with heart and bone marrow transplants. Clin Nephrol 1988; 30:248–260. 17. Myers BD, Newton L. Cyclosporine-induced chronic nephropathy: an obliterative microvascular renal injury. J Am Soc Nephrol 1991; 2:S45–S52. 18. Young BA, Burdmann EA, Johnson RJ, et al. Cellular proliferation and macrophage influx precede interstitial fibrosis in cyclosporine nephrotoxicity. Kidney Int 1985; 48:439–448.
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19. Pichler RH, Franceschini N, Young BA, et al. Pathogenesis of cyclosporine nephropathy: roles of angiotensin II and osteopontin. J Am Soc Nephrol 1995; 6:1186–1196. 20. Viera JM Jr, Noronha IL, Malheiros DM, et al. Cyclosporine-induced interstitial fibrosis and arteriolar TGF-beta expression with preserved renal blood flow. Transplantation 1999; 68:1746–1753. 21. Zhong Z, Arteel GE, Connor HD, et al. Cyclosporine A increases hypoxia and free radical production in rat kidneys: prevention by dietary glycine. Am J Physiol 1998; 275:F595–F604. 22. Jennings P, Koppelstaetter C, Aydin S, et al. Cyclosporine A induces senescence in renal tubular epithelial cells. Am J Physiol 2007; 293:F831–F838. 23. Naesens M, Kuypers DRJ, Sarwal M. Calcineurin inhibitor nephrotoxicity. Clin J Am Soc Nephrol 2009; 4:481–508. 24. Nankivell BJ, Burrows RJ, Fung CL-S, et al. The natural history of chronic allograft nephropathy. N Engl J Med 2003; 349:2326–2333. 25. Snanoudj R, Royal V, Elie C, et al. Specificity of histological markers of longterm CNI nephrotoxicity in kidney-transplant recipients under low-dose cyclosporine therapy. Am J Transplant 2011; 11:2635–2646. 26. Abrass CK, Berfield AK, Stehman-Breen C, et al. Unique changes in interstitial extracellular matrix composition are associated with rejection and cyclosporine toxicity in human renal allograft biopsies. Am J Kidney Dis 1999; 33:11– 20. 27. Koop K, Bakker RC, Eikmans M, et al. Differentiation between chronic rejection and chronic cyclosporine toxicity by analysis of renal cortical mRNA. Kidney Int 2004; 66:2038–2046. 28. Anglicheau D, Muthukumar T, Hummel A, et al. Discovery and validation of a & molecular signature for the noninvasive diagnosis of human renal allograft fibrosis. Transplantation 2012; 93:1136–1146. This study analyzed a number of mRNAs in urinary cells and found several to be significantly associated with the presence of interstitial fibrosis in corresponding renal allograft biopsies. A model composed of levels of urinary cell mRNAs for vimentin, the Na–K–Cl cotransporter NKCC2, E-cadherin, and 18S ribosomal RNA predicted renal allograft fibrosis with a sensitivity of 77–94% and a specificity of 84–88%, although this model could not distinguish between different grades of fibrosis. 29. 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. 30. Scian MJ, Maluf DJ, David KG, et al. MicroRNA profiles in allograft tissues and paired urines associated with chronic allograft dysfunction with IF/TA. Am J Transplant 2011; 11:2110–2122. 31. Ben-Dov IZ, Muthukumar T, Morozov P, et al. MicroRNA sequence profiles of & human kidney allografts with or without tubulointerstitial fibrosis. Transplantation 2012; 94:1086–1094. This study identified several microRNAs, microRNA clusters, and microRNA sequence families having two-fold to ten-fold higher expression in biopsies with interstitial fibrosis and tubular atrophy compared with histologically unremarkable protocol biopsies, and validated these differences on a second set of biopsies.
Volume 23 Number 3 May 2014
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Chronic allograft nephropathy or interstitial fibrosis and tubular atrophy: what is in a name? Mark Haas
Purpose of review Chronic allograft nephropathy has fallen into disfavor as a morphologic term to describe parenchymal scarring in the renal allograft, with a recommendation that this be replaced by the more descriptive term ‘interstitial fibrosis and tubular atrophy’. However, neither term addresses the underlying cause of the scarring. This review focuses on whether all interstitial fibrosis and tubular atrophy in the renal allograft has the same implications for long-term graft survival, and whether there are specific features of interstitial fibrosis and tubular atrophy that can be used to identify its underlying cause. Recent findings Results from a number of studies indicate that interstitial fibrosis and tubular atrophy, when associated with interstitial inflammation, is a strong predictor of graft loss, much more so than interstitial fibrosis and tubular atrophy alone. Most notably, findings from the multicenter Long-Term Deterioration of Kidney Allograft Function study, designed to identify the causes of late allograft dysfunction, showed interstitial inflammation in the areas of interstitial fibrosis and tubular atrophy (i-IF/TA) was predictive of reduced time to graft failure, even after adjustment for serum creatinine. In addition, the presence of i-IF/TA correlates with increased acute kidney injury gene transcripts. However, neither interstitial fibrosis and tubular atrophy nor i-IF/TA is associated with any specific cause of chronic graft injury. Summary Although (i-IF/TA), especially when widespread, is clearly associated with reduced renal allograft survival and molecular markers of active graft injury and repair, there is presently no reliable way, using either morphology alone, immunohistochemistry, or molecular techniques, to differentiate i-IF/TA (or interstitial fibrosis and tubular atrophy alone) resulting from different causes. Keywords chronic rejection, interstitial inflammation, kidney injury, renal transplantation
INTRODUCTION Despite improved immunosuppressive regimens that have significantly reduced the acute rejection rates during the first year after renal transplantation, there has been little or no improvement in the longterm graft outcomes resulting from the continuing problem of late graft loss [1,2,3 ,4]. The treatment of late graft dysfunction has been limited in large part because there are many potential causes of such dysfunction that, unlike the case with early graft injury, often cannot be fully distinguished on a renal allograft biopsy. The term ‘chronic allograft nephropathy’, or CAN, was introduced with the original Banff working classification for renal allograft disease (Banff ’93) and defined as mild (grade I), moderate (grade II), or severe (grade III) tubular atrophy and interstitial fibrosis, often seen in the presence of &&
mononuclear cell inflammation [5]. CAN was distinguished from chronic rejection, the latter being suggested by arterial intimal fibrosis of new onset and transplant glomerulopathy [5]. Still, in the 12 years that followed the publication of the original Banff classification, there were over 550 PubMed citations using the term ‘CAN’, creating the misconception that CAN is an allograft-specific disease, rather than just a term for parenchymal Department of Pathology and Laboratory Medicine, Cedars-Sinai Medical Center, Los Angeles, California, USA Correspondence to Mark Haas, MD, PhD, Department of Pathology and Laboratory Medicine, Cedars-Sinai Medical Center, 8700 Beverly Boulevard, Los Angeles, CA 90048, USA. Tel: +1 310 248 6695; fax: +1 310 423 5881; e-mail: [email protected] Curr Opin Nephrol Hypertens 2014, 23:245–250 DOI:10.1097/01.mnh.0000444811.26884.2d
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Renal immunology and pathology
KEY POINTS The use of the term ‘interstitial fibrosis and tubular atrophy’ is preferable to ‘CAN’, although neither term is indicative of specific underlying disease. Interstitial inflammation in the areas of i-IF/TA represents a lesion that is clearly more deleterious to the graft than is bland interstitial fibrosis and tubular atrophy. At present, there is no reliable way, using either immunohistochemical or molecular techniques, to differentiate interstitial fibrosis and tubular atrophy (with or without associated inflammation) resulting from different causes. One must rely on the clinical history, serologic markers, and established morphologic findings to most accurately determine the cause of interstitial fibrosis and tubular atrophy.
scarring resulting from one or more different causes, some of which may be seen in native kidneys as well as allografts [6]. Furthermore, the use of CAN as a quasi-specific diagnosis may inhibit efforts to identify specific causes of graft function that may still be amenable to treatment. Finally, it has been shown that a primary or secondary diagnosis of CAN on renal allograft biopsies taken for new onset of late dysfunction does not correlate with graft survival [7]. It is for these reasons that the Banff group, at their 2005 biennial conference, recommended discontinuation of the term ‘CAN’, and replacement by the more descriptive (but morphologically equivalent, according to the original Banff schema) term tubular atrophy and interstitial fibrosis [5]. It should be recognized, however, that interstitial fibrosis and tubular atrophy is no more or less disease-specific than CAN, only less apt to be misinterpreted by some clinicians.
DOES ALL INTERSTITIAL FIBROSIS AND TUBULAR ATROPHY HAVE THE SAME PROGNOSTIC SIGNIFICANCE? Clearly, some morphologic lesions seen on renal allograft biopsies performed for late dysfunction are strongly associated with, although not entirely specific for, individual causes of such dysfunction. Examples include the association of transplant glomerulopathy [8–10] and of chronic arteriopathy not present at the time of transplantation [10,11] with chronic antibody-mediated rejection (AMR), although there are multiple other such examples that are beyond the scope of this article. 246
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However, beyond that associated with a specific diagnosis, two important questions remain regarding interstitial fibrosis and tubular atrophy. First, are qualitative differences in the types of interstitial fibrosis and tubular atrophy, such as that with and without associated interstitial inflammation (Fig. 1), associated with differences in prognosis for long-term graft function? Second, is it possible by examining interstitial fibrosis and tubular atrophy using morphology alone, or in combination with immunohistochemical and molecular methods, to accurately discern the underlying cause of the scarring? The widely used Banff criteria for the diagnosis of acute cell-mediated rejection do not consider interstitial inflammation in scarred areas and tubulitis involving atrophic tubules [12]. However, when Mengel et al. [13] compared the predictive value for graft survival of the Banff i score, which grades the extent of interstitial inflammation in nonscarred areas of the renal cortex, with that of a total-i (or ti) score grading inflammation in both scarred and nonscarred cortex with a similar semi-quantitative scale as the i score, they found that the ti score was a far better predictor of graft survival in a series of 104 renal allografts biopsied for cause. Furthermore, when only biopsies with at least mild interstitial fibrosis and tubular atrophy were considered, the i score was not correlated with graft survival, whereas the ti score was [13]. Finally, the ti score, but not the i score, correlated significantly with the level of expression of a set of cytotoxic T-cell-associated transcripts [13]. The above findings were confirmed and extended by the multicenter Long-Term Deterioration of Kidney Allograft Function (DeKAF) study [14]. Mannon et al. [14] examined the effects of both interstitial inflammation and tubulitis in the areas of interstitial fibrosis and tubular atrophy in 337 renal allograft biopsies performed for new onset of late (median 5.7 years after transplantation) graft dysfunction. These investigators found a strong association between the extent of i-IF/TA (termed ‘iatr’ in this study) and death-censored graft survival (Fig. 2). This effect remained significant when adjusted for tubular atrophy (Banff ct score), interstitial fibrosis (i score), serum creatinine (SCr), C4d, and donor-specific antibodies (DSAs). Tubulitis in the areas of interstitial fibrosis and tubular atrophy was also significantly associated with reduced graft survival, although less strongly than i-IF/TA [14]. Molecular studies of 171 renal allograft biopsies with a wide variety of lesions performed for late (>1 year after transplantation) graft dysfunction showed that high levels of expression of tissue injury Volume 23 Number 3 May 2014
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Fibrosis in the renal allograft Haas
(a)
(b)
FIGURE 1. (Panel a) Bland interstitial fibrosis and tubular atrophy (IF and TA) and (panel b) IF and TA associated with inflammation (i-IF/TA). The interstitial inflammation is composed mainly of lymphocytes with some admixed plasma cells, as is quite typical for i-IF/TA. Periodic acid-Schiff (PAS) stain, original magnification 200 (both panels).
and repair response-associated transcripts (IRRATs) independently predicted graft loss in a multivariate analysis, including estimated glomerular filtration rate and proteinuria at the time of biopsy and renal scarring (Banff ci score) [15 ]. In this same study, among 188 grafts with biopsies showing no rejection (even borderline), 31 were lost; 22 of these had IRRAT gene expression in the top tertile and 20 of the latter also had i-IF/TA. Finally, in these latter 188 grafts, i-IF/TA, but not interstitial fibrosis and tubular atrophy alone, was associated with a mean &&
IRRAT signal that was significantly higher than that in biopsies without interstitial fibrosis and tubular atrophy [15 ]. These data confirm the morphologic findings [13,14] that i-IF/TA is associated with active parenchymal injury. However, it was noted that multiple disease processes, including those related to rejection, infection, and others that could not be specifically identified morphologically, all were capable of triggering the injury–repair response (IRRAT) signal that was associated with an increased risk of graft loss [15 ]. &&
&&
100 90
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80 70 60 50 40 30
Iatr = 0 Iatr = 1 Iatr = 2 Iatr = 3
20 10
Logrank = 30.39
p = 0.0000
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FIGURE 2. Death-censored renal allograft survival as a function of increasing severity of i-IF/TA (iatr), semiquantitatively scored as absent (0), mild (1), moderate (2), and severe (3). i-IF/TA, inflammation in the areas of interstitial fibrosis and tubular atrophy. Data from Mannon et al. [14], with permission from John Wiley and Sons. 1062-4821 ß 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins
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Renal immunology and pathology
CAN INTERSTITIAL FIBROSIS AND TUBULAR ATROPHY OF DIFFERENT CAUSES BE DISTINGUISHED BY MORPHOLOGIC OR MOLECULAR METHODS? The earliest attempts to associate histologic and immunohistologic patterns of interstitial fibrosis and tubular atrophy with a specific cause involved the diagnosis of chronic calcineurin inhibitor (CNI), particularly cyclosporine (CSA) nephrotoxicity. A ‘striped’ pattern of interstitial fibrosis and tubular atrophy in the renal cortex was observed in the native kidneys of human recipients of cardiac allografts treated with CSA and in an animal model of CSA nephrotoxity [16–18], suggesting this lesion to be relatively specific for CSA nephrotoxicity as opposed to chronic rejection. The cause of this pattern of interstitial fibrosis and tubular atrophy is not clear, although animal studies indicate that it may occur independently from structurally apparent arteriolopathy [19,20]. Localized hypoxia resulting from vasoconstriction induced by CSA (or tacrolimus) may lead to the formation of free radicals and reactive oxygen species that cause tubular cell injury [21–23]. In animal models, the interstitial fibrosis and tubular atrophy is preceded by an influx of macrophages and increased TGF-beta expression [18,20]. In a study of 120 diabetic recipients of renal allografts (kidney–pancreas transplants in all but one) who underwent protocol biopsies at regular intervals for 10 years after transplantation, Nankivell et al. [24] found that 90 and 100% of the 5-year and 10-year biopsies, respectively, showed arteriolar hyalinosis, and 68 and 87%, respectively, showed striped fibrosis. This led the authors to conclude that chronic CNI nephrotoxicity was almost universal by 10 years after transplantation and is a major cause of late renal allograft dysfunction. However, data from the DeKAF study [7] found evidence of CNI toxicity in only 30% of renal allograft biopsies taken a median of 5.7 years (mean 7.5 years) after transplantation. Furthermore, in a retrospective study comparing 10-year protocol biopsy results in patients who did and did not receive a CNI (CSA), it was found that although arteriolar and tubulointerstitial lesions commonly attributed to CNI nephrotoxicity were seen significantly more often (and with greater severity) in CNItreated patients, these lesions were not sufficiently specific to diagnose CNI nephrotoxicity [25]. In a recent review, Naesens et al. [23] list potential alternative causes of striped interstitial fibrosis and tubular atrophy to include ischemia–reperfusion injury, chronic bacterial and viral infections, chronic obstructive nephropathy, and chronic 248
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ischemia because of renal arterial lesions and size discrepancy in pediatric renal transplantation. Abrass et al. [26] used an immunohistologic approach to differentiate interstitial fibrosis related to rejection from that related to CNI nephrotoxicity. They found that whereas rejection led to new expression of the alpha-3 chain of type IV collagen (COL4A3) and laminin beta-2 along cortical tubular basement membranes, CSA nephrotoxicity was associated with generalized interstitial staining for collagen types I and III. However, in a later study [27] analyzing renal cortical collagen types I and III, COL4A3, and laminin beta-2 mRNA levels in biopsies showing chronic rejection and chronic CSA toxicity, no significant differences were seen between the latter two groups, although the levels of COL4A3 and laminin beta-2 mRNAs in both groups were higher than those in 6-month protocol biopsies from grafts with stable function. The findings for laminin beta-2 were also confirmed by immunohistochemistry [27]. Anglicheau et al. [28 ] reported that a model composed of levels of urinary cell mRNAs for vimentin, the Na–K–Cl cotransporter NKCC2, E-cadherin, and 18S ribosomal RNA predicted renal allograft fibrosis with a sensitivity of 77–94% and a specificity of 84–88%. The mean composite score was significantly higher in urine samples from patients with interstitial fibrosis and tubular atrophy with interstitial inflammation compared with samples from patients with interstitial fibrosis and tubular atrophy without inflammation; however, the model did not differentiate between mild, moderate, or severe fibrosis [28 ]. The authors did not test whether composite scores varied with different causes of interstitial fibrosis and tubular atrophy, and excluded biopsies with evidence of active rejection, BK virus nephropathy, and CNI nephrotoxicity from the control (no interstitial fibrosis and tubular atrophy) groups. Other potential noninvasive diagnostic markers for interstitial fibrosis and tubular atrophy within renal allograft biopsies are metzincins, including matrix metalloproteases (MMPs), and metzincinrelated proteins that are involved in remodeling of extracellular matrix [29]. Rodder et al. [29] found from microarray analysis of renal allograft biopsies that gene sets for both metzincins and metzincinrelated proteins showed progressively increased expression with increasing severity of interstitial fibrosis and tubular atrophy. Furthermore, serum levels of MMP-2 and MMP-7 were significantly higher in patients with renal allografts showing interstitial fibrosis and tubular atrophy than in those having normal allograft function and biopsies without interstitial fibrosis and tubular atrophy. &
&
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Fibrosis in the renal allograft Haas
However, it was not examined whether expression of these proteins differed with different causes of interstitial fibrosis and tubular atrophy. A number of investigators have examined microRNA (miRNA) profiles of urine and renal biopsy tissue from renal allograft recipients with and without interstitial fibrosis and tubular atrophy. Scian et al. [30] found 56 miRNAs that were differentially expressed in renal allograft biopsies with or without interstitial fibrosis and tubular atrophy. They further examined a set of five miRNAs with a large degree of differential expression in these two groups of biopsies in a second (validation) set of patients with and without interstitial fibrosis and tubular atrophy. In addition to confirming differential expression of these miRNAs in biopsies with and without interstitial fibrosis and tubular atrophy, three of the miRNAs were differentially expressed in urine samples from patients with and without interstitial fibrosis and tubular atrophy in their allograft biopsies [29]. Ben Dov et al. [31 ] identified several miRNAs, miRNA clusters, and miRNA sequence families having two-fold to ten-fold higher expression in biopsies with interstitial fibrosis and tubular atrophy compared with histologically unremarkable protocol biopsies. These miRNAs were initially identified in a discovery set of 4 biopsies with interstitial fibrosis and tubular atrophy and 4 with normal histology, and validated on a second set of 18 biopsies (10 interstitial fibrosis and tubular atrophy, and 8 controls). However, expression of these miRNAs was not different in biopsies showing interstitial fibrosis and tubular atrophy with and without chronic AMR, although a separate miRNA that was not significantly overexpressed in interstitial fibrosis and tubular atrophy had a two-fold higher expression in interstitial fibrosis and tubular atrophy alone versus interstitial fibrosis and tubular atrophy with chronic AMR [30]. It thus remains to be determined whether molecular analysis of renal allograft biopsies or urine will prove to be of use in differentiating interstitial fibrosis and tubular atrophy of different causes. &
CONCLUSION Use of the term ‘chronic allograft nephropathy’ (CAN) is best avoided, mainly because it implies a type of allograft-specific injury, when in fact this is not (and has never been) the case. Interstitial fibrosis and tubular atrophy better portrays the nonspecific nature of this lesion, although this term provides no information regarding the mechanism and in itself is not a useful diagnosis. i-IF/TA, although not contributory toward a diagnosis of acute cell-mediated rejection by the current Banff criteria, represents a lesion that is clearly more deleterious to the graft
than bland interstitial fibrosis and tubular atrophy (without associated inflammation). Compared with interstitial fibrosis and tubular atrophy alone, i-IF/ TA is associated with decreased graft survival, especially when widespread, and with greater expression of molecular markers of active tissue injury and repair. However, as is the case in interstitial fibrosis and tubular atrophy alone, i-IF/TA is not specific for any given cause (e.g., chronic cell-mediated or antibody-mediated rejection, CNI nephrotoxicity, BK virus nephropathy, glomerulonephritis, etc.). At present, there is no reliable way, using either immunohistochemistry or molecular techniques, to differentiate interstitial fibrosis and tubular atrophy (with or without associated inflammation) resulting from different causes. One must rely on the clinical history, serologic markers (e.g., DSA), and established morphologic findings (e.g., transplant glomerulopathy, vascular lesions, viral inclusions, C4d, immunofluorescence and electron microscopic studies of glomeruli, etc.) to most accurately establish the cause of the interstitial fibrosis and tubular atrophy. Just as in native kidney diseases, interstitial fibrosis and tubular atrophy, particularly when moderate to severe, appears to potentiate the rate of graft loss resulting from a number of types of graft injury, including cell-mediated and antibodymediated rejection, BK virus nephropathy, and recurrent or de novo glomerular diseases. The lack of a significant effect of interstitial fibrosis and tubular atrophy on graft loss from multivariate analysis in some studies that include molecular parameters is likely related to overlap between interstitial fibrosis and tubular atrophy and one or more of the molecular signals also included in the analysis. Acknowledgements None. Conflicts of interest There are no conflicts of interest.
REFERENCES AND RECOMMENDED READING Papers of particular interest, published within the annual period of review, have been highlighted as: & of special interest && of outstanding interest 1. Hariharan S, Johnson CP, Bresnahan BA, et al. Improved graft survival after renal transplantation in the United States. N Engl J Med 2000; 342:605–612. 2. Pascual M, Theruvath T, Kawai T, et al. Strategies to improve long-term outcomes after renal transplantation. N Engl J Med 2002; 346:580–590. 3. Sellares J, de Freitas DG, Mengel M, et al. Understanding the causes of kidney && transplant failure: the dominant role of antibody-mediated rejection and nonadherence. Am J Transplant 2012; 12:388–399. A study of 315 renal allografts, 60 of which failed. The two most common individual causes of graft failure were chronic or chronic and active rejection (64%; mostly antibody mediated) and recurrent or de novo glomerulonephritis (18%). Nearly half of graft losses resulting from rejection were associated with nonadherence with immunosuppressive medications.
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Renal immunology and pathology 4. Lamb KE, Lodhi S, Meier-Kriesche H-U. Long-term renal allograft survival in the United States: a critical reappraisal. Am J Transplant 2011; 11:450–462. 5. Solez K, Axelsen RA, Benediktsson H, et al. International standardization of criteria for the histologic diagnosis of renal allograft rejection: The Banff working classification of kidney transplant pathology. Kidney Int 1993; 44:411–422. 6. Solez K, Colvin RB, Racusen LC, et al. Banff ’05 meeting report: differential diagnosis of chronic allograft injury and elimination of chronic allograft nephropathy (‘CAN’). Am J Transplant 2007; 7:518–526. 7. Gourishankar S, Leduc R, Connett J, et al. Pathological and clinical characterization of the ‘troubled transplant’: data from the DeKAF study. Am J Transplant 2010; 10:324–330. 8. Gloor JM, Sethi S, Stegall MD, et al. Transplant glomerulopathy: subclinical incidence and association with alloantibody. Am J Transplant 2007; 7:2124– 2132. 9. Sis B, Campbell PM, Muller T, et al. Transplant glomerulopathy, late antibodymediated rejection and the ABCD tetrad in kidney allograft biopsies for cause. Am J Transplant 2007; 7:1743–1752. 10. Mauiyyedi S, Pelle PD, Saidman S, et al. Chronic humoral rejection: identification of antibody-mediated chronic allograft rejection by C4d deposits in peritubular capillaries. J Am Soc Nephrol 2001; 12:574–582. 11. Hill GS, Nochy D, Bruneval P, et al. Donor-specific antibodies accelerate arteriosclerosis after kidney transplantation. J Am Soc Nephrol 2011; 22:975–983. 12. Racusen LC, Solez K, Colvin RB, et al. The Banff 97 working classification of renal allograft pathology. Kidney Int 1999; 55:713–723. 13. Mengel M, Reeve J, Bunnag S, et al. Scoring total inflammation is superior to the current Banff inflammation score in predicting outcome and the degree of molecular disturbance in renal allografts. Am J Transplant 2009; 9:1859–1867. 14. Mannon RB, Matas AJ, Grande J, et al. Inflammation in areas of tubular atrophy in kidney allograft biopsies: a potent predictor of allograft failure. Am J Transplant 2010; 10:2066–2073. 15. Famulski KS, Reeve J, de Freitas DG, et al. Kidney transplants with progres&& sing chronic disease express high levels of acute kidney injury transcripts. Am J Transplant 2013; 13:634–644. A study of expression of genes associated with tissue injury and repair (IRRATs) in a large series of indication renal allograft biopsies, including biopsies showing Tcell-mediated and antibody-mediated rejection and a number of nonrejection lesions. IRRAT gene expression was a strong predictor of future graft loss, and in biopsies without rejection were strongly correlated with inflammation in the areas of fibrosis. 16. Nizze H, Mihatsch MJ, Zollinger HU, et al. Cyclosporine-associated nephropathy in patients with heart and bone marrow transplants. Clin Nephrol 1988; 30:248–260. 17. Myers BD, Newton L. Cyclosporine-induced chronic nephropathy: an obliterative microvascular renal injury. J Am Soc Nephrol 1991; 2:S45–S52. 18. Young BA, Burdmann EA, Johnson RJ, et al. Cellular proliferation and macrophage influx precede interstitial fibrosis in cyclosporine nephrotoxicity. Kidney Int 1985; 48:439–448.
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19. Pichler RH, Franceschini N, Young BA, et al. Pathogenesis of cyclosporine nephropathy: roles of angiotensin II and osteopontin. J Am Soc Nephrol 1995; 6:1186–1196. 20. Viera JM Jr, Noronha IL, Malheiros DM, et al. Cyclosporine-induced interstitial fibrosis and arteriolar TGF-beta expression with preserved renal blood flow. Transplantation 1999; 68:1746–1753. 21. Zhong Z, Arteel GE, Connor HD, et al. Cyclosporine A increases hypoxia and free radical production in rat kidneys: prevention by dietary glycine. Am J Physiol 1998; 275:F595–F604. 22. Jennings P, Koppelstaetter C, Aydin S, et al. Cyclosporine A induces senescence in renal tubular epithelial cells. Am J Physiol 2007; 293:F831–F838. 23. Naesens M, Kuypers DRJ, Sarwal M. Calcineurin inhibitor nephrotoxicity. Clin J Am Soc Nephrol 2009; 4:481–508. 24. Nankivell BJ, Burrows RJ, Fung CL-S, et al. The natural history of chronic allograft nephropathy. N Engl J Med 2003; 349:2326–2333. 25. Snanoudj R, Royal V, Elie C, et al. Specificity of histological markers of longterm CNI nephrotoxicity in kidney-transplant recipients under low-dose cyclosporine therapy. Am J Transplant 2011; 11:2635–2646. 26. Abrass CK, Berfield AK, Stehman-Breen C, et al. Unique changes in interstitial extracellular matrix composition are associated with rejection and cyclosporine toxicity in human renal allograft biopsies. Am J Kidney Dis 1999; 33:11– 20. 27. Koop K, Bakker RC, Eikmans M, et al. Differentiation between chronic rejection and chronic cyclosporine toxicity by analysis of renal cortical mRNA. Kidney Int 2004; 66:2038–2046. 28. Anglicheau D, Muthukumar T, Hummel A, et al. Discovery and validation of a & molecular signature for the noninvasive diagnosis of human renal allograft fibrosis. Transplantation 2012; 93:1136–1146. This study analyzed a number of mRNAs in urinary cells and found several to be significantly associated with the presence of interstitial fibrosis in corresponding renal allograft biopsies. A model composed of levels of urinary cell mRNAs for vimentin, the Na–K–Cl cotransporter NKCC2, E-cadherin, and 18S ribosomal RNA predicted renal allograft fibrosis with a sensitivity of 77–94% and a specificity of 84–88%, although this model could not distinguish between different grades of fibrosis. 29. 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. 30. Scian MJ, Maluf DJ, David KG, et al. MicroRNA profiles in allograft tissues and paired urines associated with chronic allograft dysfunction with IF/TA. Am J Transplant 2011; 11:2110–2122. 31. Ben-Dov IZ, Muthukumar T, Morozov P, et al. MicroRNA sequence profiles of & human kidney allografts with or without tubulointerstitial fibrosis. Transplantation 2012; 94:1086–1094. This study identified several microRNAs, microRNA clusters, and microRNA sequence families having two-fold to ten-fold higher expression in biopsies with interstitial fibrosis and tubular atrophy compared with histologically unremarkable protocol biopsies, and validated these differences on a second set of biopsies.
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