The Histology of Solitary Renal Allografts at 1 and 5 Years After Transplantation

Transcription

1 American Journal of Transplantation 2011; 11: Wiley Periodicals Inc. C 2010 CSIRO C 2010 The Authors Journal compilation C 2010 The American Society of Transplantation and the American Society of Transplant Surgeons doi: /j x The Histology of Solitary Renal Allografts at 1 and 5 Years After Transplantation M. D. Stegall, W. D. Park, T. S. Larson, J. M. Gloor, L. D. Cornell, S. Sethi, P. G. Dean, M.Prieto,H.Amer,S.Textor,T.Schwaband F. G. Cosio von Liebig Transplant Center, Division of Transplantation Surgery, Division of Nephrology and Hypertension, Department of Anatomic Pathology, Mayo Clinic, Rochester, MN *Corresponding author: Mark D. Stegall, stegall.mark@mayo.edu Previous studies suggest that the majority of renal allografts are affected by progressive, severe chronic histologic injury, yet studies using current protocols are lacking. The goal of this study was to examine the prevalence and progression of histologic changes using protocol allograft biopsies at 1 and 5 years after solitary kidney transplantation in patients transplanted between 1998 and Chronic histologic changes generally were mild at both 1 and 5 years and were similar in deceased and living donor kidneys. The overall prevalence of moderate or severe fibrosis was 13% (60/447) at 1 year and 17% (60/343) at 5 years. In a subgroup of 296 patients who underwent both 1- and 5-year biopsies, mild fibrosis present at 1 year progressed to more severe forms at 5 years in 23% of allografts. The prevalence of moderate or severe arteriolar hyalinosis was similar in tacrolimus and calcineurin inhibitor-free immunosuppression. These results in the recent era of transplantation demonstrate fewer, less severe and less progressive chronic histologic changes in the first 5 years after transplantation than previously reported. Key words: Fibrosis, kidney transplantation, longterm outcomes, protocol renal allograft biopsies Abbreviations: bx, Biopsy; DD, deceased donor; DWF, deceased with function; GFR, glomerular filtration rate; LD, living donor; Mod-Sev, moderate or severe; MDRD, Modification of Diet in Renal Disease; Srl, Sirolimus; Tac, Tacrolimus. Received 01 June 2010, revised 24 August 2010 and accepted for publication 08 September 2010 Introduction Chronic, progressive, histologic changes such as interstitial fibrosis, tubular atrophy and vasculopathy are thought to affect the majority of renal allografts (1 4). A pivotal study by Nankivell et al. (1) outlined the natural history of chronic allograft nephropathy in 120 patients, 119 of which were recipients of bladder-drained simultaneous pancreas kidney transplants between 1987 and In these patients, maintained primarily on cyclosporine-based immunosuppression, 66% of protocol allograft biopsies obtained at 5 years demonstrated moderate-to-severe interstitial fibrosis (Banff scores of ci2 or 3 scores) and 90.3% showed arteriolar hyalinosis. On the basis of these findings, the authors described two phases of chronic allograft nephropathy including an early fibrogenic phase attributed to ischemia reperfusion injury and acute rejection, and a late phase with fibrosis and arteriolar hyalinosis attributed to cyclosporine nephrotoxicity. Other studies appear to support this concept that solitary renal allografts develop chronic injury over time. For example, in protocol biopsies obtained in the first 2 years after transplantation, a high prevalence of mild fibrosis routinely has been observed (5,6). In addition, renal allograft biopsies obtained in the setting of chronic graft dysfunction (so called for cause biopsies) commonly showed severe injury in the majority of cases (7 9). However, it is important to note that there have been no comprehensive protocol biopsy studies of solitary kidney transplants that either have confirmed or refuted the 5-year data of Nankivell et al. (1). There is also a paucity of data regarding late histologic findings in patients treated with current management and immunosuppressive regimens. The current study aimed to assess the prevalence and progression of renal allograft histology in the first 5 years after solitary kidney transplantation using protocol biopsies in patients transplanted between 1998 and These data demonstrate that severe chronic histologic changes are uncommon in the first 5 years after kidney transplantation and that mild fibrosis does not progress to more severe forms in the majority of allografts by 5 years. Material and Methods Study design and patients This study examined histologic changes in the first 5 years after solitary kidney transplantation in protocol biopsies obtained at 1 and 5 years after transplantation. Under a protocol approved by the Institutional Review Board of the Mayo Foundation and Clinic, renal allograft recipients underwent surveillance protocol biopsy at 1 and 5 years after transplantation. 698

2 Histology of Solitary Renal Allografts After Transplantation Solitary Kidney Transplants (10/98-4/04, n=853) Functioning Grafts at 1 year (n=797) Lost graft <1 year (n=56) Functioning Grafts at 5 years (n=578, 73%) Lost to FU (n=78, 10%) Lost graft between 1-5 years (n=141, 17%) Figure 1: Outcome and biopsies obtained in solitary kidney transplant recipients. The study biopsies included any biopsy obtained at 1 or 5 years (all biopsy group) and a subset of these patients who had both 1- and 5-year protocol biopsy (paired biopsy group). ALL BIOPSY GROUP 1 year biopsy 5 year biopsy (n=447, 77%) (n=343, 59%) Paired 1 and 5 year Biopsy Group (n=296, 51%) Immunosuppression n=208 Tacrolimus 20 Sirolimus 68 Mixed Donor Type n=234 Living 62 Deceased Death with Function (n=74, 9%) Lost Graft, alive (n=67, 8%) For this study, we included all adult recipients of solitary kidney transplants performed at Mayo Clinic, Rochester, MN, between October 1998 and April We excluded (1) pediatric patients (<18 years); (2) recipients who had a positive T or B cell flow cytometric cross-match at baseline against their living donor or who were ABO blood group incompatible; (3) recipients of simultaneous kidney and other solid-organ transplant, including pancreas kidney, liver kidney and heart kidney; (4) recipients who did not give consent for the use of the biopsy for research and (5) recipients who did not have functioning graft at 5 years after transplant. Immunosuppression Recipients were maintained on triple-therapy immunosuppression stratifying them into three groups: (1) tacrolimus (Astellas, Deerfield, IL; dose adjusted to achieve a 12 h serum trough level of 6 8 ng/ml which was maintained during the study period); mycophenolate mofetil (Roche, Nutley, NJ; 2 g per day initially and then dose-adjusted for leucopenia or diarrhea); prednisone (1 g on the day of transplantation and then tapered to 5 mg/day by 3 months); (2) sirolimus (Pfizer, New York NY; dosed to achieve a 12 h trough serum level of 8 10 ng/ml which was maintained during the study period); mycophenolate mofetil and prednisone as above and (3) mixed immunosuppression, that is, patients who were maintained on cyclosporine alone (Novartis, Basel, Switzerland; dosed to achieve a 12 h trough level of 200 ng/ml; n = 7) or patients started on either tacrolimus or sirolimus and then switched to other regimens for clinical reasons (usually inability to tolerate medicine due to complications or side effects). For example, 36 patients (53%) received both Tac and Srl, primarily due to wound healing complications on Srl therapy. The majority of patients (81%) transplanted at Mayo Clinic during this era received induction antilymphocyte therapy with Thymoglobulin (Genzyme, Cambridge, MA; 1.5 mg/kg per day on day 0, 1, 2 and 4 with respect to transplantation) (9). Biopsies and scoring Biopsies were obtained using ultrasound guidance with an 18 gauge Biopty gun (Bard, Murray Hill, NJ). The Mayo Clinic Renal Pathology laboratory paraffin embedded each biopsy and each block was serially sectioned and stained with a panel of conventional histological stains (hematoxylin and eosin [H&E], periodic acid Schiff, Masson s trichrome and methenamine silver). At least two slides for each stain were created, with each slide containing multiple serial sections. Each biopsy was deemed adequate for interpretation by a Mayo Clinic renal pathologist and scored using the Banff 97 classification (10). This semiquantitative scoring system classifies and grades acute and chronic changes in the kidney including glomeruli, tubules, interstitium, arteries and arterioles. Scores range from 0 to 3 with higher scores indicating more severe abnormalities. Given the retrospective nature of this study, none of the biopsies underwent a repeat reading. Clinical outcomes Graft loss was defined as return to dialysis. Renal function (glomerular filtration rate) was estimated from serum creatinine using the modified Modification of Diet in Renal Disease (MDRD) equation for all groups (11). Statistical analyses Results are expressed as means ± SD. The proportions of nominal data were tested using chi-square (Pearson) test. Continuous variables were tested using Student s t-test for parametric data and Wilcoxon test for nonparametric data. To test for variations in Banff score categories (as nominal data), we used the Pearson chi-square test for the unpaired biopsy data and the McNemar s test for the paired biopsy data (where none = 1 and not none = 2). For the patients with paired 1- and 5-year biopsies, univariate and multivariate logistic regression analyses were used to analyze clinical variables associated with ci 2.0 at 5 years. This involved 47 variables including patient demographics, graft function, graft histology and posttransplant complications (see Table S1). Only variables determined to be significant in the univariate analyses were included in the multivariate model. When highly overlapping variables were significant (i.e., 12 m chronicity score and 12 m ci score), only one was allowed in the model. A p value of <0.05 was considered statistically significant. The JMP statistical software system was (SAS, Cary, NC) used to perform calculations. Study groups The overall outcomes and the biopsies obtained in solitary kidney transplants performed between October 1998 and April 2004 are presented in Figure 1. During the time period, 853 solitary kidney transplants met inclusion criteria 76% (n = 651) were recipients of living donor kidneys and American Journal of Transplantation 2011; 11:

3 Stegall et al. Table 1: Demographics of adult solitary kidney transplant recipients who had functioning grafts 5 years after transplantation Demographics summary Patient characteristics Missing Bx Paired Bx p-value 1 Tac-treated Sri-treated p-value 1 # of patients Recipient age Avg ± stdev 50.5 ± ± ± ± Range (min max) ( ) ( ) ( ) ( ) Recipient gender % Male/female 53%/47% 56%/44% %/43% 50%/50% Recipient race % Caucasian 93% 95% 95% 95% Type of transplant % DD vs LD 23%/77% 21%/79% %/78% 30%/70% # Other transplants % >1 kidney txps 12% (n = 35) 10% (n = 30) % (n = 20) 20% (n = 4) % Other organ txps 16% (n = 44) 9% (n = 28) % (n = 20) 0% (n = 0) % Pancreas txps 11% (n = 31) 4% (n = 12) % (n = 10) 0% (n = 0) Donor age Avg ± stdev 40.2 ± ± ± ± Range (min max) ( ) (6.7 75) (7.3 75) ( ) Donor gender % Male/female 44%/56% 51%/49% %/48% 60%/40% Donor race % Caucasian 99% 99% 98% 100% Complications <5 yr acute rejection 22% (n = 62) 19% (n = 56) % (n = 35) 10% (n = 2) <5 yrbk+ 9% (n = 24) 13% (n = 37) % (n = 19) 5% (n = 1) Graft status Active 82% (n = 232) 93% (n = 274) % (n = 198) 90% (n = 18) DWF 8% (n = 22) 4% (n = 11) 3% (n = 6) 10% (n = 2) Failed 10% (n = 28) 4% (n = 11) 2% (n = 4) 0% (n = 0) Lost to FU 0% (n = 0) 0% (n = 0) 0% (n = 0) 0% (n = 0) Lenqth of follow-up Avg ± stdev 2602 ± ± ± ± Range (min max) ( ) ( ) ( ) ( ) DD = deceased donor; LD = living donor; DWF = deceased with function; lost to FU; lost to follow-up. 1 Continuous variables were tested using student s t-test, nominal variables were tested using a chi-square test. 24% (n = 202) were recipients of deceased donor kidneys (see Table S2 for primary reason for renal transplantation). Actual 5-year death-censored graft survival was 87% (450/519) for living donor recipients and 78% (128/164) for deceased donors. Fifty-six patients (6.6%) lost their graft in the first year and were excluded from further analysis. Of the 797 patients with a functioning graft at 1 year, 219 (27%) were not available for 5-year follow-up including 78 lost to follow-up and 141 who lost their allografts between 1 and 5 years (74 died with a functioning graft by 5 years and 67 lost their graft for other reasons). The causes for graft loss for patients who did not reach 5 years are reported in Table S3 using the same classifications described in more detail in El-Zoghby et al. (12). At 5 years, 578 patients had a functioning graft and were still being followed. Of these 447 (77.3%) had a 1-year and 343 (59.3%) had a 5-year protocol biopsy. Overall, 282 missed at least one biopsy time point, whereas 296 had both paired biopsies which allowed an analysis of the progression of changes in individual patients. In the paired biopsy group, 208 patients were maintained on tacrolimus-based immunosuppression for the entire 5 years, 20 were maintained on calcineurin inhibitor-free/sirolimus-based immunosuppression and 68 had received either cyclosporine-based or had changed from their initial immunosuppression due to clinical reasons. Patients not biopsied Because many patients did not undergo protocol biopsy at 1 and 5 years, we performed several analyses in an attempt to determine whether the omission of these biopsies would significantly affect our final conclusions. First, we found that patients with relatively poor outcomes did not comprise a large percentage of possible study patients. When death with function was excluded, only 67 patients (8%) experienced graft loss between 1 and 5 years after transplantation. In addition, with the exception of cg score, we found that biopsies obtained at 1 year in patients who either lost function (death or other causes) or were lost to follow-up before 5 years were similar to 1-year biopsies from patients who reached 5 years (Table S4). The causes of graft loss between 1 and 5 years were also similar to the causes of graft loss that occurred after 5 years (Table S3). Therefore, we concluded that the patients excluded prior to 5 years were not appreciably different from the patients who reached 5 years. Second, we performed two analyses that demonstrated that patients who did not undergo both 1- and 5-year protocol biopsies were not dramatically different from those who had paired biopsies. Forty-nine percent (282/578) of patients who had a functioning graft had at least one missing biopsy and 51% (296/578) had both 1- and 5-year biopsy. The patients with missing biopsies were similar to those with paired biopsies with respect to nearly 700 American Journal of Transplantation 2011; 11:

4 Histology of Solitary Renal Allografts After Transplantation Figure 2: Renal function at 5 years after transplantation. The figure demonstrates that the renal function of patients who underwent both paired 1 and 5 years biopsies (paired Bx) was similar to patients who missed at least one or both biopsies (missing Bx). In the paired biopsy group, renal function in recipients of a deceased donor kidney had higher renal function when compared with recipients of a living donor kidney. Finally, in the paired biopsy group, renal function in tacrolimus-treated (TAC-treated) was similar to that of patients treated with calcineurin inhibitor-free immunosuppression (Srl-treated). all demographic variables (Table 1) and causes for graft loss after 5 years (Table S3). The only significant differences were with regard to transplants of another organ (primarily pancreas) and lower post-5-year graft survival. However, the missing biopsy group (including those never biopsied) had similar renal function at 5 years to those with paired biopsies (Figure 2) so the demographic differences did not appear to adversely impact graft function at 5 years. Finally, only 14 patients with a missing 5-year protocol biopsy were biopsied for cause (usually an elevated serum creatinine) within 1 year of the 5- year time point. Given the small number, the exclusion of these for cause biopsies from the overall study did not appreciably alter the 5-year protocol biopsy data and these for cause biopsies were thus excluded from the current protocol biopsy study. These results led us to conclude that protocol biopsies represented the vast majority of patients biopsied, and the missing Bx and paired Bx patients were demographically and functionally similar at 5 years. Reasons for not obtaining a 5-year biopsy We assessed reasons for lack of a biopsy at 5 years in a subgroup of 250 patients transplanted in Only two patients were lost to follow-up. In this group, 78% (194/248) had a functioning graft 134 (69%) returned for follow-up and 60 (31%) did not. The reasons given in a telephone interview for not returning included homebound (n = 2), financial (n = 12), transferred care to another transplant program (n = 16) and unwilling to return (n = 28). Of the 134 that returned, biopsies were obtained in 85% (n = 114). Reasons for not biopsying the other 20 included (1) anticoagulation therapy (n = 9), (2) patient refused (n = 6), (3) biopsy not ordered (n = 4) and (4) biopsy not technically feasible (n = 1). glomerulopathy of any severity was extremely rare, occurring in only 2% of biopsies at 1 year and 9% of biopsies at 5 years. By chi-square, the overall prevalence of all chronic histologic lesions except tubular atrophy was shown to be increased at 5 years compared with 1 year. For a subset of 5-year protocol biopsies, C4d (immunofluorescence) was performed. Of the 111 biopsies tested, seven were considered positive in peritubular capillaries, with three scoring C4d Histology at 1 and 5 years: paired biopsies Biopsy findings in the paired biopsy group at 1 and 5 years were similar to the overall group (Table 2B). Most histologic lesions were mild, and the prevalence of moderate/severe histologic changes at both 1 and 5 years was less than 20% in all categories including fibrosis and hyalinosis. Similar to the overall group, a matched pair analysis shows that all chronic lesions, except the ct score, changed significantly in severity from 1 year to 5 years in the paired biopsy group. For example, the prevalence of any degree of arteriolar hyalinosis increased from 22% to 58% in the tacrolimus group and from 15% to 68% in the sirolimus-treated group. However, most of these lesions were mild with the prevalence of moderate or severe arteriolar hyalinosis at 5 years being 19% in the tacrolimus group and 5% in the sirolimus group. Results Histology at 1 and 5 years: all biopsies In the year biopsies, interstitial fibrosis was minimal (ci0) in 47%, mild (ci1) in 40% and moderate (ci2) or severe (ci3) in 13% (Table 2A). In the year biopsies, the interstitial fibrosis scores were slightly higher: ci0 = 38%, ci1 = 45% and ci2/ci3 = 17%. Arteriolar hyalinosis was generally mild or absent with moderate/severe lesions occurring in only 3% at 1 year and 19% at 5 years. Transplant Progression of histologic changes from 1 to 5 years after transplantation An important aspect of the current theory of chronic renal allograft damage relates to the progression of interstitial fibrosis. The paired 1- and 5-year biopsies allowed for an assessment of the degree of progression in several ways. First, the overall prevalence of moderate/severe fibrosis in the paired biopsies in tacrolimus-treated patients was 13% at 1 year and 16% at 5 years (Table 3). In the paired sirolimus-treated biopsies, the prevalence of American Journal of Transplantation 2011; 11:

5 Stegall et al. Table 2: Chronic histologic scores in 1- and 5-year protocol biopsies. A summary of Banff scores for each of the chronic histologic lesions is shown for the all patients group (Panel A) and the paired biopsy patients (Panel B). With the exception of the ct score, each Banff score changed significantly from 1 to 5 years All 1 yr All 5 yr Paired Bx (n = 296) A. Banff summary 1 Bx (n = 447) Bx (n = 343) p-value 2 B. 1yr 5yr p-value 3 ci category None 47% (n = 209) 38% (n = 130) 46% (n = 136) 38% (n = 112) Mild 40% (n = 178) 45% (n = 153) % (n = 115) 44% (n = 129) Mod-sev 13% (n = 60) 17% (n = 60) 15% (n = 45) 19% (n = 55) ah category 4 None 80% (n = 337) 40% (n = 136) 81% (n = 227) 41% (n = 119) Mild 17% (n = 71) 41% (n = 137) < % (n = 47) 40% (n = 117) < Mod-sev 3% (n = 14) 19% (n = 65) 2% (n = 7) 19% (n = 56) cg category None 98% (n = 437) 92% (n = 313) 98% (n = 290) 92% (n = 273) Mild 2% (n = 9) 3% (n = 9) < % (n = 5) 2% (n = 5) Mod-sev 0% (n = 1) 6% (n = 20) 0% (n = 1) 6% (n = 18) ct category None 27% (n = 120) 28% (n = 95) 27% (n = 79) 28% (n = 84) Mild 60% (n = 267) 54% (n = 184) % (n = 174) 52% (n = 154) Mod-sev 13% (n = 59) 18% (n = 63) 15% (n = 43) 20% (n = 58) cv category None 58% (n = 258) 42% (n = 142) 57% (n = 170) 41% (n = 120) Mild 37% (n = 164) 42% (n = 143) < % (n =111) 43% (n = 127) < Mod-sev 5% (n = 23) 16% (n = 56) 5% (n = 15) 17% (n = 49) 1 Banff category definitions: None = 0; Mild <2.0; Mod-Sev p-values were determined for each Banff score category using the Pearson chi-square test. 3 p-values were determined for each Banff score category using the McNemar s test (1 = none, 2 = not none). 4 Not all patients had complete Banff scores recorded for the 1 & 5 yr bx, so the sum of each category may be lower than the total number of patients. moderate/severe fibrosis was also low: 0% at 1 year and 20% at 5 years. A more detailed analysis of the different interstitial fibrosis scores in individual patients at 1 and 5 years revealed that biopsies with at least mild fibrosis (ci 1) at 1 year were unlikely to show progression to more severe forms by 5 years (Figure 3). For example, of the 74 biopsies in tacrolimus-treated patients scored as having mild fibrosis (ci1) on the 1-year biopsy, only 23% (n = 17) progressed to more severe forms by 5 years and 39% (n = 29) were scored as having no fibrosis (Figure 3). Similarly, more than half of the cases of moderate fibrosis (ci2) at 1 year were scored as mild or no fibrosis at 5years. The 1-year tacrolimus level was not significantly different in patients who between 1 and 5 years had a ci score that increased (9.0 ± 2.7), decreased (8.6 ± 2.8) or had no change (9.2 ± 3.9). It is also noteworthy that of the 100 tacrolimus-treated patients with at least mild fibrosis at 1 year, 13 patients also had concomitant mild inflammation (Banff i score >0). Three of these patients had an increase in fibrosis at 5 years, whereas the majority decreased (n = 6) or remained unchanged (n = 4). Histology at 1 and 5 years: living versus deceased donors The histologic findings at 1 and 5 years were similar in living and deceased donors (Table 4) including the incidence of moderate-to-severe fibrosis (24% in deceased donors vs. 17% in living donors, p = 0.27), moderate-to-severe hyalinosis (25% in deceased donors vs. 18% in living donors, p = 0.15) and moderate-to-severe vascular lesions (23% in deceased donors vs. 15% in living donors, p = 0.35). Histology at 1 and 5 years: tacrolimus versus CNI-free immunousuppression A comparison between the tacrolimus and sirolimus groups did not show differences in the prevalence of any histologic lesion at either time point (Table 3) or 5-year renal function (Figure 1). The prevalence of moderate-to-severe hyalinosis was similar and uncommon in the two groups at 1 year (4% in tacrolimus group vs. 0% in the sirolimus group, p = 0.65) and at 5 years (19% in the tacrolimus group vs. 5% in the sirolimus group, p = 0.10). The prevalence of arteriolar hyalinosis of any score at 5 years was similar in the tacrolimus-treated and sirolimus-treated groups (58% vs. 68%, respectively). Although the number of sirolimus-treated patients was small, these data do suggest that hyalinosis may not be specifically associated with calcineurin-inhibitor treatment. 702 American Journal of Transplantation 2011; 11:

6 Histology of Solitary Renal Allografts After Transplantation Table 3: Chronic histologic scores in paired 1- and 5-year protocol biopsies from patients treated with tacrolimus (Tac-Treated) or sirolimus (Srl-Treated) as primary immunosuppression. None of the Banff categories were significantly different between these two patient groups p-value Tac-treated (n = 208) Srl-treated (n = 20) Tac vs. Srl 2 Banff summary 1 1yr 5yr 1yr 5yr 1yr 5yr ci category None 52% (n = 108) 41% (n = 86) 40% (n = 8) 35% (n = 7) Mild 36% (n = 74) 43% (n = 89) 60% (n = 12) 45% (n = 9) Mod-sev 13% (n = 26) 16% (n = 33) 0% (n = 0) 20% (n = 4) ah category 3 None 79% (n = 153) 42% (n = 86) 85% (n = 17) 32% (n = 6) Mild 18% (n = 34) 39% (n = 81) 15% (n = 3) 63% (n = 12) Mod-sev 4% (n = 7) 19% (n = 39) 0% (n = 0) 5% (n = 1) cg category None 98% (n = 203) 94% (n = 196) 100% (n = 20) 85% (n = 17) Mild 2% (n = 4) 0% (n = 1) 0% (n = 0) 0% (n = 0) Mod-sev 0% (n = 1) 5% (n = 11) 0% (n = 0) 15% (n = 3) ct category None 29% (n = 61) 33% (n = 69) 35% (n = 7) 15% (n = 3) Mild 59% (n = 123) 50% (n = 105) 65% (n = 13) 65% (n = 13) Mod-sev 12% (n = 24) 16% (n = 34) 0% (n = 0) 20% (n = 4) cv category None 57% (n = 119) 45% (n = 93) 60% (n = 12) 25% (n = 5) Mild 38% (n = 78) 40% (n = 83) 40% (n = 8) 60% (n = 12) Mod-sev 5% (n = 11) 15% (n = 32) 0% (n = 0) 15% (n = 3) 1 Banff category definitions: None = 0 Mild <2.0; Mod-Sev p-values were determined for each Banff score category using the Pearson chi-square test. 3 Not all patients had complete Banff scores recorded for the 1 & 5 yr bx, so the sum of each category may be lower than the total number of patients. Figure 3: Changes in ci scores between 1 and 5 years in paired protocol biopsies in tacrolimus-treated patients with evidence of mild (Panel A) or moderate/severe (Panel B) interstitial fibrosis (1-year score in gray, 5-year score in black). American Journal of Transplantation 2011; 11:

7 Stegall et al. Table 4: Chronic histologic scores in paired 1 and 5 year protocol biopsies were compared for patients who received deceased and living donor kidneys. None of the Banff categories were significantly different between these two patient groups Deceased donor (n = 62) Living donor (n = 234) p-value DD vs. LD 2 Banff summary 1 1yr 5yr 1yr 5yr 1yr 5yr ci category None 47% (n = 29) 40% (n = 25) 46% (n = 107) 37% (n = 87) Mild 39% (n = 24) 35% (n = 22) 39% (n = 91) 46% (n = 107) Mod-sev 15% (n = 9) 24% (n = 15) 15% (n = 36) 17% (n = 40) ah category 3 None 79% (n = 48) 46% (n = 28) 81% (n = 179) 39% (n = 91) Mild 16% (n = 10) 30% (n = 18) 17% (n = 37) 43% (n = 99) Mod-sev 5% (n = 3) 25% (n = 15) 2% (n = 4) 18% (n = 41) cg category None 100% (n = 62) 94% (n = 58) 97% (n = 228) 92% (n = 215) Mild 0% (n = 0) 2% (n = 1) 2% (n = 5) 2% (n = 4) Mod-sev 0% (n = 0) 5% (n = 3) 0% (n = 1) 6% (n = 15) ct category None 29% (n = 18) 31% (n = 19) 26% (n = 61) 28% (n = 65) Mild 58% (n = 36) 42% (n = 26) 59% (n = 138) 55% (n = 128) Mod-sev 13% (n = 8) 27% (n = 17) 15% (n = 35) 18% (n = 41) cv category None 60% (n = 37) 39% (n = 24) 57% (n = 133) 41% (n = 96) Mild 34% (n = 21) 39% (n = 24) 38% (n = 90) 44% (n = 103) Mod-sev 6% (n = 4) 23% (n = 14) 5% (n = 11) 15% (n = 35) 1 Banff category definitions: None = 0; Mild <2.0; Mod-Sev p-values were determined for each Banff score category using the Pearson chi-square test. 3 Not all patients had complete Banff scores recorded for the 1 & 5 yr bx, so the sum of each category may be lower than the total number of patients. Factors associated with moderate or severe fibrosis Of the 47 variables studied in patients with paired biopsies, 14 were identified as associated with moderate/severe fibrosis at 5 years using univariate analysis and six remained significant in a multivariate model (Table 5). These factors with odds ratios are: any complication between 1 and 5 years (7.65), delayed graft function (7.47), other organ transplant prior to the kidney transplant (3.93), 1-year chronicity score (3.21), >1 kidney transplants (3.08) and increasing donor age (1.05). The receiver operating characteristic curve of these six factors had an area under the curve (AUC) of 83%. Discussion The current study demonstrates that most renal allografts have only mild histologic injury at 1 and 5 years after transplantation and that it was uncommon for mild interstitial fibrosis present at 1 year to progress to more severe forms. The fact that 8% of grafts functioning at 1 year were lost to causes other than death between 1 and 5 years suggests that severe damage does occur, but it does not affect a large percentage of allografts. These findings contrast with a pivotal study involving recipients of simultaneous pancreas kidney transplants in an earlier era of immunosuppression which suggested that the majority of renal allografts develop progressive, severe histologic injury within 5 years of transplantation(1). This earlier study demonstrated that 66% of patients developed moderate-to-severe interstitial fibrosis by 5 years after transplantation, whereas the current study showed a prevalence of moderate-to-severe fibrosis of 17% at 5years. Also in contrast to prior studies, in the current study, mild fibrosis present at 1 year progressed to more severe forms at 5 years in only 23% of allografts and commonly was scored as no fibrosis at this later time point. The prevalence and severity of fibrosis was not dependent on the type of donor (living vs. deceased) or treatment with calcineurin inhibitors. As noted by others, the univariate analysis found polyoma virus infection (13) and acute cellular rejection (14,15) to be significantly associated with the development of moderate-to-severe fibrosis.however, these variables did not remain significant in the multivariate analysis. Likewise, tacrolimus levels, which have been associated with the development of fibrosis in the first year (16), were not found to be associated with progression of fibrosis from 1 to 5 years in this study. Advanced arteriolar hyalinosis was present in 19% of biopsies at 5 years in our study far less than the 90.3% in prior studies using cyclosporine-based immunosuppression (1). However, the prevalence of mild hyalinosis increased from 704 American Journal of Transplantation 2011; 11:

8 Histology of Solitary Renal Allografts After Transplantation Table 5: Clinical variables associated with 5 year ci score >2. Forty-seven clinical parameters were analyzed to determine the variables most associated with the presence of moderate or severe fibrosis on the 5 year protocol biopsy. The proportion of patients reaching the endpoint with and without each variable was calculated (Panel A). Odds ratio, 95% confidence interval (CI) and p-values are noted for significant variables identified by univariate analysis (Panel B). A multivariate model was created using a subset of the significant univariate variables (Panel C) A. Proportion to reach 5 yr ci 2 B. Univariate analysis C. Multivariate analysis Variable With Without Odds Odds category variable variable ratio CI 95% p-value ratio CI 95% p-value Demographics Donor age >1 kidney transplants 37% (n = 11/30) 17% (n = 44/266) Other organ pre-kidney 41% (n = 7/17) 17% (n = 48/279) Other organ transplants 36% (n = 10/28) 17% (n = 45/268) Complications <1 yr delayed graft function 44% (n = 15/34) 15% (n = 40/262) < yr acute rejection 45% (n = 9/20) 17% (n = 46/276) yrBK+ 42% (n = 10/24) 17% (n = 45/272) yr any complication 1 44% (n = 15/34) 15% (n = 40/262) < <5yrBK+ 41% (n = 15/37) 15% (n = 40/259) <5 yr any complication 2 29% (n = 36/125) 11% (n = 19/171) < Functional 1 yr estimated GFR (MDRD) < Histological 1 yr chronicity score % (n = 18/44) 15% (n = 37/252) < yr ci score 2 38% (n = 17/45) 15% (n = 38/251) _ 4 1 yr ah score 2 57% (n = 4/7) 17% (n = 47/274) _ 4 1 Includes patients with any of the following complications recorded between 1 & 5 yr: AR (n = 20), BK (24), DGF (0), recurrent disase (7), transplant glomerulopathy (13), wound complications (0) or re-operations (0). 2 Includes patients with any of the following complications recorded prior to 5 yr: AR (n = 56), BK (37), DGF (34), recurrent disase (12), transplant glomerulopathy (13), wound complications (20) or re-operations (3). 3 Chronicity score equals the sum of Banff chronic scores (ah + eg + ci + ct + cv). 4 Variable left out of multivariate analysis due to similarity to other variables included in the model. 17% to 41% from 1 to 5 years after transplantation, suggesting that hyalinosis may still be an important, progressive problem. The etiology of hyalinosis remains unclear. Commonly associated with chronic calcineurin inhibitor toxicity (17,18) in the current study, the prevalence of hyalinosis at 5 years was the same in patients treated with tacrolimus as those treated with a calcineurininhibitor-free regimen. Although these results agree with other recent reports suggesting that at 5 years tacrolimusbased regimens may be less prone to chronic nephrotoxicity than cyclosporine (19 21), it is possible that the increasing prevalence of mild hyalinosis observed at 5 years might foreshadow the development of more severe forms at later time points. Recent studies have suggested that alloantibody-mediated damage presenting as transplant glomerulopathy may be a major cause of late renal allograft damage (22,23). However, only 5% of tacrolimus-treated patients developed this lesion by 5 years, suggesting that our protocols may be effectively avoiding this cause of chronic injury. Several factors may account for the differences in histology between the current study and the study by Nankivell et al. These include the fact that the Nankivell et al. study involved simultaneous pancreas kidney transplants from an earlier immunosuppressive era that incurred a higher rate of acute cellular rejection than the solitary kidney transplant recipients in the current study. In addition, having a bladder-drained pancreas likely increased the incidence of chronic dehydration and recurrent urinary tract infections which might affect late histologic findings. Finally, the use of tacrolimus-based immunosuppression and other changes in patient management that have been adopted in the past decade may have contributed to the apparent decrease in histologic injury observed in the current cohort of patients. Regardless of the reasons for the differences, we contend that the current study which involved a much larger number of 1- and 5-year biopsies obtained in both living and deceased donor kidneys better represents the natural history of histologic changes in solitary kidney transplants today. Long-term biopsy studies are uncommon in renal transplantation most likely due to the difficulty in capturing the necessary clinical data and biopsies from patients with stable grafts. Interestingly, both the Nankivell et al. study and this study had similar biopsy capture rates which American Journal of Transplantation 2011; 11:

9 Stegall et al. resulted in groups of patients who did not undergo biopsy or data on others lost due to patient death and lack of patient follow-up. Although the omission of these patients could impact the observed rate of moderate-to-severe lesions at 5 years, this study includes year biopsies with mild or no fibrosis, so it is unlikely that the exclusion of small numbers of patients would dramatically alter the conclusions. We also recognize that, although the current study is the largest 5-year protocol biopsy study reported to date and includes both living and deceased donors, it is not clear that these data can be generalized to other patient populations including extended criteria deceased donors and African-American recipients. We conclude that these data support a revised concept of histologic injury after renal transplantation in which moderate-to-severe fibrosis and arteriolar hyalinosis may be avoided in the majority of renal allografts in the first 5 years after transplantation. In this view of chronic injury, mild fibrosis present at 1 year does not portend the development of more severe fibrosis by 5 years. Chronic, progressive histologic injury does indeed occur. However, these cases appear to be the result of specific incidents such as acute rejection, recurrent disease or polyoma virus infection. Thus, we suggest that efforts to improve longterm renal allograft function should focus on identifying and preventing specific causes of renal allograft injury rather than a general overhaul of our entire approach to patients and their immunosuppression. Although we are encouraged by these 5-year results, longer follow-up will be needed to determine whether these improved histologic trends will be maintained and will lead to increased long-term graft survival. Disclosure The authors of this manuscript have conflicts of interest to disclose as described by the American Journal of Transplantation. Dr. Stegall has received research grants from Wyeth (now Pfizer); Genzyme, Alexion Pharmaceuticals, Chester, CT; and Millenium Pharmaceuticals, Cambridge, MA. None of these specifically supported the current study. References 1. Nankivell BJ, Borrows RJ, Fung C L-S, O Connell PJ, Allean RDM, Chapman JR. The natural history of chronic allograft nephropathy. N Eng J Med 2003; 349: Cornell LD, Colvin RB. Chronic allograft nephropathy. Curr Opin Nephrol Hypertens 2005; 14: Tantravahi JR, Womer KL, Kaplan B. Why hasn t eliminating acute rejection improved graft survival? Ann Rev Med 2007; 58: Najafian B, Kasiske BL. Chronic allograft nephropathy. Curr Opin Nephrol Hypertens 2008; 17: Solez K, Vincenti F, Filo RS. Histopathologic findings from 2-year protocol biopsies from a US mulicenter kidney transplant trial comparing tacrolimus versus cyclosporine: A report of the FK-506 Kidney Transplant Study Group. Transplantation 1998; 66: Schwarz A, Mengel M, Gwinner W, Radermacher J, Hiss M, Kreipe H, Haller H. Risk factors for chronic allograft nephropathy after renal transplantation: a protocol biopsy study. Kidney Int 2005; 67: Kasiske BL, Kalil RS, Lee HS, Rao KV. Histopathologic findings associated with chronic, progressive decline in renal allograft function. Kidney Int 1991; 40: Humar A, Kerr S, Gillingham KJ. Features of acute rejection that increase risk for chronic rejection. Transplantation 1999; 68: Matas AJ, Leduc R, Rush D et al. Histologic clusters differentiate subgroups within the nonspecific diagnosis of CAN and CR: preliminary data from the DeKAF study. Am J Transplant 2010; 10: Racusen LC, Solez K, Colvin RB et al. The Banff 97 working classification of renal allograft pathology. Kidney Int 1999; 55: Levey AS, Greene T, Schluchter MD et al. Glomerular filtration rate measurements in clinical trials. Modification of Diet in Renal Diseases Study Group and the Diabetes Control and Complications Trial Research Group. J Am Soc Nephrol 1993; 4: El-Zoghby ZM, Stegall MD, Lager DJ et al. Identifying specific causes of kidney allograft loss. Am J Transplant 2009; 9: Hirsch HH, Brennan DC, Crachenberg CB et al. Polyomavirusassociated nephropathy in renal transplantation: Interdisciplinary analyses and recommendations. Transplantation 2005; 79: Rush DN, Jeffery J, Nickerson P. Subclinical acute rejection: Is it a cause of chronic rejection in renal transplantation? Transplant Rev 2000; 14: 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: Cosio FG, Amer H, Grande JP et al. Comparison of low versus high tacrolimus levels in kidney transplantation: assessment of efficacy by protocol biopsies. Transplantation 2007; 83: Nankivell BJ, Borrows RJ, Fund C L-S, O Connell PJ, Chapman JR, Allen RDM. Calcineurin inhibitor nephrotoxicity: longitudinal assessment by protocol histology. Transplantation 2004; 78: Williams D, Haragism L, Calcineurin nephrotoxicity. Adv Chronic Kidney Dis 2006; 13: Dean PG, Grande JP, Sethi S et al. Kidney transplant histology after one year of continuous therapy with sirolimus compared with tacrolimus. Transplantation 2008; 85: Ruiz JC, Compistol JM, Grinyo JM, et al. Early cyclosporine A withdrawal in kidney-transplant recipients receiving sirolimus prevents progression of chronic pathologic lesions. Transplantation 2004; 78: , 21. Lo A, Egidi MF, Gaber LW et al. Comparison of sirolimus-based calcineurin-inhibitor sparing and calcineurin-inhibitor-free regimens in cadaveric renal transplantation. Transplantation 2004; 77: Moll S, Pascual M. Humoral rejection of organ allografts. Am J Transplant 2005; 5: Gloor JM, Sethi S, Stegall MD et al. Transplant glomerulopathy: subclinical incidence and association with alloantibody. Am J Transplant 2007; 7: American Journal of Transplantation 2011; 11:

10 Histology of Solitary Renal Allografts After Transplantation Supporting Information Additional Supporting Information may be found in the online version of this article: Potential online supporting content Table S1. Clinical variables examined in uni- and multivariate analyses. Table S2. Primary diagnosis for transplant recipients. Table S3. Categories of the causes of graft loss for grafts surviving between 1 and 5 years and more than 5 years after transplant. Table S4. Comparison of 1-year histology of 5-year and 5-year patients. Please note: Wiley-Blackwell are not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article. American Journal of Transplantation 2011; 11: