Kidney transplantation is the treatment of choice for most patients with end-stage renal disease; transplantation is associated with longer life expectancy and better quality of life compared with dialysis treatment (1–4). The main limitation of transplantation is the shortage of organs; this has led to expanding the criteria for donors (5). Expanded criteria donors (ECDs) (6–8) are definite as age of more than or equal to 60 years, or between 50 and 59 years with at least two risk factors, including death by cerebrovascular accident, history of hypertension, and creatinine level of greater than 1.5 mg/dL.
Organs from ECD are associated with suboptimal posttransplant function (9, 10) or shorter graft survival (11, 7). To improve these outcomes, it is necessary to carefully select the grafts before transplantation. Previously, selection criteria included only the donor's clinical criteria (donor age, history of hypertension, serum creatinine, and cause of death), without an assessment of the structural integrity of the ECD kidney (12–15). Several studies have proposed that a preimplantation analysis of renal graft biopsies could facilitate graft selection (9, 16). Careful risk factor analyses may identify a subset of ECD that are suitable for transplantation.
Different algorithms based on selected histologic parameters have been proposed for selecting viable grafts from ECD (17–20). Most have been tested for dual transplantations (21), but they may also be useful for selecting ECD kidneys for single transplantations. Several authors have attempted to correlate histologic findings in ECD tissues with clinical graft outcomes (10, 22); unselected grafts with interstitial fibrosis, arteriosclerosis, glomerulosclerosis, or combinations of these lesions were associated with unfavorable long-term graft function in protocol biopsies (22, 23). Tan et al. showed significantly lower glomerular function and damaged structure in grafts from older cadaveric donors compared with those from younger cadaveric donors. This was due to the profoundly low number of functional glomeruli per allograft in grafts from older donors (10).
The aim of our study was to determine whether preimplantation analysis of kidney biopsies could make a clinically significant difference on outcomes in transplantations from expanded criteria kidney donors. Histologic analysis pretransplant could be an acceptable method of expanding the donor pool. We evaluated the contribution of renal senescence to graft survival by analyzing renal structure and clinical ECD criteria in allografts from cadaveric donors older than 60 years (older donors) or younger than 60 years (younger donors). Finally, we tested which pathologic findings of preimplantation kidney biopsies were most associated with transplant outcome.
Characteristics of Donors
We studied a cohort of 136 patients who received transplanted kidneys obtained from ECD who had undergone preimplantation biopsies. The donor characteristics are reported in Table 1. The donor groups were similar in all the variables studied except age, glomerular filtration rate (GFR), and gender.
Characteristics of the Recipients and the Transplantation
The recipients of the grafts from older donors were older and more frequently female compared with recipients of grafts from younger donors. The transplantation of kidneys from older donors showed more length of stay, a more prolonged cold ischemia time (Table 1), and more frequently developed delayed graft function in the posttransplantation period compared with transplantations from younger donors. Of note, there were no significant differences among recipients in the time spent on waiting list, human leukocyte antigen (HLA) mismatching, number of acute rejection episodes, or pretransplant percent of reactivity against panel antigens (PRAs; Table 1). Moreover, during the follow-up period (3-month and 1-, 3-, and 5-year follow-ups), no significant differences were found in the posttransplant GFR, estimated with the abbreviated Modification of Diet in Renal Disease formula. However, the recipients of grafts from younger donors were treated more frequently with tacrolimus (87.5%) as a calcineurin inhibitor than the recipients of grafts from older donors (62.5%; P=0.008).
Preimplantation Biopsy Findings
Preimplantation biopsy findings are shown in Table 2 for the whole group and for the different donor age groups. Significant differences between age groups were found in nearly all the histologic compartments. Glomerulosclerosis was present in 82.4% of all ECD biopsies, predominantly of grade 1 (64.4% vs. 59.4% for older and younger donors, respectively). Moderate glomerulosclerosis was significantly higher in older compared with younger donors (22.1% vs. 3.1%, respectively; P=0.002). Severe glomerulosclerosis (>20%) was not observed in biopsies from younger donors but was present in 2% of the biopsies from older donors.
Interstitial fibrosis was present in 61.8% of biopsies, predominantly of grade 1 (54.4%), but it was less frequent in biopsies from younger donors than those from older donors (34.4% vs. 70.2%, respectively; P=0.001). Only biopsies from older donors showed grade 2 severity (9.6%). Tubular atrophy was observed in 77% of all biopsies but was less frequent in biopsies from younger compared with those from older donors (46.9% vs. 73.1%, respectively; P=0.02). Grade 2 severity of tubular atrophy showed equivalent frequency in biopsies from younger (6.3%) and older (7.7%) donors. Fibrointimal thickening was observed in 50% of all biopsies. It occurred with equal frequency in biopsies from younger and older donors (50% both groups) and with similar severity (grade 1, 46.9% vs. 47.1; grade 2, 3.1% vs. 2.9%, respectively).
Hyaline arteriolar thickening was present in 53% of biopsies and was similar in biopsies from older donors compared with those from younger donors (56.9% vs. 40.6%, respectively; P=0.1). When present, the damage was always mild (grade 1). After scoring each of the five compartments, we calculated the total score, which ranged, theoretically, between 0 (no lesions) and 15 points. The median total score for the population (136 biopsies) was 4 points (1–7 points). Optimal scores (1–3 points) were observed in 47.7% of donors, 38.6% of older donors, and 70% of younger donors. Scores more than 5 points were observed in 12.6% of all donors, 15.6% of older donors, and 5% of younger donors.
Transplant Outcomes: Patient and Allograft Survival Rates
All 136 patients were followed up for a median of 4.7 years (interquartile range [IQR] 2.2–7.4 years). The cumulative patient survival rates were 95.5%, 87%, 80.2%, and 75.7% at 1, 3, 5, and 7 years, respectively, after kidney transplantation. The survival rate of patients who received grafts from younger donors was similar to those who received grafts from older donors (log-rank test, P=0.57; Fig. 1A).
The cumulative graft survival rates at 1, 3, 5, and 7 years were 83.4%, 77.7%, 72.8%, and 65.9%, respectively. The Kaplan-Meier allograft survival plots for grafts from older and younger donors are shown in Figure 1(B). Three- and 5-year graft survival rates were similar in both age groups (88.8% and 80.7%, respectively, for younger vs. 73.1% and 69%, respectively, for older donors; log-rank test P=0.13).
Associations Between Donor and Graft Characteristics and Renal Histology
We first analyzed associations between donor characteristics (age, GFR, and cardiovascular risk factors) and renal histology (summarized by the total score). Donor age showed a moderate linear correlation with histologic score (r=0.35, P<0.0001) for the whole group of biopsies. However, the isolated group of biopsies from older donors did not show a correlation between age and histologic score (r=0.12, P=0.23). We found no significant correlation between GFR and total score (r=−0.145, P=0.11). However, donors with a history of hypertension showed higher total scores compared with nonhypertensive donors (median total score=4, IQR 3–5 vs. median total score=3, IQR 2–4, respectively; P=0.03).
Allograft Survival and Glomerulosclerosis
The severity of glomerulosclerosis was associated with a lower graft survival rate. Biopsies with glomerulosclerosis below or equal to 10% showed graft survival rates of 93.6%, 90.5%, 83.2%, and 77.2% at 6 months, 1 year, 3 years, and 5 years, respectively, with a mean graft survival of 8.3 years (95% confidence interval [CI] 7.2–9.4). In comparison, biopsies with glomerulosclerosis more than 10% showed significantly worse survival rates at 3 months (61.5%) and after the first year (53.8%), with a mean survival of 3.9 years (95% CI 2.4–5.3; log-rank test P<0.001). Similar findings were observed when biopsies from the two age groups were analyzed separately (data not shown).
Allograft Survival and Total Score
Cumulative graft survival curves were plotted separately for kidneys with pretransplant histologic total scores of less than or equal to 3, 4 to 5, and more than 5 (Fig. 2). The overall graft survival rates at 1 and 5 years for transplants with total scores more than 5 were 31.76% and 31.76%, respectively; for transplants with total scores less than or equal to 3 were 96.8% and 84.7%, respectively; and for transplants with scores of 4 to 5 were 82.8% and 69%, respectively. Graft survival rates at both 1 and 5 years were significantly different between each group of pretransplant total scores: more than 5 versus less than or equal to 3 (P<0.001), more than 5 versus 4 to 5 (P<0.001), and 4 to 5 versus less than or equal to 3 (P=0.04).
Next, we sought to determine a cutoff score for predicting the best long-term graft outcome. Because the overall graft failure rates were lower for transplants from donors with scores more than 5 compared with those with scores less than or equal to 5, we chose a cutoff score of 5. The mean graft survival for scores less than or equal to 5 was 8.2 years (95% CI 7.2–9.3), and the mean graft survival for scores more than 5 was 2.6 years (95% CI 0.8–4.4; log-rank test P<0.001).
Graft Survival Factors From Multivariate Adjusted Models
Different Cox regression models were used to consider first, classic clinical characteristics and second, a case mix model. The first model included age of donor, dialysis exposure time (time on waiting list), PRA more than 30%, acute rejection episodes, HLA match, cold ischemic time, and gender match. Acute rejection episode and donor age were independent predictors of graft survival (hazard ratio [HR] 2.14, 95% CI 1.16–3.93, P=0.01; HR 1.13, 95% CI 1.04–1.22, P=0.04, respectively). When donor cardiovascular risk factors were included in this multivariate analysis, only donor hypertension predicted graft failure (HR 2.8, 95% CI 1.10–7.14, P=0.03).
The case-mix model (Table 3) included clinical and histologic parameters of donor age, donor cardiovascular risk factors (history of diabetes mellitus, hypertension, and death by cerebrovascular accident), dialysis exposure time (time on waiting list), PRA more than 30%, acute rejection episodes, HLA mismatch, cold ischemia time, percent glomerulosclerosis, and total histologic score. A total histologic score more than 5 was the only independent predictor of graft failure in this model (HR 6.95; 95% CI 1.57–30.73).
When the case-mix model included only data for biopsies from older donors, cold ischemia time and total score more than 5 were independent predictors of graft survival (Table 3). The total score more than 5 had an HR of 5.23 (95% CI 1.07–25.02, P=0.04), which confirmed it as an independent predictor for survival of grafts from ECD older than 60 years.
This study aimed to determine associations between graft survival, clinical data, preimplantation histologic scores, and outcome of recipients of grafts from ECD. We demonstrated that preimplantation allograft histology assessments could predict the long-term outcomes of kidney transplants from ECD more effectively than clinical data in this study population.
Previously, when a biopsy was not feasible, acceptable results were obtained in older donor kidney transplantations with a scoring system based on donor clinical and biologic variables (24, 25). However, considerable individual variability was observed in the magnitude of age-related histologic changes, estimated GFR, and medical conditions such as hypertension and diabetes.
Previously, donor age was shown to predict graft survival (26) but was not as useful for further selection of grafts within a subset of older donors, because of the wide variation in renal function and structure among older individuals (10). In this study, we carefully analyzed the effects of donor age by separately evaluating biopsies from donors older and younger than 60 years. To evaluate the contribution of renal senescence to graft survival, we analyzed degenerative damage in four histologic compartments, including glomerulosclerosis, interstitial fibrosis, tubular atrophy, and arteriolar hyalinosis. Studies that included kidneys from older donors or adjusted for donor age in multivariate analyses showed mixed results (11, 17, 21, 27). Our study only included single kidney transplantations but confirmed the effectiveness of a histopathologic scoring system in predicting allograft survival. Recently, similar studies have also shown that degenerative changes in different renal compartments could independently predict transplant outcome (28, 29); however, those histopathologic scoring systems were more difficult for practitioners to learn and use. This study presented a simple histologic scoring system that effectively predicted graft function and survival. In our opinion, adding histologic criteria to evaluations of ECD will improve graft outcome by facilitating optimal allocation of marginal donors and, furthermore, might expand the donor organ pool for transplantation.
Our scoring system indicated that recipients of grafts with total scores more than 5 had significantly lower graft survival rates than those with scores less than or equal to 5 (2.6 vs. 8.2 years mean graft survival, HR 6.95). Interestingly, we found that kidneys with more than 10% glomerulosclerosis had lower graft survival rates, but the multivariate analysis indicated that glomerulosclerosis was not an independent predictor of graft failure. However, Anglicheau et al. (28) found that the highest predictor of low GFR at 1 year posttransplantation was a composite score that included donor serum creatinine levels, donor hypertension, and glomerulosclerosis more than or equal to 10%, although only 40% of preimplantation biopsies had glomerulosclerosis less than or equal to 10%. Other authors observed that the percentage of glomerulosclerosis in donor kidney biopsies did not correlate well with graft survival and function; thus, they concluded that this percentage should not be used as the sole criterion for discarding cadaveric kidneys (29, 30).
The limitation of study is that preimplantation biopsy is already used in our center to exclude kidneys with greater degrees of glomerulosclerosis. The transplanted kidneys in this cohort with greater degrees of glomerulosclerosis were more carefully selected, because such kidneys were much less frequently transplanted (24.1% of grafts from ECD older than 60 years showed greater than 10% glomerulosclerosis). Most of expanded criteria kidneys with more than 20% glomerulosclerosis are discarded in the United States; discrimination within these intervals or among degrees of glomerulosclerosis greater than 20% is not possible (30–32). This should be kept in mind when applying scoring systems clinically, because they are usually only applicable in situations similar to the population from which they are derived.
In conclusion, the simplicity of our scoring system could facilitate adoption in clinical practice by eliminating the need to consider features that are relatively less important. Our findings suggest that preimplantation histologic assessments of ECD have important prognostic value for predicting graft survival and deciding whether to accept or reject ECD tissues.
MATERIALS AND METHODS
Long-term graft survival was determined in a cohort of patients who received single kidney transplants from ECD. Before transplantation, we performed histologic evaluations of the donor kidneys. This study included all renal transplantations performed in our center with kidneys from ECD between January 1997 and March 2008. ECDs were defined as (1) donors aged between 50 and 59 years with at least two of the following risk factors: a history of hypertension, diabetes, death by cerebrovascular accident, impaired renal function (evidenced by progressively increasing serum creatinine or decreasing urine output) or (2) donors aged 60 years or older, independent of the presence or absence of risk factors. The selection of the recipients was made taking into consideration the recipient's age and length of time waiting for transplantation.
We included a total of 136 transplantations. Transplants were obtained from cadaveric expanded criteria kidney donors who had undergone a preimplantation biopsy. To determine the contribution of renal senescence to graft survival, we compared biopsies obtained from “younger donors” (n=32 donors younger than 60 years) and “older donors” (n=104 donors older than 60 years).
After the study was approved by the institutional ethics committee, clinical and demographic data of the recipients and donors were collected. The donor, recipient, and graft characteristics are reported in Table 1. All data were recorded prospectively in our database.
Patient follow-ups began on the date of renal transplantation and ended on the date of the patient's death, return to dialysis, or the end of the observation period (November 10, 2008). We ensured at least 12 months of follow-up. Immunosuppression regimens included a combination of prednisolone, a calcineurin inhibitor, and azathioprine or mycophenolate (recent protocols). Delayed graft function was defined as the need for dialysis within the first week posttransplant. Renal allograft loss was defined as the need to return to dialysis. All acute rejection episodes were confirmed by biopsy and graded according to the Banff '97 criteria (18, 33). Posttransplant renal allograft function was evaluated by calculating the GFR with the abbreviated Modification of Diet in Renal Disease formula (34).
Kidneys were considered suitable for transplantation after macroscopic evaluation and a favorable histologic assessment. At the moment of transplantation, a preimplantation wedge biopsy was quickly processed to frozen slices to assess graft viability. All samples were graded according to an a priori modified scoring form (17–19) (Table 2). This kidney biopsy-based scoring system ranged from a minimum of 0 (indicating the absence of renal lesions) to a maximum of 15 points (indicating the presence of marked changes in the renal parenchyma). We have weighted our parameters according to the prevailing criteria of chronicity suggested in the Banff scheme plus the criteria used in the study by Remuzzi et al., which uses vascular narrowing fibrosis and glomerulosclerosis as independent parameters (18–21). The degree of glomerulosclerosis was graded from 0 to 3, depending on the percent of glomeruli that were globally sclerotic. The degree of interstitial fibrosis, tubular atrophy, arteriolsclerosis, and arteriolar hyalinosis were graded from 0 to 3, based on definitions suggested by the Banff Schema on allograft pathology (17, 18, 20). The graft was accepted for implantation when the score was less than or equal to 7. Wedge biopsies were at least 10-mm wide and 5-mm depth. Fifty glomeruli per wedge were obtained. Each pretransplant kidney biopsy was evaluated with both frozen section and permanent section histology. Initially, hematoxylin-eosin–stained frozen sections were examined by the pathologist on call. Frozen sections were used for quick assessment of the viability of the graft. After transplantation, paraffin-embedded permanent sections were processed and stained with hematoxylin-eosin, periodic acid-Schiff, and Masson's trichrome stains, and were examined by two pathologists who specialized in transplantation. Permanent section histology was used for the analysis presented in this study. A complete histologic examination included an evaluation of the presence of glomerulosclerosis, interstitial fibrosis, chronic interstitial inflammation, tubular atrophy, and vascular hyalinosis or sclerosis (18, 20).
Categorical variables were expressed as percents and were compared with the chi-square test and the Fisher's exact correction. Quantitative variables were expressed as mean±standard deviations, unless they did not follow a normal distribution. In that case, they were expressed as the median and interquartile range. Variables were compared with the Wilcoxon's rank-sum test, the Kruskal–Wallis test, or the analysis of variance, as appropriate. A multivariate analysis of clinical covariates and the baseline histologic score as potential risk factors that predicted graft function at 1, 3, and 5 years posttransplant were explored with a proportional Cox regression model. The Kaplan-Meier method was used for cumulative survival curves, and the results were compared with the log-rank test. All P values less than 0.05 were considered significant. All statistical analysis was performed with SPSS version 12.0 for Windows.
The authors thank Patricia García Morillo-Velarde for her assistance in database management.
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