**3. Results**

#### *3.1. Kidney Procurement Cohort and Rate of Kidney Discard*

During the study period, 25,154 kidneys were recovered from deceased donors with KDPI > 85%. A total of 3014 kidneys recovered for dual-kidney transplant and 134 kidneys from donors with a body weight < 20 kg were excluded. A total of 22,006 kidneys with KDPI > 85% were included in the kidney procurement cohort. Of these kidneys, 58.0% had 0–10% GS, 13.5% had 11–20% GS, 19.7% had >20% GS, and 8.8% had no kidney biopsy performed (Figure S1). Overall, the rate of kidney discard was 48.5%; 33.6% in 0–10% GS, 68.9% in 11–20% GS, and 77.4% in >20% GS. 49.8% kidneys were discarded in the no kidney biopsy group.

#### *3.2. Kidney Transplant Recipient Cohort*

In this cohort of 22,006 deceased donor kidneys with KDPI > 85%, 10,662 kidneys were discarded. After excluding 1032 recipients with prior kidney transplants or undergoing multi-organ transplant, a total of 10,312 recipients with donor KDPI > 85% were included in the post-transplant outcome analysis. Of these patients, 75.6% had 0–10% GS, 11.9% had 11–20% GS, 4.9% had >20% GS, and 7.6% had no kidney biopsy performed (Figure S2). The median (IQR) number of glomeruli in each kidney biopsy was 47 (IQR: 28, 69). There was no association between KDPI and percent of GS (*p* = 0.70). The donor, recipient, and transplant-related characteristics stratified by percent of GS are shown in Table 1.


**Table 1.** Characteristics of donors, recipients, and transplant according to percent GSin transplanted allograft.

GS, glomerulosclerosis; HCV, hepatitis C virus; BMI, body mass index; PRA, panel reactive antibody; HLA, human leukocyte antigen.

#### *3.3. Baseline Characteristics Based on Percentages of Glomerulosclerosis*

Table 2 summarizes and compares donor, recipient, and transplant-related characteristics between 0–10% and >10% GS allograft groups. Kidneys donors with >10% GS had a higher prevalence of female

sex, diabetes and hypertension. Kidney donors with 0–10% GS had a higher prevalence of donation after cardiac death and positive hepatitis C antibody. Recipients of kidneys with GS > 10% were older and had longer dialysis vintage, whereas recipients of kidneys with 0–10% GS had higher panel reactive antibodies. Kidney transplants with >10% GS had more HLA DR mismatch, cold ischemic time, and thymoglobulin induction. Kidney transplants with 0–10% GS had more transplants without induction therapy.

**Table 2.** Comparison of donors, recipients, and transplant characteristics between GS 0–10% and GS > 10% transplanted allografts.


GS, glomerulosclerosis; HCV, hepatitis C virus; BMI, body mass index; PRA, panel reactive antibody; HLA, human leukocyte antigen.

#### *3.4. Post-Transplant Outcomes Based on Percentages of Glomerulosclerosis*

The median (IQR) follow-up was 4.87 (2.90, 7.02) years after kidney transplant. During follow-up, 3015 (29.2%) patients had allograft failure, and 4433 (43.0%) patients died. A total of 1443 (14.0%) patients had acute rejection within one year, and 3436 (33.3%) patients had delayed graft function. Figure 1 compares death-censored graft survival between 0–10% and >10% GS. Graft survival rate at 5 years was 75.8% for 0–10% GS and 70.9% for >10% GS (*p* < 0.001).

**Figure 1.** Kaplan–Meier death-censored graft survival curves between 0–10% and >10% allograft glomerulosclerosis (GS) groups.

In unadjusted analysis, kidneys with >10% GS were associated with a 24% higher risk of graft failure compared to kidneys with 0–10% GS (HR 1.24; 95% CI 1.13–1.36, *p* < 0.001). After adjusting for baseline donor, recipient, and transplant-related factors, kidneys with >10% GS remained significantly associated with a 27% higher risk of graft failure compared to kidneys with 0–10% GS (HR 1.27; 95% CI 1.15–1.40, *p* < 0.001) (Table S2). Of note, there was no difference in death-censored graft survival between 11–20% GS and >20% GS (Figure 2 and Table S1). There was no significant difference in patient survival (HR 1.03; 95% CI 0.96–1.12, *p* = 0.40), rate of acute rejection at 1-year (HR 1.13; 95% CI 0.97–1.31, *p* = 0.11), and rate of delayed graft function (HR 1.10; 95% CI 0.98–1.23, *p* = 0.11) between 0–10% GS and >10% GS (Table S2).

We examined the graft outcomes of >85% KDPI kidney with a low degree of GS, compared with 71–85% KDPI kidneys. The death-censored graft survival at 5 years in >85% KDPI kidneys with 0–10% GS was inferior to in 71–85% KPDI kidneys (75.8% vs. 81.2%; *p* < 0.001), as shown in Figure 3.

#### *3.5. Characteristics and Outcomes of Kidneys with No Biopsy Performed*

Kidneys donors with no biopsy performed were younger, more were female, and had a greater prevalence of positive hepatitis C antibody, but had a lower prevalence of diabetes, hypertension, body weight, donation after cardiac death, use of machine perfusion, and expanded criteria donation when compared with kidneys with 0–10% GS (Table 1). Graft survival rate at 5 years was comparable between 0% and 10% GS and the no biopsy group (75.8% vs. 74.8%; *p* = 0.62), as shown in Figure 2.

**Figure 2.** Kaplan–Meier death-censored graft survival curves according to percent glomerulosclerosis (GS) in allografts.

**Figure 3.** Kaplan–Meier death-censored graft survival curves between the kidney donor profile index (KDPI) 71–85% group and the KDPI >85% with 0–10% percent glomerulosclerosis (GS) group.

## **4. Discussion**

Over 700,000 patients in the United States have ESKD, with the United States having the secondhighest incidence rate of treated ESKD in the world [30]. Despite an improvement in dialysis care over the last 15 years, the overall survival on dialysis remains dismal with 22% at one year, 43% at three years and 58% at five years [31]. The risk of death is reduced by up to 66% with kidney transplantation [31]. A major limitation to increasing the number of kidney transplantations is the number of donors. It is thus of paramount importance to decrease the discard rates of high KDPI kidneys, which is estimated to be as high as 50% [8,32].

Our study showed that procurement biopsies are becoming increasingly common in marginal deceased donors in the United States. Ninety-one percent of KDPI >85% kidneys were biopsied on procurement during this study period compared to 85% between 2000 and 2003 in the United States [33]. The utility of procurement biopsies has been debated as they can delay decisions, require high resources, prolong duration of cold ischemia, and lead to unnecessary kidney discard [21,22]. Furthermore, the reliability of GS degree in predicting graft outcomes has been questioned [8,32]. While several studies have reported increased delayed graft function risk, leading to poor outcomes in kidneys with GS > 20% [19,25,28,34,35], and other studies have conversely reported similar prognoses in kidneys with GS > 20% compared to kidneys with lower GS [24,26,27,29]. Banff guidelines for procurement biopsies therefore discourage the use of rigidly defined histologic cutoffs for organ decision and allocation [19].

Using the UNOS database, we demonstrated that GS > 10% is an independent prognostic factor for graft failure in >85% KDPI kidneys, with an adjusted 1.28-fold increased risk of graft failure at 5 years when compared to kidneys with 0–10% GS. The findings of our study sugges<sup>t</sup> that the use of GS percentage in procurement biopsy of >85% KDPI kidneys may improve risk stratification for recipient allograft survival. While GS > 10% was associated with a higher risk of graft failure in >85% KDPI kidneys, we did not find a di fference in death-censored graft survival between allografts with 11–20% GS and >20% GS. This may sugges<sup>t</sup> that >10% GS in procurement biopsies can potentially be utilized as a cuto ff for risk prediction in clinical practice. Given that the presence of GS > 10% in >85% KDPI kidneys had no significant impact on delayed graft function rate, acute rejection, or patient survival, the underlying explanation for higher graft failure in GS > 10% kidneys is likely due to the progressive kidney aging process in a kidney with less residual function. As the phenotype of GS is associated with podocyte detachment and a reduced number of functioning and viable glomeruli, this leads to increasing ESKD prevalence [36,37]. It has been estimated that an allocation strategy based on pretransplant donor biopsy would increase the incidence of marginal KDPI (80% to 100%) renal transplants by over 20%, which would translate into an overall increase of 4% for the entire pool of donors [38]. Our study supports the clinical utility of the pretransplant biopsy.

This data should not discourage the use of >85% KDPI kidneys with >10% GS. There is an organ shortage with a growing number of individuals who develop ESKD every year [39] combined with a non-proportional limited supply of potential donors [32]. Overall, one-year post-transplant outcomes have improved since 2007, when the Centers for Medicare and Medicaid Services (CMS) solid organ transplant regulation was first implemented [40]. However, there is still a lack of long term graft and survival outcomes [41–44]. Although transplantation with KDPI > 85% kidneys might be associated with an increased delayed graft function rate and reduced graft survival [45], it is clearly evident based on the lower mortality rate that recipients benefit from transplantation of high-KDPI kidneys when compared with those who wait for low-KDPI kidneys [46,47]. Thus, instead of discarding >85% KDPI kidneys with >10% GS due to a higher risk of allograft loss, future studies are needed to identify techniques and strategies to improve the use and outcome of these "marginal" transplantable kidneys safely. Certain strategies are already being used, such as dual transplantation (both kidneys from one donor into the same recipient) [38,48–52] or creation of a protocol designed to timely identify and match suitable patient characteristics with these "marginal" kidneys (e.g., balancing the number of viable nephrons supplied within the graft versus the metabolic demand of the recipient [32]). For example, a >85% KDPI kidney with >10% GS recovered from a female donor with a low BMI may not be the

best option for a male candidate with a BMI>35 kg/m<sup>2</sup> [53]; further studies are needed to identify other patient and donor characteristics that would yield optimal outcomes.

Although our study aimed to assess the impact of GS degree on >85% KDPI graft outcomes, the findings of our study cannot be generalized to lower KDPI kidneys. We did compare graft outcomes between KDPI >85% kidneys with 0–10% GS to the overall KDPI 71–85% kidneys. This demonstrated that graft outcomes of KDPI >85% kidneys with 0–10% GS were inferior to KDPI 71–85% kidneys, suggesting a stronger impact of KDPI-related factors on graft outcomes over the percentage of GS on procurement biopsies. As KDPI is comprised of several clinically important donor characteristics that impact outcomes [11], it is hypothesized that these characteristics would similarly have an impact on biopsy pathology that is not limited to GS. Thus, GS percentage should not be used in isolation from other biopsy findings for individualized organ acceptance decisions. In addition, the impact GS on graft outcomes of lower KDPI scores remains unclear, since many lower KDPI kidneys are not biopsied [17–19].

Although our study using the UNOS database is among the largest cohorts investigating procurement biopsies with KDPI > 85%, there are some major limitations. First, there is a lack of uniform criteria for procuring, processing and interpreting procurement graft biopsies [19,54]. Core needle biopsies (during reperfusion) are usually superior to wedge biopsies (during procurement), as wedge biopsies primarily obtain sub capsular tissue, which can overestimate the amount of GS [24,26,32]. Specimens are frozen sections as opposed to paraffin-embedded tissue obtained for regular kidney biopsies or biopsies at reperfusion [21,32]. Procurement biopsies are also often interpreted by on-call general pathologists rather than nephro-pathologists. Unfortunately, the numbers of glomeruli in samples or type of pathologist were not reported in the registry. Thus, more studies aimed at optimizing assessment of procurement biopsy samples to optimally allocate organs are needed. Second, data on other important biopsy parameters in the UNOS database, such as interstitial fibrosis, tubular atrophy, and arteriosclerosis, were limited. Only 30 patients in our cohort had available reports on the degree of interstitial fibrosis, tubular atrophy, or arteriosclerosis. Therefore, future studies evaluating the predictive role of a complete histological evaluation [55], including glomerular, tubular, interstitial, and vascular compartments of >85% KDPI kidneys, are required. Furthermore, GS percentage was reported in the database as 0–10%, 11–20%, and >20%. Thus, kidney transplant outcomes using a higher cut-off of GS percentage could not be evaluated and required future studies. Furthermore, given the differences between procurement biopsies and reperfusion biopsies [18], the findings of our study cannot be generalized to reperfusion biopsies for >85% KDPI kidneys. Finally, the registry may be subjected to selection bias. Kidneys from donors that did not undergo biopsy tended to have less unfavorable clinical characteristics, than those with biopsy as demonstrated in our study (kidney donors in the no biopsy group were younger and had a lower prevalence of diabetes and hypertension), and thus had comparable graft survival rate when compared to the 0–10% GS group, but superior to the >10% GS group. Kidneys with a higher degree of GS were likely to be more carefully selected for unreported factors, including other biopsy characteristics. Alternatively, the kidney discards in each GS percentage cohort may have been impacted by other non-reported factors that influenced study outcomes.

In conclusion, we demonstrated that procurement biopsies for >85% KDPI kidneys are very commonly obtained in the United States, at a rate of 91.2%. A higher percentage of GS in >85% KDPI kidney biopsies are associated with an increased discard rate. Among KDPI >85% kidneys, GS >10% is an independent risk factor for allograft failure. However, graft survival from 0–10% GS kidneys is still inferior to kidneys with KDPI 71–85%, suggesting a stronger impact of KDPI on graft outcomes. Instead of discarding kidneys, future studies are needed to identify strategies to optimally utilize these "marginal" kidneys safely.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2077-0383/9/5/1469/s1, Figure S1: Procurement cohort, Figure S2: Kidney transplant recipient cohort, Table S1: Univariable and multivariable cox regression analyses for post-transplant outcomes between Glomerulosclerosis >20% vs. 11−20%, Table S2: Univariable and multivariable cox regression analyses for post-transplant outcomes between Glomerulosclerosis >10% vs. 0–10%.

**Author Contributions:** Conceptualization, W.C., C.T., P.K.V., S.K., L.S.C., E.I., J.F. and N.L.; Data curation, W.C. and N.L.; Formal analysis, W.C., A.C. and N.L.; Investigation, W.C., C.T. and N.L.; Methodology, W.C., C.T. and N.L.; Project administration, T.B.; Resources, T.B. and N.L.; Software, N.L.; Supervision, C.T., F.L.K., M.A.M., T.B., S.A.S., S.K., L.S.C., E.I., J.F. and N.L.; Validation, W.C., C.T., A.C., P.H., F.L.K., M.A.M., S.A.S. and N.L.; Visualization, W.C. and C.T.; Writing—original draft, W.C.; Writing—review & editing, W.C., C.T., P.K.V., A.C., P.H., F.L.K., M.A.M., T.B., S.A.S., S.K., L.S.C., E.I., J.F. and N.L. W.C., C.T., P.K.V., A.C., P.H., F.L.K., M.A.M., T.B., S.A.S., S.K., L.S.C., E.I., J.F. and N.L. All authors have read and agreed to the published version of the manuscript.

**Acknowledgments:** None. All authors had access to the data and played essential roles in writing of the manuscript.

**Conflicts of Interest:** The authors declare no conflict of interest.
