American Journal of Transplantation 2015; 15: 2143–2151 Wiley Periodicals Inc.

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Copyright 2015 The American Society of Transplantation and the American Society of Transplant Surgeons doi: 10.1111/ajt.13260

Transplanting Kidneys From Deceased Donors With Severe Acute Kidney Injury R. L. Heilman1,*, M. L. Smith2, S. M. Kurian3, J. Huskey1, R. K. Batra4, H. A. Chakkera1, N. N. Katariya4, H. Khamash1, A. Moss4, D. R. Salomon3 and K. S. Reddy4 1

Department of Medicine, Mayo Clinic, Phoenix, AZ Departments of Laboratory Medicine and Pathology, Mayo Clinic, Phoenix, AZ 3 Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, CA 4 Department of Surgery, Mayo Clinic, Phoenix, AZ
Corresponding author: Raymond L. Heilman, [email protected] 2

kidney disease profile index; LC/MS, liquid chromatography/mass spectrometry; LOS, length of hospital stay; MMF, mycophenolate mofetil; OPO, organ procurement organization; PRA, panel reactive index; RMA, robust multichip average; r-ATG, rabbit-antithymocyte globulin; SCD, standard criteria donor Received 14 December 2014, revised 21 January 2015 and accepted for publication 05 February 2015

Background With increasing disparity between the number of patients waiting for a kidney transplant and the donor pool, transplant centers have been expanding the criteria for acceptability of organs and donors with acute kidney injury (AKI) are one such group. Several single center studies have demonstrated good 1-year graft survival with utilizing kidneys from select donors with AKI (1–6). However, most of these reports include small number of patients and the selection criteria for donors with AKI in previous reported series are highly variable. In addition, the severity of AKI is not always well defined. We presume that they include a significant proportion of kidneys from donors with less severe AKI.

Our aim was to determine outcomes with transplanting kidneys from deceased donors with acute kidney injury, defined as a donor with terminal serum creatinine 2.0 mg/dL, or a donor requiring acute renal replacement therapy. We included all patients who received deceased donor kidney transplant from June 2004 to October 2013. There were 162 AKI donor transplant recipients (21% of deceased donor transplants): 139 in the standard criteria donor (SCD) and 23 in the expanded criteria donor (ECD) cohort. 71% of the AKI donors had stage 3 (severe AKI), based on acute kidney injury network (AKIN) staging. Protocol biopsies were done at 1, 4, and 12 months posttransplant. One and four month formalin-fixed paraffin embedded (FFPE) biopsies from 48 patients (24 AKI donors, 24 non-AKI) underwent global gene expression profiling using DNA microarrays (96 arrays). DGF was more common in the AKI group but eGFR, graft survival at 1 year and proportion with IF/TA>2 at 1 year were similar for the two groups. At 1 month, there were 898 differentially expressed genes in the AKI group (p-value 2.0 compared to only 2.1% and 4.9% in the other two groups (7). Transplant rates for the recovered kidneys in these groups were 92%, 84%, and 71%, respectively. Importantly, the rate of graft loss was not increased in recipients of kidneys from donors with terminal creatinine >2.0.

Abbreviations: AKI, acute kidney injury; AKIN, acute kidney injury network; ATI, acute tubular injury; BMI, body mass index; CIT, cold ischemia time; CKD, chronic kidney disease; DCD, donation after cardiac death; DGF, delayed graft function; ECD, expanded criteria donor; eGFR, estimated glomerular filtration rate; FDR, false detection rate; FFPE, formalin-fixed paraffin embedded; GC/MS, gas chromatography/mass spectrometry; HLA, human lymphocyte antigen; IF/TA, interstitial fibrosis and tubular atrophy; KDPI,

We report here an experience with 162 kidney transplants using kidneys from deceased donors with AKI (defined as terminal creatinine 2.0 mg/dL, or the donor requiring acute renal replacement therapy), one of the largest studies to date. The severity of the AKI was classified using the acute kidney injury network (AKIN) criteria (8). The aims of this study were to determine the clinical outcomes including renal function and pathology findings on protocol biopsies done at 1, 4, and 12 months posttransplant. Global 2143

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gene expression profiling was done on formalin-fixed paraffin embedded blocks (FFPE) at 1 and 4 months to assess levels of tissue injury and inflammation at the molecular level in parallel.

Patients and Methods This study was approved by the Mayo Clinic Institutional Review Board as a retrospective, single center case control study of patients receiving a deceased donor kidney transplant between June 2004 and October 2013. Patients who received dual organ transplants were excluded. The AKI group included all kidneys from donor with terminal creatinine 2.0 mg/dL, or a donor requiring acute renal replacement therapy. The control group included all kidneys transplanted from deceased donors with terminal creatinine 60 years old or donor >50 years old with at least two of the following criteria: death from cerebral vascular accident, history of hypertension or creatinine >1.5 mg/dL (9). All other donors were classified as SCD. Delayed graft function (DGF) was defined as a recipient requiring dialysis within the first 7 days after transplantation. The severity of AKI was classified using the AKIN criteria with stage 3 (terminal creatinine >3 times the baseline value, i.e. the donor admission serum creatinine or 4 mg/dL or a donor requiring renal replacement therapy) being the most severe (8). Selection criteria for accepting AKI kidneys at our program evolved over the period of this study. Initially, we excluded AKI kidneys from ECD donors, donation after cardiac death (DCD) donors, donors with an elevated initial serum creatinine or donors with history of hypertension or diabetes. Over time, our criteria evolved to only exclude kidneys with poor perfusion pump parameters (i.e. terminal flow 0.40) or when the pre-implantation biopsy revealed cortical necrosis or moderate or severe chronic changes. We compared transplants done in the more recent period to the earlier cohort to determine whether liberalizing the selection criteria had any impact. In most cases, preprocurement biopsies were reviewed at our center by a team including a transplant surgeon, nephrologist, and pathologist. A repeat biopsy was obtained if the biopsy sample was not adequate. Most of the AKI kidneys were pumped with the exception of some young donor kidneys without any chronic changes by biopsy. Recipients were selected according to UNOS match run with no other special selection. All recipients gave informed consent. All patients received induction immunosuppression. Prior to 2011, patients received induction with rabbit-antithymocyte globulin (r-ATG). After 2011, induction was with alemtuzumab. Patients over age 65 received basiliximab, which did not change during the study period. Patients receiving induction with the depleting agents had complete withdrawal of corticosteroids by posttransplant day 5 while those receiving basiliximab induction continued maintenance corticosteroids. Maintenance immunosuppression was typically with tacrolimus and mycophenolate mofetil (MMF). Tacrolimus was started on posttransplant day 1 or 2, irrespective of DGF. Goals for trough tacrolimus levels were 8–10 ng/mL for the first month and then 6–8 ng/mL. At our center, we discharge patients on day 3 irrespective of delayed graft function (DGF) and monitor them closely in the outpatient setting. Patients with ongoing DGF will have a repeat Doppler ultrasound and biopsy of the allograft at about 2 weeks posttransplant. Our clinical protocol includes postreperfusion biopsy on all deceased donors. All the available postreperfusion biopsies for the AKI donor group and a similar number of reperfusion biopsies from the control group matched for date of transplantation were reread by one pathologist blinded to study

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group assignment. The biopsies were classified using Banff 2007 (10) criteria and for acute tubular injury (ATI) score (from 0 to 4) and presence of glomerular thrombi. Our clinical protocol includes surveillance biopsies at 1, 4, and 12 months after kidney transplantation. Banff 2007 criteria were used to quantify acute and chronic histologic findings (10). We compared the chronic allograft changes between groups by calculating the fraction of biopsies at each time point with the sum of Banff interstitial fibrosis score (ci score, scale 0–3) plus the Banff tubular atrophy score (ct score, scale 0–3) greater than 2 (IF/TA>2). In order to determine the utilization rate for AKI kidneys offered, reasons for not utilizing and opportunities to increase utilization rates, we reviewed all kidney offers to our center during the period from July 2012 to June 2013. We reviewed the donor history, anatomy, pathology, and other details from UNET to determine potentially usable kidneys according to our selection criteria and documented the reasons for not utilizing the remaining.

Methods for Gene Expression Profiling One and four month formalin-fixed paraffin embedded (FFPE) biopsies from 48 kidney transplant recipients (24 AKI donors, 24 controls; 96 total samples) were profiled using Affymetrix HT HG-U133 Plus PM plate arrays on a GeneTitan MC instrument. All AKI donor samples were randomly selected from the SCD cohort with adequate FFPE samples available. AKI donor and controls samples were matched by date of transplantation. Total RNA was extracted from four 20-m sections using Ambion RecoverAll Total Nucleic Acid Kit and processed for chip hybridization using the new Affymetrix SensationPlusTM FFPE Amplification and 30 IVT Labeling kit according to the manufacturer’s protocol. Quantile data normalization by RMA and analysis was performed using Partek Genomics Suite for FDR-corrected differential gene expression by ANOVA. Pathway mapping was performed using Ingenuity Pathway Analysis (IPA). The .CEL file data for the 96 arrays described in this study are available through the NIH GEO repository, accession # GSE61739.

Statistical analysis Descriptive analysis was done using t-tests for continuous variables and Chisquares for categorical variables. Continuous variables are displayed as the mean  standard deviation. Data that were heavily skewed were compared by nonparametric tests. All p-values are two sided. Survival analysis and comparisons of risk of rejection between groups were done using the Kaplan–Meier method and a log-rank test. The data were analyzed using C 2010 SAS Institute Inc.). JMP Pro 9.0.1 (

Results The study cohort was comprised of 162 AKI donor transplant recipients: 139 in the SCD group (23% of SCD kidney transplants) and 23 in the ECD group (14% of ECD kidney transplants). The control group included 609 non-AKI donor transplant recipients: 472 SCD kidney recipients and 137 ECD kidney recipients. For the SCD cohort, the AKI donors were more likely to be male and of Black race, less likely to be from DCD donors, have longer cold ischemia time (CIT) and were less likely to be from donors with stroke as the cause of death (Table 1). American Journal of Transplantation 2015; 15: 2143–2151

Acute Kidney Injury Donors Table 1: Baseline donor and recipient characteristics SCD cohort

Donor age Donor gender male Donor race black DCD donor Imported kidney Pulsatile pump KDPI score Death from stroke Cold ischemic time (hours) Recipient age Recipient female Recipient race black Recipient BMI Diabetic pretransplant Preemptive transplant Previous transplant HLA mismatch PRA>20%

ECD cohort

AKI-group (n ¼ 139)

Non-AKI group (n ¼ 472)

p-value

AKI-group (n ¼ 23)

Non-AKI group (n ¼ 137)

p-value

32.3  13.2 78% 12% 3% 48% 66% 42  21 9% 20.4  8.0 52.9  12.9 42% 8% 30.5  5.6 45% 11% 12% 3.9  1.6 21%

34.5  15.4 61% 7% 16% 28% 22% 39  22 24% 15.9  7.3 53.6  12.8 43% 11% 29.3  5.9 39% 15% 15% 3.5  2.0 29%

0.12