Serum aminoacylase-1 is a novel biomarker with potential prognostic utility for long-term outcome in patients with delayed graft function following renal transplantation
Authors: Matthew P. Welberry Smith, Alexandre Zougman, David A. Cairns, Michelle Wilson, Tobias Wind, Steven L. Wood, Douglas Thompson, Michael P. Messenger, Andrew Mooney, Peter J. Selby, Andrew J.P. Lewington, Rosamonde E. Banks
Journal: Kidney International (2013)

Early identification and prognostic stratification of delayed graft function following renal transplantation has significant potential to improve outcome. Mass spectrometry analysis of serum samples, before and on day 2 post transplant from five patients with delayed graft function and five with an uncomplicated transplant, identified aminoacylase-1 (ACY-1) as a potential outcome biomarker. Following assay development, analysis of longitudinal samples from an initial validation cohort of 55 patients confirmed that the ACY-1 level on day 1 or 2 was a moderate predictor of delayed graft function, similar to serum creatinine, complementing the strongest predictor cystatin C. A further validation cohort of 194 patients confirmed this association with area under ROC curves (95% CI) for day 1 serum (138 patients) of 0.74 (0.67–0.85) for ACY-1, 0.9 (0.84–0.95) for cystatin C, and 0.93 (0.88–0.97) for both combined. Significant differences in serum ACY-1 levels were apparent between delayed, slow, and immediate graft function. Analysis of long-term follow-up for 54 patients with delayed graft function showed a highly significant association between day 1 or 3 serum ACY-1 and dialysis-free survival, mainly associated with the donor–brain–dead transplant type. Thus, proteomic analysis provides novel insights into the potential clinical utility of serum ACY-1 levels immediately post transplantation, enabling subdivision of patients with delayed graft function in terms of long-term outcome. Our study requires independent confirmation. Early identification and prognostic stratification of delayed graft function following renal transplantation has significant potential to improve outcome. Mass spectrometry analysis of serum samples, before and on day 2 post transplant from five patients with delayed graft function and five with an uncomplicated transplant, identified aminoacylase-1 (ACY-1) as a potential outcome biomarker. Following assay development, analysis of longitudinal samples from an initial validation cohort of 55 patients confirmed that the ACY-1 level on day 1 or 2 was a moderate predictor of delayed graft function, similar to serum creatinine, complementing the strongest predictor cystatin C. A further validation cohort of 194 patients confirmed this association with area under ROC curves (95% CI) for day 1 serum (138 patients) of 0.74 (0.67–0.85) for ACY-1, 0.9 (0.84–0.95) for cystatin C, and 0.93 (0.88–0.97) for both combined. Significant differences in serum ACY-1 levels were apparent between delayed, slow, and immediate graft function. Analysis of long-term follow-up for 54 patients with delayed graft function showed a highly significant association between day 1 or 3 serum ACY-1 and dialysis-free survival, mainly associated with the donor–brain–dead transplant type. Thus, proteomic analysis provides novel insights into the potential clinical utility of serum ACY-1 levels immediately post transplantation, enabling subdivision of patients with delayed graft function in terms of long-term outcome. Our study requires independent confirmation. Renal transplantation provides clear benefits for patients with end-stage kidney disease,1.Port F.K. Wolfe R.A. Mauger E.A. et al.Comparison of survival probabilities for dialysis patients vs cadaveric renal transplant recipients.JAMA. 1993; 270: 1339-1343Crossref PubMed Scopus (617) Google Scholar,2.Wolfe R.A. Ashby V.B. Milford E.L. et al.Comparison of mortality in all patients on dialysis, patients on dialysis awaiting transplantation, and recipients of a first cadaveric transplant.N Engl J Med. 1999; 341: 1725-1730Crossref PubMed Scopus (3975) Google Scholar and significant cost savings compared with dialysis.3.de Wit G.A. Ramsteijn P.G. de Charro F.T. Economic evaluation of end stage renal disease treatment.Health Policy. 1998; 44: 215-232Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar,4.Kontodimopoulos N. Niakas D. An estimate of lifelong costs and QALYs in renal replacement therapy based on patients' life expectancy.Health Policy. 2008; 86: 85-96Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar In 2010, 16,151 renal transplants were performed in the United States (http://optn.transplant.hrsa.gov), and 2687 in the United Kingdom (http://www.uktransplant.org.uk). However, early complications can significantly impact clinical and economic outcomes, such as delayed graft function (DGF) that affects ∼20% of patients in the United States.5.Tapiawala S.N. Tinckam K.J. Cardella C.J. et al.Delayed graft function and the risk for death with a functioning graft.J Am Soc Nephrol. 2010; 21: 153-161Crossref PubMed Scopus (143) Google Scholar A number of definitions of DGF have been proposed6.Akkina S.K. Connaire J.J. Israni A.K. et al.Similar outcomes with different rates of delayed graft function may reflect center practice, not center performance.Am J Transplant. 2009; 9: 1460-1466Crossref PubMed Scopus (43) Google Scholar, 7.Roels L. Coosemans W. Donck J. et al.Inferior outcome of cadaveric kidneys preserved for more than 24h in histidine-tryptophan-ketoglutarate solution. Leuven Collaborative Group for Transplantation.Transplantation. 1998; 66: 1660-1664Crossref PubMed Scopus (53) Google Scholar, 8.Chang S.H. Russ G.R. Chadban S.J. et al.Trends in kidney transplantation in Australia and New Zealand, 1993-2004.Transplantation. 2007; 84: 611-618Crossref PubMed Scopus (58) Google Scholar with one commonly used being the need for dialysis in the first week after renal transplantation, other than for isolated hyperkalemia. Although there are parallels with acute kidney injury, the pathology underlying DGF is complex with contributions from donor-derived factors, such as donor age and duration of ischemia, and recipient factors such as ischemia–reperfusion injury (IRI), immunological responses, and immunosuppressant medications.9.Siedlecki A. Irish W. Brennan D.C. Delayed graft function in the kidney transplant.Am J Transplant. 2011; 11: 2279-2296Crossref PubMed Scopus (512) Google Scholar Acute tubular necrosis secondary to IRI is the predominant histological finding but acute cellular or humoral rejection may occur concurrently, and other pathologies are sometimes apparent histologically, e.g., calcineurin inhibitor toxicity. Increasing use of organs donated after circulatory death (DCD) and from extended criteria donors10.Keitel E. Michelon T. dos Santos A.F. et al.Renal transplants using expanded cadaver donor criteria.Ann Transplant. 2004; 9: 23-24PubMed Google Scholar has corresponded with an increase in the incidence of DGF. DGF increases the risk of graft failure, patient death, and death-censored graft failure by two- to three-fold,11.Halloran P.F. Aprile M.A. Farewell V. et al.Early function as the principal correlate of graft survival. A multivariate analysis of 200 cadaveric renal transplants treated with a protocol incorporating antilymphocyte globulin and cyclosporine.Transplantation. 1988; 46: 223-228Crossref PubMed Scopus (204) Google Scholar,12.Snyder J.J. Kasiske B.L. Gilbertson D.T. et al.A comparison of transplant outcomes in peritoneal and hemodialysis patients.Kidney Int. 2002; 62: 1423-1430Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar and is associated with a number of complications that contribute to reduced longer-term graft survival, such as a poor transplant function at 1 year, arterial hypertension, and acute rejection.13.Sola R. Alarcon A. Jimenez C. et al.The influence of delayed graft function.Nephrol Dial Transplant. 2004; 19: iii32-iii37PubMed Google Scholar Overall, DGF has been associated with a 41% increased risk of graft loss at just over 3 years.14.Yarlagadda S.G. Coca S.G. Formica Jr., R.N. et al.Association between delayed graft function and allograft and patient survival: a systematic review and meta-analysis.Nephrol Dial Transplant. 2009; 24: 1039-1047Crossref PubMed Scopus (539) Google Scholar Early identification, stratification, and increased understanding of DGF has significant potential to improve patient management and outcomes,15.Muhlberger I. Perco P. Fechete R. et al.Biomarkers in renal transplantation ischemia reperfusion injury.Transplantation. 2009; 88: S14-S19Crossref PubMed Scopus (36) Google Scholar allowing fluid volume status optimization, timely appropriate dialysis, tailoring of therapies, and avoidance of unnecessary investigation and treatment. There is increasing excitement about the potential of clinical proteomics in identifying new biomarkers with clinical impact,16.Smith M.P. Banks R.E. Wood S.L. et al.Application of proteomic analysis to the study of renal diseases.Nat Rev Nephrol. 2009; 5: 701-712Crossref PubMed Scopus (31) Google Scholar complementing promising markers emerging from genomic-based studies. Urinary markers currently under investigation in renal transplantation include interleukin 18 and neutrophil gelatinase lipocalin,17.Hall I.E. Yarlagadda S.G. Coca S.G. et al.IL-18 and urinary NGAL predict dialysis and graft recovery after kidney transplantation.J Am Soc Nephrol. 2010; 21: 189-197Crossref PubMed Scopus (260) Google Scholar with tissue-associated markers including ICAM-1 and VCAM.15.Muhlberger I. Perco P. Fechete R. et al.Biomarkers in renal transplantation ischemia reperfusion injury.Transplantation. 2009; 88: S14-S19Crossref PubMed Scopus (36) Google Scholar,18.Schwarz C. Regele H. Steininger R. et al.The contribution of adhesion molecule expression in donor kidney biopsies to early allograft dysfunction.Transplantation. 2001; 71: 1666-1670Crossref PubMed Scopus (55) Google Scholar Unfortunately, in the majority of cases of DGF, urine is not produced or may be mixed with residual native renal output confounding analysis of any results, and biopsied tissue is often only available once DGF is established. Although serum neutrophil gelatinase lipocalin and interleukin 18 have not shown promise,19.Hall I.E. Doshi M.D. Poggio E.D. et al.A comparison of alternative serum biomarkers with creatinine for predicting allograft function after kidney transplantation.Transplantation. 2011; 91: 48-56Crossref PubMed Scopus (42) Google Scholar blood-borne biomarkers would be ideal being readily accessible and routinely used in hospital laboratories. However, biomarker discovery with serum or plasma is challenging with only 22 proteins comprising ∼99% of the total protein mass, and the wide dynamic range of protein abundances spanning >10 orders of magnitude.20.Anderson N.L. Anderson N.G. The human plasma proteome: history, character, and diagnostic prospects.Mol Cell Proteomics. 2002; 1: 845-867Crossref PubMed Scopus (3556) Google Scholar In this study (Figure 1), we have compared serum proteins pre- and postoperatively from patients undergoing renal transplantation, with and without DGF, using our previously optimized immunodepletion followed by label-free single-dimensional liquid chromatography-tandem mass spectrometry analysis strategy.21.Smith M.P. Wood S.L. Zougman A. et al.A systematic analysis of the effects of increasing degrees of serum immunodepletion in terms of depth of coverage and other key aspects in top-down and bottom-up proteomic analyses.Proteomics Clin Appl. 2011; 5: 561Crossref Google Scholar A key candidate marker of DGF was identified as aminoacylase-1 (ACY-1). Following assay development and validation, allowing the measurement of ACY-1 in serum for the first time, the predictive use of ACY-1 for DGF as early as day 1 post transplant was confirmed. Using follow-up data from a cohort of 194 transplant patients where as expected DGF was associated with poor prognosis, serum ACY-1 day 1 post transplant was shown to further subdivide the 54 patients with DGF in terms of their long-term outcome. Examination of the patient groups (Figure 1, Table 1) shows similar characteristics of cohorts 1 (the discovery and initial validation group) and 2 (the larger validation group with long-term outcome data), with the exceptions of proportion of DCD transplants and the induction regimen, reflecting changing clinical practice. DGF was diagnosed in 31.9% of patients in cohort 1 and 28.4% of patients in cohort 2. Mean age and cold ischemic time (CIT) were significantly higher in the DGF groups in each cohort, as was warm ischemic time (WIT) in cohort 2. The five DGF and five non-DGF patients used for the initial proteomic discovery had no evidence of calcineurin inhibitor toxicity or acute rejection and were matched as closely as possible in terms of mean age, ethnicity mix, CIT, WIT, and mean HLA mismatches at the A, B, and DR loci, and immunosuppression regimens, differing slightly in donor type (five DCD in the DGF group compared with three DCD/two donation after brain death (DBD) in the non-DGF group).Table 1Demographic and clinical details for the DGF and non-DGF patients in cohorts 1 and 2, following renal transplantation at the St James's University Hospital in LeedsNumber of patients, and subgroupsCohort 1Cohort 2DGFNon-DGFDGFNon-DGFn=15 totaln=32 totaln=55 totaln=1396 Biopsied: 4 acute rejection and 2 ATN8 Biopsied: 6 acute rejection and 2 mild tubular injuryP-value40 Biopsied: 26 ATN, 8 rejection, and 6 both ATN and acute rejection16 Biopsied: 11 ATN, and 5 ATN and acute rejectionP-value9 Not biopsied: 5 calcineurin inhibitor toxicity and 4 assumed ATN24 Not biopsied: 12 uncomplicated, 4 post transplant UTIs, and 8 calcineurin inhibitor toxicityNA15 Not biopsied: all assumed ATN123 Not biopsied: uncomplicated transplantsNACause of ESRD (%) Chronic pyelonephritis0.018.80.57314.512.20.719 Diabetes6.73.112.710.1 Glomerulonephritis33.331.332.730.9 Hypertension6.712.55.510.8 Inherited26.712.514.510.8 Other20.015.67.35.0 Unknown6.76.312.720.1Ethnicity (%) Asian6.76.30.99720.08.60.113 Black/African/Caribbean6.76.31.81.4 Caucasian86.787.558.261.9 Unknown0.00.018.228.1Transplant number (%) 186.778.10.80780.090.60.247 2–413.321.918.27.9 Unknown001.81.4Pre-emptive (%) No100.078.10.12896.492.80.338 Yes0.021.91.86.5 Unknown001.80.7Mean time (months) on dialysis at transplantation±s.d.aExcludes pre-emptive transplants, for second and subsequent transplants, total time on dialysis aggregated;32.8±20.732.2±25.00.94154.6±40.436.5±36.4<0.001Mean age (years)±s.d.52.4±12.140.6±14.10.00750±13.946.2±14.90.046Gender % Male73.359.30.54737.062.60.002Transplant types % DBD20.028.10.006 (0.123)#P-value if LD excluded as rare in DGF.57.461.9<0.001 % DCD80.034.338.913.7 % LD0.037.53.724.5Induction (%) Alemtuzumab26.731.20.98300NA Basiliximab73.368.7100100 Maintenance steroids used (%)26.721.90.99414.810.10.527Mean ischemic time±s.d. Cold (h:min)16:06±03:1110:24±06:080.00216:42±04:4813:48±08:180.019 Warm (min)40±1339±90.78249.2±19.136.9±13.6<0.001Mean total HLA mismatch±sd3.3±1.02.6±1.80.1272.4±1.32.0±1.40.096Abbreviations: ATN, acute tubular necrosis; DBD, donations after brain death; DCD, donations after circulatory death; DGF, delayed graft function; ESRD, end-stage renal disease; HLA, human leukocyte antigen; LD, live donor; NA, not applicable; UTI, urinary tract infection.P-values are provided for differences between the DGF and non-DGF groups in each cohort.Details of biopsies performed, and other postoperative events (e.g., UTIs) were collected along with serum creatinine, tacrolimus concentrations, C-reactive protein concentrations, and urinary protein/creatinine ratios. In addition, eight live donors were included in the study to check for the effects of surgery on marker levels (mean age 44.0±12.3, 37.5% male).a Excludes pre-emptive transplants, for second and subsequent transplants, total time on dialysis aggregated;# P-value if LD excluded as rare in DGF. Open table in a new tab Abbreviations: ATN, acute tubular necrosis; DBD, donations after brain death; DCD, donations after circulatory death; DGF, delayed graft function; ESRD, end-stage renal disease; HLA, human leukocyte antigen; LD, live donor; NA, not applicable; UTI, urinary tract infection. P-values are provided for differences between the DGF and non-DGF groups in each cohort. Details of biopsies performed, and other postoperative events (e.g., UTIs) were collected along with serum creatinine, tacrolimus concentrations, C-reactive protein concentrations, and urinary protein/creatinine ratios. In addition, eight live donors were included in the study to check for the effects of surgery on marker levels (mean age 44.0±12.3, 37.5% male). Across all 20 samples (10 patients, two time points) used for the initial mass spectrometry screen, 553 proteins with at least two peptides (at least one of which was unique) were identified and relative quantification determined (all proteins listed and quantification data shown for selected proteins in Supplementary Data—Proteins 1 and 2). On the basis of the statistical significance (P<0.05), 34 candidates differentiated between DGF and non-DGF groups either preoperatively, postoperatively, or by pattern of change. These included cystatin C demonstrating proof-of-principle. ACY-1 was prioritized for further investigation, being undetectable preoperatively but increasing markedly postoperatively, particularly in the DGF group (Figure 2a). Download .doc (.45 MB) Help with doc files Supplementary Data 1 Download .xls (.08 MB) Help with xls files Supplementary Data 2 Using our in-house ELISA, data for serum ACY-1 broadly supported the mass spectrometry data for the discovery samples (Figure 2). ACY-1 was undetectable (<15.6ng/ml) in all pre-transplant samples in this set, and in cohort 1 as a whole 43/47 patients had undetectable ACY-1 pre-transplant with the remainder being <50ng/ml and with no significant pre-transplant difference between DGF and non-DGF groups. Of the 636 longitudinal samples analyzed, ACY-1 was detected in only 230 samples, predominantly in the 5 days post transplant. Similarly live donors exhibited no ACY-1 concentrations (>50ng/ml) post surgery with 10/12 having undetectable ACY-1 concentrations, indicating that serum ACY-1 concentrations were not affected by surgery per se. Examples of serum ACY-1 profiles with different clinical courses are shown in Figure 3 (full data for all patients including additional clinical events and measurements is in Supplementary Data—Profiles). A peak in ACY-1 concentration (>200ng/ml) was observed at ≤4 days post transplantation in 10/15 patients with DGF (66.7%) but in only 6/32 (18.8%) non-DGF transplants, three of whom had an uncomplicated clinical course immediately post transplant. Longitudinal profiles of ACY-1 concentration did not follow the trends seen in any of serum creatinine, cystatin C, C-reactive protein, tacrolimus, or urinary protein/creatinine ratio, with the exception of a peak in ACY-1 in one patient suffering from tacrolimus toxicity with an extremely high concentration of tacrolimus (trough level 33ng/ml, intended range 9–14ng/ml). Importantly, there was no distinct peak in serum ACY-1 in patients at the time of positive mid-stream urinary cultures (n=19; ACY-1<15.6–88.3ng/ml), episodes of postoperative dialysis, or at the time of biopsy-proven rejection, with the exception of one patient where ACY-1 concentration peaked at 260ng/ml 4 days before biopsy, becoming undetectable by the time of the biopsy. In cohort 2 (the larger final validation set), of the 138 day 1 samples, 30 patients had undetectable serum ACY-1 concentrations (4/42 (9.5%) DGF patients, 26/96 (27.1%) non-DGF), with a similar pattern being seen (18.8% vs. 42.6%, respectively) for day 3 values also (data not shown). In both cohorts, day 1 concentrations of ACY-1 were significantly different between the DGF and non-DGF groups although overlapping (Figure 4a and b). When non-DGF patients from the larger cohort 2 were categorized on the basis of creatinine reduction ratio (CRR=(day 0 creatinine minus day 7 creatinine)/day 0 creatinine),17.Hall I.E. Yarlagadda S.G. Coca S.G. et al.IL-18 and urinary NGAL predict dialysis and graft recovery after kidney transplantation.J Am Soc Nephrol. 2010; 21: 189-197Crossref PubMed Scopus (260) Google Scholar,22.Johnston O. O'Kelly P. Spencer S. et al.Reduced graft function (with or without dialysis) vs immediate graft function—a comparison of long-term renal allograft survival.Nephrol Dial Transplant. 2006; 21: 2270-2274Crossref PubMed Scopus (101) Google Scholar significant differences in day 1 serum ACY-1 (Figure 4c) between patients categorized as slow graft function (CRR<0.7) and immediate graft function (CRR≥0.7) were seen with an increasing trend from immediate graft function to slow graft function to DGF, demonstrating a relationship between the rate of graft function improvement and ACY-1. Similar results were also seen for day 3 serum ACY-1 concentrations (data not shown). For initial exploratory examination of the predictive utility in cohort 1, ACY-1 and cystatin C concentrations for days 1 or 2 were combined as one time point (n=35). The area under the receiver operating characteristic (ROC) curve (AUC) for day 1/2 ACY-1 predicting DGF was 0.74, with corresponding figures for creatinine and cystatin C of 0.79 and 0.92, respectively. ACY-1 and cystatin C combined improved the AUC to 0.94 (Figure 5a). In cohort 2, the AUC for day 1 ACY-1 was 0.77 with corresponding figures for creatinine and cystatin C of 0.75 and 0.9, respectively, with the latter improving to 0.93 if combined with ACY-1 (Figure 5b). Data-derived optimum cut-points for day 1 serum ACY-1 and cystatin C in cohort 2 (Table 2) show high specificity and sensitivity, respectively. The optimum cut-point from the combination of both through a logistic regression model provide higher sensitivity and similar specificity, as is apparent through the increased Youden index23.Youden W.J. Index for rating diagnostic tests.Cancer. 1950; 3: 32-35Crossref PubMed Scopus (7227) Google Scholar (0.71 vs. 0.43 and 0.64; Table 2).Table 2Estimates and 95% confidence intervals for measures of diagnostic accuracy for ACY-1, cystatin C, serum creatinine, and combined serum ACY-1 and cystatin C for optimal cut-offs as determined in cohort 2 data for day 1 measurements (for serum Cr the additional condition that specificity was >50% was applied)EstimateLCIUCIACY-1 (cut-off 200ng/ml) Sensitivity54.7639.7169.81 Specificity88.5482.1794.91 PPV67.6551.9283.37 NPV81.7374.3089.16 OR9.353.9022.40 Youden index0.43Cystatin C (cut-off 3.3μg/ml) Sensitivity87.1876.6997.67 Specificity76.4067.5885.23 PPV61.8248.9874.66 NPV93.1587.3698.95 OR22.027.6463.47 Youden index0.64Serum creatinine (cut-off 550μmol/l) Sensitivity79.6368.8990.37 Specificity55.5646.8864.23 PPV43.4333.6753.20 NPV86.4278.9693.88 OR4.892.3110.35 Youden index0.35ACY-1 + cystatin C (cut-off probability 0.2) Sensitivity92.3183.94100 Specificity78.6570.1487.16 PPV65.4552.8978.02 NPV95.8991.34100 OR44.2112.26159.37 Youden index0.71Abbreviations: ACY-1, aminoacylase-1; LCI, lower confidence interval; NPV, negative predictive value; OR, odds ratio relative to diagnostic cut-off; PPV, positive predictive value; UCI, upper confidence interval. Open table in a new tab Abbreviations: ACY-1, aminoacylase-1; LCI, lower confidence interval; NPV, negative predictive value; OR, odds ratio relative to diagnostic cut-off; PPV, positive predictive value; UCI, upper confidence interval. Using the larger final validation cohort 2, significant associations were observed between serum ACY-1 concentration and biopsy-proven acute tubular necrosis, transplant type, age at transplantation, CIT, total WIT, and day 1 serum creatinine and cystatin C (Table 3). On univariate analysis, day 1 serum ACY-1, cystatin C and creatinine concentrations, and transplant type, WIT and total HLA mismatch were all significantly associated with the development of DGF (Table 4). A multivariable logistic regression model for the prediction of DGF was developed incorporating day 1 serum ACY-1, creatinine and cystatin C concentrations, patient age and gender, transplant type (DBD, DCD), HLA mismatch, CIT, WIT, and initial steroid use. Day 1 serum ACY-1 was significant using the likelihood ratio test (P=0.013; Table 4) and maintained a high, if reduced, odds ratio compared with univariate analysis. Cystatin C was the only other independent predictor of DGF in this model (P<0.0001).Table 3Associations between day 1 serum ACY-1 concentration and relevant variables in cohort 2 (n=138 with day 1 samples)CharacteristicSubgroupNMedian (range)P-value*Spearman's rank correlation coefficient and P-value if a single continuous variable, or median (range) and P-value from Wilcoxon–Mann–Whitney test or Kruskal–Wallis test if comparing two or three subgroups.GenderM9043.4 (15.6, 7324.5)F4874.5 (15.6, 7207.2)0.1277Age at transplantation (years)—1380.300.0003Transplant typeDBD7948.3 (15.6, 5905.3)DCD29365.6 (29.3, 7324.5)LD3015.6 (15.6, 156.1)<0.0001CIT (min)—1380.250.0033Total WIT (min)—1380.53<0.0001Total HLA mismatch0, 1, and 28250.7 (15.6, 5905.3)3, 4, 5, and 65676.2 (15.6, 7324.5)0.1261Initial steroid useY1572.3 (15.6, 1094.2)N12353.2 (15.6, 7324.5)0.9397DGFY42253.7 (15.6, 7324.5)N9633.6 (15.6, 864.1)<0.0001Biopsy proven ARY1166.3 (15.6, 1094.2)N12753.4 (15.6, 7324.5)0.6354Biopsy proven ATNY29143.9 (15.6, 7324.5)N10944.0 (15.6, 1225.1)0.0007Both ATN and AR on biopsyY4140.8 (18.8, 511.1)N13453.7 (15.6, 7324.5)0.5965Serum creatinine day 1 (μmol/l)—1300.250.0050Serum cystatin C day 1 (μg/ml)—1280.41<0.0001Abbreviations: ACY-1, aminoacylase-1; AR, acute rejection; ATN, acute tubular necrosis; CIT, cold ischemic time; DBD, donation after brain death; DCD, donation after cardiac death; DGF, delayed graft function; F, female; HLA, human leukocyte antigen; LD, live donor; M, male; N, no; WIT, warm ischemic time; Y, yes.'Initial steroid use' refers to whether patients received maintenance oral prednisolone as part of their initial daily immunosuppression regime. Biopsy results—a total of 32 of these 138 patients had biopsies, and therefore the N category for the biopsy results includes those who did not undergo a biopsy.* Spearman's rank correlation coefficient and P-value if a single continuous variable, or median (range) and P-value from Wilcoxon–Mann–Whitney test or Kruskal–Wallis test if comparing two or three subgroups. Open table in a new tab Table 4Univariate and multivariable analysis of factors associated with delayed graft function in cohort 2 as determined by logistic regressionFactorLevelUnivariateMultivariableOdds ratio95% CIP-valueOdds ratio95% CIP-valueGenderMale11Female0.901(0.456, 1.780)0.76450.888(0.223, 3.377)0.8612Age at transplantation (years)—1.015(0.992, 1.039)0.20851.000(0.959, 1.043)0.9871Transplant typeDBD11DCD3.066(1.457, 6.453)0.00320.730(0.086, 6.181)0.7724CIT (min)—1(0.998, 1.001)0.57201.000(0.997, 1.003)0.8496Total WIT (min)—1.040(1.017, 1.064)0.00071.043(0.991, 1.097)0.1040Total HLA mismatch0, 1, and 2113, 4, 5, and 62.500(1.255, 4.979)0.00911.679(0.415, 6.798)0.4676Initial steroid useN11Y1.239(0.424, 3.618)0.69492.465(0.290, 20.99)0.4089Serum ACY-1 day 1 (per increase 1μg/ml)—31.17(3.400, 285.7)0.002313.82(1.321, 434.9)0.0115Serum creatinine day 1 (per increase 1μmol/l)—1.004(1.002, 1.006)<0.00011.001(0.997, 1.004)0.7526Serum cystatin C day 1 (per increase 1μg/ml)—5.536(2.775, 11.05)<0.00016.865(2.678, 17.60)<0.0001Abbreviations: ACY-1, aminoacylase-1; CI, confidence interval; CIT, cold ischemic time; DBD, donation after brain death; DCD, donation after cardiac death; HLA, human leukocyte antigen; N, no; WIT, warm ischemic time; Y, yes.'Initial steroid use' refers to whether patients received maintenance oral prednisolone as part of their initial daily immunosuppression regime. P-values from Wald test for all variables other than multivariable ACY-1 day 1, where likelihood ratio test P-value is more appropriate because of larger relative odds ratio. Open table in a new tab Abbreviations: ACY-1, aminoacylase-1; AR, acute rejection; ATN, acute tubular necrosis; CIT, cold ischemic time; DBD, donation after brain death; DCD, donation after cardiac death; DGF, delayed graft function; F, female; HLA, human leukocyte antigen; LD, live donor; M, male; N, no; WIT, warm ischemic time; Y, yes. 'Initial steroid use' refers to whether patients received maintenance oral prednisolone as part of their initial daily immunosuppression regime. Biopsy results—a total of 32 of these 138 patients had biopsies, and therefore the N category for the biopsy results includes those who did not undergo a biopsy. Abbreviations: ACY-1, aminoacylase-1; CI, confidence interval; CIT, cold ischemic time; DBD, donation after brain death; DCD, donation after cardiac death; HLA, human leukocyte antigen; N, no; WIT, warm ischemic time; Y, yes. 'Initial steroid use' refers to whether patients received maintenance oral prednisolone as part of their initial daily immunosuppression regime. P-values from Wald test for all variables other than multivariable ACY-1 day 1, where likelihood ratio test P-value is more appropriate because of larger relative odds ratio. A risk assessment plot for this full multivariable model (new model) and the model without ACY-1 (reference model) are shown in Supplementary Figure S1 online for patients with and without DGF. Neither model showed evidence of a lack of goodness of fit