Serum aminoacylase-1 is a novel biomarker with potential prognostic utility for long-term outcome in patients with delayed graft function following renal transplantation

Abstract

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.

Figure 1

Schematic showing the study design using samples undergoing renal transplantation at the St James's University Hospital in Leeds. Following initial discovery of aminoacylase-1 (ACY-1) as a novel potential serum marker for delayed graft function (DGF) using mass spectrometry, this was validated using an immunoassay for ACY-1, and the performance as a predictive marker assessed. Using serum samples from a further independent cohort of patients, these results were confirmed and additionally the association with long-term outcome in those patients with DGF was examined.

Figure 2

Serum aminoacylase-1 (ACY-1) concentrations preoperatively and on day 2 postoperatively in the delayed graft function (DGF) and non-DGF groups used for initial biomarker discovery (five patients per group). ACY-1 as measured by (a) mass spectrometry label-free intensity and (b) subsequent enzyme-linked immunosorbent assay for the same samples. LFQ, label-free quantification. A–J indicate different patients.

Figure 3

Examples of profiles for serum aminoacylase-1 (ACY-1), creatinine (Cr), and cystatin C concentrations longitudinally following renal transplantation in three patients with different clinical courses. (a) Uncomplicated transplant, (b) delayed graft function (a marked peak in ACY-1 concentration >200 ng/ml was seen in 10/15 patients with DGF (66.7%) but only 6/32 (18.8%) non-DGF patients), (c) acute rejection (AR; *) followed by two urinary tract infections (UTIs; ↓). The pre-transplant concentrations are shown as day 0. Full profiles for all cohort 1 patients and associated clinical events and measurements are provided in Supplementary Data—Profiles.

Figure 4

Serum aminoacylase-1 (ACY-1) concentrations on day 1 post transplant in the delayed graft function (DGF) and non-delayed graft function groups. (a) Cohort 1 and (b) cohort 2, and (c) on day 1 post renal transplant in patients in cohort 2 with DGF, slow graft function (SGF), and immediate graft function (IGF). Tukey's box plots show median values and interquartile ranges with significant differences between the groups indicated as determined by the Mann–Whitney test.

Figure 5

Receiver operating characteristic curves for the prediction of delayed graft function, each showing serum aminoacylase-1 (ACY-1), creatinine (SCr), cystatin C (CystC), and ACY-1 combined with cystatin C. (a) Cohort 1 results—days 1/2 post transplant (n=47 but n=35 for ACY-1 and CystC); (b) Cohort 2 results—day 1 post transplant (n=194 but n=138 for ACY-1 and 128 for CystC). AUC, area under the curve; CI, confidence interval.

Figure 6

Serum aminoacylase-1 (ACY-1) concentrations (log scale) on day 1 or 3 post transplant for delayed graft function (DGF) and non-DGF patients in cohort 2, separated by donor type. Medians are indicated by the horizontal bar. Within the non-DGF group, significant differences were seen between each donor type (P<0.001) with median values of 35.2, 107, and 15.6 ng/ml for donation after brain death (DBD), donation after cardiac death (DCD), and live donor (LD), respectively, and similarly within the DGF group between DBD and DCD groups with median values of 70.3 and 483.2 ng/ml. Comparing DGF and non-DGF groups, significantly higher ACY-1 concentrations were seen in patients receiving transplants from both DBD (P=0.023) and DCD (P<0.001).

Figure 7

Kaplan–Meier estimates of survival function for dialysis-free survival post renal transplant in cohort 2 separated by salient characteristics, where median follow-up was 5.93 years with a range of 0.02–7.90 years. (a) Delayed graft function (DGF) and (b) non-DGF patients separated by serum concentrations of aminoacylase-1 (ACY-1) on day 1 post transplant (or day 3 if no day 1 measurement was available). Numbers of events were: 1/28 (3.6%) with ACY-1⩾200 and 9/24 (37.5%) with ACY-1<200 for DGF patients, and 2/16 (12.5%) with ACY-1⩾200 and 5/89 (5.6%) with ACY-1<200 for non-DGF patients. (c) DGF and (d) non-DGF patients separated by donor type (donation after cardiac death (DCD) and donation after brain death (DBD)). Numbers of events were: 1/21 (4.8%) for DCD and 9/31 (29%) for DBD within the DGF patients, and 1/19 (5.3%) for DCD and 6/86 (7%) for DBD in the non-DGF group. (e) DGF patients with donor type DBD separated by ACY-1 concentration (as above). Numbers of events were: 0/9 (0%) for DBD/ACY-1⩾200 and 9/22 (40.9%) for DBD/ACY-1<200. Reasons for return to dialysis included recurrent focal segmental glomerular fibrosis, vascular rejection, and chronic scarring on biopsy.