Characterization of the perfusate proteome of human donor kidneys

Abstract

Background: Preservation of deceased donor kidneys by hypothermic machine perfusion results in superior transplant outcomes as compared with static cold storage and provides the opportunity to measure biomarkers of cellular injury in perfusate samples. Identification of biomarkers predicting early graft dysfunction so far has met with limited success. Methods: Two-dimensional difference gel electrophoresis and mass spectrometry were used to explore the proteome of perfusate samples from machine-perfused human donor kidneys (N ¼ 18) and to discover novel biomarkers of ischaemic acute kidney injury. Results: Thirty-two protein spots were successfully identified, representing 19 unique proteins that were derived from renal tissue and from residual plasma in the renal microcirculation. Two unidentified protein spots were significantly upregulated, whereas one protein spot -identified as haptoglobin -was significantly down-regulated in the perfusate of ischaemically injured kidneys from donors after cardiac death as compared with kidneys from brain-dead donors who had not suffered warm ischaemic injury. Furthermore, two protein spots were up-regulated in kidneys that never functioned after transplantation, whereas one spot was up-regulated -identified as a1-antitrypsin -in kidneys with delayed graft function. Conclusions: We provide the first description of the renal perfusate proteome and present preliminary evidence of differentially expressed biomarkers in human donor kidneys with different levels of acute ischaemic injury. Their diagnostic value for the selection of marginal kidneys in clinical transplantation should be determined in future studies.

Associations of Perfusate Biomarkers and Pump Parameters With Delayed Graft Function and Deceased Donor Kidney Allograft Function

“…Total GST, heart‐type fatty acid–binding protein, redox‐active iron and NGAL were not associated with primary nonfunction, and no measured biomarkers were associated with 1‐year graft failure. Snoeijs et al used 1‐h perfusate samples from 18 kidneys to demonstrate that α1‐antritrypsin was upregulated in kidneys that developed DGF . Guy et al performed one‐dimensional proton‐nuclear magnetic resonance spectroscopy on 45‐min and 4‐h perfusate samples from 26 kidneys to show that 28 different metabolites varied in concentration throughout HMP, whereas certain metabolites (leucine, inosine, gluconate, and glucose) predicted DGF, with areas under the curve ranging between 0.732 and 0.895 .…”

Proteomic Analysis of Perfusate from Machine Cold Perfusion of Transplant Kidneys: Insights Into Protection from Injury

“…Few studies have been done looking at the proteins in the perfusate of kidneys preserved with machine cold perfusion. One group [ 29 ] compared the perfusates of kidneys from uncontrolled DCDD, controlled DCDD, and DNDD kidneys using 2-dimensional gel electrophoresis, and identified 19 proteins that differed between the groups. They also identified A1AT, albumin, and immunoglobulins as being significantly different between their 3 groups.…”

Machine perfusion viability testing

“…Although perfusate concentration was associated with transplant outcome, its predictive value for PNF was poor (AURC 0.503) [22 & ]. However, their diagnostic value in viability testing has to be determined in future studies [25]. In DCD kidneys, it was associated with an increased risk of PNF and DGF (OR 1.001; 95% CI 1.000-1.002; P ¼ 0.003 and OR 1.003; 95% CI 1.001-1.004; P ¼ 0.002, respectively).…”