Proteomic analysis of machine perfusion solution from brain dead donor kidneys reveals that elevated complement, cytoskeleton and lipid metabolism proteins are associated with 1-year outcome

Abstract

Assessment of donor kidney quality is based on clinical scores or requires biopsies for histological assessment. Noninvasive strategies to identify and predict graft outcome at an early stage are, therefore, needed. We evaluated the perfusate of donation after brain death (DBD) kidneys during nonoxygenated hypothermic machine perfusion (HMP). In particular, we compared perfusate protein profiles of good outcome (GO) and suboptimal outcome (SO) 1-year post-transplantation. Samples taken 15 min after the start HMP (T1) and before the termination of HMP (T2) were analysed using quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS). Hierarchical clustering of the 100 most abundant proteins showed discrimination between grafts with a GO and SO at T1. Elevated levels of proteins involved in classical complement cascades at both T1 and T2 and a reduced abundance of lipid metabolism at T1 and of cytoskeletal proteins at T2 in GO versus SO was observed. ATP-citrate synthase and fatty acid-binding protein 5 (T1) and immunoglobulin heavy variable 2-26 and desmoplakin (T2) showed 91% and 86% predictive values, respectively, for transplant outcome. Taken together, DBD kidney HMP perfusate profiles can distinguish between outcome 1-year post-transplantation. Furthermore, it provides insights into mechanisms that could play a role in post-transplant outcomes.

Keywords: biomarker discovery; complement activation; donation after brain death; hypothermic machine perfusion; kidney preservation; proteomics.

Figure 1

Experimental design and workflow. (a) Overview of kidney perfusate sample collection. Perfusate samples were selected on the basis of 1‐year transplant outcomes. Good outcome (GO) was defined as kidney function with an estimated glomerular filtration rate (eGFR) ≥ 45 ml/min/1.73 m² and a serum creatinine level of ≤ 120 µmol/l, whereas suboptimal outcome (SO) was defined as kidneys with an eGFR ≤ 45 ml/min/1.73 m² and a serum creatinine level of ≥ 120 µmol/l. Samples from each kidney were taken at 2 time points: 15 min after the start of HMP (T1) and at the end of HMP (T2). (b) Workflow for proteomics analysis. A total of 44 samples were analysed (11 kidneys per group, 22 samples per outcome). Perfusate samples were chemically depleted and loaded onto a gel for SDS‐PAGE. Albumin bands were removed, and samples were prepared for in‐gel digestion, desalted and analysed using LC‐MS/MS. Raw peaks and spectra were searched against Homo sapiens database and data were analysed. eGFR, estimated glomerular filtration rate; LC‐MS/MS, liquid chromatography–tandem mass spectrometry. (Image created using biorender.com).

Figure 2

Quantitative proteomic analysis of perfusate samples after 15 min of HMP (T1). (a) Heat map and hierarchical clustering of 100 most abundant proteins at T1. (b) STRING analysis of 100 most abundant proteins at T1. Nodes circled with blue represent proteins located in extracellular regions (FDR: 1.39E‐40) and red represent functional enrichment of the KEGG complement and coagulation pathway (FDR: 2.31E‐32). (c) Volcano plot showing differential protein expression of 498 identified proteins at T1 between good outcome (GO) and suboptimal outcome (SO) at 1‐year post‐transplantation. X‐axis demonstrates protein level difference indicated by log2 fold change, and Y‐axis demonstrates statistical significance indicated by ‐log10 (P‐value). A ‐log10 (P‐value) of > 1.3, and a fold change of > 0 was considered significant. Blue dots represent significant downregulated proteins. Red dots represent significantly upregulated proteins. Green dots represent proteins identified using prediction models. (d) STRING pathway analysis of significantly upregulated proteins at T1. Nodes circled with blue represent proteins located in extracellular regions (FDR: 9.01E‐7) and red represent functional enrichment of the KEGG complement and coagulation pathway (FDR: 1.36E‐9). (e) STRING analysis of significantly downregulated proteins at T1. Nodes circled with purple represent proteins involved in PPAR signalling (FDR: 0.004). FDR, false discovery rate.

Figure 3

Quantitative proteomic analysis of perfusate samples taken before the termination of HMP (T2). (a) Heat map and hierarchical clustering of 100 most abundant proteins at T2. (b) STRING analysis of 100 most abundant proteins at T2. Nodes circled with blue represent proteins located in extracellular regions (FDR: 2.28E‐41) and red represent functional enrichment of the KEGG complement and coagulation pathway (FDR: 4.2E‐34). (c) Volcano plot showing differential protein expression of 498 identified proteins at T2 between good outcome (GO) and suboptimal outcome (SO) at 1‐year post‐transplantation. X‐axis demonstrates protein level difference indicated by log2 fold change; Y‐axis demonstrates statistical significance indicated by ‐log10 (P‐value). A ‐log10 (P‐value) of > 1.3, and a fold change of > 0 was considered significant. Blue dots represent significant downregulated proteins. Red dots represent significantly upregulated proteins. Green dots represent proteins identified using prediction models. (d) STRING pathway analysis of significantly upregulated proteins at T2. Nodes circled with red represent functional enrichment of the KEGG complement and coagulation pathway (FDR 1.21e‐8) and purple represents proteins involved in complement activation of the classical pathway (FDR: 1.6E‐6). (e) STRING analysis of significantly downregulated proteins at T2. Nodes circled with turquoise represent functional enrichment GO component cytoskeleton (FDR 1.33e‐5) and green represents proteins affiliated with the immune system (FDR: 4.28E‐5). FDR, false discovery rate.

Figure 4

Receiver‐operated (ROC) curves for the prediction models based on T1 and T2. (a) Perfusate analysis at T1 shows a 91% predictive value for ATP‐citrate synthase (ALCY) and fatty acid‐binding protein 5 (FABP5). (b) Perfusate analysis at T2 shows an 86% predictive value for immunoglobulin heavy variable 2‐26 (IGHV2‐26) and desmoplakin (DSP).