Integration of Proteomics and Metabolomics to Unravel the Mechanism of Ischemia Reperfusion Injury in Donor Kidneys for Transplantation.

Abstract

Increased acceptance of higher risk donor organs for transplantation, results in an increased risk of compromised outcomes after kidney transplantation. These organs are exposed to ischemia/reperfusion injury (IRI) which has detrimental effects on organ function and survival. Understanding the underlying pathophysiological events, at the molecular level, that occur following IRI is still not well defined. In this study, we used a rat model employing proteomcis and metabolomics approaches to evaluate the molecular changes associated with IRI. IRI was induced by clamping left kidney for 45 min in male Fischer rats, allowing for 4 hours of reperfusion (n=6). The right kidney served as an internal control, and healthy rodents used as additional controls (n=2). Renal cortical samples, containing the proximal convoluted tubules were micro-dissected for proteomics and metabolomics studies. Liquid chromatography tandem mass spectrometry based label free quantitative proteomics was employed to measure tryptic digested kidney lysates. NMR spectroscopy was used to measure aqueous fractions extracted from cortex of the same samples. In total, 1390 proteins were identified with 64 proteins shortlisted (>2 fold change, ANOVA P<0.05) between IRI and control groups. Among 64 differentially expressed proteins, 4 up-regulated proteins were associated with acute renal failure. Proteins associated with decreased transmembrane potential and depolarization of mitochondria were up-regulated as were enzymes involved in amino acid and fatty acid metabolism. 1H-NMR spectra were acquired and 29 metabolites identified. Principle component analysis using all spectra showed clear separation of IRI against controls. Amongst 29 identified metabolites, creatinine and leucine were up regulated in IRI vs healthy controls, consistent with acute renal injury and dysregulation of amino-acid metabolism. A decrease of glucose and AMP, NAD+, and increased lactate may indicate dependence on glycolysis and mitochondrial dysfunction during IRI. In summary, this study demonstrates successful use of proteomics and metabolomics to evaluate IRI in a rat model revealing specific molecular signatures. Preventing acute renal injury by preserving mitochondrial function through pre or post conditioning will lead to improved organ quality and improve transplant outcome.