Proteomic profiling of discarded human marginal kidneys undergoing hypothermic oxygenated perfusion: a comparison of treatment with versus without MSC-derived extracellular vesicles

Mesenchymal stromal cell-derived extracellular vesicles (EVs) have emerged as a promising strategy to enhance organ preservation during hypothermic perfusion. In this study, we evaluated their effects on human kidneys discarded from transplantation due to marginal donor status and high histological damage score. Eight kidneys were perfused for 240 min using either hypothermic oxygenated perfusion alone (HOPE; n = 4) or HOPE supplemented with EVs (HOPE+EVs; n = 4). Tissue samples were collected at baseline (T0) and after perfusion (T240) for mass spectrometry-based proteomics, and key findings were validated by immunohistochemistry and real-time PCR. EVs supplementation promoted a pro-survival and anti-senescence cellular state, characterized by the upregulation of antioxidant/pro-survival kinases (OSR1 and AKT2), downregulation of senescence-associated regulators (CDK6 and GSK3β), enhancement of cytoskeletal stability, and metabolic reprogramming. These molecular changes translated into tangible tissue-level improvements, including reduced expression of the cellular senescence marker (p16), increased proliferation (Ki-67), and improved epithelial integrity (E-cadherin). Our findings demonstrate that EV treatment enhances marginal kidney resilience, offering critical mechanistic insights into a novel strategy to improve organ quality for transplantation. Trial registration ClinicalTrials.gov ID:NCT03837197.