Proteomic evidence of anti senescent reconditioning by mesenchymal stromal cell derived extracellular vesicles in human marginal kidneys during hypothermic oxygenated perfusion
Sonia Spinelli1, Marilena Gregorini2, Maria Antonietta Grignano2, Stefania Bruno3, Simona Granata4, Gianluigi Zaza4, Giuliana Germinario5, Emma Diletta Stea2, Tefik Islami2, Palma Minutillo2, Eleonora Francesca Pattonieri2, Gabriele Ceccarelli6, Enrico Verrina1, Matteo Ravaioli5, Teresa Rampino2, Maurizio Bruschi1,7.
1Laboratory of Molecular Nephrology, IRCCS Istituto Giannina Gaslini, Genoa, Italy; 2Department of Nephrology, Dialysis and Transplantation, IRCCS Policlinico San Matteo, Pavia, Italy; 3Department of Medical Sciences and Molecular Biotechnology Center, University of Torino, Torino, Italy; 4Division of Nephrology, Dialysis and Transplantation, Annunziata Hospital, Cosenza, Italy; 5Department of General Surgery and Transplantation, IRCCS Azienda Ospedaliero-Universitaria di Bologna, Bologna, Italy; 6Human Anatomy Unit, Department of Public Health, Experimental and Forensic Medicine, University of Pavia, Pavia, Italy; 7Department of Experimental Medicine (DIMES), University of Genoa, Genoa, Italy
Background:
Kidneys from extended criteria donors (ECDs) are increasingly utilized to address organ shortages, but their elevated susceptibility to ischemia-reperfusion injury (IRI) poses a significant challenge. Cellular senescence and structural deterioration are key contributors to this vulnerability. Hypothermic oxygenated perfusion (HOPE) has shown promise in improving graft quality, and the addition of mesenchymal stromal cell-derived extracellular vesicles (EVs) may further enhance protective and regenerative outcomes.
Methods:
Eight human ECD kidneys were perfused ex vivo for 240 minutes using HOPE, with four kidneys receiving stromal cell-derived EVs and four serving as HOPE-only controls. Biopsies were collected at baseline, 180, and 240 minutes. Renal tissue and EVs cargo were analyzed via high-resolution mass spectrometry. Differential protein expression and pathway enrichment analyses were performed.
Results:
Proteomic analysis identified 2,424 proteins, of which 710 displayed significant time-dependent variation. At 240 minutes, 27 proteins were differentially expressed between the HOPE and HOPE+EVs groups, indicating a specific molecular effect of EVs treatment. Gene ontology enrichment revealed modulation of 21 biological processes, including pathways associated with senescence, oxidative stress, energy metabolism, cytoskeletal organization, and immune response. Proteomic characterization of the EVs confirmed the presence of several regulatory protein kinases linked to these pathways. Immunohistochemistry confirmed reduced p16(INK4a) expression and increased Ki67 in HOPE+EVs-treated kidneys, consistent with anti-senescent and regenerative effects.
Conclusions:
EVs derived from mesenchymal stromal cells induce a distinct proteomic reprogramming in marginal human kidneys during HOPE, promoting anti-senescent, antioxidant, and pro-regenerative responses. These effects appear to be mediated by EV-associated proteins. Stromal EVs represent a promising therapeutic strategy to recondition ECD kidneys before transplantation. Further in vivo validation is warranted.
[1] Extracellular Vesicles
[2] Mesenchymal Stromal Cells
[3] Hypothermic Oxygenated Perfusion
[4] Proteomics
[5] Senescence
[6] Ischemia-Reperfusion Injury
[7] Kidney Transplantation
[8] Extended Criteria Donors
[9] Regeneration