Abstract
Normothermic ex-vivo kidney perfusion (NEVKP) results in significantly improved graft function in porcine auto-transplant models of donation after circulatory death injury compared with static cold storage (SCS); however, the molecular mechanisms underlying these beneficial effects remain unclear. We performed an unbiased proteomics analysis of 28 kidney biopsies obtained at three time points from pig kidneys subjected to 30 min of warm ischemia, followed by 8 h of NEVKP or SCS, and auto-transplantation. 70/6593 proteins quantified were differentially expressed between NEVKP and SCS groups (false discovery rate < 0.05). Proteins increased in NEVKP mediated key metabolic processes including fatty acid ß-oxidation, the tricarboxylic acid cycle, and oxidative phosphorylation. Comparison of our findings with external datasets of ischemia-reperfusion and other models of kidney injury confirmed that 47 of our proteins represent a common signature of kidney injury reversed or attenuated by NEVKP. We validated key metabolic proteins (electron transfer flavoprotein subunit beta and carnitine O-palmitoyltransferase 2, mitochondrial) by immunoblotting. Transcription factor databases identified members of the peroxisome proliferator-activated receptors (PPAR) family of transcription factors as the upstream regulators of our dataset, and we confirmed increased expression of PPARA, PPARD, and RXRA in NEVKP with reverse transcription polymerase chain reaction. The proteome-level changes observed in NEVKP mediate critical metabolic pathways. These effects may be coordinated by PPAR-family transcription factors and may represent novel therapeutic targets in ischemia-reperfusion injury.
Highlights
Quantitative proteomics of Normothermic ex-vivo kidney perfusion (NEVKP) and cold storage pig kidneys at three time points. Proteins increased in NEVKP are associated with key steps of mitochondrial metabolism. NEVKP attenuates proteins increased in the kidney response to injury in prior studies. In silico and mRNA analyses suggest peroxisome proliferator-activated receptors (PPAR)-family members as likely regulators in NEVKP
As previously reported by our group [41], NEVKP-preserved grafts demonstrated superior kidney function after heterotopic auto-transplantation compared with static cold storage (SCS)-preserved grafts, with significantly lower serum creatinine (SCr) postoperatively in the NEVKP group compared with the SCS (Fig. 1B) (F-test, p < 2.23 × 10−15)
PPARA, PPARD, and RXRA showed significantly increased expression in NEVKP compared with SCS at POD3
Summary
In Brief The molecular changes associated with normothermic ex-vivo kidney perfusion (NEVKP) compared with static cold storage were studied using discovery proteomics in a porcine model. Normothermic ex-vivo kidney perfusion (NEVKP) results in significantly improved graft function in porcine autotransplant models of donation after circulatory death injury compared with static cold storage (SCS); the molecular mechanisms underlying these beneficial effects remain unclear. The proteome-level changes observed in NEVKP mediate critical metabolic pathways These effects may be coordinated by PPARfamily transcription factors and may represent novel therapeutic targets in ischemia-reperfusion injury. We hypothesized that NEVKP would induce key alterations in the renal proteome compared with SCS in a DCD model and that identifying these changes would provide insights into the molecular mechanisms associated with superior graft function in this setting.
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