Abstract
Ischemia-reperfusion injury (IRI) is a hallmark for tissue injury in donation after circulatory death (DCD) kidneys. The implementation of hypothermic machine perfusion (HMP) provides a platform for improved preservation of DCD kidneys. Doxycycline administration has shown protective effects during IRI. Therefore, we explored the impact of doxycycline on proteolytic degradation mechanisms and the urinary proteome of perfused kidney grafts. Porcine kidneys underwent 30 min of warm ischemia, 24 h of oxygenated HMP (control/doxycycline) and 240 min of ex vivo reperfusion. A proteomic analysis revealed distinctive clustering profiles between urine samples collected at T15 min and T240 min. High-efficiency undecanal-based N-termini (HUNTER) kidney tissue degradomics revealed significantly more proteolytic activity in the control group at T-10. At T240, significantly more proteolytic activity was observed in the doxycycline group, indicating that doxycycline alters protein degradation during HMP. In conclusion, doxycycline administration during HMP led to significant proteomic and proteolytic differences and protective effects by attenuating urinary NGAL levels. Ultimately, we unraveled metabolic, and complement and coagulation pathways that undergo alterations during machine perfusion and that could be targeted to attenuate IRI induced injury.
Highlights
Renal transplantation is the most effective treatment for patients suffering from endstage renal disease [1]
Global analysis resulted in 2955 identified peptides that were matched to 303 unique proteins at a 1% false discovery rate (FDR)
Urinary proteomics was performed on collected ultra-filtrate during ex vivo reperfusion using a label-free quantitative proteomics workflow
Summary
Renal transplantation is the most effective treatment for patients suffering from endstage renal disease [1]. Reperfusion is essential for the reintroduction of oxygen, reperfusion itself causes additional injury due to massive mitochondrial production of reactive oxygen species (ROS), ATP depletion, cytoskeletal dysfunction and intracellular Ca2+ accumulation, which subsequently leads to activation of various injury pathways [7,8,9]
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