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POTENTIAL PROTECTIVE EFFECTS OF DOXYCYCLINE ON RENAL PROTEOME AND DEGRADOME DURING HYPOTHERMIC MACHINE PERFUSION AND REPERFUSION OF PORCINE KIDNEYS

Introduction: Donation after circulatory death (DCD) kidneys are exposed to ischemia/reperfusion injury (IRI), making them more susceptible to fibrosis and chronic failure. In the Netherlands, ex vivo hypothermic machine perfusion (HMP) is the standard way of preserving kidneys for clinical transplant. HMP provides the opportunity to add therapies to a functioning organ, allowing you to directly manipulate and improve its condition. Doxycycline shows to have protective effects during renal IRI, however this has not been tested in a transplant setting. Here we explore the impact of doxycycline on proteolytic degradation mechanisms and urinary proteome of perfused kidney grafts. Our aim was to protect DCD kidneys from IRI using doxycycline during HMP and observe its effect on kidney function and the renal proteome and degradome. Materials and Methods: Porcine kidneys received 30 minutes of warm ischemia, 24 hours of oxygenated HMP with or without doxycycline, and 4 hours of ex vivo reperfusion. We performed a high-efficiency undecanalbased N termini enrichment workflow combined with nano-flow reversed phase chromatography-tandem mass spectrometry to identify the degradome. A proteomic analysis was performed on urine samples collected during reperfusion using high-throughput label-free quantitation mass spectrometry. Results: 717 peptides and 265 proteins were identified as the renal degradome, and 304 proteins as the urinary proteome. Supplementation of doxycycline during HMP led to less protein degradation (FIG. 1). Degradation in the control group post-reperfusion occurred mostly in metabolic pathways and are most probably caused by range of cathepsins, matrix metalloprotease 2 and Meprin α and β. Urinary analysis also showed significant upregulation of proteins related to metabolic pathways in the control (FIG 2). For example, LDH and superoxide dismutase were both significantly upregulated (FIG 2B). Degradation in the doxycycline group occurred mostly in proteins related to metabolic pathways. However, proteins related to complement and coagulation cascades were mostly up-regulated in the urinary proteome, in particular complement C1s subcomponent and coagulation factor V (FIG 2A). Conclusion: The addition of doxycycline during HMP led to significant proteomic differences, indicating that doxycycline potentially works under hypothermic conditions. Doxycycline possibly protects the kidney against disruption of metabolic processes during HMP, but can activate complement and coagulation cascades during reperfusion. These identified protein profiles could play an important role in protein remodelling and wound healing after IRI. Doxycycline could be a potential pharmacological intervention strategy to attenuate protein degradation in DCD kidneys during HMP.