Potential protective effects of doxycycline on renal degradome during hypothermic machine perfusion and reperfusion

L.L. van Leeuwen, L.H. Venema, H.G.D. Leuvenink, B.M. Kessler

Wednesday 4 march 2020

14:10 - 14:20h at Joep Nicolas zaal

Parallel session: Parallel sessie IV – Basale abstracts

Background: Donation after circulatory death donor kidneys (DCD) are used to enlarge the donor pool, however these kidneys are exposed to ischemia/reperfusion injury (IRI), making them more susceptible to chronic failure. In the Netherlands, hypothermic machine perfusion (HMP) is the standard way of preserving kidneys for clinical transplant. HMP allows you to add therapies to a functioning organ. Doxycycline shows to have protective effects during renal IRI. Therefore, our aim was to protect DCD kidneys from IRI using doxycycline during HMP and observe its effect on kidney function and the renal degradome.

Methods: Porcine kidneys (n=7) received 30min of warm ischemia, 24h of HMP with or without doxycycline, and 4h of ex vivo reperfusion. Renal cortex samples were obtained after HMP and after reperfusion. We performed a high-efficiency undecanal-based N termini enrichment workflow combined with nano-flow reversed phase chromatography-tandem mass spectrometry to identify the degradome. KEGG pathway analysis of proteins significantly up- or downregulated was performed using STRING 11 and the Merops protease database was used for matching proteolytic events to the most probable responsible protease.

Results: 717 peptides and 265 proteins were identified as the renal degradome. The addition of doxycycline during HMP led to significantly less protein degradation after HMP and after reperfusion compared to control. After 24h of HMP, 17 proteins were significantly more degraded and 3 proteins were less degraded in the control group compared to the group with added doxycycline. 4h of reperfusion led to 38 proteins that were significantly more degraded and 7 proteins that were less degraded in the control group compared to the doxycycline group. Degradation after reperfusion in the control group occurred mostly in metabolic pathways and more specifically in oxidative phosphorylation. These proteolytic events are most probably caused by a range of cathepsins, matrix metalloprotease 2 and Meprin α and β. No significant differences in renal function were observed during reperfusion.

Conclusions: Addition of doxycycline during HMP protects the kidney against protein degradation and disruption of metabolic processes during HMP and reperfusion. These identified degradation profiles could play an important role in provoking short- and long-term graft failure caused by IRI. Doxycycline could be a potential pharmacological intervention strategy to attenuate protein degradation during HMP of DCD kidneys.