The effect of ischemia on protein degradation during renal transplantation

L.L. van Leeuwen, B.M. Kessler, R.J. Ploeg, H.G.D. Leuvenink

Wednesday 13 march 2019

14:18 - 14:20h at Koningin Máximazaal

Categories: Basaal, Parallelsessie

Parallel session: Parallelsessie III - Basaal I

Background: Degradation mechanisms underlying transplant related renal ischemia are still not fully understood, and the proteolytic events causing this protein degradation are still undefined. Proteomic profiling with the use of mass spectrometry has been widely adapted in medical research, including the field of transplantation. N-terminal amine isotopic labelling of substrates(TAILS) is a specialized technique that is used for protease substrate discovery and N-terminome analysis. To date, this method has only scarcely been used for clinical research. The main objectives of this study were to test and optimize a TAILS to gain insights in the degradation mechanisms during warm ischemia and static cold storage.

Methods: We used a TMT-TAILS based workflow to characterize the N-terminome during different periods of warm ischemia time (WIT) and cold ischemia time (CIT) of human kidneys.

Results: 1218 peptides and 691 proteins were identified as the WIT N-terminome, and 2511 peptides and 1649 proteins were identified as the CIT N-terminome.We found 29 proteins that were significantly up or downregulated during medium and long warm ischemia compared to short warm ischemia. 19 of these proteins are located in extracellular regions, and 5 of these proteins play a role in the PI3K-Akt signalling pathway.Moreover, our results show that proteins of the extracellular matrix are significantly degraded during warm ischemia. Based on both our findings, and previous findings, these proteins are mostly degraded by proteases that are known for the degradation and remodeling of the extracellular matrix, namely matrix metalloproteinase (MMP) 2 and 9. Nonetheless, we identified that many proteins involved in oxidative phosphorylation, reduction of H202, and many cytoskeletal proteins are degraded during warm and cold ischemia. We also identified cleavage of calpain activity and degradation of its activator calpastatin.

Conclusions: Taken all together, these identified degradation mechanisms and proteolytic events could play an important role in the loss of kidney function caused by ischemia.Thereby offering pharmacological intervention strategies to modulate proteolytic pathways to attenuate proteolytic tissue degradation during renal ischemia, storage and preservation.