199 Effectiveness of hypothermic machine perfusion and static cold storage as delivery methods of a novel RIPK1 inhibitor into pig kidneys and its effects on ischaemia-reperfusion injury

Abstract Introduction Ischaemia-reperfusion injury (IRI) is inevitable in transplantation. Necroptosis is a form of cell death in kidney IRI and phosphorylation of RIPK1 plays a key role in this pathway. This study investigated the effectiveness of delivering GNE684 -a RIPK1 kinase inhibitor- during static cold storage (SCS) and hypothermic machine perfusion (HMP) in an ex-vivo model of kidney IRI. Methods Pig kidneys underwent 1h warm ischaemia and were retrieved. In the SCS model, kidney pairs (n=6) were randomised to treatment with UW+GNE684 via the renal artery (5µM) or control. Kidneys underwent 7h SCS in UW±GNE684 followed by 4h ex-vivo normothermic reperfusion with whole blood (NR). In the HMP model, kidney pairs (n=6) were randomised to HMP alone or treatment with UW-MPS+GNE684 (5µM) for 12h followed by 8h NR. Results In both SCS and HMP groups increasing levels of cytokines and injury markers (e.g. LDH, AST, IL-6) were observed without significant differences between control and treatment groups. HMP led to higher GNE684 tissue concentrations post preservation compared to SCS (3143±385.9 vs 1036.22±453.49ng/ml;P<0.0001) and during NR (987.5±507.6 vs 213.7±37.37ng/ml at t=4h;P=0.013) and levels stayed steady until t=8h NR. GNE684 in both models reduced pRIPK1 at 1h NR but the effect persisted at 4h NR in the HMP model. Discussion This study showed the superiority of HMP over SCS as a delivery method of GNE684 to ischaemically injured pig kidneys. GNE684 did not have marked effects on inflammation or IRI but did inhibit RIPK1 activation in the necroptosis pathway.