Necroptosis plays a role in kidney injury following ischemia-reperfusion

Abstract

Pefanis, Aspasia1,2,3; Bongoni, Anjan K1; McRae, Jennifer L1; Salvaris, Evelyn J1; Fisicaro, Nella1; Murphy, James M4,5,6; Ierino, Francesco L2,3; Cowan, Peter J1,2*

Necroptosis plays a role in kidney injury following ischemia-reperfusion 1. Immunology Research Centre, St Vincent’s Hospital, Melbourne, Victoria, Australia 2. Department of Medicine, The University of Melbourne, Victoria, Australia 3. Department of Nephrology, St Vincent’s Hospital, Melbourne, Victoria, Australia 4. Walter and Eliza Hall Institute of Medical Research, Parkville, Australia 5. Department of Medical Biology, The University of Melbourne, Parkville, Australia 6. Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, VIC Australia

Aims Ischemia-reperfusion injury (IRI) occurs during kidney transplantation and is associated with delayed graft function, rejection and kidney fibrosis. Necroptosis, a pathway of regulated necrosis, is triggered by recruitment of the intracellular kinases RIPK1 and RIPK3 and activation of the pseudokinase MLKL resulting in cell death by plasma membrane rupture.

The aims of this study were to (1) investigate the role of necroptosis in a mouse model of kidney IRI, using knockout mice and small molecule inhibitors, and (2) determine whether inhibition of RIPK1 or RIPK3 after IRI interferes with the progression to kidney fibrosis.

Methods 1) 10-12 week old male MLKL KO mice and wild type (WT) littermates underwent right nephrectomy followed by left kidney ischemia. Samples were collected at 24 h to assess renal injury, inflammation and necroptosis. In a separate experiment WT mice were treated either before or after IR with the RIPK1 inhibitor Nec-1s, the RIPK3 inhibitor GSK872, or vehicle. 2) In this fibrosis model, WT mice were treated with Nec-1s, GSK872 or vehicle daily on days 3-9 following IR, with samples collected at 28 days to evaluate fibrosis.

Results MLKL KO mice were protected from IRI, with lower creatinine (47.13 ± 6.26 vs 80.78 ± 10.45; p=0.002) and less tubular injury (16.79 ± 2.80 vs 26.95 ± 3.68; p=0.048) compared to WT littermates. WT mice receiving Nec-1s before (p<0.01) or after IR (p<0.05) showed reduced kidney injury compared to vehicle controls, whereas GSK872 was not protective. In the fibrosis model, GSK872 (p<0.01) or Nec-1s (p<0.01) treatment reduces kidney fibrosis at 28 days compared to vehicle control.

Conclusion Our data support a role for necroptosis in acute kidney injury and fibrosis following IR, and identifies inhibition of the necroptosis pathway as a potential therapeutic strategy.

Impact This study enhances understanding of the molecular mechanisms of kidney IRI, and hold significant implications for novel targeted therapies to improve kidney transplant outcomes.