A PEFANIS1,2, J MCRAE1, A BONGONI1, U NACHBUR2,3, J MURPHY2,3, F IERINO1,2, P COWAN1,2
1St Vincent’s Hospital, Melbourne, 2The University of Melbourne, 3Walter and Eliza Institute
Background: Acute kidney injury secondary to ischemia-reperfusion injury (IRI) is a common clinical event which contributes to significant patient morbidity and healthcare costs. IRI results from temporary reduction in renal blood flow and subsequent reperfusion. This is followed by an inflammatory state resulting in cell death by apoptosis or necrosis. Necroptosis, a form of regulated necrosis, is triggered by death receptor-mediated recruitment of the kinases RIPK1 and RIPK3 and activation of the pseudokinase MLKL. Active MLKL causes cell death by plasma membrane permeabilisation, driving “necroinflammation”.
Aims: To investigate the role of necroptosis in a mouse model of renal IRI using (i) knockout (KO) mice and (ii) small molecule inhibitors targeting the necroptosis pathway.
Methods: 10-12 week old male mice underwent right nephrectomy followed by 18 minutes left renal ischemia. Samples were collected at 24 hours post reperfusion to assess renal injury, inflammation and necroptosis pathway markers. MLKL KO mice were compared to wild type (WT) littermate controls. A separate cohort of WT mice were treated with a RIPK1 inhibitor (Nec-1s) prior to ischemia.
Results: Expression of necroptotic (RIPK1, RIPK3 and MLKL) and inflammatory (IL-6, IL-1β, IL-33, TNFα) genes was significantly upregulated in renal IRI. MLKL KO mice demonstrated renal protection compared to WT controls, measured by a lower creatinine (µmol/L) (47.13±6.28 vs 80.78±10.45, p=0.007) and less tubular injury (%) (16.79±2.80 vs 26.95±3.68, p=0.048) . Mice receiving Nec-1s had reduced creatinine compared to vehicle controls (115.5±21.24 vs 201.0±17.03, p=0.015).
Conclusions: Our data support a role for necroptosis and necroinflammation in renal IRI. Inhibiting necroptosis pathway components such as RIPK1 and MLKL may provide a future effective therapeutic strategy to minimise kidney injury following renal IRI.
Sia Pefanis is a consultant nephrologist at St Vincent’s Hospital and Barwon Health. She obtained her medical degree from The University of Melbourne, prior to completing a Masters of Public Health at the University of Queensland. Sia is currently undertaking a PhD at St Vincent’s Hospital Immunology Research Centre in the Department of Medicine at The University of Melbourne. She was awarded an NHMRC postgraduate scholarship investigating the role of regulated necrosis in renal ischemia reperfusion injury under the supervision of Professor Peter Cowan and Professor Frank Ierino.