Two independent pathways of regulated necrosis mediate ischemia–reperfusion injury

TLDR

Regulated necrosis can arise via cyclophilin‑D–mediated mitochondrial permeability transition or receptor‑interacting protein kinase‑1–mediated necroptosis, but it is unknown whether these converge into a single pathway or represent distinct routes. In vivo studies of cisplatin‑induced acute kidney injury and hyperacute TNF‑shock in mice revealed that cyclophilin‑D–mediated MPT is mechanistically separate from RIPK1/RIPK3‑driven necroptosis. RIPK3‑deficient mice were protected from ischemia‑reperfusion injury, with stronger protection than CypD‑deficient mice; double knockouts and combined pharmacologic inhibition confirmed that two independent regulated‑necrosis pathways coexist and that targeting both may improve treatment of ischemic injury.

Abstract

Regulated necrosis (RN) may result from cyclophilin (Cyp)D-mediated mitochondrial permeability transition (MPT) and receptor-interacting protein kinase (RIPK)1-mediated necroptosis, but it is currently unclear whether there is one common pathway in which CypD and RIPK1 act in or whether separate RN pathways exist. Here, we demonstrate that necroptosis in ischemia-reperfusion injury (IRI) in mice occurs as primary organ damage, independent of the immune system, and that mice deficient for RIPK3, the essential downstream partner of RIPK1 in necroptosis, are protected from IRI. Protection of RIPK3-knockout mice was significantly stronger than of CypD-deficient mice. Mechanistically, in vivo analysis of cisplatin-induced acute kidney injury and hyperacute TNF-shock models in mice suggested the distinctness of CypD-mediated MPT from RIPK1/RIPK3-mediated necroptosis. We, therefore, generated CypD-RIPK3 double-deficient mice that are viable and fertile without an overt phenotype and that survived prolonged IRI, which was lethal to each single knockout. Combined application of the RIPK1 inhibitor necrostatin-1 and the MPT inhibitor sanglifehrin A confirmed the results with mutant mice. The data demonstrate the pathophysiological coexistence and corelevance of two separate pathways of RN in IRI and suggest that combination therapy targeting distinct RN pathways can be beneficial in the treatment of ischemic injury.