Necrostatin-1 attenuates renal ischemia/reperfusion injury by inhibiting necroptosis, oxidative stress, and inflammation via mediation of HIF-1α/miR-26a/TRPC6/PARP1 signaling

Abstract: Necroptosis, oxidative stress, and inflammation are major contributors to the pathogenesis of ischemic acute kidney injury. Necrostatin-1 (Nec-1), an inhibitor of the kinase domain of receptor-interacting protein kinase-1 (RIP1), has been reported to regulate renal ischemia/reperfusion (I/R) injury; however, its underlying mechanism of action remains unclear. HK-2 cells were used to create an in vitro I/R model, in which the cells were subjected to hypoxia, followed by 2 h, 6 h, and 12 h of reoxygenation. For the in vivo study, a rat model of renal I/R was established in which samples of rat blood serum and kidney tissue were harvested after reperfusion to assess renal function and detect histological changes. Cell viability and necroptosis were analyzed using the CCK-8 assay and flow cytometry, respectively. The expression levels of molecules associated with necroptosis, oxidative stress, and inflammation were determined by real-time PCR, western blotting, immunofluorescence staining, and ELISA. Luciferase and chromatin immunoprecipitation (ChIP) assays were performed to confirm the relevant downstream signaling pathway. We found that pretreatment with Nec-1 significantly decreased HIF-1α and miR-26a expression, as well as the levels of factors associated with necroptosis (RIP1, RIP3, and Sirtuin-2), oxidative stress (MDA, NADP + /NADPH ratio), and inflammation (IL-1β, IL-10, and TNF-α) in I/R injury cells and the rat model. However, these effects could be reversed by miR-26a overexpression or TRPC6 knockdown. Mechanistic studies demonstrated that hypoxia inducible factor-1α (HIF-1α) directly binds to the promoter region of miR-26a and that TRPC6 is a potential target gene for miR-26a. Our findings indicate that Nec-1 can effectively protect against renal I/R injury by inhibiting necroptosis, oxidative stress, and inflammation, and may exert its effects through mediation of the HIF-1α/miR-26a/TRPC6/PARP1 signaling pathway.