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 and 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, 6, 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 Cell Counting Kit (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 hypoxia-inducible factor-1α (HIF-1α) and miR-26a expression, as well as the levels of factors associated with necroptosis (RIP1, RIP3, and Sirtuin-2), oxidative stress (malondialdehyde [MDA], NADP+/NADPH ratio), and inflammation (interleukin [IL]-1β, IL-10, and tumor necrosis factor alpha [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 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.
Highlights
Renal ischemia and reperfusion (I/R) injury, the major cause of acute renal failure, typically occurs after renal transplantation or surgery, and is one of the most serious and common health problems seen in the clinic.[1,2] The pathogenic mechanism underlying renal I/R injury is complicated and involves energy metabolism dysfunction,[3] tubular necrosis and apoptosis,[4] inflammation,[5,6] and oxidative stress.[7]
Nec-1 Treatment Increased Cell Viability and Reduced the Number of Necrotic HK-2 Cells under Conditions of hypoxia and reoxygenation (H/R) Injury First, a Cell Counting Kit (CCK)-8 assay was performed to assess the cytotoxicity of Nec-1, which is an inhibitor of the kinase domain of receptor-interacting protein kinase-1 (RIP1)
Nec-1 Treatment Affected the Levels of HIF-1a, miR-26a, The pcDNA3.1TRPC6 (TRPC6), Necroptosis, Oxidative Stress, and InflammationRelated Molecules in HK-2 Cells after H/R Injury We further detected the levels of hypoxia-inducible factor-1a (HIF-1a) and miR-26a, and found that the increases in HIF-1a (Figure 2A) and miR-26a expression (Figure 1S, p < 0.05) induced by H/R injury were inhibited by Nec-1 treatment in a time-dependent manner (p < 0.05)
Summary
Renal ischemia and reperfusion (I/R) injury, the major cause of acute renal failure, typically occurs after renal transplantation or surgery, and is one of the most serious and common health problems seen in the clinic.[1,2] The pathogenic mechanism underlying renal I/R injury is complicated and involves energy metabolism dysfunction,[3] tubular necrosis and apoptosis,[4] inflammation,[5,6] and oxidative stress.[7] The anti-malarial drug, hydroxychloroquine, was shown to markedly reduce the levels of pro-inflammatory cytokines (IL-1b, IL-6, and tumor necrosis factor alpha [TNF-a]) because it exerts its anti-inflammatory effects in the treatment of acute kidney injury.[8] I/R results in excessive reactive oxygen species (ROS) production and antioxidant levels.[9] Administration of a MnSOD mimetic was shown to reduce the severity of a renal I/R injury and increase the expression of antioxidant enzymes.[10] gaining a better understanding of the mechanisms of necrosis, inflammation, and oxidative stress that underlie I/R-induced injury would aid in developing new and effective therapies for acute renal failure
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