Activation Of NRF2/ARE Signaling Pathway Protects Against Palmitic Acid-induced Renal Tubular Epithelial Cells Damage Via Amelioratingmtros-mediated Mitochondrial Dysfunction And NLRP3 Inflammasome Activation

Background: Chronic kidney disease(CKD)is often accompanied by lipid metabolism disturbance.Elevated levels of triglycerides,cholesterol and free fatty acids(FFA)in serum are important independent risk factors for the occurrence and progression of CKD.Previous studies have shown that lipid-induced renal tubular epithelial cell injury play an important role in the pathogenesis of CKD,and the specific mechanism of which was involved in mitochondrial dysfunction and the activation of inflammation.Nuclear factor E2-related factor 2(Nrf2),known as a redox-sensitive transcription factor,is responsible for regulating the cellular redox homeostasis by binding to the antioxidant response element(ARE).Accumulating evidence has demonstrated that NRF2/ARE pathway plays a major role in the pathogenesis of many diseases including acute kidney injury(AKI),diabetic nephropathy(DN)and lupus nephritis.However,therole and mechanism of the NRF2/ARE pathway in CKD patients with lipid metabolism disturbance,especially in high lipid-mediated renal tubular epithelial cell injury,have not been fully elucidated.Objective: The purpose of this project is to investigate the role of the NRF2/ARE antioxidant signaling pathways and its regulation on the mitochondrial functions and NLRP3 inflammasome activation in hyperlipidemia-induced injury and apoptosis of renal tubular epithelial cells(TECs).Methods: Human proximal renal tubular epithelial cells were treated with PA(300 μmol/L),the content of lipid accumulation in renal tubular epithelial cells was detected by oil red O,Nile red,and BODIPY staining.Cell viability was detected by CCK-8 assay kit,the cytoskeleton was observed by phalloidin staining,and the cell cycle and apoptosis were detected by flow cytometry.Mitochondrial morphology was observed by Mitotracker staining,mitochondrial membrane potential was monitored by JC-1 staining,and the levels of intracellular and mitochondrial-derived reactive oxygen species was detected by fluorescent probe DCFH-DA and MitoSOX staining.Furthermore,the expression of mitochondrial apoptotic pathway-related proteins,NRF2/ARE pathway-related proteins Nrf2,HO-1,NQO-1,and NLRP3 inflammasome-associated proteins NLRP3,caspase1,and IL-18 were detected by western blot analysis.Nrf2 nuclear translocation was demonstrated by immunofluorescence and western blotof HK-2 cells after nuclear fractionation.Pretreatment with mitochondrial antioxidant Mito tempol,using siRNA to knock-down or tBHQ to activated the expression of Nrf2,to explore the effect of which on PA induced mitochondrial damage,mtROS production,NLRP3 inflammasome activation,and cell injury in HK-2 cells.Meanwhile,an animal model of high-fat induced kidney injury was treated with tBHQ to verify the above mechanism.Results: PA induced the accumulation of lipid,decreased cell viability,caused cytoskeleton damage,and increased cell apoptosis in HK-2 cells compared with the control group.PA induced significant mitochondrial fragmentation,decreased the mitochondrial membrane potential,and increased the intracellular ROS and mtROS production.PA mediated cell apoptosis through activation of the mitochondrial intrinsic apoptotic pathway,which accompanied with a dose-dependent upregulation of Bax and Cytochrome C,cleaved caspase-3,and cleaved poly(ADP-ribose)polymerase(PARP),while concomitant with a decreased expression in Bcl-2 and caspase9.PA activated the NRF2/ARE signaling pathway,which is manifested with a dos-and time-dependent increase in the expression of Nrf2,HO-1 and NQO-1 proteins,and an increased Nrf2 nuclear translocation in HK-2 cells.PA stimulation activated NLRP3 inflammasome,which is manifested by increased protein expression of NLRP3,Caspase1,and IL-18.Furthermore,silencing Nrf2 furtherenhanced PA-induced mitoROS production,accelerated the loss of PA-induced mitochondrial membrane potential,aggravated the activation of NLRP3 inflammasome and cell injury.However,pretreatment with Mito tempol or tBHQ significantly reduced PA-induced mtROS production,attenuated mitochondrial injury,inhibited the activation of NLRP3 inflammasome,and alleviated cell injury.Additionally,tBHQ treatment significantly activated NRF2/ARE signaling pathway,attenuated mitochondrial damage,inhibited the activation of NLRP3 inflammasome and mitigated cell apoptosis in kidney of HFD-induced obese rats.Conclusion: High lipids induced mitochondrial dysfunction,mitochondrial ROS generation,the activation of the NRF2/ARE signaling pathway and NLRP3 inflammasome,and cell injury in renal tubular epithelial cells.MtROS was involved in PA-induced mitochondrial dysfunction,the activation of NLRP3 inflammasome and cellular injury in renal tubular epithelial cells.NRF2/ARE mediated antioxidant response prevented TECs injury and tissue damage after high lipid intervention through decreasing mtROS production,and subsequently preserving mitochondrial function,inhibiting the activation of the NLRP3 inflammasome,attenuating cytoskeleton damage and cell apoptosis.