The Role Of Nrf2-ARE Signaling Pathway In Oxalate-Induced Renal Tubular Epithelial Cell Injury And Renal Stone Formation

1.Expression of proteins related to nuclear factor erythroid 2-related factor 2-antioxidant response element signaling pathway in oxalate-induced renal tubular epithelial cell injury modelObjective: To clarify the expression of proteins associated with nuclear factor erythroid 2-related factor 2-antioxidant response element(Nrf2-ARE)signaling pathway in renal tubular epithelial cells exposure to oxalate at different concentrations.Methods: The normal rat kidney-52 epithelial(NRK-52E)injury model was induced by oxalate.The cell viability of NRK-52 E cells was evaluated using the CCK-8 kit.The toxicity of NRK-52 E cell membrane damage was tested using the lactate dehydrogenase(LDH)kit.The lipid peroxidative damage of NRK-52 E cells was assessed with the malondialdehyde(MDA)kit.DCFH-DA and Mito SOX Red fluorescent probes were chosen to label intracellular and mitochondrial reactive oxygen species(ROS),respectively.Chemiluminescence assay was used to detect the activities of superoxide dismutase(SOD)and catalase(CAT)in NRK-52 E cells.The expression levels of proteins(NRF2,HO1,and NQO1)related to the Nrf2-ARE signaling pathway were assessed by western blot.Results: Findings demonstrated that a high concentration of oxalate distinctly inhibited the cell viability and increased LDH release,which presented the characteristics of concentration/time dependence.Exposed to a high concentration of oxalate,intracellular and mitochondrial ROS generation was significantly increased and intracellular MDA generation was also significantly increased,while intracellular SOD and CAT activities were significantly weakened.The expression levels of total intracellular Nrf2,HO1,NQO1,and nuclear NRF2 proteins decreased with the increase of oxalate concentration.Conclusion: High concentration oxalate induces oxidative stress injury of renal tubular epithelial cells.When renal tubular epithelial cells are exposed to oxalate,the expression of major proteins related to the Nrf2-ARE signaling pathway is inhibited and the activities of related antioxidant enzymes downstream of Nrf2 are reduced.2.Role and mechanism of Nrf2-ARE signaling pathway in oxalateinduced renal tubular epithelial cell damageObjective: To investigate the role and mechanism of the Nrf2-ARE signaling pathway in oxalate-induced renal tubular epithelial cell damage.Methods: NRK-52 E cell lines stably knocked out and overexpressed Nrf2 were constructed by lentiviral transfection,respectively.Oxalate(800 μmol/L)was administered to induce damage to normal NRK-52 E cells and Nrf2 knockout and overexpressed NRK-52 E cells.The lactate dehydrogenase(LDH)kit was applied to assess NRK-52 E cell injury.Intracellular and mitochondrial reactive oxygen species(ROS)generation was analyzed by flow cytometry.The apoptosis of NRK-52 E cells was detected by Annexin V-APC/DAPI fluorescence staining.The lipid peroxidative damage of NRK-52 E cells was assessed with the malondialdehyde(MDA)kit.ATP production was detected by the ATP kit.Chemiluminescence assay was used to detect the activities of superoxide dismutase(SOD)and catalase(CAT)in NRK-52 E cells.The expressions of NRF2,HO1,NQO1,p-p65,p65,p-IKBα,IKBα,and OPN were assessed using the western blot.Cell-crystal adhesion test was chosen to determine the adhesion ability of cells to calcium oxalate crystal.Results: In the oxalate-induced NRK-52 E cell injury model,oxalate inhibited the expression levels of NRF2 and its downstream antioxidant proteins HO1 and NQO1.Knockdown of Nrf2 expression further inhibited the expression of antioxidant proteins HO1 and NQO1,while overexpression of Nrf2 reversed this effect.Inhibition of the Nrf2-ARE signaling pathway significantly promoted the release of LDH,MDA production,and apoptosis caused by oxalate,while activating the Nrf2-ARE signaling pathway reversed the above-mentioned damage effects.Inhibition of the Nrf2-ARE signaling pathway significantly increased intracellular and mitochondrial ROS generation in NRK-52 E cells,decreased ATP synthesis,and decreased intracellular SOD and CAT activities.Conversely,activation of the Nrf2-ARE signaling pathway decreased intracellular and intramitochondrial ROS generation,increased ATP synthesis,and boosted intracellular SOD and CAT activities.Meanwhile,the Nrf2-ARE signaling pathway regulated the activation of NF-κB,regulated the expression level of OPN,and regulated cell-crystal adhesion.Physiological or low concentrations of oxalate caused oxidative stress injury in Nrf2-knockout NRK-52 E cells.Conclusion: Nrf2-ARE signaling pathway plays an important role in the oxidative stress injury of renal tubular epithelial cells exposure to oxalate,which may participate in regulating mitochondrial function,expression of proteins associated with stone formation,and cell-crystal adhesion.After inhibition of the Nrf2-ARE signaling pathway,physiological or low concentrations of oxalate can also cause oxidative stress injury of renal tubular epithelial cells.3.Effects of regulating Nrf2-ARE signaling pathway on renal oxidative stress injury and renal calcium deposition in hyperoxaluria ratsObjective: To investigate the effects of regulating the Nrf2-ARE signaling pathway on renal oxidative stress injury and renal calcium deposition in hyperoxaluria rats.Methods: The renal Nrf2 knockout model was established by subcapsular injection of adeno-associated virus 9(AAV9),and the expression of renal Nrf2 was activated by Nrf2 activator-dimethyl fumarate(DMF).The hyperoxaluria model of SD rats was induced by intraperitoneal injection of glyoxylic acid monohydrate.The level of ROS in renal tissue was detected by DHE fluorescent probe.MDA test was used to detect lipid peroxidation products in renal tissue and urine.The activities of SOD and CAT in renal tissue were detected by chemiluminescence.Von Kossa staining was used to observe the calcium deposition in the kidney.The expressions of NRF2,HO1,NQO1,p-p65,p65,OPN,and MCP1 in kidneys were detected by western blot.Results: ROS and the content of MDA in renal tissue of the hyperoxaluria rat model were significantly increased.Inhibiting the Nrf2-ARE signal pathway significantly increased the ROS generation and the content of MDA in renal tissue,while activating the Nrf2-ARE signaling pathway significantly decreased the ROS level and the generation of MDA in tissue.Meanwhile,the Nrf2-ARE signaling pathway was involved in regulating the activation of the important signaling pathway(NF-κB)related to stone formation.Activation of the Nrf2-ARE signaling pathway inhibited the expression level of proteins related to stone formation.The significant calcium salt deposition at the junction of renal endothelium and medulla in these rat models was seen.Inhibiting the Nrf2-ARE signal pathway significantly increased the deposition of calcium salt in the kidney,while activating the Nrf2-ARE signaling pathway significantly reduced that in renal tissue.Conclusion: Nrf2-ARE signaling pathway is involved in regulating renal oxidative stress injury in hyperoxaluria rats.Activating the Nrf2-ARE signaling pathway can inhibit the expression level of inflammatory factors and proteins associated with stone formation,and decrease the deposition of calcium in hyperoxaluria rats.