| Objective: The renal tubular epithelium contains a large number of mitochondria that ensure its reabsorption and excretion for energy supply.In pathological conditions oxidative stress can damage mitochondria and lead to renal tubular interstitial damage.Oxidative stress leads to mitochondrial damage mainly through the following mechanisms: disruption of mitochondrial enzymatic activity;damage to mitochondrial membrane lipids and structural and functional integrity of the inner mitochondrial membrane;blocking the process of mitochondrial oxidative phosphorylation,resulting in reduced ATP production;and direct damage to mitochondrial DNA.damaged mitochondria exacerbate oxidative stress in the body,creating a vicious cycle.The KEAP1/NRF2 pathway is by far one of the most important antioxidant regulatory pathways in the organism,which exerts a protective effect on tissue cells by initiating the transcriptional process of antioxidant and detoxification enzyme genes.However,there are few studies on how this pathway affects oxidative stress and mitochondrial autophagy in Human kidney-2(HK-2)cells in the setting of hyperuricemia(HUA).In this study,we intend to investigate the effects of HUA on HK-2 cells,as well as the effects and possible regulatory mechanisms of the KEAP1/NRF2 pathway on oxidative stress and mitochondrial autophagy in HK-2 cells in the setting of HUA,to elucidate its role in the development and progression of uric acid nephropathy,and to provide new theoretical support for the prevention and treatment of HUA and its associated diseases.Methods:1.HK-2 cells were cultured in vitro,and different concentrations of UA(0,4,8,12,16 mg/dl)and different concentrations of the NRF2 agonist sulforaphane(SFN)(0,4,8,12,16 μM)were set to treat HK-2 cells for 24 h.The cell viability of each group of cells was assayed using the CCK-8kit to determine The optimal UA and SFN concentrations were used for subsequent experiments.2.The cells were grouped according to the experimental requirements: CTL group(0 mg/dl UA),UA group(12 mg/dl UA),UA+SFN group(12 mg/dl UA+8 μM SFN),UA+NAC group(12 mg/dl UA+reactive oxygen species inhibitor NAC),and the interventions were carried out for 24 hours according to the requirements of each group,using the Reactive oxygen species(ROS)assay kit.The ROS level of cells in each group was measured using the Reactive oxygen species(ROS)assay kit for 24 hours.3.The Mitochondrial membrane potential(MMP)assay kit was used to measure the MMP level in each group of cells after 24 hours of intervention according to the requirements of each group.4.The intervention was performed for 24 hours according to the requirements of each subgroup,and the cell death level of each group was detected using calcein staining.5.The expression levels of pathway protein NRF2,oxidative stress-related protein HO-1,mitochondrial autophagy-related protein PINK1,and LC3 were detected by Western blot method for 24 hours of intervention according to the requirements of each subgroup,respectively.Results:1.Based on the results of CCK-8 kit,the optimal concentration of UA was determined to be 12mg/dl and the optimal concentration of SFN was 8 μM.2.The results of cellular ROS level assay showed that the ROS level of HK-2 cells increased in HUA environment and decreased after pretreatment with SFN and NAC compared with UA group.3.The results of cellular MMP level assay showed that the MMP level of HK-2 cells decreased in the HUA environment and increased after pretreatment with SFN and NAC compared with the UA group.4.The results of cell death level detection showed that HUA induced a large number of HK-2 cells to die,and SFN and NAC could reduce the number of cell death.5.The results of Western blot assay showed that the expression of mitochondrial autophagy-related proteins PINK1,LC3-II and pathway proteins NRF2 and HO-1 protein increased in HK-2 cells under the effect of HUA;the expression levels of proteins NRF2,HO-1,PINK1 and LC3-II decreased after the use of reactive oxygen species inhibitor NAC to inhibit ROS levels;the expression levels of NRF2,HO-1,PINK1,and LC3-II protein expression levels were increased after treatment of HK-2 cells with the NRF2 agonist SFN.Conclusion: In this study,we found that HUA causes damage to HK-2 cells and confirmed that HUA activates the KEAP1/NRF2 pathway and mitochondrial autophagy by inducing oxidative stress in HK-2 cells.The KEAP1/NRF2 pathway has antioxidant protective effects and positive regulation of mitochondrial autophagy in HK-2 cells under HUA environment. |
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