DsbA-L Protects Against Tubular Damage In Diabetic Kidney Disease By Retaining Peroxisome Function

Background:Diabetic kidney disease（DKD）is a common secondary chronic kidney disease and the main cause of end-stage renal disease.In recent years,researchers pay more and more attention to oxidative stress.Studies have shown that oxidative stress is closely related to the development of DKD.Peroxisomes are present in all cell types except red blood cells but are particularly abundant in liver and kidney cells.It has been proved that peroxisomes can inactivate toxic substances such as hydrogen peroxide（H₂O₂）and protect cells from damage caused by these toxic substances.On the other hand,the increase of intracellular reactive oxygen species（ROS）and abnormal lipid metabolism caused by peroxisome defect or dysfunction aggravate kidney damage.In addition,catalase is located in the peroxisome and plays an important role in the oxidation-reduction process.We and other researchers have recently discovered that disulfide-bond-A oxidoreductase-like protein（DsbA-L）,glutathione S-transferase kappa 1（GSTK1）,may have an important detoxification effect on ROS and lipid peroxides,and is highly expressed in peroxisomes of renal tubular epithelial cells.Whether DsbA-L improves peroxisomal function through catalase and reduces oxidative stress in DKD is unclear currently.Therefore,the effect and possible mechanism of DsbA-L on catalase and peroxisome in DKD were discussed.Objective:To observe the effect of DsbA-L on peroxisome and oxidative damage in DKD tubular epithelial cells.To explore whether DsbA-L can improve peroxisomal function in DKD through catalase.Methods:1.Animal experiments:Mouse with DsbA-L gene-specific knockout in renal proximal tubule cells(DsbA-L^ptKO)was generated and identified.DKD mouse model was established by intraperitoneal injection of streptozotocin（STZ）and high-fat feeding.The mice were divided into the DsbA-L^ctrl group,DsbA-L^ptKO group,DsbA-L^ctrl+DKD group,and DsbA-L^ptKO+DKD group.After 16 weeks of STZ injection,urine samples were collected for biochemical testing.After the mice were sacrificed,kidney tissues were taken to observe the pathological changes.At the same time,Western blot,immunohistochemistry,immunofluorescence,and other methods were used to observe the expression of DsbA-L,the number and phenotype of peroxisomes and renal oxidative stress-related indexes in the renal cortex.2.Cell experiments:The human proximal tubular epithelial cell line（HK-2 cells）and Hy Per-PTS1 stable cell line were treated with high glucose（HG）and palmitic acid（PA）,DsbA-L Plasmid,and catalase inhibitor 3-Amino-1,2,4-triazole（3-AT）.The expression and changes of DsbA-L,the number of peroxisomes,the change of peroxisomal phenotype,and oxidative stress-related indexes were detected by Western blot and immunofluorescence.Results:1.Compared with the DsbA-L^ctrl group,the expression of DsbA-L in the DsbA-L^ctrl+DKD group was significantly decreased,the renal tubular damage was significantly aggravated,the oxidative stress and apoptosis were increased,the number of peroxisomes was significantly decreased,“ghost”peroxisomal phenotype where cells show defective catalase import but have peroxisomal bodies positive for peroxisomal membrane protein 70（PMP70）was increased.The above changes were more obvious in the DsbA-L^ptKO+DKD group.2.The number of DsbA-L and the function of peroxisomes were significantly decreased,the“ghost”peroxisomal phenotype was increased,the H₂O₂ of peroxisomes was significantly increased after intervened with HG and PA in HK-2.Overexpression of DsbA-L could improve the above changes,while the effect of DsbA-L can be prevented by 3-AT.Conclusion:DsbA-L may ameliorate DKD tubular oxidative damage by regulating catalase activity and improving peroxisome function,thereby reducing oxidative stress and apoptosis in renal tubular epithelial cells.