The Role And Mechanism Of Phosphorylation And SUMOylation Of STAT1 In Renal Tubular Epithelial Cell Injury In Diabetes

Objective:Diabetic kidney disease(DKD),a complication of diabetes,is a common chronic kidney disease and a main cause of end-stage renal disease.One of the pathological manifestations of DKD is tubulointerstitial lesion(TIL),and TIL is closely associated with progressive renal function decline.In this study,diabetes mice and human renal tubular epithelial cells(HK2)were taken as the research objects.Through observing the phosphorylation and SUMOylation of STAT1 and the expression of early tubular injury indicators,we explored the role and mechanism of STAT1 phosphorylation and SUMOylation in the damage of renal tubular epithelial cells in diabetic kidney disease.Methods:1.Effect of STAT1 signal activation on the damage of renal tubular epithelial cells in diabetes mice.The mice were divided into control group,diabetes group,fludarabine administration group(STAT1 activity inhibitor)and solvent control group.Western blot was used to detect STAT1 protein and its phosphorylation.Western blot and immunohistochemistry were used to detect the expression of kidney injury molecule-1(Kim-1)and neutrophil gelatinase-associated lipocalin(NGAL)in renal tissue.2.Effect of high glucose on HK2 cell injury.After high glucose or mannitol treating for 0 h,6 h,12 h,and 24 h,the cells and supernatant were collected.The expression of Kim-1 and NGAL were measured by western blot and the content of N-acetyl-β-D-glucosidase(NAG)in the cell supernatant was measured by ELISA.3.High glucose induced phosphorylation of STAT1 in HK2 cells:Cells,stimulating with high glucose for 0 h,3 h,6 h,12 h and 24 h,were collected and the expression of STAT1 and p-STAT1(Try701/Ser727)was detected by western blot.4.Effect of STAT1 protein and its phosphorylation modification on renal tubular epithelial cell injury.Cells were treated with fludarabine(STAT1 activity inhibitor)or transfected with STAT1 siRNA,STAT1-YE-GFP(STAT1 Try701 site-sustained activation plasmid carrying GFP tag)or STAT1-SD-GFP(STAT1 Ser727 site-sustained activation plasmid carrying GFP tag).The expression of Kim-1 and NGAL was detected by western blot,and the content of NAG in the cell supernatant was measured by ELISA.5.SUMOylation of STAT1 in HK2 cells under high glucose conditions.HK2 cells were randomly divided into control group and high glucose group.The SUMOylation of STAT1 and the combination of STAT1 with PIAS proteins were detected by Co-IP.6.Effect of SUMOylation of STAT1 on the damage of HK2 cells.After silencing the endogenous STAT1 with siRNA transfection,HK2 cells were transfected with STAT1-WT-GPF(wild type STAT1 plasmid carrying GFP tag)or STAT1-KR-GPF(mutant plasmid of STAT1 SUMO modification site carrying GFP tag)plasmid.After high glucose stimulation,the expression of Kim-1 and NGAL was detected by western blot.And the content of NAG in cell supernatant was detected by ELISA.7.Effect of phosphorylation of STAT1 on its SUMOylation in HK2 cells:HK2 cells were transfected with STAT1-WT-GFP,STAT1-YE-GFP or STAT1-SD-GFP plasmid respectively.Co-IP was used to detect the binding of STAT1 and SUMO1.8.Effect of SUMOylation of STAT1 on its phosphorylation in HK2 cells.After the endogenous STAT1 knockdown,the cells were transfected with STAT1-WT-GFP or STAT1-KR-GFP plasmid.The expression of p-STAT1(Try701/Ser727)was detected by western blot and the combination of STAT1 and JAK2 was detected by Co-IP.Results:1.Fludarabine inhibited the expression of STAT1 and p-STAT1,and alleviated the damage of renal tubular epithelial cells in diabetes.The expression of Kim-1,NGAL and p-STAT1(Try701/Ser727)in renal tissue of diabetes mice significantly increased.However,fludarabine treatment reduced the level of STAT1 and p-STAT1(Try701/Ser727)and inhibited the expression of Kim-1 and NGAL.2.High glucose induced damage in HK2 cells.The increase of Kim-1,NGAL and NAG after high glucose stimulation appeared at 6 h,12 h and 3 h respectively.Mannitol did not affect the expression of Kim-1 and NGAL.Results suggested that high glucose rather than hypertonicity caused cell damage.3.High glucose induced phosphorylation modification of STAT1 in HK2 cells.Western blot results showed that compared with 0 h,the level of pSTAT1(Try701/Ser727)was upregulated at different time points stimulated by high glucose.4.STAT1 protein induced HK2 cells damage,while its phosphorylation alleviates cell injury.The results showed that the expression of STAT1 and pSTAT1(Try701/Ser727)was reduced and the expression of Kim-1,NGAL and NAG was restored after fludarabine treatment.STAT1 siRNA downregulated also the expression of them.However,STAT1 overexpression induced the expression of Kim-1,NGAL and NAG.And compared with those in STAT1-WT-GFP plasmid-transfected cells,the expression of Kim-1,NGAL and NAG decreased in cells transfected with STAT1-YE-GFP or STAT1-SD-GFP plasmid.5.Upregulation of SUMOylation of STAT1 in high glucose-stimulated HK2 cells.Co-IP results showed that high glucose enhanced the binding of STAT1 with SUMO1 or SUMO4,but did not affect its binding with SUMO2/3.Moreover,PIAS1,PIAS3 and PIAS4 may mediate the SUMOylation of STAT1.6.Effect of SUMOylation of STAT1 on HK2 cell injury.Western blot and ELISA showed that under high glucose conditions,the expression of Kim1,NGAL and NAG decreased in STAT1-KR-GFP plasmid transfected-cells compared with those in STAT1-WT-GFP transfected-cells.7.Phosphorylation modification of STAT1 enhanced the SUMO1 modification of STAT1.Co-IP results showed that compared with STAT1WT-GFP plasmid,STAT1-YE-GFP or STAT1-SD-GFP plasmid increased the combination of STAT1 and SUMO1 in HK2 cells.8.Inhibiting the SUMOylation of STAT1 enhanced the phosphorylation modification of STAT1.Western blot showed that high glucose induced higher phosphorylation level of STAT1 mutated with SUMO site than wild type STAT1.In addition,Co-IP results showed that the combination of STAT1 and JAK2 increased when the SUMO site of STAT1 mutated.Conclusions:1.In high glucose environment,the phosphorylation and SUMOylation of STAT1 increased,but its phosphorylation alleviated the injury of renal tubular epithelial cells,while SUMOylation aggravated the injury.2.The phosphorylation modification of ST AT1 promoted its SUMOylation,which in turn inhibited its phosphorylation modification.The two modifications interacted each other to affect renal tubular epithelial cell damage in high glucose environment.