Regulation Of Srebp1 Expression In Renal Tubular Cells Of Diabetes Mellitus By FBXW7

Objective: Diabetic nephropathy is a chronic complication of diabetes mellitus and SREBP1 is a key regulator of lipid metabolism in diabetic kidneys.FBXW7 is an E3 ubiquitin ligase involved in the regulation of SREBP1 in tumor tissues.However,it is not clear whether FBXW7 is also involved in the regulation of SREBP1 in diabetic nephropathy.Therefore,in this study,diabetic mice and human renal tubular epithelial cells cultured in vitro were chosen to explore the expression of FBXW7 and its effect on the expression of SREBP1 in diabetic nephropathy.Methods:1.Diabetic mice model and HKC cells were used to detect the expression of FBXW7 and SREBP1 at the levels of protein and mRNA by immunohistochemistry,immunofluorescence,Western blot and Real-time PCR.2.The expression of FBXW7 and SREBP1 at the levels of protein and mRNA were detected by Western blot,immunocytochemistry and Real-time PCR in HKC cells transfected with FBXW7 recombinant expression plasmid.3.HKC cells were transfected with FBXW7 RNA interference plasmid.The expression of FBXW7 and SREBP1 were detected by Western blot,immunocytochemistry and Real-time PCR.Results:1.Expression of FBXW7 in renal tissues of diabetic mice and HKC cellsThe expression of FBXW7 was located in the cytoplasm of renal tubular cells and glomeruli of diabetic and normal mice.The expression of FBXW7 in diabetic mice was lower than that in normal mice.SREBP1 was distributed in the cytoplasm of renal tubular epithelium of diabetic mice and normal mice.The expression of SREBP1 in diabetic group was higher than that in normalgroup.The expression of FBXW7 protein and mRNA were significantly decreased in high glucose-stimulated HKC cells than those of normal glucose group at each time point.The most decrease in FBXW7 protein was found at72 h after high glucose stimulation.Compared with normal glucose group,FBXW7 protein was reduced by 70% at 72 h high glucose group.Additionally,the most decrease in FBXW7 mRNA reached at 12 h after high glucose treatment.Immunofluorescence assay showed that the expression of FBXW7 was mainly in the cytoplasm,and the fluorescence intensity in the high glucose group was lower than that in the normal glucose group.On the contrary,high glucose significantly upregulated the expressions of precursor segment and mature segment of SREBP1 protein,and reached the peak at 48 h after stimulation.2.The effect of upregulation of FBXW7 on SREBP1 expression in HKC cellsCompared with the control plasmid group,the transfection of FBXW7 plasmid significantly up-regulated the expression of FBXW7 in HKC cells,and respectively down-regulated the expressions of precursor segment of SREBP1 and mature segment of SREBP1 by 91% and 62%.3.The effect of knockdowning FBXW7 on the expression of SREBP1 in HKC cellsCompared with pGenesil-1-NC plasmid group,pGenesil-1-FBXW7-1and pGenesil-1-FBXW7-2 plasmid respectively decreased FBXW7 proteinby 29% and 41%.Whereas,precursor segment and mature segment ofSREBP1 were evidently increased with the transfection ofp Genesil-1-FBXW7-1 or pGenesil-1-FBXW7-2 plasmid.Conclusion:1.The expression of FBXW7 in renal tubular epithelial cells of diabetic mice was decreased and the expression of SREBP1 was increased.2.High glucose could induce the expression of FBXW7 in human renal tubular epithelial cells and decrease the expression of SREBP1.3.Up-regulation of FBXW7 in renal tubular epithelial cells decreased the expression of SREBP1,and down-regulation of FBXW7 in renal tubular epithelial cells increased the expression of SREBP1.