| Background:Diabetic nephropathy(DN)is a major microvascular complication of both type 1 and type 2 diabetes that progresses to end-stage renal disease.Persistent microalbuminuria is widely used as a biomarker of early DN,which indicates a progressive decline in renal function.Podocytes are terminally differentiated cells that are unable to proliferate,and which form the glomerular filtration barrier,together with endothelial cells and the glomerular basement membrane.Micro RNAs(mi RNAs)are single-stranded,small,noncoding RNAs(21~25 nucleotides)that regulate gene expression by binding to the m RNAs of protein-coding genes to inhibit their translation.Cumulative studies suggest that mi RNAs are involved in the pathogenesis of various diseases,including kidney disease.The BCL2 gene family and its related protein bcl-2 were the first apoptosis-related genes to be studied.This study aimed to explore whether mi R-134-5p can participate in the pathological process of DN by regulating the expression of bcl-2 protein in vitro and in vivo.Methods:1.Twenty male mice(eight weeks of age),including ten C57BL/Ks J db/db mice as the experimental group and ten C57BL/Ks J db/m mice as the normal control group.After 20 weeks of persistent hyperglycemia,the mice were sacrificed by cervical dislocation and all kidney tissues were immediately isolated.The collected kidney samples were stained with HE and PAS to observe changes in the glomerular,mesangial matrix and capillary basement membrane.At the same time,immunohistochemistry and immunofluorescence were used to detect the changes of bcl-2 expression in nephridial tissue and the expression level of mouse kidney podocyte marker protein,nephrin.2.Microarray mi RNA analysis was used to show differentially expressed mi RNAs.3.Real-time quantitative PCR was used to detect the expression of mi R-134-5p in immortalized human podocytes at different time points induced by high glucose.4.After transfection of anti-mi R-134-5p in podocytes treated with high-glucose(HG,30 m M D-glucose)and overexpression of mi R-134-5p in podocytes treated with normal glucose(NG,5 m M D-glucose),the relative m RNA level of nephrin and the expression of mi R-134-5p were analyzed by q RT-PCR;The expression level of nephrin and apoptosis-related protein cleaved caspase3 were analyzed by western blot;Flow cytometry analysis was used to measure podocyte apoptosis.5.We used double luciferase reporter assay to verify whether mi R-134-5p directly targets the bcl-2 3’ untranslated region(3’-UTR);After transfection with anti-mi R-ctrl or anti-mi R-134-5p in HG-treated podocytes and mi R-ctrl or mi R-134-5p in NG-treated podocytes,the expression level of bcl-2 was analyzed by western blot.6.The expression levels of nephrin and bcl-2 were measured by western blot after co-transfection with anti-mi R-ctrl or anti-mi R-134-5p,and sh-Bcl2 expression plasmid in HG-treated podocytes and mi R-ctrl or mi R-134-5p,and Bcl-2 expression plasmid in NG-treated podocytes,together or separately.Apoptosis was measured by western blot and flow cytometry analysisResults:1.HG induced the up-regulated expression of mi R-134-5p in podocytes.2.Mi R-134-5p promotes apoptosis in podocytes.3.The luciferase assay verified that the direct target of mi R-134-5p is bcl-2.4.Inhibition of bcl-2 attenuates the anti-apoptotic effect of anti-mi R-134-5p and decreases nephrin expression,vice versa.Conclusions:1.High glucose induces upregulation of mi R-134-5p in podocytes and promotes apoptosis.2.Mi R-134-5p promotes podocyte apoptosis by targeting bcl-2. |
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