Role Of LncRNA Dlx6-os1 In Proteinuria And Podocyte Injury In Diabetic Nephropathy Mice

Background:Diabetic nephropathy is one of the most serious microvascular complications of diabetes and a major cause of end-stage renal disease.At present,the pathogenesis of diabetic nephropathy is complicated,and no exact pathogenesis is found.Studies in recent years have shown that in a series of kidney tissue damage caused by hyperglycemia,the damage of podocytes and the reduction of the number of albuminuria are considered to be the main cause of diabetic nephropathy.Long non-coding RNA(LncRNA)is a non-coding RNA of greater than 200 nucleotides in length.It is a research hotspot in recent years and has a wide range of functions,which can regulate gene expression from various aspects such as cell differentiation,cell cycle and epigenetic.An important pathway by which Wnt signaling mediates podocyte injury.Glycogen synthase kinase(GSK-3?)is one of the key molecules of Wnt signaling pathway and plays a crucial role in podocyte injury.Our previous experimental results showed that the expression of GSK-3?was increased in podocytes cultured under high glucose conditions,and the expression of podocyte injury protein was increased and podocytes were damaged.However,the role of LncRNA Dlx6-os1 elevation in the development of proteinuria and podocyte injury is unclearObjective:1.Defining the expression of LncRNA Dlx6-os1 in renal tissues of mice with type 2 diabetic nephropathy2.To investigate the role of LncRNA Dlx6-os1 in proteinuria and podocyte injury in mice with type 2 diabetic nephropathyMethod:Male db/db with genetic background of C57BLKs/J and db/m mice born in littermates were randomly numbered,(no intervention group)db/m 14w,db/m 22w,db/db 14w,db/db22w,(over-expressed lentivirus group)db/m+saline,db/m+con-LV,db/m+Dlx6-os1 4w,db/m+Dlx6-os1 12w(shRNA lentivirus group)db/db+saline Db/db+con-shRNA,db/db+Dlx6-os1 shRNA 4w,db/db+Dlx6-os1 shRNA 12w,adaptive feeding for one week,weekly monitoring of mouse blood glucose,dot albumin.In the presence of microalbuminuria in mice,db/db group was injected with LncRNA Dlx6-os1 shRNA lentivirus(titer 1×10~8 TU/only)with podocyte-specific marker NPHS2,and the control group was injected with normal saline and no.The lentivirus(titer 1×10~8 TU/only)was injected into the db/m group at the same time with the overexpression of LncRNA Dlx6-os1(titer 1×10~8 TU/only)lentivirus with podocyte-specific marker NPHS2.The control group was injected with normal saline and non-sense lentivirus(titer 1×10~8 TU/only),and some mice were sacrificed at 0,4,and 12 weeks after lentivirus injection,and blood,urine,and kidney were collected.Organize specimens.The pathological changes of renal tissues were observed by PAS staining and electron microscopy.The expression levels of Podocin,GSK-3?,P-GSK-3?(s9),IL-17,MCP-1 and B7-1 were detected by western blotting.The expression levels of IL-17,Claudin-1 and B7-1 were detected,and the expression level of LncRNA Dlx6-os1 and podocyte marker protein podocin was detected by RNA in situ hybridization(FISH).Result:1.In the db/m of type 2 diabetic nephropathy model,the expression of LncRNA Dlx6-os1 was up-regulated,FISH observed a decrease in podocyte marker protein expression,and LncRNA Dlx6-os1 was increased in glomeruli.PAS staining and electron microscopy showed that mesangial cells in the glomeruli of mice overexpressing virus injection showed mesangial cell proliferation,mesangial matrix hyperplasia,and electron microscopic observation showed that the basement membrane of mouse glomeruli after over-expression of virus injection was compared with the control group.Significantly thickened,the foot process was widely fused,western blotting showed that the podocyte marker protein podocin expression was down-regulated,and the expression of inflammatory damage proteins IL-17,MCP-1,B7-1 increased,immunofluorescence IL-17,Claudin-1,B7-1 The expression is up-regulated.2.In the mice with diabetic nephropathy with microalbuminuria,shRNA lentivirus was injected to down-regulate the expression of LncRNA Dlx6-os1,and FISH observed a decrease in LncRNA Dlx6-os1 expression.PAS staining and electron microscopy showed that the glomerular mesangial matrix hyperplasia was alleviated.Electron microscopy showed that the glycosaminoglycans injected with shRNA lentivirus reduced the glomerular basement membrane thickening and improved the foot process fusion.Western blotting showed The expression of podocyte marker protein podocin was up-regulated,the expression of inflammatory injury proteins IL-17,MCP-1 and B7-1 was decreased,the expression of immunofluorescence podocyte marker proteins Podocin and Synaptopodin was up-regulated,and the expressions of IL-17,mcp-1 and B7-1 were down-regulated.Conclusion:1.Up-regulation of LncRNA Dlx6-os1 expression in db/m of type 2 diabetic nephropathy model mice,leading to kidney damage and proteinuria.2.In the type 2 diabetic nephropathy model mouse db/db,down-regulate the expression of LncRNA Dlx6-os1,protect the kidney structure and reduce proteinuria.3.GSK-3?may play a regulatory role in the transcription of LncRNA Dlx6-os1during the development of diabetic nephropathy proteinuria;provide experimental evidence for Dlx6-os1 as a biomarker for DN early renal damage and a new therapeutic target.