The Mechanism Of PPAR? Affecting Renal Fibrosis In Diabetic Kidney Disease By Regulating The Expression Of SFRP5

Objective:The aim is to study whether PPAR?can affect the activity of Wnt/?-catenin pathway by regulating the expression of SFRP5 from the level of in vivo and vitro,and then affect the development of DKD renal fibrosis.Methods:18clean male Sprague-Dawley?SD?rats were randomly divided into normal control?NC?group,diabetic kidney disease?DKD?group and pioglitazone?PIO?group.Rats of DKD group and PIO group were injected with 2%STZ 55mg/kg by tail vein to reproduce rat model of diabetic kidney.In the NC group,an equal volume of citric acid-sodium citrate buffer was injected into the tail vein.The pioglitazone was administered by gavage?1mg/kg/d?at 7 weeks after the successful modeling in the PIO group.At the end of the 12th week,rats in each group were sacrificed.Blood samples were taken from femoral arteries to detect biochemical indicators.HE and Masson staining were used to observe the morphological changes of kidney tissue in each group.Western blot analysis was used to detect the expression of PPAR?,SFRP5,p-GSK3?^ser9,GSK3?,?-catenin,Collagen?,Collagen?and nuclear protein?-catenin in rat kidney,NRK-52E cells were cultured and divided into normal glucose?NG,G=5.5m M?group,high glucose?HG,G=30m M?group,and pioglitazone treatment?HG+PIO,G=30m M,PIO=64?M?group.Western blot was used to detect the expression of PPAR?,SFRP5,p-GSK3?^ser9,GSK3?,?-catenin,Collagen III,Collagen?,and nucleoprotein?-catenin in NRK-52E cells.Immunofluorescence revealed the expression change and distribution of?-catenin in NRK-52E cells.NRK-52E cells were transfected with PPAR?overexpression and knockdown plasmids,respectively.Dividing into NG group,HG group,high glucose+empty vector group?HG+empty?and high glucose+PPAR?overexpression?HG+PPAR?overexpression?group;and NG group,normal glucose+empty vector group?NG+empty?and normal glucose+PPAR?knock-down group?NG+PPAR?knock-down?.Western blot was used to detect the expression of PPAR?,SFRP5,p-GSK3?^ser9,GSK3?,?-catenin,Collagen?and Collagen?in NRK-52E cells of each group.Results:1.The biochemical results showed that compared with the NC group,blood glucose,BUN,and 24h UAlb increased in the DKD group?P<0.05?;Compared with the DKD group,BUN and 24h UAlb decreased?P<0.05?,but there was no significant change in blood glucose in the PIO group.2.Pathological test:The HE staining showed that the glomerular capillary expanded in the DKD group compared with the NC group,the glomerular cavity was narrowed,the renal tubules appeared hollow degeneration,and the brush border of the epithelial cells fell off.The pathological changes in the PIO group were improved compared with the DKD group.Masson staining of DKD group showed significant blue staining of the glomeruli and tubules and collagen volume fraction?CVF?increased?P<0.05?compared with the NC group,PIO group has fewer blue staining and CVF decreased?P<0.05?than DKD group.3.Changes of renal tissues and NRK-52E cells after pioglitazone intervention by Western blot:Compared with the normal control group,the expression of PPAR?and SFRP5 decreased?P<0.05?,?-catenin,p-GSK3?^ser9,Collagen?and Collagen?increased in DKD group and HG group?P<0.05?.?-catenin expression were increased in nucleoproteins?P<0.05?.Compared with the DKD group and HG group,the expression of PPAR?and SFRP5 increased in the PIO group?P<0.05?,but the expressions of?-catenin,p-GSK3?^ser9,Collagen?and Collagen?,and?-catenin in nucleoproteins decreased in the PIO group?P<0.05?.GSK3?expression did not change significantly in each group.4.Immunofluorescence experiment:It can be seen that the expression of?-catenin in cell nucleus in the HG group is higher than that in the NG group,but the expression of?-catenin in PIO group is lower than that in the HG group.5.Cell transfection with PPAR?overexpressed plasmids:Western blot showed that the expression of PPAR?increased in the HG+PPAR?overexpression group,which was about 190%of that than the empty vector group;Compared with the HG+empty vector group,the expression of SFRP5 was increased in the PPAR?overexpression group?P<0.05?,but the expressions of p-GSK3?^ser9,Collagen?and Collagen?were decreased?P<0.05?,the expressions of GSK3?were not significantly changed.6.Cell transfection with PPAR?knockdown plasmids:Western blot showed that the expression of PPAR?was significantly reduced in the NG+PPAR?knockdown group,which was about 44%of that than the empty group.Compared with the NG+empty vector group,the expression of SFRP5 was also reduced in the NG+PPAR?knockdown group?P<0.05?,?-catenin,p-GSK3?^ser9,Collagen III and Collagen?expression increased?P<0.05?,and GSK3?expression did not change significantly.Conclusions:Overexpression of PPAR?in kidney tissue of DKD rats and NRK-52E cells cultured in high glucose environment can up-regulate the expression of the Wnt/?-catenin pathway inhibitor SFRP5,inhibit the nuclear translocation of?-catenin and the activation of Wnt/?-catenin signaling pathway,reducing the production and deposition of ECM,thereby delaying the occurrence and development of DKD renal fibrosis.