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

Objective:The aim is to study whether PPARγcan affect the activity of Wnt/β-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 PPARγ,SFRP5,p-GSK3β^ser9,GSK3β,β-catenin,CollagenⅢ,CollagenⅣand nuclear proteinβ-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=64μM）group.Western blot was used to detect the expression of PPARγ,SFRP5,p-GSK3β^ser9,GSK3β,β-catenin,Collagen III,CollagenⅣ,and nucleoproteinβ-catenin in NRK-52E cells.Immunofluorescence revealed the expression change and distribution ofβ-catenin in NRK-52E cells.NRK-52E cells were transfected with PPARγoverexpression and knockdown plasmids,respectively.Dividing into NG group,HG group,high glucose+empty vector group（HG+empty）and high glucose+PPARγoverexpression（HG+PPARγoverexpression）group;and NG group,normal glucose+empty vector group（NG+empty）and normal glucose+PPARγknock-down group（NG+PPARγknock-down）.Western blot was used to detect the expression of PPARγ,SFRP5,p-GSK3β^ser9,GSK3β,β-catenin,CollagenⅢand CollagenⅣin 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 PPARγand SFRP5 decreased（P<0.05）,β-catenin,p-GSK3β^ser9,CollagenⅢand CollagenⅣincreased in DKD group and HG group（P<0.05）.β-catenin expression were increased in nucleoproteins（P<0.05）.Compared with the DKD group and HG group,the expression of PPARγand SFRP5 increased in the PIO group（P<0.05）,but the expressions ofβ-catenin,p-GSK3β^ser9,CollagenⅢand CollagenⅣ,andβ-catenin in nucleoproteins decreased in the PIO group（P<0.05）.GSK3βexpression did not change significantly in each group.4.Immunofluorescence experiment:It can be seen that the expression ofβ-catenin in cell nucleus in the HG group is higher than that in the NG group,but the expression ofβ-catenin in PIO group is lower than that in the HG group.5.Cell transfection with PPARγoverexpressed plasmids:Western blot showed that the expression of PPARγincreased in the HG+PPARγoverexpression 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 PPARγoverexpression group（P<0.05）,but the expressions of p-GSK3β^ser9,CollagenⅢand CollagenⅣwere decreased（P<0.05）,the expressions of GSK3βwere not significantly changed.6.Cell transfection with PPARγknockdown plasmids:Western blot showed that the expression of PPARγwas significantly reduced in the NG+PPARγknockdown 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+PPARγknockdown group（P<0.05）,β-catenin,p-GSK3β^ser9,Collagen III and CollagenⅣexpression increased（P<0.05）,and GSK3βexpression did not change significantly.Conclusions:Overexpression of PPARγin kidney tissue of DKD rats and NRK-52E cells cultured in high glucose environment can up-regulate the expression of the Wnt/β-catenin pathway inhibitor SFRP5,inhibit the nuclear translocation ofβ-catenin and the activation of Wnt/β-catenin signaling pathway,reducing the production and deposition of ECM,thereby delaying the occurrence and development of DKD renal fibrosis.