Mechanism Of DNA Methyltransferase 3B Regulating The Expression Of Secretory Frizzled-relatd Protein 1 In Diabetic Nephropathy In Mice

Objective:To observe the expression of DNA methyltransferase 3B（DNMT3B）,secreted frizzled-relatd protein 1（SFRP1）,Wnt/β-catenin signaling pathway and extracellular matrix（ECM）in renal tissue of diabetic mice and renal tubular epithelial cells（RTEC）stimulated by high glucose,and to observe the regulation effect of DNMT3b on SFRP1 protein expression was observed,and to detect the methylation frequency of SFRP1 DNA promoter in renal tissue of diabetic mice and RTEC under high glucose condition,and to investigate the role and mechanism of DNMT3B and SFRP1 in the occurrence of tubulointerstitial fibrosis in diabetic nephropathy.Methods:（1）The renal pathological changes were observed by HE,Masson,PAS and PASM staining in type 1 and type 2 diabetic mice.The expression of DNMT3B,SFRP1 andβ-catenin in renal tissues of mice was observed by immunohistochemical staining.（2）Western blot and q RT-PCR were used to detect the protein and m RNA expression changes of DNMT3B,SFRP1,Wnt/β-catenin pathway related molecules,E-cadherin,extracellular matrix（ECM）in renal tissues of diabetic mice in different group and RTEC compared with normal C57BL mice and NG-stimulated RTEC.（3）The empty vector or DNMT3B knockdown（DNMT3B-sh）or DNMT3B overexpression plasmid（DNMT3B-OE）were transfected respectively after RTEC was cultured in the HG environment.The protein and m RNA expression changes of DNMT3B,SFRP1,Wnt/β-catenin pathway related proteins and EMC were detected by Western blot and q RT-PCR.（4）Bisulfite sequencing PCR（BSP）method was used to detect the methylation levels of sfrp1 DNA promoter in cells after DNA was extracted from kidney tissue and cell DNA after RTEC stimulation with normal glucose（NG）and high glucose（HG）.Results:（1）Compared with the NC group,the blood glucose,BUN,24h UAlb were increased in the diabetic group（P<0.05）;HE,Masson,PAS and PASM staining results showed that the mesangial area of the glomeruli was increased,the epithelial cells of the renal tubules showed vacuolar degeneration and granular degeneration,and the renal interstitium showed focal edema,which compared with the NC group.（2）The m RNA and protein expressions of SFRP1 and E-cadherin in renal tissue and HG-stimulated RTEC of diabetic mice were decreased（P<0.05）,nevertheless,The m RNA and protein expressions of DNMT3B,β-catenin,p-GSK3β^ser9,CollagenⅢand Fibronectin were increased（P<0.05）;However,the expression of GSK3βhad no significant change in each group.（3）Compared with the HG+Vector group,the protein and m RNA expressions of SFRP1 and E-cadherin in the HG+DNMT3B-OE group were significantly increased（P<0.05）;the protein expression of DNMT3B,β-catenin,p-GSK3β^ser9,CollagenⅢ,and Fibronectin were remarkablely reduced yet（P<0.05）,whereas,the expression of GSK3βhad no significant change.（4）The BSP results showed that the methylation levels of some Cp G sites（12th,18th,23rd,24th,and 40th）in sfrp1 DNA of RTEC with high glucose stimulation were significantly higher in the high glucose stimulation group than in the normal glucose stimulation group（P<0.05）.Methylation levels of some Cp G sites（12th,16th,21st,25th,29th,32nd,and 40th）in renal tissue DNA of diabetic mice were increased compared with normal controls.Conclusions:The decreased expression of SFRP1 in kidney tissue of DM mice and RTEC cultured in high glucose may be related to the increased the expression of DNMT3B under high glucose condition,which promoted the methylation frequency of some Cp G sites near the transcription starting point of sfrp1 gene promoter region.（2）The protein and m RNA expression of DNMT3B were increased in kidney tissue of DM mice and RTEC cultured with high glucose,which lead to the down-regulation of SFRP1expression,and decreased the inhibition of Wnt/β-catenin signaling pathway.The abnormal activation of Wnt signaling pathway promoted the progression of diabetic renal fibrosis.