| Objective: To evaluate the pathophysiological effects of zinc transporter 8(ZnT8)and its disfunction on the occurrence and development of epithelial-to-mesenchymal transition(EMT)and to analyze its regulatory pathway in the kidneys of diabetic mice(C57BL)and normal rat kidney tubular epithelial cells(NRK-52E).Methods: To explore the effect of ZnT8 on the development of EMT in kidney tissues of various types of diabetic mice,inflammation-related factors and EMT-related indicators in kidney tissues of mice,experimental animals such as STZ-induced C57BL/6J mice,db/db mice,ZnT8-KO mice,ZnT8-KO-STZ mice and ZnT8-KO-db/db mice were used to detect the expression of transforming growth factor ?1(TGF-?1),interleukin-6(IL-6),tumor necrosis factor ?(TNF-?),vimentin,E-cadherin and other related factors in the kidney tissues of mice by ELISA,immunofluorescence staining and Western blot respectively.HZnT8-EGFP and si RNA were used to transiently transfect and interfere with NRK-52 E respectively to establish ZnT8 overexpression model and ZnT8 silencing model.High glucose(HG,30 m M)was used to induce NRK-52 E cells for EMT.To explore the effect of ZnT8 on the occurrence and development of EMT and the distribution of free zinc ions in NRK-52 E cells induced by HG,and to detect whether ZnT8 can regulate EMT by regulating TGF-?1/Smad signaling pathway,the expression of ZnT8,vimentin,E-cadherin,TGF-?1,Smad2/3 and p-Smad2/3 in NRK-52 E cells were detected by immunofluorescence staining,Western blot and zinc TSQ staining respectively.Results: 1.The values of various biochemical and metabolic indexes of diabetic mice in each group were significantly different from those in the normal control group,and the changes were most obvious in ZnT8-KO-STZ mice and ZnT8-KO-db/db mice.Compared with non-diabetic control group(C57BL/6J)and ZnT8-KO mice,STZ-induced diabetic mice,ZnT8-KO-STZ mice,db/db mice,and ZnT8-KO-db/db mice appeared weight loss,increased diet and drinking,increased urine output,increased fasting blood glucose(FBG),increased serum creatinine(Cr),blood urea nitrogen(BUN),and urinary albumin to creatinine ratio(UACR),of which ZnT8-KO-STZ mice and ZnT8-KO-db/db mice showed the most significant changes in various indicators.2.The expression levels of three inflammatory factors in diabetic mice in each group increased,and the expression levels of ZnT8-KO-STZ mice and ZnT8-KO-db/db mice changed in parallel.Compared with non-diabetic control group and ZnT8-KO mice,the expression levels of TGF-?1,IL-6 and TNF-? in the serum of STZ-induced diabetic mice,ZnT8-KO-STZ mice,db/db mice and ZnT8-KO-db/db mice were significantly increased,and the expression levels of them in ZnT8-KO-STZ mice and ZnT8-KO-db/db mice were similar.3.The expression levels of EMT indicators in diabetic mice in each group were significantly different from those in the normal control group,and the changes were most obvious in ZnT8-KO-STZ mice and ZnT8-KO-db/db mice.Immunofluorescence detection showed that compared with the non-diabetic control group,the expression of aquaporin-1(AQP1)in each group was basically unchanged,the expression of vimentin was increased in STZ-induced diabetic mice and db/db mice and was significantly increased in ZnT8-KO-STZ and ZnT8-KOdb/db mice,and the expression of E-cadherin was reduced in STZ-induced diabetic mice and db/db mice and was significantly reduced in ZnT8-KO-STZ mice and ZnT8-KO-db/db mice.4.Regulating the expression of ZnT8 can significantly change the distribution of zinc ions in NRK-52 E cells.TSQ staining results showed that compared with the control group,overexpression of ZnT8 significantly increased the amount of zinc ion expression in cells,while ZnT8 silencing can significantly reduce the expression of zinc ions in cells,and this change trend is not controlled by HG induction.5.Regulating the expression of ZnT8 can significantly change the degree of HG-induced EMT in NRK-52 E cells.Immunofluorescence staining showed that compared with the control group,the cell morphology became fibroblast-like,and the expression of vimentin was increased while the expression of E-cadherin was decreased after HG treatment.Compared with the control group,the EMT trend of h ZnT8 group induced by HG was slowed down,and the EMT trend of ZnT8 RNAi group induced by HG was increased obviously.6.ZnT8 may inhibit HG-induced EMT in NRK-52 E cells by regulating the TGF-?/Smad signaling pathway.Western blot results showed that compared with the control group,HG-induced ZnT8 overexpression inhibited Smad2/3 phosphorylation,while ZnT8 silencing stimulated their phosphorylation.Conclusion: 1.ZnT8 has protective effect on EMT in kidney tissues of T1 DM and T2 DM mice.2.ZnT8 has a protective effect on EMT in NRK-52 E cells induced by HG.3.ZnT8 can inhibit the occurrence of EMT in NRK-52 E cells induced by HG by regulating the TGF-?1/Smad signaling pathway. |
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