Protective Effect Of ZnT7 On High Glucose-induced Epithelial-to-mesenchymal Transition In Renal Tubular Epithelial Cells

Objective:To investigate the effect of ZnT7 on EMT of renal tubular epithelial cells（RTEC）in diabetic nephropathy（DN）.Methods:A dual-fluorescent staining protocol was used for detection of ZnT7 in a normal rat kidney tubular epithelial cell line（NRK-52E cells）.EMT in the cells was induced with high glucose（HG,30 mM）.Transfection of hZnT7-EGFP and interfering RNA were applied in NRK-52E cells for determination of ZnT7 over-expression and silence,respectively.Activities of ZnT7,the MAPK/ERK and TGF-β/Smad pathways were analyzed with Western blot.Results:1.Intracellular Zn²⁺levels in HG-stimulated NRK-52E cells.Normal and HG-stimulated NRK-52E cells at concentration of 30 mM for 12 h,24 h,48 h,and 72 h,respectively.TSQ fluorescence staining showed that the cellular Zn²⁺levels in HG-stimulated NRK-52E cells after 24 h were significantly decreased compared with what in non-stimulated NRK-52E cells.2.ZnT7 activities in HG-stimulated NRK-52E cells.Under non-stimulated and HG conditions,Western blot demonstrated that ZnT7expression was increased in HG-stimulated NRK-52E cells after 48 h.3.Dual immunofluorescence of ZnT7 and TGN38.Fluorescence staining demonstrated ZnT7 was mainly localized in the perinuclear region and Golgi apparatus of the cells.4.ZnT7activity and Zn²⁺in the perinuclear region.A dispersed pattern of Zn staining was seen in the perinuclear region of the cells of HG,ZnT7 RNAi and HG/ZnT7 RNAi groups.The highest score of Zn fluorescence staining was detected in hZnT7 group.The fluorescent intensity in the HG/ZnT7 RNAi group was weaker than in any other group.5.Effect of ZnT7 on HG-induced EMT.In contrast,over-expressing ZnT7 in NRK-52E cells significantly decreased HG-induced EMT,as evidenced by the reduced up-regulation ofα-SMA and vimentin,and ameliorated expression of E-cadherin.6.Effect of ZnT7 on the TGF-β/Smad Pathway.The levels of p-Smad2/3 were significantly increased in HG group compared with the control group,which robust-enhanced in HG/ZnT7 RNAi group.Conversely,over-expression of ZnT7 significantly inhibited HG-induced levels of p-Smad2/3.However,the co-treatment with SB43152effectively inhibited Smad2 and Smad3 phosphorylation and HG-induced EMT.7.Effect of ZnT7 on the MAPK Pathway.We observed a robust increase of the phospho-p38,and phospho-ERK in the HG-treated NRK-52E cells compared with the control group.Conversely,over-expression ZnT7 significantly inhibited HG-induced expression of phospho-ERK.However,pretreatment of these cells with PD98059 decreased the HG-induced EMT.Conclusion:The present study provides evidence that ZnT7 has the protective effect on EMT of RTEC in DN and suggests that the inhibition of HG-induced EMT may be involved in the MAPK/ERK and TGF-β/Smad pathways.