SLC30A7 Has Anti-oxidant Stress Effects In High Glucose-induced Apoptosis Via The NFE2L2/HMOX1 Signal Transduction Pathway

Objective: Apoptosis and oxidant stress are known to be involved in the pathogenesis of diabetic kidney disease(DKD).We have previously reported that zinc transporter 7 in the SLC30 family(Zn T7,SLC30A7)can inhibit the apoptosis of rat peritoneal mesothelial cells under high glucose(HG)conditions.In the current study,we aimed to investigate whether SLC30A7 had effect for antioxidant stress in renal tubular epithelial cells under HG.Methods: SLC30A7 in HG-induced apoptosis in a normal rat kidney tubular epithelial cell line(NRK-52 E cells)/kidneys of STZ-induced diabetic mice was examined and the activity of nuclear factor erythroid 2-related factor 2(NFE2L2)was further analyzed by using real time RT-PCR,si RNA and Western blot protocols.Results:1.Decreased serum zinc level and kidney injury in diabetic mice.Compared to those in control group(n=8),STZ-induced diabetic mice(n=8)exhibited typical diabetic symptoms,such as drink,diet and urine increases,and kidney weight/body weight ratio(KW/BW)in diabetic mice were significantly higher.Moreover,diabetic mice exhibited slightly decreased levels of zinc in serum and kidney compared with the control group mice.2.SLC30A7 and NFE2L2 expression levels in renal proximal tubular cells of diabetic mice.Immunofluorescence staining detected that the expression of SLC30A7 was localized in the perinuclear region of renal proximal tubular cells(RPTC)in diabetic mice,which was consistent with its localization in the Golgi apparatus.Subsequently,we used immunofluorescence staining to co-localize the anti-aquaporin-1(APQ1)of SLC30A7 and RPTC,and also detected that the expression level of NFE2L2 in diabetic mice increased compared with the blank control group.3.RPTCs apoptosis in diabetic mice.The percentage of TUNEL-positive RPTCs was significantly higher in diabetic mice than in control mice.Western blot analysis revealed increases in caspase3 and caspase9 protein expression in cortex tissues of STZ-induced diabetic mice compared with the control mice.4.Intracellular free Zn and SLC30A7 expression levels in HG-stimulated NRK-52 E cells.AAS was used to determine the concentration of Zn ions in the NRK-52 E cells.We measured intracellular free zinc levels after culture at normal(5.5 mmol/L)or HG(30 m M)for 24 h,48 h,72 h and 96 h,respectively.Exposed to HG after 24 h leading to decreased in the levels of free intracellular Zn compared with non-stimulated NRK-52 E cells.To understand the cyto-protectant role of SLC30A7 in NRK-52 E cells and to further elucidate the mechanisms responsible for tubular epithelial cells apoptosis in DKD,we firstly analyses of SLC30A7 expression in response to HG.SLC30A7 protein expression was increased in HG-stimulated NRK-52 E cells after 48 h.Mannitol(44.4 m M)did not change the expression of SLC30A7,which suggested that it was not the high osmolarity,but HG that increased SLC30A7 expression in the NRK-52 E cells.5.Influence of SLC30A7 on HG-induced apoptosis in NRK-52 E cells.The cell viability was detected using MTT analysis,after exposure to HG and it was found that the cell viability of HG/SLC30A7/si RNA group was lower than that of HG group.We found that the apoptosis rate of the HG/ Zn T7/si RNA group was significantly increased compared with the HG group by using TUNEL and PI double staining.6.ROS production and NFE2L2/ HMOX1 expression after exposed to HG in NRK-52 E cells.We first measured the HG-induced intracellular ROS levels in NRK-52 E cells with DCF-DA staining.In conjunction with HG treatment for 0,12,24,48,and 72 h,resulted in a substantial increase of ROS production in the cells.In addition,RT-PCR was used to detect the expression levels of NFE2L2 and HMOX1 m RNA in the HG treatment group.We found that the levels of NFE2L2 and HMOX1 m RNA increased with time when HG was cultured,which proved that NFE2L2 is involved in the oxidative stress induced by HG in DKD.7.Reduction in NFE2L2 expression decreased SLC30A7 expression in HG-induced apoptosis in NRK-52 E cells.si RNA knockdown of the NFE2L2 gene at various time-points and the levels of NFE2L2,HMOX1,and SLC30A7 m RNA levels were detected using real time-PCR analysis after si RNA-NFE2L2 transfection.The expression levels of NFE2L2,HMOX1,and SLC30A7 were decreased after si RNA-NFE2L2 transfection at 24,48,72 and 96 h respectively.These results suggested that SLC30A7 might protect tubular epithelial cells from HGinduced apoptosis may through activation of NFE2L2 pathway.Conclusion: The current study provides new evidence that SLC30A7 has anti-oxidant stress effects in HG-induced apoptosis via the NFE2L2/HMOX1 signal transduction pathway.