1,25-(OH)_(2)-VitD3通过抑制Snail1-SMAD3/SMAD4复合物形成减轻糖尿病肾病肾小管间质纤维化

1,25-(OH)_(2)-VitD3 attenuates renal tubulointerstitial fibrosis in diabetic kidney disease by inhibiting Snail1-SMAD3/SMAD4 complex formation

目的研究1,25-(OH)2-VitD3(VitD3)对糖尿病肾病肾小管间质纤维化的影响.方法NRK-52E肾小管上皮细胞分为对照组(5.5 mmol/L葡萄糖培养基处理)、高糖组(25 mmol/L葡萄糖的培养基处理)和高糖加VitD3组(25 mmol/L葡萄糖培养基联合10-8 mol/L VitD3).实时定量PCR和Western blot法分别检测NRK-52E细胞Snail家族转录抑制物1(Snail1)、SMAD家族成员3(SMAD3)、SMAD4、α平滑肌肌动蛋白(α-SMA)和上皮钙黏蛋白(E-cadherin)的mRNA和蛋白表达;免疫荧光细胞化学染色检测Snail1、SMAD3、SMAD4的表达及定位;通过染色质免疫沉淀检测Snail1与SMAD3/SMAD4形成的复合物与柯萨奇病毒-腺病毒受体(CAR)的启动子结合情况;荧光素酶报告检测Snail1、SMAD3/SMAD4和E-cadherin的相互作用;采用小干扰RNA(siRNA)抑制细胞Snail1、SMAD4的表达后,通过实时定量PCR检测E-cadherin的mRNA表达.SD大鼠随机分为对照组、DKD组和VitD3处理组.DKD组和VitD3处理组通过注射链脲佐菌素(STZ)先制备DKD模型,DKD造模成功后,VitD3处理组予60ng/kgVitD3灌胃,对照组和DKD组给予生理盐水灌胃;DKD组和VitD3处理组同时皮下注射胰岛素(1~2)U/kg控制血糖,持续8周.采用实时定量PCR和Western blot法分别检测肾组织中Snail1、SMAD3、SMAD4、α-SMA、E-cadherin的mRNA和蛋白水平,免疫组织化学检测肾组织Snail1、SMAD3、SMAD4、α-SMA和E-cadherin的表达及定位.结果与对照组相比,高糖处理的NRK-52E细胞及DKD肾组织Snail1、SMAD3、SMAD4和α-SMA的mRNA和蛋白表达均上调,E-cadherin表达下调;给予VitD3干预后,DKD模型中Snail1、SMAD3、SMAD4、α-SMA和E-cadherin的表达水平恢复到与对照组接近.染色质免疫沉淀提示Snail1、SMAD3/SMAD4与CAR的启动子Ⅳ结合,而VitD3能够阻止Snail1、SMAD3/SMAD4与CAR的启动子Ⅳ结合;荧光素酶报告检测证实Snail1、SMAD3/SMAD4和E-cadherin的互作关系;用siRNA抑制Snail1、SMAD4的mRNA后,上调高糖诱导下E-cadherin的表达.结论VitD3可抑制Snail1-SMAD3/SMAD4的复合物的形成,减轻糖尿病肾病肾小管间质纤维化.

Objective To investigate the effect of 1,25-(OH)_(2)-VitD3(VitD3)on renal tubuleinterstitial fibrosis in diabetic kidneydisease.Methods NRK-52E renal tubular epithelial cells were divided into control group(5.5 mmol/L glucose medium treatment),high glucose group(25 mmol/L glucose medium treatment)and high glucose with added VitD3 group(25 mmol/L glucose medium combined with 10^(-8) mmol/L VitD3).The mRNA and protein expression of Snai1,SMAD3,SMAD4,a-SMA and E-cadherin in NRK-52E cells were detected by real-time quantitative PCR and Western blot analysis respectively.The expression and localization of Snail,SMAD3 and SMAD4 were detected by immunofluorescence cytochemical staining.The binding of Snaill with SMAD3/SMAD4 complex to the promoter of Coxsackie-adenovirus receptor(CAR)was detected by chromatin immunoprecipitation.The interaction among Snaill,SMAD3/SMAD4 and E-cadherin were detected by luciferase assay.Small interfering RNA(siRNA)was used to inhibit the expression of Snaill and SMAD4,,and the expression of mRNA of E-cadherin was detected by real-time quantitative PCR.SD rats were randomly divided into control group,DKD group and VitD3-treated group.DKD model was established by injection of streptozotocin(STZ)in DKD group and VitD3-treated group.After DKD modeling,VitD3-treated group was given VitD3(60 ng/kg)intragastric administration.Control group and DKD group were given normal saline intragastric administration.In the DKD group and VitD3-treated group,insulin(1-2 U/kg)was injected subcutaneously to control blood glucose for 8 weeks.The mRNA and protein levels of Snail,SMAD3,SMAD4,a-SMA and E-cadherin in renal tissues were detected by real-time quantitative PCR and Western blot analysis respectively.Immunohistochemistry was used to detect the expression and localization of Snaill,SMAD3,SMAD4,α-SMA and E-cadherin in renal tissue.Results Compared with the control group,the mRNA and protein expressions of Snail1,SMAD3,SMAD4 and a-SMA in NRK-52E cells cultured with high glucose and in DKD renal tissues were up-regulated,while E-cadherin expression was down-regulated.After the intervention of VitD3,the expression levels of Snaill,SMAD3,SMAD4,a-SMA and E-cadherin in the DKD model improved to be close to those in the control group.Chromatin immunoprecipitation showed that Snaill and SMAD3/SMAD4 bound to CAR promoter IV,while VitD3 prevented Snail and SMAD3/SMAD4 from binding to CAR promoter IV.Luciferase assay confirmed the interaction among Snaill,SMAD3/SMAD4 and E-cadherin.After the mRNA of Snail and SMAD4 was inhibited by siRNA,the expression of E-cadherin induced by high glucose was up-regulated.Conclusion VitD3 could inhibit the formation of Snail-SMAD3/SMAD4 complex and alleviate the renal tubulointerstitial fibrosis in DKD.