Smad锚着蛋白在糖尿病肾病肾小管上皮细胞转分化中的作用及机制研究

Role of SARA in renal tubular epithelial to mesenchymal transition in diabetic nephropathyand its associated mechanism

目的研究Smad锚着蛋白（SARA）在高葡萄糖诱导的HK-2细胞转分化中的作用及相关机制。方法用高浓度葡萄糖（30mmol／LD．葡萄糖）刺激HK-2细胞，分别采用细胞免疫荧光、Western印迹及实时定量PCR等方法检测HK-2细胞波形蛋白（vimentin）、紧密连接蛋白（ZO-1）、SARA、转化生长因子p1（TGF-β1）、Smad2及Smad3的表达。分别转染全长SARA质粒[SARA（wT）]及敲除SBD结构域的SARA质粒[SARA（dSBD）]，检测转染后高糖诱导的HK-2细胞Vimentin、ZO-1、Smad2、Smad3、p-Smad2、p-Smad3表达的变化。结果高糖刺激时，HK-2细胞出现转分化；ZO．1蛋白和mRNA表达呈时间依赖性下调；vimentin蛋白和mRNA表达呈时间依赖性上调，而SARA蛋白和mRNA表达呈时间依赖性下调；TGF-61、Smad3蛋白和mRNA表达呈时间依赖性上调；Smad2mRNA表达呈时间依赖性上调，而其蛋白表达呈时间依赖性下调；Smad2和Smad3磷酸化，Smad3活化时间更长。与高糖刺激组相比，转染全长SARA质粒[SARA（wT）]过表达SARA使HK-2细胞ZO-1表达上调（P〈0．05）；vimentin表达下调（P〈0．05）；Smad2蛋白表达上调，但mRNA表达无明显变化；Smad2活化时间延长。转染SARA（dSBD）质粒对高糖诱导的HK-2细胞ZO．1、vimentin、Smad2的表达无影响，对Smad2和Smad3的磷酸化亦无明显影响。结论高糖诱导的HK．2细胞转分化过程中，TGF-β1信号通路活化，SARA的表达下调。过表达SARA可能通过上调Smad2的蛋白表达，延长Smad2活化时间，从而抑制TGF-β1信号传导，进而抑制高糖诱导的HK-2细胞转分化进程。

Objective To determine the effect of smad anchor for receptor activation （SARA） on renal tubular epithelial to mesenehymal transtion （EMT） induced by high glucose and to investigate the associated mechanism. Methods HK-2 cells were exposed to high glucose （30 mmol/L）. HK-2 cells were transfected with the plasmids of wild-type SARA [SARA （WT）] or SARA mutant [SARA with SBD deletion, called SARA （dSBD）] and then was stimulated by high glucose. The gene expression was assayed by real-time PCR and the protein expression wasdetected by Western blotting. Results During the process of high glucose-induced EMT of HK- 2 ceUs, the gene and protein expression of SARA were down-regulated. The expression of TGF-β1 and Smad3 increased after stimulation of high glucose in HK-2. However, the Smad2 mRNA expression increased while its protein expression was down-regulated in a time-dependent manner. Smad2 and Smad3 were activated by high glucose stimulation and Smad3 kept activation for longer time than Smad2. Compared with high glucose group, over-expression of SARA by transfection of SARA （WT） up-regulated the expression of zona occludens（ZO）l and down-regulated the expression of vimentin （P〈0.05）. However, SARA （dSBD） had no such effects on above expressions. The Smad2 protein expression increased along with the over-expression of SARA. Meanwhile, over- expression of SARA prolonged the activation time of Smad2 and shortened the activation time of Smad3. Conclusions TGF-β1 signaling is activated and SARA expression is down-regulated during the process of high glucose-induced EMT in HK-2 cells. Over-expression of SARA can inhibit the EMT via increase of Smad2 protein expression and longer activation time of Smad2.