内质网应激相关凋亡途径参与单侧输尿管梗阻大鼠肾间质纤维化  

Endoplasmic reticulum stress-related apoptosis pathway is involved in unilateral ureteralobstruction-induced renal interstitial fibrosis in rats

作　　者：阮颖新[1] 张鹏宇[2] 林珊[1] 李春媚[3] 刘素雁[3] 

机构地区：[1]天津医科大学总医院肾内科阮颖新、林珊,300052 [2]天津医科大学肿瘤医院检验科天津市肿瘤防治重点实验室张鹏字,300052 [3]哈尔滨医科大学第二临床医学院肾内科李春媚、刘素雁,300052

出　　处：《中华肾脏病杂志》2011年第5期357-362,共6页Chinese Journal of Nephrology

基　　金：国家自然科学基金（30800529）;黑龙江省卫生厅科研课题（2007-305）

摘　　要：目的探讨内质网应激（ERS）相关凋亡途径在单侧输尿管梗阻（UUO）大鼠肾间质纤维化发生、发展中的作用。方法健康雄性Wistar大鼠25只，按随机数字表法分为UUO模型组（n=18）和假手术组（n=7），UUO模型组行左侧输尿管结扎术，假手术组仅分离输尿管不结扎，分别于术后3d、7d、14d处死各组大鼠，行HE和Masson染色，观察肾脏病理变化；比色法测定肾组织羟脯氨酸（HYP）含量；免疫组化法检测仅平滑肌肌动蛋白（d—SMA）；原位末端标记法（TUNEL）与DNA电泳观察肾小管问质细胞凋亡情况；RT．PCR法检测梗阻侧肾组织ERS相关分子葡萄糖调节蛋白78（GRP78）mRNA表达变化；Western印迹法分析凋亡相关蛋白半胱氨酸天门冬氨酸蛋白酶3（caspase．3）和GRP78的蛋白表达变化。结果与假手术组比较，UUO模型组肾脏病理改变加重，。肾间质纤维化程度随梗阻时间延长逐渐加重，肾组织HYP含量显著升高（P〈0．05），肾组织仅一SMA也在肾小管间质细胞广泛表达，TUNEL染色及DNA琼脂糖凝胶电泳提示大量的肾小管间质细胞凋亡。UUO模型组GRP78mRNA表达于术后3d即发生显著上调，而蛋白表达在术后7d开始出现显著变化，与假手术组差异均有统计学意义（均P〈0．叭）；在此后观察期间内GRP78mRNA和蛋白均持续高水平表达。模型组大鼠肾组织caspase-3的蛋白表达在UUO术后3d即有显著上调（P〈0．05），且随着梗阻时间延长进行性升高，于术后7d、14d增多更为显著，与假手术组差异均有统计学意义（P〈0．05）。相关分析显示GRP78蛋白表达与肾组织HYP含量和caspase-3蛋白表达均呈正相关（r=0．657，P〈0．01；r=0．714，P〈0．01）。结论UUO早期即可诱导ERS标志蛋白表达变化，触发ERS。长期ERS可诱导肾小管间质细胞凋亡；caspase-3介导的ERS相关凋亡途径可能参与了肾间质纤维化过程。Objective To investigate the effect of endoplasmic reticulum stress （ERS）associated apoptosis on renal interstitial fibrosis induced by unilateral ureteral obstruction （UUO） in rats. Methods Eighteen healthy male Wistar rats undergoing UUO were sacrificed at 3,7,14 daysafter operation. Additional seven rats underwent sham operation. Histological changes were observed by HE and Masson staining. Immunohistochemistry was performed on renal tissue for α-smooth muscle actin （α-SMA）. Chromatometry was used to detect the content of hydroxyproline. Apoptosis cells were determined by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling （TUNEL） and the electrophoresis analysis of genome DNA. The mRNA expression of glucoseregulated protein 78 （GRP78）, which are important markers of ERS, were detected by RT-PCR. Western blotting was used to assess the protein level of GRP78 and cysteinyl aspartate specific proteinase-3 （caspase-3）. Results Compared with sham operation group, the degree of renal interstitial and the level of hydroxyproline content of UUO group increased significantly （P〈0.05）. lmmunohistochemistry staining indicated that ct-SMA extensively expressed in renal tubular and interstitial cells. The apoptotic ceils in the renal tubular and interstitium were continuously increased from day 3 to the end of experiment of UUO group. As early as 3 days after surgery, the mRNA level of GRP78 in UUO group increased compared with sham operation group （P〈0.01）, while the protein expression increased on day 7 after surgery （P〈0.01）. Prolonged ERS triggered apoptosis, the protein expression of caspase-3 increased significantly on day 3 after surgery （P〈 0.05）, and the expression sustained high level during the experiment afterwards. There was a positive correlation between GRP78 protein expression and hydroxyproline content （r=0.657, P〈 0.01） as well as caspase-3 protein expression （r=0.714, P〈0.01）. Conclusions UUO induces a significant up-regulat

关 键 词：输尿管梗阻 纤维化 细胞凋亡 内质网应激 

分 类 号：R285.5[医药卫生—中药学]