CAJ | 학술논문

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3 d、7 d、14 d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α平滑肌肌动蛋白(α-SMA);原位末端标记法(TUNEL)与DNA电泳观察肾小管间质细胞凋亡情况;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α-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GRP78 mRNA表达于术后3 d即发生显著上调,而蛋白表达在术后7 d开始出现显著变化,与假手术组差异均有统计学意义(均P＜0.01);在此后观察期间内GRF78 mRNA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3 d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7 d、14 d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.
목적 탐토내질망응격(ERS)상관조망도경재단측수뇨관경조(UUO)대서신간질섬유화발생、발전중적작용.방법 건강웅성Wistar대서25지,안수궤수자표법분위UUO모형조(n=18)화가수술조(n=7),UUO모형조행좌측수뇨관결찰술,가수술조부분리수뇨관불결찰,분별우술후3 d、7 d、14 d처사각조대서,행HE화Masson염색,관찰신장병리변화;비색법측정신조직간포안산(HYP)함량;면역조화법검측α평활기기동단백(α-SMA);원위말단표기법(TUNEL)여DNA전영관찰신소관간질세포조망정황;RT-PCR법검측경조측신조직ERS상관분자포도당조절단백78(GRP78)mRNA표체변화;Western인적법분석조망상관단백반광안산천문동안산단백매3(caspase-3)화GRP78적단백표체변화.결과 여가수술조비교,UUO모형조신장병리개변가중,신간질섬유화정도수경조시간연장축점가중,신조직HYP함량현저승고(P＜0.05).신조직α-SMA야재신소관간질세포엄범표체,TUNEL염색급DNA경지당응효전영제시대량적신소관간질세포조망.UUO모형조GRP78 mRNA표체우술후3 d즉발생현저상조,이단백표체재술후7 d개시출현현저변화,여가수술조차이균유통계학의의(균P＜0.01);재차후관찰기간내GRF78 mRNA화단백균지속고수평표체.모형조대서신조직caspase-3적단백표체재UUO술후3 d즉유현저상조(P＜0.05),차수착경조시간연장진행성승고,우술후7 d、14 d증다경위현저,여가수술조차이균유통계학의의(P＜0.05).상관분석현시GRP78단백표체여신조직HYP함량화caspase-3단백표체균정정상관(r=0.657,P＜0.01;r=0.714,P＜0.01).결론 UUO조기즉가유도ERS표지단백표체변화,촉발ERS.장기ERS가유도신소관간질세포조망;caspase-3개도적ERS상관조망도경가능삼여료신간질섬유화과정.
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 days after 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 glucose-regulated 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). Immunohistochemistry staining indicated that a-SMA extensively expressed in renal tubular and interstitial cells. The apoptotic cells 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-regulation in endoplasmic reticulum molecular chaperones at early stage, indicating that ERS response is activated in the rat kidney. Prolonged ERS can lead to renal tubular and interstitial cell apoptosis, and caspase-3-mediated ERS associated apoptosis may contribute to the fibrosis.