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中华妇幼临床医学杂志(电子版) ›› 2009, Vol. 05 ›› Issue (04) : 331 -337. doi: 10.3877/cma.j.issn.1673-5250.2009.04.101

论著

Podocin与nephrin在多柔比星诱导肾病大鼠模型肾皮质部的表达及共定位分布
温捷, 于力, 温跃强, 郝志宏, 王丽娜, 陈蓉燕   
  1. 510180 广州,广州医学院附属广州市第一人民医院儿科
  • 出版日期:2009-08-01

Expression and Co-Localization of Nephrin and Podocin in Adriamycin-Induced Nephrotic Rats

Jie WEN, Li YU, Yue-qiang WEN, Zhi-hong HAO, Li-na WANG, Rong-yan CHEN   

  1. Department of Pediatrics, Affiliated Guangzhou First Municipal People's Hospital of Guangzhou Medical College, Guangzhou 510180, China
  • Published:2009-08-01
  • Supported by:
    * Project No.5000424, supported by the Natural Science Foundation Project of Guangdong Province; project No.2006Z3–E0231, supported by the Science and Technology Research Project of Guangzhou; project No.2007–Z–78–1, supported by the Science and Technology Research Project of Panyu District
引用本文:

温捷, 于力, 温跃强, 郝志宏, 王丽娜, 陈蓉燕. Podocin与nephrin在多柔比星诱导肾病大鼠模型肾皮质部的表达及共定位分布[J]. 中华妇幼临床医学杂志(电子版), 2009, 05(04): 331-337.

Jie WEN, Li YU, Yue-qiang WEN, Zhi-hong HAO, Li-na WANG, Rong-yan CHEN. Expression and Co-Localization of Nephrin and Podocin in Adriamycin-Induced Nephrotic Rats[J]. Chinese Journal of Obstetrics & Gynecology and Pediatrics(Electronic Edition), 2009, 05(04): 331-337.

目的

观察podocin与nephrin在多柔比星诱导肾病(adriamycin–inducing nephrotic,ADN)大鼠模型病变过程中的表达及共定位分布,探讨podocin和nephrin在肾病病变过程中的作用机制。

方法

将60只雄性Spraque–Dawley(SD)大鼠分为模型组(尾静脉注射6.5 mg/kg多柔比星,n=30)和对照组(尾静脉注射0.8 mL生理盐水,n=30)。分别于造模后的第1、第2、第4、第6和第8周末处死大鼠,并观察各项指标的变化:①24 h尿蛋白定量,血浆白蛋白、血胆固醇、血肌酐和血尿素氮水平;②两组上述各时间点取肾皮质进行光镜和电镜检测;③采用双标准曲线法进行荧光定量PCR检测肾皮质部podocin mRNA和nephrin mRNA表达;④应用双重免疫荧光法观察肾小球podocin和nephrin的共定位分布。

结果

①模型组大鼠出现大量蛋白尿、低白蛋白血症、高脂血症,肾功能减退,表现为典型的肾病综合征;模型组病理改变由微小病变逐渐发展至局灶节段性肾小球硬化,电镜下可见足细胞在病变早期出现足突增宽、融合,晚期足突消失、细胞核固缩;②模型组podocin mRNA表达第1周末时升高95.8%(P<0.01),第2周末时升高252.5%(P<0.01)且达高峰,第4周末时开始下降27.6%(P<0.05),第6和第8周末分别下降52.9%和48.0%,与对照组比较,差异有显著意义(P<0.01)。模型组nephrin mRNA表达第1周末时升高140.8%(P<0.05)且达高峰,第2周末时持续升高80.4%(P<0.05),第4、第6和第8周末时分别下降39.2%,47.5%和52.5%,与对照组比较,差异有显著意义(P<0.01)。③模型组免疫荧光染色可见podocin与nephrin在大鼠肾小球内分布,且随病变进展而增加,由正常的沿肾小球血管襻呈均匀线性分布转变为呈不连续分布,而且重叠程度减少。

结论

Podocin和nephrin在多柔比星肾病大鼠肾病进展过程中出现表达及分布异常,导致两者不能形成正常的复合体结构,这可能是蛋白尿发生的重要分子机制。

Objective

To observe the expression and distribution of nephrin and podocin in adriamycin-inducing nephrotic (ADN) rats, and explore the possible mechanisms of nephrin and podocin during the progression of nephrotic syndrome(NS).

Methods

Sixty Spraque-Dawley(SD) rats were divided into ADN model group(injected 6.5 mg/kg adriamycin in tail vein, n=30)and control group (injected 0.8 mL saline solution in tail vein, n=30). After the nephropathy model was established, rats were killed at the end of 1st, 2nd, 4th, 6th, 8th week. The changes of the following indicators were observed: ① 24-hour urinary protein, serum albumin, serum cholesterol, serum creatinine and serum urea nitrogen were detected; ② The characters of renal cortex detected by optical microscope and electron microscope at different time points were observed; ③ The expression of podocin mRNA and nephrin mRNA in cortex of kidney were examined by real time RT-PCR; ④ The co-localization of nephrin and podocin in glomerular was observed by dual-labeling immunofluorescence.

Results

①ADN model group came out massive proteinuria, hypoalbuminemia, hypercholesterolaemia, renal failure, and presented as nephrotic syndrome. ADN model group developed from minimal change diseases to focal segmental glomerulosclerosis. The ultrastructure of podocyte showed that foot process conflurence in the early stage, foot process disappeared and chromatin raritas in the end stage. ② ADN model group compared with control group: podocin mRNA expression of AND model group increased 95.8% (P<0.01)at the end of the first week, increased 252.5%( P<0.01) at the end of the second week and reached the peak value, then began to decrease 27.6%(P<0.05)at the end of the fourth week, and decreased 52.9%(P<0.01)at the end of the sixth week, and decreased 48.0%(P<0.01)at the end of the eighth week. Nephrin mRNA expression of ADN model group increased 140.8% (P<0.05) at the end of the first week and reached the peak value, then increased 80.4%( P<0.05)at the end of the second week, decreased 39.2%(P<0.01)at the end of the fourth week, decreased 47.5%(P<0.01)at the end of the sixth week, decreased 52.5%(P<0.01)at the end of the eighth week. ③In ADN model group, the distribution of nephrin and podocin gradually shifted from a linear-like pattern along the capillary loops of glomerulus to a discontinuous coarse granular pattern, and the degree of overlapping localization decreased.

Conclusion

The abnormal expression and distribution of nephrin and podocin during the progression of adriamycin nephropathy lead nephrin and podocin can not form normal complex, but it may be the important molecular mechanism of proteinuria.

图1 两组肾组织病理学变化[图A、图B、图C、图D分别为对照组及模型组第4、第6、第8周末的肾小球组织结构(光镜×400);图E、图F、图G、图H分别为对照组及模型组第4、第6、第8周末的足细胞及足突超微结构(电镜×15 000)]
图2 Podocin、nephrin和β-actin琼脂糖凝胶电泳图
图3 两组nephrin mRNA表达变化
图4 Podocin和nephrin在肾小球的共定位分布(免疫荧光×400)[图A、图D、图G、图J、图M分别为对照组,模型组第2、第4、第6、第8周末podocin和nephrin在肾小球共定位分布;图B、图E、图H、图K、图N分别为对照组,模型组第2、第4、第6、第8周末nephrin在肾小球的分布;图C、图F、图I、图L、图O分别为对照组,模型组第2、第4、第6、第8周末podocin在肾小球的分布]
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