摘要
目的动态观察足细胞裂孔隔膜复合体分子nephrin、podocin、CD2AP及α-actinin-4 在阿霉素肾病大鼠蛋白尿发生发展中的表达变化,探讨其在蛋白尿发生发展中的分子行为及其机制。方法尾静脉注射阿霉素建立阿霉素肾病大鼠模型,3、7、14、28 d每组处死6只大鼠留取肾脏标本。应用间接免疫荧光染色、实时定量PCR、Western印迹分别检测各个时间点 nephrin、podocin、CD2AP、α-actinin-4的分布、mRNA和蛋白表达量的变化。结果 (1)14 d肾病组大鼠24 h尿蛋白显著高于对照组(P<0．01),增高持续到28 d。(2)透射电镜显示14 d肾病组足突不同程度变宽,28 d足突弥漫性融合。(3)与对照组相比,从第7天开始肾病组nephrin和 podocin染色从沿肾小球毛细血管袢线样分布向不连续粗颗粒样的分布模式转变;CD2AP节段染色增强区逐渐扩大,有的区域呈斑片状和连续线状增强;α-actinin-4从沿肾小球毛细血管袢均匀的点线样分布向不均匀粗颗粒的分布转变,而且随时间进展这种转变逐渐加重。(4)与对照组相比,肾病组于7 d时nephrin mRNA表达显著增高(P<0．01);podocin mRNA表达14 d时显著增高(P<0．05),直至28 d(P<0．05);CD2AP mRNA表达28 d时显著增高(P<0．05)。(5)与对照组相比,肾病组nephrin蛋白表达28 d时显著增高(P<0．05);podocin蛋白表达于7 d时显著增高(P<0．05),而28 d时又显著降低(P<0．05);CD2AP蛋白表达于14 d时显著增高(P< 0．05),直至28 d(P<0．05)。(6)α-actinin-4 mRNA与蛋白表达在实验过程中未出现明显变化。结论 nephrin、podocin和CD2AP的表达增加及分布异常是导致阿霉素肾病大鼠蛋白尿发生发展的分子机制,而分子表达的增加是足细胞抵抗损伤的一种代偿反应。
Objective To dynamically observe the expression of slit diaphragm complex molecules, including nephrin, podocin, CD2AP, and cytoskeleton protein α-actinin-4, in adriamycin-induced nephrotic （ADN） rats, and to further explore the molecular behavior of podocyte proteins during the occurrence and development of proteinuria and their possible mechanisms. Methods Adriamycin nephropathy was induced by a single tail intravenous injection of adriamycin. Renal tissue samples were collected at day 3, 7, 14, and 28, respectively. The distribution, mRNA expression and protein expression of nephrin, podocin, CD2AP and ct-actinin-4 were examined by indirect i mmunofluorescence, real-time PCR and Western blotting, respectively. Results （1） After the adriamycin injection, a significant increment of the 24-hour urinary protein was observed at day 14 and persisted up to day 28 （P 〈 0.01）. （2） In ADN rats, the foot processes broadened to a different extent at day 14, and the diffuse fusion and effacement of foot processes were observed at day 28. （3） From the 7th day to the 28th day after adriamycin injection, nephrin and podocin staining gradually shifted from a linear-like pattern along the capillary loops of glomerulus to a discontinuous coarse granular pattern. Similarly, CD2AP shifted from an even GBM-hke pattern to a coarse granular pattern, and -αactinin-4 changed from a dot linear-like pattern along capillary loops to a coarse granular pattern. （4） In ADN rats, nephrin mRNA expression was up-regulated at day 7 （P 〈 0.01 ） and returned to normal level at day 14 and 28. Podocin and CD2AP constantly increased from day 3 and reached a significant level at day 14 and day 28, respectively （P 〈 0.05）. （5） The protein expression of nephrin started to increase at day 7 after adriamycin injection and was markedly elevated up to day 28 （P 〈 0.05 ）. Likewise, compared with the control group, CD2AP protein expression of the ADN rats prominently increased at day 14 and persisted to day 28 （P 〈 0.05）. Interestingly, after the injection of adriamycin, podocin protein expression was dramatically upregulated at day 7 （P 〈 0.05）, and thereafter recovered again, whereas it was significantly downregulated at day 28 （P 〈 0.05）. （6） Alpha-actinin-4 mRNA and protein expression showed no change at the time studied. Conclusion The increased expression of nephrin, podocin and CD2AP and their abnormal distributions are the molecular mechanism that leads to the occurrence and development of proteinuria in ADN rats. The enhanced expression may be a compensatory reaction of podocyte to injury.
出处
《中华肾脏病杂志》
CAS
CSCD
北大核心
2006年第1期27-32,共6页
Chinese Journal of Nephrology
基金
国家自然科学基金资助项目(30170992)教育部留学回国人员科研启动基金(2003-14)
