血管内皮生长因子表达降低导致多柔比星肾病大鼠蛋白尿发生

Reduction of Vascular Endothelial Growth Factor Expression Induced Proteinuria in Adriamycin Nephrotic Rats

目的在经典多柔比星肾病大鼠模型中动态观察其蛋白尿发生前后血管内皮生长因子(VEGF)分布和表达的时相变化,及其与足细胞裂孔隔膜关键分子nephrin磷酸化水平之间的关系,以深入了解VEGF在蛋白尿发生机制中的作用。方法1.留取正常大鼠(对照组)及多柔比星肾病大鼠模型(肾病组)3、7、14和28d肾皮质标本。2.应用免疫组织化学染色法观察对照组及28d肾病组大鼠肾组织VEGF分布。3.提取对照组及3、7、14和28d肾病组大鼠肾皮质总RNA,应用反转录获得cDNA,实时定量RT-PCR检测其VEGFmRNA表达。4.提取对照组及3、7、14和28d肾病组大鼠肾皮质总蛋白,应用免疫蛋白印迹检测其VEGF蛋白表达。5.提取对照组及28d肾病组大鼠肾皮质总蛋白,应用免疫沉淀分析其nephrin酪氨酸磷酸化水平。6.采用SPSS10.0软件进行统计学分析,各时间点肾病组与对照组间比较采用t检验。结果1.免疫组织化学染色显示对照组大鼠肾小球VEGF的染色较深,主要分布于肾小球足细胞及近端小管上皮细胞,系膜区亦有少许表达。在28d肾病组大鼠,VEGF在肾小球足细胞和系膜区的染色明显减弱。2.与对照组比较,3、7、14及28d各时间点肾病组大鼠肾皮质VEGF的mRNA表达无显著性改变(Pa>0.05)。3.与对照组比较,肾病组大鼠肾皮质VEGF蛋白表达于注射多柔比星后7d显著降低(P<0.05),且在14d和28d2个时间点亦显著低于对照组(Pa<0.01)。4.与对照组比较,28d肾病组大鼠肾皮质nephrin磷酸化水平显著降低(P<0.05)。结论VEGF表达降低可能参与了多柔比星肾病大鼠蛋白尿的发生,VEGF与裂孔隔膜关键分子nephrin的磷酸化水平可能存在联系,它们相互协调、相互作用,共同参与维护肾小球滤过屏障结构和功能的完整。

Objective To detect the temporal and spacial changes of vascular endothelial growth factor（VEGF） expression in the occurrence and development of proteinuria in adriamyein nephritic rats and explore preliminarily the possible relationship between VEGF and podocyte slit diaphragm molecule nephfin phosphorylation in order to further elucidate the mechanisms of proteinuria. Methods 1. The samples of renal tissue were obtained from the adriamycin nephritic rats at day 3,7,14 and 28 after injections of adriamyein for immunohistochemistry staining and total RNA and protein extractions. 2. The distributions of VEGF were observed in the controls and the adfiamycin nephritic rats at day 28 by using the immunohistochemistry staining. 3. Total renal cortex RNA were extracted from the controls and the adriamycin nephritic rats at day 3,7,14 and 28 ,and were used to make eDNA by using reverse transcription （RT） reactions. Then,the mRNA expression of VEGF was detected by using the real time quantitative RT - PCR. 4. Total renal cortex protein were extracted from the controls and the adriamycin nephritic rats at day 3,7,14 and 28 ,and the protein expression of VEGF was detected by using Western blot. 5. Total renal cortex protein were extracted from the controls and the adriamycin nephritic rats at day 28, and the level of nephrin tyrosinase phosphorylation was evaluated by using immunoprecipitation. 6. Data were analyzed by SPSS 10.0 software and performed by using t - test. Results 1. VEGF was displayed mainly in the podocyte and proximal tubule epithelium, and also was detected weakly in the mesangium. Compared with the controls, the staining intensity of VEGF in the glomeruli of the adriamycin nephritic rats decreased evidently at day 28.2. In the adriamyein nephritic rats, VEGF mRNA expression did not show significant changes （ P 〉 0.05 ） at any observed time points as compared with the controls. 3. From day 7 after the adriamyein injeetion,VEGF protein reduced significantly （P 〈 0.05 ） until day 14 and 28 as compared with the controls （Pa 〈 0. 01 ）. 4. At day 28 after the adriamyein injection, the level of nephrin tyrosinase phosphorylation decreased evidently （ P 〈 0.05 ） as compared with the controls. Conclusions The reduction of VEGF protein might be involved into the occurrence and development of proteinuria in the adriamycin nephritic rats. There might be some relationship between VEGF and nephrin tyrosinase phosphorylation, which might be very important for maintaining the integrity of the structure and function of glomerular filtration barrier.