泼尼松对阿霉素肾病大鼠肾组织FAK/Pyk2表达的影响

Effects of prednisone on renal FAK and Pyk2 expressions in rats with adriamycin-induced nephritis

目的观察泼尼松对阿霉素肾病大鼠FAK、Pyk2表达的影响。方法利用随机数字表将SD大鼠分为正常组、模型组、泼尼松组,建立阿霉素肾病大鼠模型,于阿霉素注射后第7天干预组以泼尼松10 mg/(kg·d)灌胃,于实验不同时间点测定24 h尿蛋白定量,观察肾组织电镜结果,实时荧光定量PCR检测FAK、Pyk2、Nephrin表达水平的变化,Western blot检测肾组织FAK、Pyk2蛋白及磷酸化蛋白,免疫组织化学法检测Nephrin表达水平。结果⑥与正常组比较,模型组尿蛋白升高(P<0.01);⑥与模型组比较,治疗组尿蛋白降低(P<0.01);⑥模型组电镜可见足细胞足突广泛融合,免疫组化示治疗组Nephrin较模型组有改善(P<0.05);⑥第21、35天,模型组、治疗组Nephrin mRNA表达较正常组低,FAK mRNA表达较正常组升高(P<0.01),治疗组Nephrin mRNA表达较模型组上升(P<0.05);⑥第21、35天,模型组FAK总蛋白及其磷酸化蛋白表达、P-FAK/FAK、P-Pyk2/Pyk2较正常组高(P<0.01),治疗组FAK总蛋白及其磷酸化蛋白表达、P-FAK/FAK、P-Pyk2/Pyk2较模型组降低(P<0.01);⑥多元相关分析显示,尿蛋白与FAKmRNA、FAK、pFAK、Pyk2mRNA及pPyk2/Pyk2比值呈正相关(r=0.819、0.750、0.838、0.762、0.934,P<0.01)。结论阿霉素可能通过激活FAK、Pyk2磷酸化导致肾病的发生;泼尼松能抑制FAK、Pyk2激活,降低阿霉素肾病大鼠蛋白尿,改善足突病变,从而保护肾小球滤过屏障。

Objective To investigate the effects of prednisone on the expressions of FAK and Pyk2 in the kidneys of rats with adriamydn-induced nephritis. Methods Thirty SD rats were randomized into normal control group, adriamycin-induced nephritic model group, and prednisone treatment group （n=10）. Prednisone was administered at 10 mg/kg once daily in nephritic rats starting since the 7th day after adriamycin injection. Twenty-four-hour proteinuria was measured in the rats at different time points, and renal tissue histology was examined using transmission electron microscope. The expression levels of Pyk2, FAK and nephrin mRNA in the renal tissue were detected tested by RT-PCR, and the protein expressions of FAK, Pyk2, phosphorylated Pyk2 and phosphorylated FAK-Tyr397 were detected by Western blotting; immunohistochemistry was used for detecting nephrin protein expression in the kidney. Results Compared with the normal control group, the rats with adriamycin-induced nephritis showed significantly increased proteinuria （P〈0.01）, which was obviously lowered by prednisone treatment （P〈0.01）. Transmission electron microscopy revealed extensive fusion of the foot processes of the podocytes in the model group. Prednisone treatment promoted nephrin expression in the kidney （P〈0.05）. Compared with the control group, the model and prednisone treated groups showed significantly lowered nephrin mRNA expression （P〈0.01） but increased FAK mRNA expression （P〈0.01）, but prednisone-treated group had a higher nephrin mRNA expression than the model group （P〈0.05）. The model group exhibited significantly increased expressions of FAK total and phosphorylated proteins, P-FAK/FAK, and P-Pyk2/Pyk2 （P〈0.01）, which were all lowered in the treatment group （P〈0.01）. Correlation analysis suggested that the expressions of FAK mRNA, FAK, pFAK, Pyk2 mRNA and pPyk2/Pyk2 were positively correlated with proteinuria （r=0.819, 0.750, 0.838, 0.762, 0.934, respectively, P〈0.01）. Conclusions Adriamycin increases phosphorylated FAK and Pyk2 expressions to mediate kidney injury in rats. Prednisone inhibits Pyk2 and FAK activation, decreases proteinuria, and alleviates podocyte lesions to protect the glomerular filtration barrier.