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目的研究足细胞内环腺苷酸(c AMP)信号对足细胞标志蛋白Podocalyxin、细胞内氯离子通道蛋白5(CLIC5)及相关的埃兹蛋白-根蛋白-膜突蛋白(ERM)表达的影响。方法构建阿霉素(ADR)肾病小鼠模型,部分小鼠使用腺苷酸环化酶激活剂Forskolin治疗(ADR组和ADR+Forskolin组)。考马斯亮蓝染色法测定尿液中蛋白质含量;电子显微镜观察足突宽度;免疫荧光染色和Western blotting检测足细胞中磷酸化ERM(P-ERM)、CLIC5和Podocalyxin的表达。结果与ADR组比较,ADR+Forskolin组小鼠尿白蛋白含量明显减少,足突增宽显著改善(P<0.05)。Western blotting和免疫荧光染色结果显示:与ADR组比较,ADR+Forskolin组小鼠足细胞内P-ERM、Podocalyxin和CLIC5蛋白表达及共定位均显著增加。Western blotting检测结果显示:体外以嘌呤霉素氨基核苷(PAN)孵育足细胞72 h,可使P-ERM和CLIC5的表达显著减少(P<0.01);Epac信号激动剂2Me-c AMP预处理可防止PAN诱导的足细胞P-ERM低表达,而PKA信号激动剂p CPT-c AMP预处理可防止PAN诱导的CLIC5表达减少。结论 Forskolin可减轻ADR肾炎小鼠的白蛋白尿,改善足突融合,缓解Podocalyxin/CLIC5/ERM蛋白复合体低表达。c AMP分别通过PKA和Epac信号通路防止PAN诱导的CLIC5和P-ERM表达降低。
Objective To investigate the effects of cAMP signaling on podocyte marker protein Podocalyxin, intracellular chloride ion channel 5 (CLIC5) and related ezrin-membrane protein (ERM) expression . Methods A mouse model of adriamycin (ADR) nephropathy was established. Some mice were treated with adenosine cyclase activator Forskolin (ADR group and ADR + Forskolin group). The protein content in urine was determined by Coomassie Brilliant Blue staining. The width of the foot process was observed by electron microscopy. The expressions of phosphorylated ERM (P-ERM), CLIC5 and Podocalyxin in podocytes were detected by immunofluorescence staining and Western blotting. Results Compared with ADR group, the content of urinary albumin in ADR + Forskolin group decreased significantly and the width of foot process was significantly improved (P <0.05). Western blotting and immunofluorescence staining showed that compared with ADR group, the expression and co-localization of P-ERM, Podocalyxin and CLIC5 in podocytes of ADR + Forskolin group increased significantly. The results of Western blotting showed that the expression of P-ERM and CLIC5 was significantly decreased after incubation of podocytes with puromycin aminonucleoside (PAN) for 72 h in vitro (P <0.01); Epac signal agonist 2Me-c AMP pretreatment Can prevent PAN-induced low expression of P-ERM in podocytes, whereas pCPT-c AMP, a PKA signal agonist, prevents the decrease of CLIC5 expression induced by PAN. Conclusion Forskolin can reduce albuminuria in ADR nephritis mice, improve the process of foot process fusion, and alleviate the low expression of Podocalyxin / CLIC5 / ERM protein complex. c AMP prevented the decrease of PAN-induced CLIC5 and P-ERM expression through PKA and Epac signaling pathways, respectively.