Rac1及Cdc42在肾上腺髓质肽(AM)缓解嘌呤霉素氨基核苷(PAN)所诱导足细胞损伤中作用机制的初步探讨

A preliminary study on Rac1 and Cdc42 in puromycin aminonucleoside(PAN)-induced podocyte injury treated with adrenomedullin(AM)

目的观察肾上腺髓质肽(adrenomedullin,AM)对嘌呤霉素氨基核苷(puromycin aminonucleoside,PAN)所诱导受损足细胞的作用以及Rac1/Cdc42表达变化,初步探讨其可能机制。方法将小鼠肾足细胞株分为对照组、PAN处理组(100μg/mL)、PAN联合AM处理组(10-7mol/L)及PAN联合AM和蛋白激酶A(PKA)抑制剂H89(10-6mol/L)处理组。经一次性腹腔注射PAN(150 mg/kg)建立大鼠足细胞损伤模型,每天经尾静脉注射AM蛋白(66μg/kg)进行干预。SDS-PAGE法检测尿蛋白水平,电镜观察足细胞足突宽度变化,双重免疫荧光染色及/或Western blot观察足细胞特异性骨架蛋白(synaptopodin、nephrin)、Rac1及Cdc42的蛋白表达水平,实时定量PCR(qRT-PCR)检测synaptopodin、nephrin的mRNA表达,谷胱甘肽巯基转移酶-拉下实验(GST-pull down assay)法检测Rac1及Cdc42活性变化。结果PAN在体内、外均显著降低synaptopodin、nephrin的蛋白或/及mRNA表达水平,且显著升高desmin表达;大鼠PAN肾病模型的尿蛋白水平显著升高,足突宽度显著增加;PAN显著降低Rac1、Cdc42总蛋白及活性水平。上述由PAN所介导的效应被AM显著抑制;H89显著阻断AM的体外作用。结论AM主要通过PKA通路促进受损足细胞的修复,该保护机制与AM调控Rac1/Cdc42表达密切相关。

Objective To investigate the effect of adrenomedullin(AM)on injured podocytes induced by puromycin aminonucleoside(PAN)and its modulation on Rac1/Cdc42,and to explore the possible mechanisms.Methods Murine podocytes were divided into control group and groups that treated with PAN(100μg/mL),PAN combined with AM(10-7 mol/L),and PAN combined with both AM and a PKA inhibitor H89(10-6 mol/L),respectively.A rat podocyte-injured model was established by a single intraperitoneal injection of PAN(150 mg/kg body wt)and treated daily with AM protein(66μg/kg body wt)via tail vein injection.Urine protein excretion was evaluated by SDS-PAGE.Foot process length was detected by electron microscopy.Protein levels and mRNA expression of podocyte specific cytoskeletal proteins including synaptopodin and nephrin were investigated by double-immunofluorescence or Western blot,and qRT-PCR respectively.Rac1 and Cdc42 were detected by Western blot for their protein levels and by GST-pull down assay for their activities.Results PAN significantly decreased the protein levels or(and)mRNA expressions of synaptopodin and nephrin,and significantly increased desmin expression both in vitro and in vivo.PAN treatment significantly increased urinary protein excretion and the width of foot processes.Rac1 and Cdc42 protein levels and activities were decreased by PAN.The effects of PAN were mostly blocked by AM administration,which were dramatically mitigated by H89 in vitro,a PKA inhibitor.Conclusion AM alleviates PAN-induced podocye injury partially through enhancing the expressions of Rac1 and Cdc42,which may be mainly mediated via a PKA-dependent pathway.