Dong Nan, Meng Lixia, Xue Ruqun, Yu Meng, Zhao Zhonghua, Liu Xueguang
Department of Pathology, School of Basic Medical Sciences, Fudan University, 131 Dongan Road, Shanghai, 200032, China.
Department of Clinical Medicine, Fudan University, 131 Dongan Road, Shanghai, 200032, China.
Int Urol Nephrol. 2017 Aug;49(8):1489-1506. doi: 10.1007/s11255-017-1622-y. Epub 2017 May 20.
Podocyte injury is a key event in proteinuric kidney disease and eventually glomerular scarring. While adrenomedullin (AM), a potent vasodilatory peptide, has been reported to confer renoprotection in several experimental models of kidney diseases, its effect on injured podocytes and the related mechanism is still largely unknown.
Employing Western blotting analysis, immunoprecipitation and immunofluorescence, we investigated the effects of AM on the expressions of podocyte cytoskeletal proteins and Rho-family small GTPases (Rho GTPases) in puromycin aminonucleoside (PAN)-induced podocyte injury, both in cultured podocytes and in PAN nephrosis rats. Urinary protein excretion and the morphologic changes of kidney in PAN nephrosis rats were evaluated. Glutathione-S-transferase pull-down assay was applied for Rho GTPases activity.
PAN induced massive albuminuria and morphologic injury, which were significantly mitigated by AM administration. AM significantly antagonized not only the PAN-decreased expressions of synaptopodin, nephrin, CD2AP and podocin, but also the PAN-disrupted interactions between synaptopodin-RhoA, nephrin-CD2AP, and CD2AP-Rac1-cortactin. These effects of AM in cultured podocytes were mostly significantly blocked by H89, a PKA inhibitor. AM dramatically upregulated the PAN-induced Rho GTPases protein expressions and their activities. PAN increased the expressions of endogenous AM and its receptor RAMP2 which was furthermore upregulated by AM administration.
AM alleviated podocyte injury induced by PAN both in cell culture and in PAN nephrosis. The beneficial effects of AM on podocytes can be attributable to direct modulation of podocyte cytoskeletal proteins and Rho GTPases, mainly via a PKA-dependent pathway.
足细胞损伤是蛋白尿性肾病及最终肾小球瘢痕形成的关键事件。虽然肾上腺髓质素（AM）是一种有效的血管舒张肽，已报道其在几种肾脏疾病实验模型中具有肾脏保护作用，但其对损伤足细胞的作用及相关机制仍 largely 未知。
采用蛋白质印迹分析、免疫沉淀和免疫荧光技术，我们研究了 AM 对嘌呤霉素氨基核苷（PAN）诱导的足细胞损伤中足细胞细胞骨架蛋白和 Rho 家族小 GTP 酶（Rho GTP 酶）表达的影响，包括在培养的足细胞和 PAN 肾病大鼠中。评估了 PAN 肾病大鼠的尿蛋白排泄和肾脏形态学变化。应用谷胱甘肽 - S - 转移酶下拉试验检测 Rho GTP 酶活性。
PAN 诱导大量蛋白尿和形态学损伤，AM 给药可显著减轻这些损伤。AM 不仅显著拮抗 PAN 降低的突触足蛋白、nephrin、CD2AP 和 podocin 的表达，还拮抗 PAN 破坏的突触足蛋白 - RhoA、nephrin - CD2AP 和 CD2AP - Rac1 - cortactin 之间的相互作用。AM 在培养的足细胞中的这些作用大多被 PKA 抑制剂 H89 显著阻断。AM 显著上调 PAN 诱导的 Rho GTP 酶蛋白表达及其活性。PAN 增加内源性 AM 及其受体 RAMP2 的表达，AM 给药进一步上调其表达。
AM 在细胞培养和 PAN 肾病中均减轻了 PAN 诱导的足细胞损伤。AM 对足细胞的有益作用可归因于主要通过 PKA 依赖性途径直接调节足细胞细胞骨架蛋白和 Rho GTP 酶。
