上海交通大学学报（医学版）

上海交通大学学报（医学版）

• 论著（基础研究） • 上一篇    下一篇

激活足细胞环腺苷酸信号调节细胞磷酸化ERM和CLIC5的表达

陈晓欢1，阿依加肯·卡司木马力1，陶 花2，李肖瑛2，魏 凯2，顾乐怡1,2   

  1. 1.新疆维吾尔自治区喀什地区第二人民医院肾病科， 喀什 844000； 2.上海交通大学 医学院附属仁济医院肾脏科,分子细胞（肾病）实验室， 上海 200162
  • 出版日期:2014-11-28 发布日期:2014-12-02
  • 通讯作者: 顾乐怡, 电子信箱: guleyi@aliyun.com。
  • 作者简介:陈晓欢（1980—）， 女， 主治医师， 硕士生; 电子信箱: 1165625697@qq.com。
  • 基金资助:

    国家自然科学基金（30971363, 81270781）；上海市自然科学基金（08ZR1413200）

Activation of cAMP signaling for regulating phosphorylation of ERM and expression of CLIC5 of podocytes

CHEN Xiao-huan1, AYIJIAKEN Kasmumali1, TAO Hua2, LI Xiao-ying2, WEI Kai2, GU Le-yi1,2   

  1. 1.Renal Division, Kashgar Second People's Hospital, Xinjiang Uygur Autonomous Region， Kashi 844000, China; 2.Department of Nephrology & Molecular Cell Laboratory for Kidney Disease, Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200162, China
  • Online:2014-11-28 Published:2014-12-02
  • Supported by:

    National Natural Science Foundation of Chian, 30971363, 81270781; Natural Scinece Foundation of Shanghai, 08ZR1413200

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摘要：

目的 研究足细胞内环腺苷酸（cAMP）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-cAMP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pCPT-cAMP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。cAMP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。

关键词: 蛋白尿, 足细胞, 环腺苷酸, 细胞骨架

Abstract:

Objective To investigate the effects of cyclic adenosine monophosphate (cAMP) signal of podocytes on expressions of podocyte marker protein Podocalyxin, chloride intracellular channel protein 5 (CLIC5), and relevant ezrin/radixin/moesin (ERM). Methods The adriamycin (ADR) nephrosis mouse model was established and some mice were treated by adenylate cyclase activator Forskolin (the ADR group and ADR+Forskolin group). Urinary protein levels were detected by the coomasie blue staining. The width of foot processes was observed under the electron microscope. The expressions of phosphorylated ERM (P-ERM), CLIC5, and Podocalyxin of podocytes were detected by the immunofluorescence staining and Western blotting. Results Compared to the ADR group, the urinary albumin level of the ADR+Forskolin group was significantly lower and the width of foot processes was significantly smaller (P<0.05). The results of immunofluorescence staining and Western blotting showed that compared to the ADR group, expressions and colocalization of P-ERM, Podocalyxin, and CLIC5 protein of podocytes of the ADR+Forskolin group significantly increased. The results of Western blotting showed that expressions of P-ERM and CLIC5 of podocytes significantly decreased after being cultured by puromycin aminonucleoside (PAN) for 72h in vitro (P<0.01). Pretreatment by Epac signal agonist 2Me-cAMP could prevent the low expression of P-ERM of podocytes induced by PAN, while pretreatment by PKA signal agonist pCPT-cAMP could prevent the low expression of CLIC5 induced by PAN. Conclusion Forskolin can alleviate albuminurine, widening of foot process, and low expression of Podocalyxin/ CLIC5/ERM protein complex of mice with nephrosis. And cAMP can prevent PAN-induced low expressions of CLIC5 and P-ERM through PKA and Epac signaling pathways, respectively.

Key words: proteinuine, podocyte, cyclic adenosine monophosphate, cytoskeleton