Department of Physiology, Medical College of Wisconsin, 8701 Watertown Plank Rd., Milwaukee, WI 53226, USA.
FASEB J. 2013 Jul;27(7):2723-32. doi: 10.1096/fj.12-223792. Epub 2013 Apr 4.
The epithelial sodium channel (ENaC) is one of the central effectors involved in regulation of salt and water homeostasis in the kidney. To study mechanisms of ENaC regulation, we generated knockout mice lacking the insulin receptor (InsR KO) specifically in the collecting duct principal cells. Single-channel analysis in freshly isolated split-open tubules demonstrated that the InsR-KO mice have significantly lower ENaC activity compared to their wild-type (C57BL/6J) littermates when animals were fed either normal or sodium-deficient diets. Immunohistochemical and Western blot assays demonstrated no significant changes in expression of ENaC subunits in InsR-KO mice compared to wild-type littermates. Insulin treatment caused greater ENaC activity in split-open tubules isolated from wild-type mice but did not have this effect in the InsR-KO mice. Thus, these results suggest that insulin increases ENaC activity via its own receptor affecting the channel open probability. To further determine the mechanism of the action of insulin on ENaC, we used mouse mpkCCDc14 principal cells. Insulin significantly augmented amiloride-sensitive transepithelial flux in these cells. Pretreatment of the mpkCCDc14 cells with phosphatidylinositol 3-kinase (LY294002; 10 μM) or mTOR (PP242; 100 nM) inhibitors precluded this effect. This study provides new information about the importance of insulin receptors expressed in collecting duct principal cells for ENaC activity.
上皮钠通道（ENaC）是参与肾脏盐和水稳态调节的核心效应器之一。为了研究 ENaC 调节的机制，我们生成了胰岛素受体（InsR）在集合管主细胞中特异性缺失的敲除小鼠。在新鲜分离的劈开小管的单通道分析中，当动物喂食正常或低钠饮食时，InsR-KO 小鼠的 ENaC 活性明显低于其野生型（C57BL/6J）同窝仔鼠。免疫组织化学和 Western blot 检测表明，InsR-KO 小鼠与野生型同窝仔鼠相比，ENaC 亚基的表达没有明显变化。胰岛素处理可使野生型小鼠分离的劈开小管中的 ENaC 活性增加，但对 InsR-KO 小鼠没有这种作用。因此，这些结果表明胰岛素通过自身受体增加 ENaC 活性，从而影响通道的开放概率。为了进一步确定胰岛素对 ENaC 的作用机制，我们使用了小鼠 mpkCCDc14 主细胞。胰岛素显著增加了这些细胞中阿米洛利敏感的跨上皮通量。在用磷脂酰肌醇 3-激酶（LY294002；10 μM）或 mTOR（PP242；100 nM）抑制剂预处理 mpkCCDc14 细胞后，这种作用被阻断。这项研究提供了关于在集合管主细胞中表达的胰岛素受体对 ENaC 活性重要性的新信息。
