Table2_Aldosterone-stimulated endothelial epithelial sodium channel (EnNaC) plays a role in cold exposure–induced hypertension in rats.PDF

Background: Previous studies have demonstrated that activated endothelial epithelial sodium channel (EnNaC) impairs vasodilatation, which contributes to salt-sensitive hypertension. Here, we investigate whether mesenteric artery (MA) EnNaC is involved in cold exposure–induced hypertension (CIH) and identify the underlying mechanisms in SD rats.
Methods: One group of rats was housed at room temperature and served as control. Three groups of rats were kept in a 4°C cold incubator for 10 h/day; among which two groups were administrated with either benzamil (EnNaC blocker) or eplerenone (mineralocorticoid receptor antagonist, MR). Blood pressure (BP), vasodilatation, and endothelial function were measured with tail-cuff plethysmography, isometric myograph, and Total Nitric Oxide (NO) Assay kit, respectively. A cell-attached patch-clamp technique, in split-open MA, was used to determine the role of EnNaC in CIH rats. Furthermore, the plasma aldosterone levels were detected using an ELISA kit; and Western blot analysis was used to examine the relative expression levels of Sgk1 and Nedd4-2 proteins in the MA of SD rats.
Results: We demonstrated that cold exposure increased BP, impaired vasodilatation, and caused endothelial dysfunction in rats. The activity of EnNaC significantly increased, concomitant with an increased level of plasma aldosterone and activation of Sgk1/Nedd4-2 signaling. Importantly, CIH was inhibited by either eplerenone or benzamil. It appeared that cold-induced decrease in NO production and impairment of endothelium-dependent relaxation (EDR) were significantly ameliorated by either eplerenone or benzamil in MA of CIH rats. Moreover, treatment of MAs with aldosterone resulted in an activation of EnNaC, a reduction of NO, and an impairment of EDR, which were significantly inhibited by either eplerenone or GSK650394 (Sgk1 inhibitor) or benzamil.
Conclusion: Activation of EnNaC contributes to CIH; we suggest that pharmacological inhibition of the MR/Sgk1/Nedd4-2/EnNaC axis may be a potential therapeutic strategy for CIH.

研究背景：既往研究已证实，活化的内皮上皮钠通道（endothelial epithelial sodium channel, EnNaC）可损伤血管舒张功能，进而参与盐敏感性高血压的发生发展。本研究旨在探讨肠系膜动脉（mesenteric artery, MA）内皮上皮钠通道是否参与冷暴露诱导的高血压（cold exposure–induced hypertension, CIH）的发病过程，并在SD大鼠中阐明其潜在分子机制。

实验方法：将大鼠分为常温饲养的对照组；另有三组大鼠置于4℃冷环境中每日暴露10小时，其中两个亚组分别给予苯amil（benzamil，EnNaC阻断剂）与依普利酮（eplerenone，盐皮质激素受体拮抗剂，mineralocorticoid receptor antagonist, MR）干预。分别采用尾袖容积描记法（tail-cuff plethysmography）、等长肌张力描记法（isometric myograph）及总一氧化氮（nitric oxide, NO）检测试剂盒，检测各组大鼠的血压（blood pressure, BP）、血管舒张功能与内皮功能。采用细胞贴附式膜片钳技术（cell-attached patch-clamp technique）联合分离开放的肠系膜动脉标本，明确EnNaC在冷暴露诱导高血压大鼠中的作用。此外，采用ELISA试剂盒检测血浆醛固酮水平；通过蛋白质印迹分析（Western blot analysis）检测SD大鼠肠系膜动脉中Sgk1与Nedd4-2蛋白的相对表达水平。

实验结果：本研究证实，冷暴露可升高大鼠血压、损伤血管舒张功能并诱发内皮功能障碍。此时EnNaC活性显著升高，伴随血浆醛固酮水平上升及Sgk1/Nedd4-2信号通路活化。重要的是，依普利酮或苯amil干预均可抑制冷暴露诱导的高血压。在冷暴露高血压大鼠的肠系膜动脉中，冷暴露所致的一氧化氮生成减少及内皮依赖性舒张（endothelium-dependent relaxation, EDR）损伤，均可被依普利酮或苯amil显著改善。进一步研究发现，醛固酮处理肠系膜动脉可激活EnNaC、减少一氧化氮生成并损伤内皮依赖性舒张，上述效应均可被依普利酮、GSK650394（Sgk1抑制剂）或苯amil显著阻断。

研究结论：EnNaC的活化参与了冷暴露诱导的高血压发病过程；我们提出，靶向抑制MR/Sgk1/Nedd4-2/EnNaC信号轴可能是治疗冷暴露诱导高血压的潜在药理学策略。