Aldosterone-independent regulation of ENaC by systemic salt

描述（由申请人提供）：众所周知，醛固酮影响远端肾元ENaC活性，从而调节钠平衡，从而调节血压。这一建议挑战了我们目前关于ENaC活性如何响应饮食盐摄入量变化的观点，通过确定这种调节的醛固酮独立手段。具体来说，我们已经找到证据表明，刺激型Ang II和抑制性慢激素（BK）级联之间的协调耦合是ENaC活性对饮食钠变化的适当适应所必需的。我们的初步工作有力地支持了我们的中心假设，即Ang II直接刺激enec介导的Na+-重吸收在哺乳动物醛固酮敏感远端肾元（ASDN）中，这种调节与醛固酮对ENaC的作用无关。此外，我们假设在钠限制过程中Ang II级联的激活通过限制BK信号对ENaC的抑制作用进一步增强了ENaC的活性。相反，BK信号在盐摄入量升高时抑制ENaC活性是必需的，在缺乏B2受体的小鼠中，BK信号功能障碍导致ENaC活性升高，导致Na+过度保存。重要的是，缺失B2受体的小鼠会产生盐敏感性高血压。为了验证这一假设，我们提出了3个具体目标：1)通过全身盐摄入量确定不依赖醛固酮的ENaC调节的生理重要性。明确在此过程中，Ang II对ENaC的调控作用。2)明确angii在哺乳动物ASDN中调控ENaC活性的作用机制和细胞信号通路。3)在B2 -/-小鼠中，确定Ang II和BK信号级联之间的功能偶联如何控制ENaC活性以响应系统盐的变化，并确定破坏这种调节的病理生理后果。我们的实验方法包括从天然远端肾细胞的电生理学到整个动物平衡研究，并将结合1)膜片钳测量裂口小鼠远端肾细胞中ENaC活性与western blotting/RPPA和免疫组织化学检测ENaC表达水平；2)利用分子遗传学工具评估小鼠肾Na+排泄的平衡研究，以确定醛固酮对ENaC和肾脏处理钠的不依赖调节的生理作用。从药理学的角度来看，这项工作的重要性将促使B2激动剂的发展，作为纠正远端肾元钠处理的工具，以控制血压。

DESCRIPTION (provided by applicant): It is well known that aldosterone affects ENaC activity in the distal nephron to regulate sodium balance and, consequently blood pressure. This proposal challenges our current view on how ENaC activity responds to variations in dietary salt intake by identifying aldosterone-independent means of this regulation. Specifically, we have developed evidence that coordinated coupling between stimulatory Ang II and inhibitory Bradykinin (BK) cascades is necessary for proper adaptation of ENaC activity to variations in dietary sodium. Our preliminary work strongly supports our central hypothesis that Ang II directly stimulates ENaC-mediated Na+- reabsorption in the mammalian aldosterone-sensitive distal nephron (ASDN) and this regulation is non- redundant to aldosterone actions on ENaC. In addition, we hypothesize that the activation of Ang II cascade during sodium restriction further augments ENaC activity by limiting inhibitory actions of BK signaling on ENaC. In contrast, BK signaling is necessary to suppress ENaC activity during elevated salt intake with its dysfunction in mice lacking B2 receptors causing excessive Na+ conservation as a result of elevated ENaC activity. Importantly, mice with deleted B2 receptors develop salt-sensitive hypertension. To test this hypothesis we address 3 specific aims: 1) Establish the physiological importance of aldosterone-independent regulation of ENaC by systemic salt intake. Define the role of Ang II regulation of ENaC in this process. 2) Define the mechanism of action and delineate the cellular signaling pathway of Ang II regulation of ENaC activity in mammalian ASDN. 3) Determine how functional coupling between Ang II and BK signaling cascades controls ENaC activity in response to changes in systemic salt and establish the patho-physiological consequences of disrupting this regulation in B2 -/- mice. Our experimental approach ranges from electrophysiology in native distal nephron cells to whole animal balance studies and will combine 1) patch clamp measurements of ENaC activity in the split-open murine distal nephrons with ENaC expression levels probed by western blotting/RPPA and immunohistochemistry, and 2) balance studies to assess renal Na+ excretion with molecular genetic tools in mice to define a physiological role of aldosterone-independent regulation of ENaC and sodium handling by the kidney. From a pharmacological standpoint, the importance of this work will urge the development of B2 agonists as tools for correction of sodium handling in the distal nephron to control blood pressure.