Mechanisms and relevance of ENaC regulation by EGF and Rac1

描述（由申请方提供）：血压的长期控制涉及通过精确调节醛固酮敏感性远端肾单位（ASDN）中的上皮Na+通道（ENaC）实现Na+稳态。ENaC功能障碍是导致与血容量、血压和肺液体平衡的异常调节相关的全身Na+水平紊乱的原因。我们提供的初步证据表明，表皮生长因子（EGF）可以作为ENaC配体，并假设EGF家族和Rac 1的成员调节ENaC介导的钠离子转运的ASDN和参与盐敏感性高血压的发展。EGF及其相关的EGF家族成员与ErbB受体结合，并作为负责肾脏发育，生理学和病理生理学的信号因子。在生理条件下，ErbB受体在肾脏血液动力学和电解质处理的调节中起重要作用，而在不同的病理生理状态下，ErbB活化可介导对肾脏的有益或有害作用。ErbB受体的刺激激活涉及小GTP酶，特别是Rac 1的细胞内级联反应。小G蛋白及其调节蛋白参与肾脏和心血管疾病的病理过程。我们的初步结果，包括电生理实验，使用隔离，分裂开放的ASDN，表明ENaC的调节EGF和Rac 1，可能通过收敛机制。在这些研究中使用的达尔盐敏感（SS）大鼠在高盐饮食中发展为严重的高血压。我们提供的初步数据表明，ENaC有助于SS大鼠品系的高血压的发展。此外，我们的初步数据显示，EGF浓度降低，在SS大鼠，我们建议将增强ENaC活性，钠潴留和高血压。建立在这个初步的数据和我们以前发表的研究结果，这个建议的具体目标是确定是否通过Rac 1的EGF作用是重要的生理控制肾钠处理通过调节ENaC和定义的确切机制EGF和Rac 1介导的ENaC活性的变化。电生理学，免疫组织化学，生物化学，显微镜和慢性研究在体内和体外的组合将被用于本提案中提供ENaC是如何调节的EGF家族和Rac 1的成员，以及如何在这一途径的变化有助于盐诱导的高血压在SS大鼠的机制的见解。这些研究将解决三个具体目标：1）确定和量化EGF和相关生长因子在ASDN中ENaC活性的生理调节中的作用，并建立该途径在盐敏感性高血压发展中的作用; 2）建立RhoGDI和Rac 1在ENaC调节中的生理作用，并确定Rac 1在介导EGF对ENaC的作用中的作用;和3）确定Rac 1调节ENaC活性的细胞和分子机制：WAVE是否将Rac 1调节传递给ENaC？ 公共卫生相关性：血压的控制通过肾脏中的Na+稳态发生，并且涉及对醛固酮敏感性远端肾单位（ASDN）中的上皮Na+通道（ENaC）的精确调节。目前的建议将研究表皮生长因子家族成员和小GTGFRac 1如何调节ASDN中ENaC介导的Na+转运，并参与盐敏感性高血压的发展。这项工作有可能为这一重要离子通道的控制和功能提供新的见解，并揭示与体液失衡和高血压相关的疾病的机制。

DESCRIPTION (provided by applicant): Long term control of blood pressure involves Na+ homeostasis through the precise regulation of the Epithelial Na+ Channel (ENaC) in the aldosterone-sensitive distal nephron (ASDN). ENaC dysfunction is causative for disturbances in total body Na+ levels associated with abnormal regulation of blood volume, blood pressure, and lung fluid balance. We provide preliminary evidence that epidermal growth factor (EGF) can serve as an ENaC ligand and hypothesize that members of the EGF-family and Rac1 modulate ENaC-mediated Na+ transport in the ASDN and participate in the development of salt-sensitive hypertension. EGF and its related EGF-family members bind to ErbB receptors and act as signaling factors responsible for renal development, physiology and pathophysiology. Under physiological conditions, ErbB receptors play an important role in the regulation of renal hemodynamics and electrolyte handling by the kidney, while in different pathophysiological states ErbB activation may mediate either beneficial or detrimental effects on the kidney. Stimulation of ErbB receptors activates an intracellular cascade involving small GTPases, particularly Rac1. Small G proteins and their regulatory proteins contribute to the pathology of renal and cardiovascular diseases. Our preliminary results, including electrophysiological experiments using isolated, split open ASDN, demonstrate that ENaC is regulated by EGF and Rac1, possibly through a convergent mechanism. Dahl salt-sensitive (SS) rats used in these studies develop severe hypertension on high-salt diet. We provide preliminary data indicating that ENaC contributes to the development of hypertension in the SS rat strain. Furthermore, our preliminary data reveal that EGF concentration is reduced in the SS rats, which we propose would enhance ENaC activity, sodium retention and hypertension. Building upon this preliminary data and our previously published findings, the specific objective of this proposal is to determine whether EGF acting through Rac1 is important for physiologic control of renal sodium handling through regulation of ENaC and define the precise mechanisms of EGF- and Rac1-mediated changes in ENaC activity. A combination of electrophysiological, immunohistochemical, biochemical, microscopy and chronic studies in vivo and in vitro will be used in this proposal to provide mechanistic insights on how ENaC is regulated by member of the EGF-family and Rac1 and how changes in this pathway contributes to salt induced hypertension in SS rats. These studies will address three Specific Aims: 1) To identify and quantify the role of EGF and related growth factors in the physiological regulation of ENaC activity in the ASDN and establish the role of this pathway in the development of salt-sensitive hypertension; 2) To establish the physiological role of RhoGDI and Rac1 in regulation of ENaC and determine the role of Rac1 in mediating EGF effects on ENaC; and 3) To define the cellular and molecular mechanism by which Rac1 modulates ENaC activity: do WAVEs convey Rac1 regulation to ENaC? PUBLIC HEALTH RELEVANCE: The control of blood pressure occurs via Na+ homeostasis in the kidney and involves the precise regulation of the Epithelial Na+ Channel (ENaC) in the aldosterone-sensitive distal nephron (ASDN). The current proposal will study how members of the epidermal growth factors family and small GTPase Rac1 modulate ENaC- mediated Na+ transport in the ASDN and participate in the development of salt-sensitive hypertension. This work has the potential to provide new insights into the control and function of this important ion channel and uncover mechanisms involved in diseases associated with fluid imbalance and hypertension.