Mechanisms and relevance of ENaC regulation by EGF and Rac1

描述(由申请人提供)：长期的血压控制包括通过精确调节对醛固酮敏感的远端肾单位(ASDN)的上皮性Na+通道(ENaC)来实现Na+稳态。ENAC功能障碍是导致全身Na+水平紊乱的原因，与血容量、血压和肺液平衡的异常调节有关。我们提供了表皮生长因子(EGF)可以作为ENaC配体的初步证据，并假设EGF家族和rac1家族的成员调节ENaC介导的ASDN中的Na+转运，并参与盐敏感型高血压的发展。EGF及其相关的EGF家族成员与ErbB受体结合，是参与肾脏发育、生理和病理生理的信号因子。在生理条件下，ErbB受体在调节肾脏血流动力学和肾脏对电解质的处理中起着重要作用，而在不同的病理生理状态下，ErbB受体的激活可能对肾脏产生有益或有害的影响。ErbB受体的刺激激活了细胞内涉及小GTP酶的级联反应，特别是rac1。小G蛋白及其调节蛋白参与了肾脏和心血管疾病的病理过程。我们的初步结果，包括使用分离的、分裂的开放ASDN的电生理实验，证明ENaC受EGF和rac1的调节，可能是通过一种收敛机制。这些研究中使用的Dahl盐敏感(SS)大鼠在高盐饮食下会出现严重的高血压。我们提供的初步数据表明，ENaC参与了SS大鼠高血压的发展。此外，我们的初步数据显示，SS大鼠的EGF浓度降低，我们认为这可能会增强ENaC活性、钠滞留和高血压。基于这一初步数据和我们之前发表的研究结果，本研究的具体目标是确定EGF是否通过调节ENaC对肾脏钠处理的生理调控起重要作用，并确定EGF和rac1介导的ENaC活性变化的确切机制。本研究将结合电生理学、免疫组织化学、生化、显微镜以及体内和体外的慢性研究，为ENaC如何受EGF-家族和rac1家族成员的调控以及这一途径的变化如何导致SS大鼠的盐源性高血压提供机制方面的见解。这些研究将针对三个具体目标：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，并确定这一途径在盐敏感型高血压发病中的作用；2)确定RhoGDI和rac1在ENaC调节中的生理作用，并确定rac1在介导EGF对ENaC的影响中的作用；以及3)确定rac1调节ENaC活性的细胞和分子机制：波是否将rac1调节传递给ENaC？ 与公众健康相关：血压的控制是通过肾脏中的Na+稳态进行的，并涉及对醛固酮敏感的远端肾单位(ASDN)上皮性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GTPase rac1如何调节ENaC介导的ASDN中的Na+运输，并参与盐敏感型高血压的发展。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，并揭示与液体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.