Collecting duct endothelin and sodium homeostasis

集合管(CD)内皮素系统已成为肾脏钠排泄的重要调节因子 和全身血压(BP)。CD衍生的内皮素-1(ET-1)可能具有降压作用 至少部分原因是抑制了CD上皮钠通道(ENaC)。CD来源的ET-1在 介导对细胞外液容量(ECFV)扩张的利钠反应，控制动脉血压； CDET-1系统缺陷可能与高血压有关。而CD ET的一般生物学 系统已经建立，但这个系统如何运行仍有很多未知之处。特别是， 迫切需要研究的关键成分是：1)确定ET-1是否以及如何抑制CD-Na转运； 2)确定ECFV状态如何与CD ET-1生产相关联。根据初步数据， 拟议的研究将解决以下假设：ECFV扩张增加肾小管液体流速 通过CD。增加的流量通过多囊蛋白-1和-2增加细胞内钙离子浓度。 增加Cd[Ca~(2+)]诱导信号通路，导致ET-1基因转录激活。这 增加CD ET-1的产生和分泌，导致ETB受体的自分泌激活 (ETRB)，但也可能是ETA受体(ETRA)。ET-1结合通过减少通道开放概率(Po)和可能的心尖通道数(N)而抑制ENaC。对Po的影响至少部分归因于c-src/MAPK通路的激活。该项目的具体目标包括： 目的1.验证FLOW刺激CD-ET-1产生的假说，以及这种作用是通过 激活特定的细胞信号通路、顺式作用元件和反式激活因子。 因此，我们将：a)确定FLOW对CD ET-1产生的影响；以及b)确定FLOW和细胞内钙对ET-1基因转录的偶联的细胞信号通路、顺式作用元件和反式激活因子。 目的2.验证ET-1调节CD中ENaC的假说，以及这种调节的特定细胞和分子机制。因此，我们将：a)确定ET-1是否调节天然分离的CD细胞中的ENaC Po；b)鉴定参与调节天然CD细胞ENaC的特定ET受体，并阐明将该受体耦合到通道的细胞信号通路；c)确定ET-1调节CD ENaC在肾脏钠处理的生理控制中的重要性，并利用基因靶向小鼠探索破坏这一调节的病理生理后果；以及d)确定ET-1调节ENaC功能的分子机制，包括确定使通道能够响应ET-1的ENaC的特定残基/区域。

The collecting duct (CD) endothelin system has emerged as an important regulator of renal Na excretion and systemic blood pressure (BP). CD-derived endothelin-1 (ET-1) exerts a hypotensive effect that is likely due, at least partly, to inhibition of the CD epithelial Na channel (ENaC). CD-derived ET-1 is important in mediating the natriuretic response to extracellular fluid volume (ECFV) expansion and controlling arterial BP; defects in the CD ET-1 system may contribute to hypertension. While the general biology of the CD ET system has been established, there remains much unknown about how this system functions. In particular, key components in critical need of study are: 1) determination if and how ET-1 inhibits CD Na transport; and 2) determination how ECFV status is coupled to CD ET-1 production. Based on preliminary data, the proposed studies will address the following hypotheses; ECFV expansion increases tubule fluid fiow rate through the CD. Increased fiow increases intracellular Ca concentration [Ca2+] through polycystins-1 and -2. Increased CD [Ca2+] induces signaling pathways causing transcriptional activation of the ET-1 gene. This increases CD ET-1 production and secretion resulting in autocrine activation of most likely the ETB receptor (ETRB), but also possibly the ETA receptor (ETRA). ET-1 binding leads to inhibition of the ENaC through reduction of channel open probability (Po) and possibly apical channel number (N). The effect on Po is due, at least partly, to activation of the c-src/MAPK pathway. The specific aims for this project include: Aim 1. Test the hypothesis that fiow stimulates CD ET-1 production, and that this effect is exerted by activation of specific cellular signaling pathways, cis-acting elements and trans-activating factors. Accordingly, we will: a) determine the effects of flow on CD ET-1 production; and b) determine the cellular signaling pathways, cis-acting elements and trans-activating factors coupling flow and intracellular Ca to ET-1 gene transcription. Aim 2. Test the hypothesis that ET-1 regulates ENaC in the CD, and that specific cellular and molecular mechanisms underpin this regulation. Accordingly, we will: a) determine if ET-1 regulates ENaC Po in native isolated CD cells; b) identify the specific ET receptor involved in regulating ENaC in native CD cells and elucidate the cellular signaling pathway coupling this receptor to the channel; c) define the importance of ET-1 regulation of CD ENaC in physiologic control of renal Na handling, and probe pathophysiological consequences of disrupting this regulation using gene targeted mice; and d) define the molecular mechanisms by which ET-1 modulates ENaC function, including identifying the specific residues/regions of ENaC that enable the channel to respond to ET-1.