An Aldosterone-Endothelin Feedback Mechanism on Sodium Homeostasis

描述（由申请人提供）：本项目的长期目标是了解醛固酮和内皮素-1（ET-1）之间的相互作用，以及这种相互作用作为肾钠（Na）转运反馈系统的潜在作用。这两种激素在高血压、心血管纤维化和肾硬化的发病机制中起作用。醛固酮作用于远端肾单位和集合管以增加钠重吸收和血压。最近两项临床研究（RALES和EPHESUS）的结果表明，醛固酮阻滞剂降低了发病率和死亡率，即使在已经接受血管紧张素转换酶抑制剂和β受体阻滞剂的患者中也是如此。显然，了解醛固酮的作用将提供重要的洞察几个病理生理状态。醛固酮的经典机制涉及通过盐皮质激素受体和可能的糖皮质激素受体刺激靶基因表达。在早期的工作中，我们证明，醛固酮刺激转录的ET-1基因的内髓集合管（IMCD）细胞系。在本申请中，我们已经在急性分离的大鼠IMCD和三种另外的体外收集细胞模型中验证了这一观察结果。已知肾ET-1刺激尿钠排泄和利尿。集合管特异性ET-1基因敲除小鼠表现出盐敏感性高血压。同样，多项研究表明ET-1抑制上皮Na通道（ENaC）。因此，醛固酮和ET-1对肾钠转运发挥相反作用，并通过该机制对全身血压发挥相反作用。这使我们假设醛固酮诱导的ET-1触发负反馈机制以减弱醛固酮介导的Na重吸收。本申请将表征肾中醛固酮对ET-1的诱导以及ET-1在肾Na转运调节中的作用。为了证明这种相互作用发生在动物中，一系列剂量-反应和时间过程研究将检测ET-1 mRNA和蛋白水平对醛固酮的反应。为了证明所提出的反馈机制在动物中是功能性的，我们将检查正常和集合管特异性ET-1敲除小鼠的微灌注小管中醛固酮介导的Na转运。该结果将通过在ET-1 A和B受体的药理学抑制剂存在下测定Na转运来证实。我们还将研究醛固酮对ET-1基因作用的分子机制。体外工作将通过使用DNase I超敏反应，染色质免疫沉淀和DNA亲和纯化试验来研究染色质结构和转录因子结合在醛固酮介导的ET-1调节中的作用。这些研究的结果将提供有价值的洞察醛固酮和ET-1如何相互作用，以调节钠稳态。重要的是，这些研究将有助于了解一种新的信号通路，可以解释醛固酮在心血管和肾脏疾病进展中的作用。公共卫生相关性：醛固酮是一种非常重要的控制血压的激素。几年前，我们发现这种激素在肾细胞中非常迅速地调节某些基因，这表明这些基因对控制醛固酮的作用很重要。醛固酮刺激的这些基因之一是内皮素，一种强大的血管收缩剂。在肾脏内，内皮素也促进肾脏的盐排泄，而醛固酮增强肾脏的盐潴留。本研究将探讨内皮素是否作为一种局部抑制机制和反馈控制系统对醛固酮的作用。

DESCRIPTION (provided by applicant): The long-term goal of this project is to understand the interaction between aldosterone and endothelin-1 (ET-1) and the potential role of this interaction as a feedback system on renal sodium (Na) transport. Both hormones play a role in the pathogenesis of hypertension, cardiovascular fibrosis, and nephrosclerosis. Aldosterone acts on the distal nephron and collecting duct to increase Na reabsorption and blood pressure. Results from two recent clinical studies (RALES and EPHESUS) demonstrated that aldosterone blockade reduced morbidity and mortality even in patients already receiving angiotensin- converting enzyme-inhibitors and beta blockers. Clearly, understanding aldosterone action will provide important insight into several pathophysiological states. The classical mechanism of aldosterone involves stimulation of target gene expression through the mineralocorticoid receptor, and possibly the glucocorticoid receptor. In earlier work, we demonstrated that aldosterone stimulated transcription of the ET-1 gene in an inner medullary collecting duct (IMCD) cell line. In the present application we have validated this observation in both acutely isolated rat IMCD and in three additional collecting cell models in vitro. Renal ET-1 is known to stimulate natriuresis and diuresis. Collecting-duct specific ET-1 knockout mice exhibit salt- sensitive hypertension. Likewise, multiple studies have demonstrated that ET-1 inhibits the epithelial Na channel (ENaC). Thus, aldosterone and ET-1 exert opposing actions on renal Na transport, and by this mechanism, opposing actions on systemic blood pressure. This led us to hypothesize that aldosterone- induced ET-1 triggers a negative feedback mechanism to attenuate aldosterone-mediated Na reabsorption. The present application will characterize the induction of ET-1 by aldosterone in the kidney and the role of ET-1 in the regulation of renal Na transport. To demonstrate that this interaction occurs in the animal a series of dose-response and time course studies will examine ET-1 mRNA and protein levels in response to aldosterone. To demonstrate that the proposed feedback mechanism is functional in the animal we will examine aldosterone mediated Na transport in microperfused tubules from normal and collecting duct- specific ET-1 knockout mice. The result will be corroborated by determining Na transport in the presence of pharmacological inhibitors of ET-1 A and B receptors. We will also investigate the molecular mechanism of action of aldosterone on the ET-1 gene. The in vitro work will be extended by using DNase I hypersensitivity, chromatin immunoprecipitation, and DNA affinity purification assays to study the role of chromatin structure and transcription factor binding in the aldosterone-mediated regulation of ET-1. The results of these studies will provide valuable insight into how aldosterone and ET-1 interact to modulate Na homeostasis. Importantly, these studies will lend understanding into a novel signaling pathway that may explain the role of aldosterone in the progression of cardiovascular and renal disease. PUBLIC HEALTH RELEVANCE: Aldosterone is a very important hormone that controls blood pressure. We discovered several years ago that this hormone regulates certain genes very quickly in kidney cells, which suggests that these genes are important for the control of aldosterone's action. One of these genes that aldosterone stimulates is endothelin, a powerful vasoconstrictor of blood vessels. Within the kidney endothelin also promotes salt excretion by the kidney whereas aldosterone enhances kidney salt retention. This study will examine whether endothelin acts as a local inhibitory mechanism and feedback control system on the action of aldosterone.