An Aldosterone-Endothelin Feedback Mechanism on Sodium Homeostasis

该项目的长期目标是了解醛固酮与 内皮素-1（ET-1）和这种相互作用作为肾钠（Na）反馈系统的潜在作用 运输这两种激素都在高血压、心血管纤维化和高血压的发病机制中发挥作用。 肾硬化醛固酮作用于远端肾单位和集合管，增加钠重吸收 还有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- 转化酶抑制剂和β受体阻滞剂。显然，了解醛固酮的作用将提供 对几种病理生理状态的重要见解。醛固酮的经典作用机制包括 通过盐皮质激素受体和可能的糖皮质激素刺激靶基因表达 受体的在早期的工作中，我们证明了醛固酮刺激ET-1基因的转录， 内髓集合管（IMCD）细胞系。在本申请中，我们已经验证了这一点。 在急性分离的大鼠IMCD和三个额外的体外收集细胞模型中观察到。肾 已知ET-1刺激尿钠排泄和利尿。集合管特异性ET-1敲除小鼠表现出盐- 敏感性高血压同样，多项研究表明，ET-1抑制上皮细胞Na+， 通道（ENaC）。因此，醛固酮和ET-1对肾钠转运发挥相反的作用， 机制，对抗对全身血压的作用。我们由此推测醛固酮- 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。 本申请将表征醛固酮在肾脏中对ET-1的诱导，以及醛固酮对ET-1的诱导作用。 ET-1对肾钠转运的调节为了证明这种相互作用发生在动物中， 一系列的剂量反应和时间过程研究将检测ET-1 mRNA和蛋白水平， 醛固酮为了证明所提出的反馈机制在动物中是有效的，我们将 检查正常和集合管微灌注小管中醛固酮介导的Na转运- 特异性ET-1敲除小鼠。该结果将通过确定钠转运在存在下得到证实 ET-1 A和B受体的药理学抑制剂。我们还将研究 醛固酮对ET-1基因的作用。体外工作将通过使用DNase I来扩展 超敏反应，染色质免疫沉淀和DNA亲和纯化试验，以研究 醛固酮介导的ET-1调节中的染色质结构和转录因子结合。的 这些研究结果将为了解醛固酮和ET-1如何相互作用调节Na+浓度提供有价值的信息 体内平衡重要的是，这些研究将有助于了解一种新的信号通路， 解释醛固酮在心血管和肾脏疾病进展中的作用。

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.