Renal and extrarenal toxicity of aldosterone

描述(由申请人提供)： 醛固酮是最重要的心血管毒素之一。旨在阻断它的药物可以延长心力衰竭患者的生命，减少患者的蛋白尿。然而，醛固酮是 也是一种重要的内源性激素，具有多种重要的体内平衡功能。阻断醛固酮作用的药物虽然有用，但也受到副作用的限制。这些副作用在很大程度上(尽管不完全是)与它们对肾小管的作用有关。近年来，醛固酮对肾小管的作用比以往认为的更广泛，涉及远端曲管、连接小管和集合管。在这里，我们将通过选择性地从心脏和肾脏中删除盐皮质激素受体(MR)来测试醛固酮在介导心脏和肾脏损伤中的直接作用。进一步，我们将研究醛固酮调节沿着远端曲管的硫氮化物敏感的钠-氯共转运体的活性的机制。在这里，我们将检验这样一种假设，即沿着远端曲管的醛固酮效应对其肾脏活动是必不可少的，并且醛固酮引起的肾脏损伤依赖于增强的盐分转运。我们还将测试醛固酮是如何通过SGK1向对噻嗪敏感的钠-氯共转运体发出信号的。在第二个目标中，我们将研究心脏MR在面对高醛固酮血症时在介导心脏损伤中的作用。我们将使用MR位于loxP位点两侧的动物。当这些动物与表达Cre重组酶的动物饲养时，在组织特异性启动子的控制下，它允许MR被缺失。我们已经获得了Cre重组酶在心肌细胞和肾小管特异的诱导性启动子控制下的动物，并培育了可诱导的基因敲除动物。在心脏实验中，动物将接受单肾切除，然后删除心脏MR，然后它们将服用氟可的松，并将确定心肌细胞MR缺失对心脏毒性的影响。对于肾脏MR缺失实验，MR将首先沿着整个肾单位被删除，然后特定地在远端曲管中被删除。然后将在正常情况下和肾脏损伤发生后确定动物的表型。为了研究MR对盐远端转运的作用机制，将在细胞内和体外检测醛固酮诱导蛋白SGK1对WNK/Spak信号通路的影响。这个项目具有临床意义，因为醛固酮对心血管死亡有重要作用。阻断它在治疗上很重要，但由于肾脏毒性，它是危险的。如果我们能证明心脏MR对于醛固酮的破坏性作用是必不可少的，那么治疗可以直接针对心脏的阻断，同时避免肾脏的阻断。在肾脏，如果我们能证明不依赖于血压的醛固酮效应对肾脏保护是必不可少的，那么就可以开发肾小球特异性MR调节器。此外，心血管疾病是退伍军人的头号杀手。阻断MR被认为是预防MR的关键。实现这一目标的更好和更安全的方法将增强退伍军人的健康。

DESCRIPTION (provided by applicant): Aldosterone is one of the most important cardiovascular toxins. Drugs aimed at blocking it prolong life in patients with heart failure, and reduce proteinuria in patients. Yet aldosterone is also an essential endogenous hormone, which has several important homeostatic functions. Drugs that block aldosterone actions, while useful, are also limited by side effects. These side effects are largely, although not exclusively, related to their actions on kidney tubules. Recently effects of aldosterone on kidney tubules have been suggested to be more widespread than previously believed, and involve the distal convoluted tubule, as well as the connecting tubule and collecting duct. Here, we will test for direct effects of aldosterone in mediating cardiac and renal damage by deleting mineralocorticoid receptors (MR) selectively from heart and kidney. Further, we will investigate the mechanisms by which aldosterone modulates the activity of the thiazide-sensitive Na-Cl cotransporter, along the distal convoluted tubule. Here, we will test the hypothesis that aldosterone effects along the distal convoluted tubule are essential for its renal actions, and that aldsosterone-induced renal damage depends on enhanced salt transport. We will also test how aldosterone signals to the thiazide-sensitive Na-Cl cotransporter through SGK1. In Aim #2, we will examine role of cardiac MR in mediating cardiac damage in the face of hyperaldosteronism. We will use animals in which MR is flanked by LoxP sites. When these animals are bred with animals expressing CRE recombinase, under the control of a tissue specific promoter, it permits MR to be deleted. We have obtained animals in which CRE recombinase is under the control of inducible promoters specific for cardiac myocytes and kidney tubules and have bred inducible knockout animals. For the cardiac experiments, animals will undergo uninephrectomy followed by deletion of cardiac MR. They will then consume fludrocortisone and the effects of cardiac myocyte MR deletion on cardiac toxicity will be determined. For the renal MR deletion experiments, the tubule MR will be deleted first along the entire nephron and then in the distal convoluted tubule specifically. The animal phenotypes will then be determined during normal conditions and after the generation of kidney damage. For the experiments testing mechanisms of MR action on distal salt transport, effects of the aldosterone-induced protein SGK1 on the WNK/SPAK signaling pathway will be examined in cells and in vitro. This project is clinically relevant, as aldosterone contributes importantly to cardiovascular death. Blocking it is important therapeutically, but is hazardous, owing to renal toxicity. If we can demonstrate that cardiac MR is essential for destructive effects of aldosterone, treatment can be directed at blockade in the heart, while avoiding blockade in the kidney. In the kidney, if we can demonstrate that aldosterone effects independent of blood pressure are essential for renal protection, then glomerulus specific MR modulators can be developed. Furthermore, cardiovascular disease is the number one killer of veterans. Blocking MR is considered essential for its prevention. Better and safer methods to achieve this will enhance the health of veterans.