Role of Mineralocorticoids in Hypertension

描述（由申请人提供）：醛固酮在高血压和心血管损害的发展中起重要作用。原发性醛固酮增多症是一种以肾小球肾上腺带自主分泌过多醛固酮为特征的高血压。世界各地的研究表明，原发性高血压的发病率在7-10%之间，难治性高血压患者的发病率更高。控制肾上腺肾小球带醛固酮生物合成的限速酶是CYP11B2基因醛固酮合成酶的产物。醛固酮合成酶在转录水平上受到调控，我们和其他人已经描述了多种基因对表达的转录调控有贡献。microrna是一类基因表达的调控因子，最近被发现对胰岛发育、胰岛素分泌、脂肪形成和b细胞发育等至关重要。H295细胞是一种人肾上腺癌，对醛固酮分泌剂有反应。我们证明了35种不同的microrna在H295细胞中高度表达，初步的微阵列研究表明它们的表达受醛固酮分泌物的上调或下调。我们研究了其中一种microrna mir21，发现它在大鼠肾上腺的肾小球带中表达最高，并在与血管紧张素II孵育后上调。在H295R细胞中，mir21的过表达增强了醛固酮的合成，从而响应血管紧张素II，表明mir21在醛固酮的生物合成中起调节作用。我们最近发现了一种新的miRNA，它在大鼠肾小球带中表达，来自于我们克隆的一种受血管紧张素II高度调控的非编码RNA。我们的假设是：“调节编码醛固酮合成酶的CYP11B2基因的转录和翻译以及直接或间接调节醛固酮生物合成的基因受到肾上腺肾小球特异性MicroRNAs的调节。”此外，“MicroRNAs调节肾上腺肾小球带细胞增殖和分化，导致肾小球带重塑”，以响应生理需要，如钠剥夺。为了检验这些假设，提出了以下具体目标：研究大鼠肾小球带功能芯片筛选中鉴定的mirna的表达，并研究慢性钠耗尽、血管紧张素II、ACTH输注和肾上腺退役诱导的肾上腺重塑过程中最有希望的候选mirna的调控。2. 表征在H295R肾上腺皮质癌细胞系中参与调节醛固酮合成酶和/或醛固酮生物合成表达的mirna。公共卫生相关性：醛固酮增多症是继发性高血压最常见的形式，约占世界范围内未选择的高血压患者的7-10%。除了血压升高外，超过生理需要的醛固酮水平，即使是相对轻微的，与没有过量醛固酮的类似血压升高水平相比，与更大的心血管、肾脏和脑血管病理相关。治疗仍然局限于矿皮质激素受体拮抗剂。确定参与肾上腺重塑和醛固酮产生的基因将使我们能够直接解决特发性高醛固酮症的发病机制。肾上腺皮质细胞与许多内分泌和内分泌激素驱动的器官一样，必须根据不断变化的生理环境和需要进行增殖、调节激素合成和凋亡。mirna的研究还很年轻。这类翻译调节因子的潜力在其他系统中很重要，这些系统自然经历细胞数量、大小和代谢活动的循环变化，因此在这些紊乱的病因学中，特别是在癌症中，是非常巨大的，对卫生保健具有普遍重要性。这项建议的内在力量在于调查小组的丰富经验。初步数据进一步加强了实验提议，其中包括最近发表在《内分泌学》上的一篇文章，为mirna依赖性调节醛固酮分泌提供了令人信服的证据。然而，关于研究计划的几个问题，包括拟研究的mirna的数量，以及缺乏如何集中精力的详细策略，缺乏关键试剂（评估醛固酮合成酶和11b-羟化酶蛋白表达的抗体），以及缺乏解决许多拟议实验局限性的方法，这些都降低了应用的热情。

DESCRIPTION (provided by applicant): Aldosterone plays a significant role in the development of hypertension and cardiovascular damage. Primary aldosteronism is a form of hypertension characterized by the autonomous and excessive secretion of aldosterone by the adrenal zona glomerulosa. Studies throughout the world have shown that the incidence is between 7-10% of essential hypertensives, and higher in patients with refractory hypertension. The rate limiting enzyme in the control of aldosterone biosynthesis in the zona glomerulosa of the adrenal is the product of the CYP11B2 gene, aldosterone synthase. Aldosterone synthase is regulated at the transcriptional level and we and others have described multiple genes that contribute to the transcriptional regulation of the expression. MicroRNAs are a class of regulators of gene expression that have recently been found to be crucial for pancreatic islet development, insulin secretion, adipogenesis, and B-cell development, and others. The H295 cell is a human adrenal carcinoma and responds to aldosterone secretagogues. We demonstrated that 35 different microRNAs are highly expressed in H295 cells and initial microarray studies suggest that their expression is up or down regulated by aldosterone secretagogues. We have studied mir21, one of these microRNAs, and found that it is most highly expressed in the zona glomerulosa of the rat adrenal and is upregulated by incubation with angiotensin II. Overexpression of mir21 in H295R cells enhances aldosterone synthesis in response to angiotensin II, suggesting that mir21 plays a role in the regulation of aldosterone biosynthesis. We have recently discovered a new miRNA that is expressed in the rat zona glomerulosa from a non-coding RNA that we cloned that is highly regulated by angiotensin II. Our hypotheses are: "Genes that regulate the transcription and translation of the CYP11B2 gene encoding Aldosterone Synthase and directly or indirectly modulate aldosterone biosynthesis are regulated by adrenal glomerulosa-specific MicroRNAs." In addition, "MicroRNAs regulate adrenal zona glomerulosa cellular proliferation and differentiation resulting in zona glomerulosa remodeling" in response to physiological need, such as sodium deprivation. The following specific aims are proposed to test these hypotheses: 1. Study the expression of miRNAs identified in functional microarray screens of rat zona glomerulosa and study the regulation of the most promising candidate miRNAs during adrenal remodeling induced by chronic sodium depletion, angiotensin II, ACTH infusion, and adrenal decommissioning. 2. Characterize the miRNAs that participate in the regulation of the expression of the aldosterone synthase and/or aldosterone biosynthesis in the H295R adrenal cortical carcinoma cell line. PUBLIC HEALTH RELEVANCE: Aldosteronism is the most common form of secondary hypertension, accounting for about 7-10% of unselected patients with hypertension World-wide. In addition to increasing blood pressure, levels of aldosterone in excess of physiological need, even when relatively mild, are associated with a greater cardiovascular, renal and cerebrovascular pathology than that associated with similar levels of blood pressure increase without excessive aldosterone. Treatment remains limited to mineralocorticoid receptor antagonists. Identifying genes involved in adrenal remodeling and aldosterone production will allow us to address the pathogenesis of Idiopathic Hyperaldosteronism directly. Cells of the adrenal cortex, like many endocrine and endocrine hormone-driven organs, must proliferate, regulate hormone synthesis and undergo apoptosis in response to ever-changing physiological circumstances and need. The science of miRNAs is still very young. The potential that this class of translational regulators are important in other systems that naturally undergo cyclic changes in cell number, size and metabolic activity, and thus in the etiology of derangements of these, particularly cancer, is very great and of general importance to health care. An inherent strength of the proposal lies in the extensive experience of the team of investigators. The experiments proposed are further strengthened by preliminary data, which include a recent publication in Endocrinology, that provide compelling evidence for miRNA-dependent modulation of aldosterone secretion. However, several concerns about the research plan, including the number of miRNAs proposed to be investigated coupled with the lack of a detailed strategy of how efforts will be focused, the lack of key reagents (antibodies to assess aldosterone synthase and 11b-hydroxylase protein expression), and the absence of approaches to address the limitations of many of the proposed experiments temper enthusiasm for the application.