Renal Effects of Aldosterone

这项工作的目标是确定醛固酮如何引起心血管疾病，包括 高血压和心力衰竭。盐皮质激素受体可以在小鼠体内缺失， 肾脏仅用于检查醛固酮在信号传导中的作用。这些老鼠表现出 减少钠通量通过噻嗪敏感的NaCl协同转运蛋白和上皮细胞 钠离子通道然而，当小鼠被置于 低钾饮食，表明钠通道刺激是主要的直接影响， 醛固酮拟议的实验将测试盐皮质激素受体是否沿沿着 远曲小管的第二部分，称为DCT2，调节氯化钠 运输，但这样做的占用糖皮质激素，而不是醛固酮。这将是 通过比较删除醛固酮合酶基因的影响，这导致缺乏 醛固酮的产生，具有删除盐皮质激素受体的作用。的活性 将使用膜片钳和全细胞记录来测量钠通道， 噻嗪敏感性NaCl协同转运蛋白的活性将使用噻嗪检测进行评估， 磷蛋白分析如果在受体敲除中通道转运活性较低， 合酶基因敲除这将支持我们的假设 在第二个目标中，将寻求醛固酮信号传导的新介质。我们将解剖 从有和没有盐皮质激素受体的小鼠收集导管，并消耗以下饮食： 改变电解质含量。这些收集管将经受RNAseq，以确定 转录差异可能是醛固酮效应的基础。我们会把记录 在高醛固酮状态与低醛固酮状态小鼠中不存在的差异表达 醛固酮或缺乏盐皮质激素受体。然后，我们将选择候选基因， 使用培养的上皮细胞进行测试，并最终在体内进行测试。我们将寻找那些 在高醛固酮条件下刺激钠通道所必需的。

The goal of this work is to determine how aldosterone causes cardiovascular disease, including hypertension and heart failure. Mice in which the mineralocorticoid receptor can be deleted in the kidney only will be used to examine aldosterone's role in signaling. These mice exhibit diminished sodium flux through both the thiazide-sensitive NaCl cotransporter and the epithelial sodium channel. Yet transport via the former is fully normalized when the mice are placed on low potassium diet, indicating that sodium channel stimulation is the major direct effect of aldosterone. The proposed experiments will test whether mineralocorticoid receptors along the second part of the distal convoluted tubule, known as the DCT2, modulate sodium chloride transport, but do so by occupancy with glucocorticoids, rather than by aldosterone. This will be done by comparing effects of deleting the aldosterone synthase gene, which leads to deficient aldosterone production, with the effects of deleting the mineralocorticoid receptor. The activity of the sodium channel will be measured using patch clamp and whole cell recording and the activity of the thiazide-sensitive NaCl cotransporter will be assessed using thiazide testing and phosphor-protein analysis. If channel transport activity is low in the receptor knockout, but not the synthase knockout, this will support the hypothesis. In a second aim, novel mediators of aldosterone signaling will be sought. We will dissect collecting ducts from mice with and without mineralocorticoid receptors, and consuming diets of varied electrolyte content. These collecting ducts will be subjected to RNAseq, to determine transcriptional differences that may underlie aldosterone effects. We will compare the transcripts differentially expressed in high aldosterone states versus those absent in mice with low aldosterone or lacking mineralocorticoid receptors. We will then select candidate genes to be tested using cultured epithelial cells, and ultimately in vivo. We will seek proteins that are essential for stimulating sodium channels under high aldosterone conditions.