Regulation of intercalated cell function by the mineralocorticoid receptor

7.项目总结和相关性： 虽然Na+摄入会加剧高血压，但血压也会随着Cl-摄入量的增加而升高， 独立于Na+，使得Cl-摄入和肾Cl-吸收在血压调节中至关重要。 体内醛固酮给药主要通过皮质集合管（CCD）增加NaCl摄取， 通过主要细胞介导的Na+吸收和插入细胞介导的Cl-吸收，这发生在 交换HCO 3-，主要通过Cl-/HCO 3-交换器，pendrin。Cl-如何导致高血压 特别是醛固酮如何刺激ICCl-/HCO 3-交换的机制还知之甚少。 醛固酮在主细胞中的信号传导机制已被充分确立。11 kDa HSD 2代谢细胞 糖皮质激素使醛固酮与MR（Nr 3c 2）结合，从而刺激ENaC。然而，在这方面， 公开的和初步的数据表明，醛固酮和MR的信号传导机制非常复杂， 不同的IC首先，11 kHz HSD 2没有在IC中表示。第二，MR抑制剂减少IC转运体 即使在已知缺乏醛固酮的情况下也是如此。第三，公布的初步数据显示， 血管紧张素II和高NaCl饮食可以独立于醛固酮激活MR，可能是通过 Rac 1是Rho家族中的一种GT3，调节细胞骨架重组和基因表达， 表情我们观察到Rac 1基因切除或Rac 1抑制降低了Pendrin的丰度， 血管紧张素II治疗的小鼠。我们观察到，增加NaCl摄入量会降低Cl-吸收和Cl-转运蛋白， 未处理小鼠的CCD中的丰度，但增加了 血管紧张素II处理小鼠的嵌入细胞Cl-转运蛋白。因此，我们假设IC MR激活 通过或独立于醛固酮发生。我们还假设血管紧张素II诱导的IC MR 激活通过Rac 1和MR发生，它们充当分子开关，通过该开关，NaCl摄入IC的作用 Cl-转运体丰度和功能（IC Cl-/HCO 3-交换）从抑制性变为刺激性。到 为了研究IC MR对IC转运蛋白的调节，我们开发了缺乏盐皮质激素受体的小鼠， 嵌入细胞中的Rac 1（IC MR KO和IC Rac 1 KO小鼠）。确定醛固酮和血管紧张素II 靶向闰细胞可提供高血压的额外靶点。 目的是确定：1）盐皮质激素受体是否调节嵌入细胞 转运蛋白丰度和功能，血压和盐平衡，2）如果盐皮质激素受体 刺激不依赖于醛固酮或醛固酮结合的嵌入细胞功能，和3）如果血管紧张素II 通过MR和Rac 1调节NaCl摄入对IC转运蛋白丰度和功能的影响。 我们将研究K+，醛固酮，血管紧张素II和MR对嵌入细胞转运蛋白的影响， 丰度和功能在体内和体外定量真实的时间PCR，免疫组织化学， 免疫金细胞化学，肾小管灌注在体外和整个动物研究。

7. PROJECT SUMMARY AND RELEVANCE: While Na+ intake exacerbates hypertension, blood pressure also rises with increased Cl- intake, independently of Na+, making Cl- intake and renal Cl- absorption critical in blood pressure regulation. Aldosterone administration in vivo increases NaCl uptake by the cortical collecting duct (CCD) primarily through principal cell-mediated Na+ absorption and intercalated cell-mediated Cl- absorption, which occurs in exchange for HCO3-, largely through the Cl-/HCO3- exchanger, pendrin. How Cl- contributes to hypertension and, in particular, how aldosterone stimulates IC Cl-/HCO3- exchange is poorly understood. Aldosterone’s signaling mechanism in principal cells is well established. 11 HSD2 metabolizes cellular glucocorticoids enabling aldosterone binding to the MR (Nr3c2), thereby stimulating ENaC. However, published and preliminary data show that the signaling mechanism(s) of aldosterone and the MR are very different in ICs. First, 11 HSD2 is not expressed in ICs. Second, MR inhibitors reduce IC transporter abundance even in the known absence of aldosterone. Third, published and preliminary data show angiotensin II and a high NaCl diet can activate the MR independently of aldosterone, likely through a mechanism involving Rac1, a GTPase in the Rho family, that regulates cytoskeletal reorganization and gene expression. We observed that Rac1 gene ablation or Rac1 inhibition reduces pendrin abundance in angiotensin II-treated mice. We observed that increased NaCl intake reduces Cl- absorption and Cl- transporter abundance in CCDs from untreated mice, but increases the apical plasma membrane abundance of intercalated cell Cl- transporters in angiotensin II-treated mice. We therefore hypothesize that IC MR activation occurs through or independently of aldosterone. We also hypothesize that angiotensin II-induced IC MR activation occurs through Rac1 and the MR, which act a molecular switch, by which the effect of NaCl intake IC Cl- transporter abundance and function (IC Cl-/HCO3- exchange) changes from inhibitory to stimulatory. To study IC transporter regulation by the IC MR, we developed mice lacking the mineralocorticoid receptor and Rac1 in intercalated cells (IC MR KO and IC Rac1 KO mice). Determining how aldosterone and angiotensin II target intercalated cells may provide additional targets for hypertension. Proposal Aims are to determine: 1) if the mineralocorticoid receptor modulates intercalated cell transporter abundance and function, blood pressure and salt balance, 2) if the mineralocorticoid receptor stimulates intercalated cell function independently of aldosterone or aldosterone binding and 3) If angiotensin II acts through the MR and Rac1 to modulate the effect of NaCl intake on IC transporter abundance and function. We will examine the effect of K+, aldosterone, angiotensin II and the MR on intercalated cell transporter abundance and function in vivo and in vitro with quantitative real time PCR, immunohistochemistry, immunogold cytochemistry, renal tubules perfused in vitro and in whole animal studies.