Regulation of intercalated cell function by the mineralocorticoid receptor

7.项目摘要和相关性： 虽然钠的摄入量会加剧高血压，但随着氯离子摄入量的增加，血压也会上升。 不依赖于钠，使得氯离子摄取和肾脏氯离子吸收在血压调节中起关键作用。 体内应用醛固酮主要增加皮质集合管对氯化钠的摄取 通过主细胞介导的钠吸收和插层细胞介导的氯离子吸收，发生在 与HCO3-的交换，主要通过Cl3-/HCO3-交换器，垂直环。氯离子是如何影响高血压的 尤其是，人们对醛固酮如何刺激ICCl-/HCO3-交换知之甚少。 醛固酮在主细胞中的信号机制已经很好地建立了。11热休克蛋白2在细胞内代谢 糖皮质激素使醛固酮与MR(Nr3c2)结合，从而刺激ENaC。然而， 已发表的和初步的数据表明，醛固酮和MR的信号机制(S)非常 在集成电路方面有所不同。首先，11HSD2在IC中不表达。第二，MR抑制剂减少IC转运蛋白 即使在已知的没有醛固酮的情况下也是如此。第三，公布和初步数据显示 血管紧张素II和高盐饮食可以独立于醛固酮激活MR，可能是通过 Rho家族中调节细胞骨架重组和基因的GTP酶--rac1的作用机制 表情。我们观察到，Rac1基因的去除或Rac1的抑制降低了膜蛋白丰度 血管紧张素II处理的小鼠。我们观察到，增加的氯化钠摄入量减少了氯离子的吸收和氯离子的转运 在未经处理的小鼠CCDs中的丰度，但增加了顶端质膜丰度 血管紧张素II处理的小鼠内嵌细胞氯转运体。因此我们假设IC MR激活 通过或独立于醛固酮发生的。我们还假设血管紧张素II诱导的IC MR 激活通过Rac1和MR发生，它们起着分子开关的作用，通过这种开关，食盐摄入IC的影响 CL-转运体的丰度和功能(ICCl-/HCO3-交换)由抑制性转变为刺激性。至 研究IC-MR对IC转运蛋白的调节，我们建立了缺乏盐皮质激素受体和 嵌合细胞(IC-MR-KO和IC-rac1-KO小鼠)中的rac1。测定醛固酮和血管紧张素II 靶向嵌入细胞可能为高血压提供额外的靶点。 该提案的目的是确定：1)盐皮质激素受体是否调节嵌入的细胞 转运体的丰度和功能，血压和盐平衡，2)如果盐皮质激素受体 不依赖于醛固酮或醛固酮结合而刺激嵌入细胞的功能；3)如果血管紧张素II 通过MR和RAC1调节食盐摄入对IC转运蛋白丰度和功能的影响。 我们将检测K、醛固酮、血管紧张素II和MR对嵌入细胞转运体的影响 用实时定量聚合酶链式反应、免疫组织化学、 免疫金细胞化学，体外和整个动物研究中的肾小管灌流。

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.