Regulation of Pendrin by Angiotensin II

描述(申请人提供)：B型插入细胞分泌HCO3-，并通过Cl3-/HCO3-交换器Pendrin的作用通过根尖质膜吸收Cl-。侧耳环介导的Cl-/HCO3-交换降低了动脉的pH值和HCO3-浓度，从而减轻了代谢性碱中毒。在高盐饮食和醛固酮类似物(脱氧皮质酮，DOCP)作用下，在氯化钠敏感型高血压啮齿动物模型中观察到的氯离子吸收也有助于血管体积的扩张。我们观察到，在编码侧膜蛋白基因(SLC26A4)基因突变的小鼠中，没有观察到DOCP注射和高盐饮食所预期的高血压。在SLC26A4基因缺失的小鼠中，没有醛固酮诱导的高血压很可能不仅是因为缺乏垂垂蛋白介导的氯离子摄取，而且是因为在这些突变小鼠的肾脏中观察到的上皮钠通道(ENaC)表达减少。然而，Pendrin和ENaC都是通过醛固酮依赖和非依赖机制来调节的。我们的实验室和其他研究人员已经证明，血管紧张素II在体外灌流时，通过Pendrin和ENaC之间的协同作用，刺激皮质集合管(CCD)对Na和Cl-的吸收。血管紧张素II如何刺激侧膜蛋白和ENaC的表达和功能，以及侧膜蛋白和ENaC如何相互作用以促进盐的吸收是本提案的主题。本研究的目的如下：1)确定血管紧张素II在体外刺激血管紧张素Ⅱ介导的氯离子/HCO3-交换的机制，并确定血管紧张素II后ENaC和垂蛋白如何相互作用以增加对氯化钠的吸收；2)确定血管紧张素II在体内长期调节垂蛋白的机制以及垂蛋白如何在体内调节ENaC的表达；以及3)确定垂蛋白和ENaC表达相互依赖的机制。为了实现这些目标，野生型和转基因小鼠，如SLC26A4(-/-)，总的和细胞特异性的AT1a(-/-)，组织特异性的ENaC(-/-)和利德尔综合征的小鼠模型，将使用实时定量聚合酶链式反应，光镜免疫组织化学，免疫金细胞化学和免疫印迹来研究。将在体外灌流的小鼠肾小管和培养的小鼠主细胞中研究转运。整个动物研究将进一步用于平衡研究和血压和GFR.7的测量。 项目简介：我们的实验室观察到，一种名为吊环蛋白的蛋白质介导了肾脏对氯的吸收，从而增加了血压。此外，垂丝蛋白还通过改变肾脏内转运钠的蛋白质(即上皮钠通道，ENaC)的量来调节肾脏对钠的吸收。这项研究将探索吊环蛋白如何控制肾脏对钠和氯化物的吸收。

DESCRIPTION (provided by applicant): Type B intercalated cells secrete HCO3- and absorb Cl- across the apical plasma membrane through the action of the Cl-/HCO3- exchanger, pendrin. Pendrin-mediated Cl-/HCO3- exchange reduces arterial pH and HCO3- concentration, thereby attenuating a metabolic alkalosis. Pendrin-mediated Cl- absorption also contributes to the vascular volume expansion observed in rodent models of NaCl-sensitive hypertension, such as following a high NaCl diet and aldosterone analogues (deoxycorticosterone pivalate, DOCP). We have observed that the hypertension expected with DOCP administration and a high NaCl diet is not observed in mice with genetic disruption of the gene encoding pendrin (Slc26a4). The absence of aldosterone-induced hypertension in Slc26a4 null mice occurs most likely not only from the absence of pendrin-mediated Cl- uptake but also from the reduced epithelial Na+ channel (ENaC) expression observed in the kidneys of these mutant mice. However, pendrin and ENaC are regulated through both aldosterone-dependent and -independent mechanisms. Our laboratory and others have shown that angiotensin II stimulates Na+ and Cl- absorption in the cortical collecting duct (CCD) when perfused in vitro through synergy between pendrin and ENaC. How angiotensin II stimulates pendrin and ENaC expression and function and how pendrin and ENaC interact to promote NaCl absorption is the subject of the present proposal. The aims of this proposal are the following: 1) to determine the mechanism whereby angiotensin II stimulates pendrin-mediated Cl-/HCO3- exchange in vitro and to determine how ENaC and pendrin interact following angiotensin II to increase NaCl absorption, 2) to determine the mechanism of the long-term regulation of pendrin by angiotensin II in vivo and how pendrin modulates ENaC expression in vivo and 3) to determine the mechanism for the interdependency of pendrin and ENaC expression. To accomplish these objectives wild type and genetically modified mice, such as Slc26a4 (-/-), total and cell-specific AT1a (-/-), tissue-specific ENaC (-/-) and mouse models of Liddle's Syndrome, will be studied using quantitative real time PCR, light microscopic immunohistochemistry, immunogold cytochemistry and immunoblots. Transport will be studied in mouse renal tubules perfused in vitro and in cultured mouse principal cells. Whole animal studies will be employed further in balance studies and measurements of blood pressure and GFR.7. Project Narrative: Our laboratory has observed that a protein called pendrin mediates absorption of chloride by the kidney, which increases blood pressure. In addition, pendrin also regulates absorption of sodium by the kidney by changing the amount of a protein that transports sodium within the kidney (i.e. the epithelial sodium channel, ENaC). This proposal will explore how pendrin controls absorption of sodium and chloride by the kidney.