Regulation of Pendrin by Angiotensin II

描述（由申请人提供）：B型插层细胞通过Cl-/HCO3-交换剂pendrin的作用分泌HCO3-并吸收Cl-穿过顶质膜。pendrin介导的Cl-/HCO3-交换降低了动脉pH和HCO3-浓度，从而减轻了代谢性碱中毒。pendrin介导的Cl-吸收也有助于在盐敏感性高血压啮齿动物模型中观察到的血管体积扩张，例如高盐饮食和醛固酮类似物（脱氧皮质酮，DOCP）。我们已经观察到，在pdrin （Slc26a4）基因编码基因被破坏的小鼠中，没有观察到DOCP和高盐饮食预期的高血压。Slc26a4缺失小鼠中醛固酮诱导高血压的缺失，很可能不仅是因为penddrin介导的Cl-摄取缺失，还因为这些突变小鼠肾脏中上皮Na+通道（ENaC）表达减少。然而，penddrin和ENaC通过醛固酮依赖性和非依赖性机制进行调节。我们的实验室和其他研究表明，血管紧张素II通过pendrin和ENaC的协同作用，在体外灌注时刺激皮质集管（CCD）中Na+和Cl-的吸收。血管紧张素II如何刺激pendrin和ENaC的表达和功能，以及pendrin和ENaC如何相互作用促进NaCl吸收是本研究的主题。本课题的目的是：1)确定血管紧张素II在体外刺激pendin介导的Cl-/HCO3-交换的机制，确定ENaC与pendrin在血管紧张素II后如何相互作用以增加NaCl的吸收；2)确定体内血管紧张素II对pendrin的长期调控机制以及pendrin如何调节ENaC的体内表达；3)确定pendrin与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(-/-)，总和细胞特异性AT1a(-/-)，组织特异性ENaC（-/-）和利德尔综合征小鼠模型，将使用定量实时PCR，光镜免疫组织化学，免疫金细胞化学和免疫印迹进行研究。将在体外灌注的小鼠肾小管和培养的小鼠主细胞中研究转运。全动物研究将进一步用于平衡研究和血压和gfr的测量。

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.