AT2 regulation of proximal tubular electrolyte transport

AT2 调节近端肾小管电解质转运

• 批准号: 6458448
• 负责人: Ulrich Hopfer
• 金额: $ 25.95万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-06-01 至 2002-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6458448
• 关键词: angiotensin II; angiotensin receptor; arachidonate; biological signal transduction; brush border membrane; calcium flux; cell line; chloride channels; eicosanoid metabolism; eicosanoids; electrophysiology; fluorescent dye /probe; gene targeting; genetically modified animals; hormone regulation /control mechanism; laboratory mouse; membrane transport proteins; protein tyrosine kinase; renal tubular transport; sodium bicarbonate; sodium potassium exchanging ATPase; transport proteins; video microscopy

Angiotensin II (angII) is a major hormone that regulates blood pressure and sodium retention. Its mechanisms of action are incompletely understood at the cellular level, in part because of pharmacological and molecular heterogeneity of angII receptors. Based on molecular and pharmacological data two major subclasses are distinguished: AT1 receptors are highly sensitive to biphenylimidazoles (e.g., losartan), while AT2 receptors are sensitive to tetrahydro-imidopyridines (e.g.., PD 123177, PD123319). Renal AT receptors are important fore regulation of the pressure natriuresis relationship by angiotensin. Pharmacological finger-printing of angII receptors and of angII regulation of electrolyte and fluid reabsorption in the proximal tubule indicate roles for AT1 and AT2 receptors. While the role of AT1 receptors is well established, importance of AT2 receptors has been recognized only recently. While the role of AT1 receptors is well established, importance of AT2 receptors has been recognized only recently. We hypothesize that AT2 receptors, although not the dominant type in the proximal tubule, are responsible for diminished salt and fluid reabsorption that is observed at high physiological angII concentrations. To directly test this hypothesis and study the signaling mechanisms and target transporters for AT2 receptors, we propose to isolate well differentiated proximal tubule cell lines from mouse models that lack either AT1 or AT2 receptors. These cell lines will be accomplished by crossing available AT-"knock-out" mouse models with an "immortomouse" that carries a temperature- sensitive mutant of the immortalization gene SV40 large T antigen. Proximal tubule cell lines will be isolated by expansion of microdissected tubules in culture under permissive conditions (T antigen expressed). Differentiation of these epithelial cells will be induced by a shift of cells to non-permissive conditions (T antigen suppressed). These cell lines, grown under conditions of high differentiation (confluent, electrically resistant monolayers), will be used to quantitatively assess angII effects on overall Na bicarbonate reabsorption as well as key transporters (chloride conductance, apical Na/H exchanger, basolateral Na- bicarbonate cotransporter, Na, K-ATPase, K-conductance). We will also test the novel hypothesis that a tyrosine kinase signaling pathway is involved in the inhibition of NHE3 by AT2 and eicosanoids. Transport measurements will utilize real-time, on-line recordings, based on imaging of fluorescent indicator dyes of electrophysiology. These data should provide specific information about the effects and roles of the AT2 receptor, thus complementing available information on AT1 receptors and providing a much better overall understanding of angII actions on renal Na+ metabolism.

血管紧张素II（angII）是调节血压和钠潴留的主要激素。其作用机制在细胞水平上还不完全清楚，部分原因是angII受体的药理学和分子异质性。基于分子和药理学数据，区分了两个主要的亚类：AT 1受体对联苯咪唑高度敏感（例如，氯沙坦），而AT 2受体对四氢-亚氨基吡啶敏感（例如，PD 123177、PD 123319）。肾AT受体在血管紧张素调节压力性尿钠排泄关系中起重要作用。血管紧张素Ⅱ受体和血管紧张素Ⅱ调节电解质和液体重吸收的近端小管的药理指纹表明AT 1和AT 2受体的作用。虽然AT 1受体的作用已得到充分证实，但AT 2受体的重要性仅在最近才得到认识。虽然AT 1受体的作用已得到充分证实，但AT 2受体的重要性仅在最近才得到认识。我们推测，AT 2受体，虽然不是在近端小管的主导类型，是负责减少盐和液体重吸收，在高生理AngII浓度观察。为了直接检验这一假设，并研究信号传导机制和目标转运蛋白的AT 2受体，我们建议分离分化良好的近端小管细胞系的小鼠模型，缺乏AT 1或AT 2受体。这些细胞系将通过将可用的AT-“敲除”小鼠模型与携带永生化基因SV 40大T抗原的温度敏感突变体的“永生小鼠”杂交来实现。在允许条件下（表达T抗原），通过在培养物中扩增显微切割的小管分离近端小管细胞系。这些上皮细胞的分化将通过细胞向非允许条件（T抗原抑制）的转变来诱导。这些在高分化条件下生长的细胞系（融合、电阻单层）将用于定量评估angII对总体碳酸氢钠重吸收以及关键转运蛋白（氯离子电导、顶端Na/H交换蛋白、基底外侧碳酸氢钠协同转运蛋白、Na，K-ATP酶、K-电导）的影响。我们还将测试新的假设，酪氨酸激酶信号通路参与抑制NHE 3的AT 2和类花生酸。运输测量将利用实时，在线记录，基于电生理学的荧光指示剂染料的成像。这些数据应提供有关AT 2受体的作用和作用的具体信息，从而补充AT 1受体的可用信息，并提供更好的全面了解血管紧张素II对肾脏Na+代谢的作用。