AT2 regulation of proximal tubular electrolyte transport

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尚不完全清楚，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，可以区分出两个主要亚类：AT1受体对联苯咪唑（如氯沙坦）高度敏感，而AT2受体对四氢咪啶（如氯沙坦）敏感。， pd123177, pd123319)。肾AT受体在血管紧张素调节压力钠尿关系中起着重要作用。angII受体的药理指纹图谱和angII对近端小管电解质和液体重吸收的调节表明AT1和AT2受体的作用。虽然AT1受体的作用已经确立，但AT2受体的重要性直到最近才被认识到。虽然AT1受体的作用已经确立，但AT2受体的重要性直到最近才被认识到。我们假设AT2受体虽然不是近端小管中的主要受体，但在高生理angII浓度下观察到的盐和液体重吸收减少是由AT2受体引起的。为了直接验证这一假设并研究AT2受体的信号机制和靶转运体，我们建议从缺乏AT1或AT2受体的小鼠模型中分离分化良好的近端小管细胞系。这些细胞系将通过将现有的AT“敲除”小鼠模型与携带永生基因SV40大T抗原的温度敏感突变体的“不朽小鼠”杂交来实现。近端小管细胞系将在允许条件下（T抗原表达）通过扩增微解剖小管来分离。这些上皮细胞的分化将被细胞转移到非允许条件（T抗原抑制）诱导。这些细胞系在高分化条件下生长（融合，耐电单层），将用于定量评估angII对整体碳酸氢钠重吸收的影响，以及关键的转运蛋白（氯化物电导，顶端Na/H交换剂，基底侧Na-碳酸氢钠共转运蛋白，Na， k - atp酶，k -电导）。我们还将验证酪氨酸激酶信号通路参与AT2和二十烷类蛋白抑制NHE3的新假设。传输测量将利用实时在线记录，基于电生理荧光指示染料的成像。这些数据应该提供关于AT2受体的作用和作用的具体信息，从而补充了关于AT1受体的现有信息，并提供了对angII对肾Na+代谢作用的更好的整体理解。

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.