AT2 RECEPTOR SIGNALING IN THE KIDNEY

血管紧张素II(AngII)是一种调节血压和钠滞留的主要激素。其作用机制在细胞水平上还不完全清楚，部分原因是药理和分子上的异质性是血管紧张素Ⅱ受体。根据分子和药理学数据，AT1受体对联苯咪唑(如氯沙坦)高度敏感，而AT2受体对四氢咪啶类(如PD123177、PD123319)高度敏感。肾脏AT受体在血管紧张素调节压力钠尿关系中起重要作用。血管紧张素Ⅱ受体的药理指纹和血管紧张素Ⅱ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受体的作用已经确定，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类型，但与在高生理性血管紧张素Ⅱ浓度下观察到的盐和液体重吸收减少有关。为了直接验证这一假说，并研究AT2受体的信号机制和靶向转运体，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。这些细胞系将用于研究影响碳酸氢钠转运的血管紧张素转换酶依赖信号。这种细胞的分离将通过将可用的AT-“敲除”小鼠模型与携带永生化基因SV40大T抗原的温度敏感突变体的“永生小鼠”杂交来完成。近曲小管细胞系将通过在允许的条件下(表达T抗原)在培养中扩张显微解剖的小管来分离。这些上皮细胞的分化将通过细胞转移到不允许的条件(表达T抗原)来诱导。这些细胞系生长在高分化条件下(融合的、耐电性的单层)，将用于定量评估AngII对整个碳酸氢钠重吸收以及关键转运体(氯离子电导、顶端Na/H交换器、底侧Na-碳酸氢共转运体、Na/K-ATPase、K电导)的影响。我们还将测试新的假设，即酪氨酸激酶信号通路参与了AT2和二十烷类化合物对NHE3的抑制。运输测量将利用实时、在线记录，基于荧光指示剂染料或电生理学的成像。这些数据应该提供有关AT2受体的作用和作用的具体信息，从而补充有关AT1受体的现有信息，并对Angii在肾脏Na+代谢中的作用提供更好的全面了解。

Angiotensin II (angII) is a major hormone that regulates blood pressure and sodium retention. Its mechanism of action are incompletely understood at the cellular level, in part because of pharmacological and molecular heterogeneity are 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., PD123177, PD123319). Renal AT receptors are important for 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 indicates roles for AT1 and AT2 receptors. 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 mechanism 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 used to study angII dependent signaling that affects sodium bicarbonate transport. Isolation of such cells 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 expressed). 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 or 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.