AT2 regulation of proximal tubular electrolyte transport

AT2 调节近端肾小管电解质转运

基本信息

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
项目类别：
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

项目摘要

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尚不完全清楚，部分原因是 angII 受体的药理学和分子异质性。根据分子和药理学数据，区分了两个主要亚类：AT1 受体对联苯咪唑（例如氯沙坦）高度敏感，而 AT2 受体对四氢亚胺吡啶（例如 PD 123177、PD123319）敏感。肾 AT 受体对于血管紧张素调节压力尿钠关系非常重要。 angII 受体的药理学指纹以及 angII 对近曲小管电解质和液体重吸收的调节表明了 AT1 和 AT2 受体的作用。虽然 AT1 受体的作用已得到充分证实，但 AT2 受体的重要性直到最近才被认识到。虽然 AT1 受体的作用已得到充分证实，但 AT2 受体的重要性直到最近才被认识到。我们假设 AT2 受体虽然不是近曲小管中的主要类型，但会导致在高生理 angII 浓度下观察到的盐和液体重吸收减少。为了直接检验这一假设并研究 AT2 受体的信号传导机制和靶转运蛋白，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”杂交来实现。在允许的条件下（表达 T 抗原），通过在培养物中扩增显微解剖的肾小管来分离近端肾小管细胞系。这些上皮细胞的分化将通过细胞转移到不允许的条件（T 抗原抑制）来诱导。这些细胞系在高分化条件下（汇合、抗电单层）生长，将用于定量评估 angII 对整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 交换器、基底外侧碳酸氢钠）的影响。协同转运蛋白、Na、K-ATP酶、K-电导）。我们还将测试新的假设，即酪氨酸激酶信号通路参与 AT2 和类二十烷酸对 NHE3 的抑制。传输测量将利用基于电生理学荧光指示染料成像的实时在线记录。这些数据应提供有关 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，并提供对 angII 对肾 Na+ 代谢的作用的更好的整体了解。