Salt Absorption by the THAL: Role of NKCC2 trafficking

描述(申请人提供)：细胞膜上的钠转运体的数量由转运体的动态插入、检索和循环来调节。这些过程统称为贩运。在某些形式的人类和实验性高血压中，盐滞留是由某些肾单位节段顶膜中异常高水平的钠转运蛋白引起的。粗大的升支(THAL)吸收NaCI，有助于盐分和水的平衡，从而影响血压。Thal对NaCI的吸收主要受NKCC2调节，NKCC2是一种顶端的Na/K/2CI共转运体，介导Na和Cl进入细胞。到目前为止，还不清楚NKCC2的贩运是否规范了NACI的吸收。刺激THAL吸收NACI的血管活性激素和抑制NACI吸收的金盏花素是否通过影响NKCC2的运输来调节顶膜中的NKCC2水平也尚不清楚。我们推测，在cAMP刺激和一氧化氮抑制的过程中，THAL对NACI的吸收部分是通过将NKCC2运输到顶膜来调节的。在盐敏感型高血压模型中，NKCC2插入顶膜的增强有助于盐滞留。在目标I中，我们将研究NKCC2在顶膜中的水平和NaCI的吸收是否受到NKCC2插入和恢复的调节。在AIM II中，我们将研究刺激NKCC2依赖的NaCI通过cAMP进入Thals的激素是否增加了顶膜中NKCC2的水平。在目标III中，我们将研究一氧化氮是否通过降低顶膜中NKCC2的水平来抑制依赖NKCC2的NaCI进入。最后，我们将检查盐敏感动物的TALL中基础和激素刺激的顶膜NKCC2水平是否升高。我们将使用最先进的技术来测量NKCC2在分离的大鼠Thals中的贩运和活动。这项提案的数据将增加我们对THAL如何调节正常和盐敏感动物的盐吸收的理解，并将为利尿剂的开发确定新的靶点。

DESCRIPTION (provided by applicant): The number of Na-transporters in the cell membrane is regulated by dynamic insertion, retrieval and, recycling of transporters. Collectively these processes are known as trafficking. In some forms of human and experimental hypertension salt retention is caused by abnormally high levels of Na-transporters in the apical membrane of some nephron segments. The thick ascending limb (THAL) absorbs NaCI and contributes to salt and water homeostasis, thereby influencing blood pressure. NaCI absorption by the THAL is primarily regulated by NKCC2, an apical Na/K/2CI cotransporter that mediates Na and Cl entry into the cell. To date, it is not clear whether trafficking of NKCC2 regulates NaCI absorption. It is also not clear whether vasoactive hormones that stimulate and autacoids that inhibit NaCI absorption by the THAL regulate NKCC2 levels in the apical membrane by affecting its trafficking. We hypothesize that NaCI absorption by the THAL is regulated in part by trafficking of NKCC2 into the apical membrane, in a process stimulated by cAMP and inhibited by nitric oxide. Enhanced insertion of NKCC2 into the apical membrane contributes to salt retention in models of salt-sensitive hypertension. In Aim I we will examine whether NKCC2 levels in the apical membrane and NaCI absorption are regulated by insertion and retrieval of NKCC2. In Aim II we will study whether hormones that stimulate NKCC2-dependent NaCI entry into THALs via cAMP increase NKCC2 levels in the apical membrane. In Aim III we will study whether nitric oxide inhibits NKCC2-dependent NaCI entry by decreasing NKCC2 levels in the apical membrane. Finally we will examine whether basal and hormone-stimulated NKCC2 levels in the apical membrane are enhanced in THALs from salt-sensitive animals. We will use state-of-the-art techniques to measure NKCC2 trafficking and activity in isolated rat THALs. Data from this proposal will increase our understanding of how salt absorption by the THAL is regulated in normal and salt-sensitive animals, and will identify new targets for the development of diuretics.