Kinase modulation of Na+-dependent CI-coupled transporters in mouse kidney

描述（由申请人提供）：编码肾电中性Na-Cl和Na-K-2Cl共转运蛋白的基因SLC 12 A1和SLC 12 A2中的遗传突变导致与血压变化相关的盐消耗性疾病。这两种综合征被称为Gittleman和Bartter，分别是由于远曲小管和Henle升支粗的Na+重吸收减少所致。两种Sterile 20激酶SPAK和OSR 1参与调节共转运蛋白。激酶直接结合到共转运蛋白的N-末端尾部并使其磷酸化。一项全基因组研究发现SPAK与血压升高之间存在关联，SPAK敲除小鼠表现出Gittleman样表型。令人惊讶的是，NKCC 2磷酸化在SPAK敲除动物中增加，但在磷酸化缺陷敲入SPAK小鼠中减弱，表明SPAK和OSR 1在粗升支中可能存在相互作用。该2 PD/PI应用的目的是了解导致SPAK敲除中Na-K-2Cl共转运蛋白磷酸化增加的分子细节，并确定每种激酶在调节两个小管节段中Na+重吸收中的作用。在本申请中，我们建议1）检查SPAK/OSR 1激活的机制，确定二聚化的作用，并建立SPAK-OSR 1相互作用的存在; 2）检查推定的抑制性亚型和Cab 39（一种支架钙结合蛋白）的功能; 4）建立SPAK和OSR 1在调节肾盐转运和维持血压中的作用。前两个目标将涉及在非洲爪蟾卵母细胞和哺乳动物肾细胞中使用异源表达的功能研究，以及涉及体外和体内蛋白质-蛋白质相互作用的分子研究。第三个目标将包括在不同饮食方案下对转基因小鼠进行详细的表型分析，以及开发和研究一种新型的组成型活性OSR 1基因敲入小鼠。该申请是提交PI对阳离子氯化物共转运蛋白及其调节的广泛研究的逻辑延伸，也是两个PD/PI之间正在进行的AARA资助的挑战资助合作的自然延伸。完成后，拟议的研究将阐明两种激酶的作用，并证明它们被整合到Henle粗升支和远曲小管中的精确信号网络中。

DESCRIPTION (provided by applicant): Inherited mutations in SLC12A1 and SLC12A2, the genes encoding the renal electroneutral Na-Cl and Na-K-2Cl co-transporters, result in salt wasting disorders associated with changes in blood pressure. The syndromes, called Gittleman and Bartter, are due to decreased Na+-re-absorption in distal convoluted tubule and thick ascending limb of Henle, respectively. Two Sterile20 kinase, SPAK, and OSR1, are involved in regulating the co-transporters. The kinase directly binds to the N-terminal tails of the co-transporter and phosphorylates them. A whole-genome study found an association between SPAK and increased blood pressure, and SPAK knockout mice exhibit a Gittleman-like phenotype. Surprisingly, NKCC2 phosphorylation is increased in SPAK knockout animals, but is blunted in phosphorylation-deficient knock-in SPAK mice, suggesting a possible interaction between SPAK and OSR1 in the thick ascending limb. The purpose of this 2 PD/PI application is to understand the molecular details leading to increased Na-K-2Cl co-transporter phosphorylation in the SPAK knockout and to define the role of each kinase in modulating Na+-re-absorption in the two tubule segments. In this application, we propose to 1) examine the mechanisms of SPAK/OSR1 activation, determine the role of dimerization, and establish the existence of SPAK-OSR1 interactions; 2) examine the function of putative inhibitory isoforms and of Cab39, a scaffold calcium binding protein; and 4) establish the role of SPAK and OSR1 in regulating renal salt transport and maintaining blood pressure. The first two aims will involve functional studies using heterologous expression in both Xenopus laevis oocytes and mammalian kidney cells, as well as molecular studies involving protein-protein interaction both in vitro and in vivo. The third aim will consist of detailed phenotypic analysis of genetically-modified mice under different diet regiments, and the development and study of a novel constitutively active OSR1 knock-in mouse. This application is a logical extension of extensive studies of cation-chloride co-transporters and their regulation by the submitting PI and a natural extension of a successful ongoing AARA-funded Challenge grant collaboration between the two PD/PIs. Upon completion, the proposed studies will clarify the role of two kinase and demonstrate that they are integrated into precise signaling networks in the thick ascending limb of Henle and the distal convoluted tubule.