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

描述(申请人提供)：SLC12a1和SLc12a2基因的遗传突变，编码肾脏电子中和性Na-Cl和Na-K-2Cl共转运体的基因，导致与血压变化相关的盐耗紊乱。这些综合征被称为Gittleman和Bartter，分别是由于Henle远曲小管和粗大的升支对Na+的重吸收减少。两个Sterile20激酶，Spak和OSR1，参与调节共转运蛋白。该激酶直接与辅助转运蛋白的N末端结合，并将其磷酸化。一项全基因组研究发现，Spak与血压升高之间存在关联，Spak基因敲除小鼠表现出类似Gittleman的表型。令人惊讶的是，在Spak基因敲除的动物中，NKCC2的磷酸化水平增加，但在磷酸化缺陷的Spak基因敲除小鼠中，NKCC2的磷酸化水平降低，这表明Spak和OSR1之间可能在粗大的上肢中存在相互作用。此2 pD/PI应用的目的是了解导致Spak基因敲除中Na-K-2Cl共转运体磷酸化增加的分子细节，并确定每个激酶在调节两个小管段的Na+重吸收中的作用。在这一应用中，我们建议1)研究Spak/OSR1的激活机制，确定二聚化的作用，并确定Spak-OSR1相互作用的存在；2)检测可能的抑制异构体和支架钙结合蛋白Cab39的功能；以及4)确定Spak和OSR1在调节肾盐运输和维持血压中的作用。前两个目标将涉及利用非洲爪哇卵母细胞和哺乳动物肾脏细胞中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。第三个目标将包括对不同饮食方案下的转基因小鼠进行详细的表型分析，以及开发和研究一种新的具有结构性活性的OSR1敲入小鼠。这项申请是对阳离子-氯化物共转运体及其通过提交的PI进行调控的广泛研究的合乎逻辑的延伸，也是两个PD/PI之间由AARA资助的挑战赠款成功合作的自然延伸。完成后，拟议的研究将阐明两个激酶的作用，并证明它们整合到Henle粗大的升支和远端曲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.