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

描述（由申请人提供）：SLC12A1和SLC12A2的遗传突变，编码肾电子中性Na-Cl和Na-K-2Cl共转运体的基因，导致与血压变化相关的盐消耗疾病。Gittleman综合征和Bartter综合征分别是由于远曲小管Na+再吸收减少和Henle升肢增厚所致。SPAK和OSR1两种Sterile20激酶参与调节共转运体。该激酶直接结合到共转运体的n端尾部并使其磷酸化。一项全基因组研究发现，SPAK基因与血压升高之间存在关联，而敲除SPAK基因的小鼠表现出一种小人样表型。令人惊讶的是，NKCC2磷酸化在SPAK敲除动物中增加，但在磷酸化缺陷敲除的SPAK小鼠中减弱，这表明SPAK和OSR1可能在厚升肢中相互作用。本2 PD/PI应用的目的是了解导致SPAK敲除中Na- k - 2cl共转运体磷酸化增加的分子细节，并确定每种激酶在调节两个小管段Na+再吸收中的作用。在本应用中，我们拟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)检验推定的抑制性亚型和支架钙结合蛋白Cab39的功能；4)确定SPAK和OSR1在调节肾盐转运和维持血压中的作用。前两个目标将包括利用非洲爪蟾卵母细胞和哺乳动物肾细胞的异种表达进行功能研究，以及在体外和体内进行蛋白质-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构成活性OSR1敲入小鼠。该申请是提交PI对阳离子-氯共转运体及其调控的广泛研究的合理延伸，也是两个PD/PI之间正在进行的成功的美国退休人员协会资助的挑战基金合作的自然延伸。在完成后，提出的研究将阐明两种激酶的作用，并证明它们被整合到Henle厚升肢和远曲小管的精确信号网络中。

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. PUBLIC HEALTH RELEVANCE: The proposed research will address the role and interdependency of two related kinases, SPAK and OSR1, in the modulation of transport mechanisms involved in renal salt transport and the maintenance of blood pressure. The studies will examine the molecular details of kinase activation and examine the possible interaction between the two kinases and/or their isoforms. The studies will make use of novel genetically-modified mouse models to probe the role of the kinase in two different segments of the renal tubule.