Sgk1-Mediated Effects on Sodium Transport in the Kidney

描述（由申请人提供）： 钠（Na+）通过醛固酮敏感性远端肾单位（ASDN）重吸收的调节对血压的调节至关重要，并且该系统中已知的畸变导致高血压的发作。血清和糖皮质激素激酶1，Sgk 1，一种醛固酮调节的早期反应基因，通过磷酸化和抑制泛素连接酶Nedd 4 -2，间接刺激通过上皮Na+通道ENaC的内向Na+转运。 sgk 1抑制Nedd 4 -2的机制尚不清楚，将是本研究的主要焦点。假设辅助蛋白如14-3-3蛋白介导sgk 1对远端肾单位中Nedd 4 -2的作用。将构建Nedd 4 -2的靶向突变体并用于共表达测定，以表征14-3-3蛋白在sgk 1介导的Nedd 4 -2抑制中的作用。为了确定14-3-3蛋白是否与sgk 1-磷酸化的Nedd 4 -2物理相互作用，将构建酵母双杂交系统，其中sgk 1-磷酸化的Nedd 4 -2作为“诱饵”，14-3-3蛋白作为“猎物”。集合管细胞系中的免疫共沉淀实验将用于确认体内物理相互作用。为了研究14-3-3蛋白对牛磺酸刺激的Na+电流的意义，将利用RNA干扰（RNAi）技术来抑制细胞培养物中Nedd 4 -2或14-3-3蛋白的翻译，以确定每种蛋白对内向Na+电流的影响。

DESCRIPTION (provided by applicant): The regulation of sodium (Na+) reabsorption across the aldosterone-sensitive distal nephron (ASDN) is critical to the regulation of blood pressure, and known aberrations in this system lead to the onset of hypertension. Serum and glucocorticoid kinase 1, Sgk1, an aldosterone-regulated early-response gene, indirectly stimulates inward Na+ transport via the epithelial Na+ channel, ENaC, by phosphorylating and inhibiting a ubiquitin ligase, Nedd4-2. The mechanism of sgk1 inhibition of Nedd4-2 is unknown and will be the primary focus of this investigation. The hypothesis is that accessory proteins such as 14-3-3 proteins mediate the effects of sgk1 on Nedd4-2 in the distal nephron. Targeted mutants of Nedd4-2 will be constructed and used in a co-expression assay to characterize the role of 14-3-3 proteins in sgk1-mediated inhibition of Nedd4-2. To determine if 14-3-3 proteins physically interact with sgk1-phosphorylated Nedd4-2, a yeast two-hybrid system will be constructed with sgk1-phosphorylated Nedd4-2 as 'bait', and 14-3-3 proteins as 'prey'. Co-immunoprecipitation experiments in a collecting duct cell line will be used to confirm in vivo physical interaction. To study the significance of 14-3-3 proteins on hormone-stimulated Na+ current, RNA interference (RNAi) technology will be utilized to inhibit translation of Nedd4-2, or 14-3-3 proteins in cell culture to determine the effect of each protein on inward Na+ current.