Interaction of NEDD4-2 and Aldosterone in Intercalated Cell Function

 描述(申请人提供)：在盐敏感型高血压中，盐摄入量的增加会增加血管容量，从而增加血压。然而，在盐敏感型高血压的发生中，氯离子摄入量可能与钠一样重要，甚至更重要。醛固酮通过增加顶端质膜上的氯化钠转运体插入来增加肾脏对氯化钠的吸收，从而刺激氯化钠的吸收，从而使血压升高。这种类固醇激素至少部分地通过调节肾单位对醛固酮敏感区域表达的泛素连接酶NEDD4-2来增加这些肾脏钠和氯转运体的顶端质膜丰度。在NEDD4-2调节的氯化钠转运蛋白中，研究最多的是硫氮化物敏感的氯化钠共转运蛋白NCC和上皮性钠通道ENaC。NEDD4-2与ENaC结合，ENaC使转运蛋白泛化，导致其内吞和降解。给予醛固酮后，SGK1被刺激，从而使NEDD4-2磷酸化，从而阻止它与ENaC的联系。在去除NEDD4-2基因的小鼠和具有NEDD4-2人类同源物NEDD4-L特定多态的人中观察到的高血压，部分是由于肾脏Na和Cl-转运体丰度增加所致。在皮质集合管，NEDD4-2在主细胞和间质细胞中均有表达。在嵌合细胞亚型中，NEDD4-2蛋白在B型细胞中的表达最高，这表明NEDD4-2对B型嵌合细胞I的氯离子转运有调节作用。虽然主细胞NEDD4-2的功能已经得到了很好的研究，但对NEDD4-2在嵌入细胞中的作用却知之甚少。因此，我们使用Cre-lox技术产生了嵌合细胞NEDD4-2缺失小鼠。随着嵌入细胞NEDD4-2基因的去除，我们观察到CCD的电中和氯吸收和HCO3-分泌都显著增加，平均动脉压也增加。我们推测NEDD4-2与插层细胞的氯离子转运体如Pendrin和ClC-5结合，从而导致它们的泛素化、内吞和降解。我们进一步假设，醛固酮通过减少这些转运蛋白与NEDD4-2的联系，增加了CCD对氯的吸收。这一建议将剖析嵌入细胞NEDD4-2通过嵌入细胞改变氯离子转运的机制。该方案的目的是确定以下内容：1)醛固酮是否通过刺激Cl-/HCO3-和Cl-/H交换来增加CCDCl-的吸收；2)醛固酮是否通过NEDD4-2作用于CCD4-2，从而增加CCDC对Cl-的吸收；3)NEDD4-2靶向于CCDC中哪些嵌入细胞的氯离子转运体。为了实现这些目标，我们将通过实时定量聚合酶链式反应、免疫组织化学和免疫荧光、免疫金细胞化学、免疫印迹、电生理学和体外肾小管转运研究，检测醛固酮和NEDD4-2对小鼠体内和体外插层细胞转运体丰度和功能的影响。

 DESCRIPTION (provided by applicant): In salt-sensitive hypertension, an increase in NaCl intake raises vascular volume and therefore blood pressure. However, Cl- intake may be as much or more important than Na+ in the generation of salt-sensitive hypertension. Aldosterone increases renal NaCl absorption by increasing NaCl transporter insertion in the apical plasma membrane, which stimulates NaCl absorption, thereby raising blood pressure. This steroid hormone increases apical plasma membrane abundance of these renal Na+ and Cl- transporters, at least in part, through regulation of the ubiquitin ligase, NEDD4-2, expressed in the aldosterone-sensitive region of the nephron. Of the NEDD4-2-regulated NaCl transporters, the best studied are the thiazide-sensitive NaCl cotransporter, NCC, and the epithelial Na+ channel, ENaC. NEDD4-2 binds to ENaC, which ubiquitinates the transporter, resulting in its endocytosis and degradation. With aldosterone administration, Sgk1 is stimulated, thereby phosphorylating NEDD4-2, which prevents its association with ENaC. The hypertension observed in mice with NEDD4-2 gene ablation and in people with certain polymorphisms of NEDD4-L, the human homologue of rodent NEDD4-2, occurs, in part, from the increased renal Na+ and Cl- transporter abundance. In the cortical collecting duct (CCD) NEDD4-2 is expressed in both principal and intercalated cells. Within intercalated cell subtypes, NEDD4-2 protein expression is highest in type B cells, suggesting that Cl- transport in type B intercalated cells i modulated by NEDD4-2. While principal cell NEDD4-2 function has been well studied, little is known about NEDD4-2 in intercalated cells. As such, we generated intercalated cell NEDD4-2 null mice using Cre-lox technology. With intercalated cell NEDD4-2 gene ablation, we observed a substantial increase in both electroneutral Cl- absorption and HCO3- secretion in the CCD and increased mean arterial blood pressure. We hypothesize that NEDD4-2 associates with intercalated cell Cl- transporters such as pendrin and ClC-5, which leads to their ubiquitination, endocytosis and degradation. We hypothesize further that aldosterone increases Cl- absorption in the CCD, in part, by reducing the association of these transporters with NEDD4-2. This proposal will dissect the mechanism whereby intercalated cell NEDD4-2 alters Cl- transport by intercalated cells. Aims of the proposal are to determine the following: 1) If aldosterone increases CCD Cl- absorption by stimulating Cl- /HCO3- and Cl-/H+ exchange in tandem, 2) if aldosterone acts through NEDD4-2 to increase Cl- absorption by intercalated cells of the CCD and 3) what intercalated cell Cl- transporters are targeted by NEDD4-2 in the CCD. To accomplish these objectives, we will examine the effect of aldosterone and NEDD4-2 on intercalated cell transporter abundance and function in mice both in vivo and in vitro, using quantitative real time PCR, immunohistochemistry and immunofluorescence, immunogold cytochemistry, immunoblots, electrophysiology and transport studies in renal tubules perfused in vitro.