Interaction of NEDD4-2 and Aldosterone in Intercalated Cell Function

 描述（由申请人提供）：在盐敏感性高血压中，NaCl摄入量的增加会增加血管容积，从而升高血压。然而，在盐敏感性高血压的产生中，Cl-摄入可能与Na+一样重要或更重要。醛固酮通过增加顶端质膜中的NaCl转运蛋白插入来增加肾脏NaCl吸收，这刺激NaCl吸收，从而升高血压。这种类固醇激素增加这些肾Na+和Cl-转运蛋白的顶端质膜丰度，至少部分是通过调节在肾单位的醛固酮敏感区域中表达的泛素连接酶NEDD 4 -2。在NEDD 4 -2调节的NaCl转运蛋白中，研究得最好的是噻嗪敏感性NaCl协同转运蛋白NCC和上皮Na+通道ENaC。NEDD 4 -2与ENaC结合，ENaC使转运蛋白泛素化，导致其内吞和降解。醛固酮给药后，Sgk 1被刺激，从而磷酸化NEDD 4 -2，阻止其与ENaC结合。在具有NEDD 4 -2基因消融的小鼠和具有NEDD 4-L（啮齿动物NEDD 4 -2的人类同源物）的某些多态性的人中观察到的高血压部分地发生于肾Na+和Cl-转运体丰度增加。在皮质集合管（CCD）中，NEDD 4 -2在主细胞和闰细胞中表达。在闰细胞亚型中，NEDD 4 -2蛋白表达在B型细胞中最高，表明B型闰细胞中的Cl-转运受NEDD 4 -2调节。虽然主细胞NEDD 4 -2的功能已经得到了很好的研究，但对闰细胞中的NEDD 4 -2知之甚少。因此，我们使用Cre-lox技术产生了嵌入细胞NEDD 4 -2缺失小鼠。插入细胞NEDD 4 -2基因切除后，我们观察到CCD的电中性Cl-吸收和HCO 3-分泌显著增加，平均动脉血压升高。我们推测NEDD 4 -2与插入细胞Cl-转运蛋白如pendrin和ClC-5相关，这导致它们的泛素化、内吞和降解。我们进一步假设，醛固酮增加氯离子吸收的CCD，在一定程度上，通过减少这些转运蛋白与NEDD 4 -2。本研究将对NEDD 4 -2改变插入细胞Cl-转运的机制进行深入的探讨。该提案的目的是确定以下内容：1）醛固酮是否通过刺激Cl- /HCO 3-和Cl-/H+交换来增加CCD的Cl-吸收，2）醛固酮是否通过NEDD 4 -2作用以增加CCD的嵌入细胞的Cl-吸收，以及3）CCD中NEDD 4 -2靶向什么样的嵌入细胞Cl-转运蛋白。为了实现这些目标，我们将使用定量真实的时间PCR、免疫组织化学和免疫荧光、免疫金细胞化学、免疫印迹、电生理学和在体外灌注的肾小管中的转运研究来检查醛固酮和NEDD 4 -2对小鼠体内和体外的嵌入细胞转运蛋白丰度和功能的影响。

 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.