Interaction of NEDD4-2 and Aldosterone in Intercalated Cell Function

NEDD4-2 和醛固酮在闰细胞功能中的相互作用

• 批准号: 9284447
• 负责人: SUSAN MARIE WALL
• 金额: $ 33.13万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-08-17 至 2020-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9284447
• 关键词: Ablation; Aldosterone; Animals; Anions; Apical; B-Lymphocytes; Bicarbonates; Binding; Blood Pressure; Blood Vessels; Cardiovascular Diseases; Cell membrane; Cell physiology; Cells; Data; Duct (organ) structure; Electrophysiology (science); Endocytosis; Epidemic; Generations; Genes; Genetic Polymorphism; Homologous Gene; Human; Hypertension; Immunofluorescence Immunologic; Immunohistochemistry; Immunoprecipitation; In Vitro; Industrialization; Intake; Intercalated Cell; Kidney; Kidney Diseases; Knockout Mice; Mediating; Minority; Mus; Nephrons; Pathogenesis; Phosphorylation; Regulation; Renal tubule structure; Rodent; Role; Sodium Chloride; Time; Ubiquitination; absorption; blood pressure regulation; cardiovascular risk factor; cell type; cytochemistry; epithelial Na+ channel; hypertension treatment; in vivo; novel; prevent; protein expression; public health relevance; recombinase-mediated cassette exchange; response; salt sensitive hypertension; steroid hormone; thiazide; ubiquitin ligase; uptake

 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.

 描述（由适用提供）：在盐敏感的高血压中，NaCl摄入量的增加会增加血管量，从而增加血压。但是，在产生盐敏感的高血压中，CL-摄入量可能比Na+更重要。醛固酮通过增加刺激NaCl吸收的顶端质膜中NaCl转运蛋白插入来增加肾脏NaCl的吸收，从而增加血压。这种类固醇的果烯会增加这些肾脏Na+和Cl-转运蛋白的顶端质膜抽象，至少部分通过调节在侄子醛固酮敏感区域中表达的泛素连接酶NEDD4-2。在NEDD4-2调节的NaCl转运蛋白中，最好的研究是对噻嗪类敏感的NaCl共转运蛋白NCC和上皮Na+通道ENAC。 NEDD4-2与ENAC结合，ENAC泛素化转运蛋白，导致其内吞和降解。使用醛固酮给药，刺激了SGK1，从而磷酸化NEDD4-2，从而阻止了其与ENAC的关联。在具有NEDD4-2基因消融的小鼠中观察到的高血压，以及具有NEDD4-L的某些多态性的人，NEDD4-L（啮齿动物NEDD4-2的人类同源物），部分原因是肾脏Na+和Cl- Transporter的增加发生在增加。在皮质收集管（CCD）中，nedd4-2在主细胞和插入细胞中均表达。在插入的细胞亚型中，NEDD4-2蛋白表达在B型细胞中最高，这表明B型中的Cl-插入式细胞I中的CL传输由NEDD4-2调节。虽然主细胞NEDD4-2功能已经很好地研究了，但对插入细胞中的NEDD4-2知之甚少。因此，我们使用Cre-Lox技术生成了插入的细胞NEDD4-2无效小鼠。随着插入的细胞NEDD4-2基因的消融，我们观察到CCD中电up- cl-吸收和HCO3分泌的大幅增加，并增加了平均动脉血压。我们假设NEDD4-2与Pendrin和clc-5等插入的细胞转运蛋白相关，后者导致它们的泛素化，内吞和降解。我们进一步假设，提案的醛固酮增加的目标是确定以下方面：1）如果醛固酮通过刺激Cl- /hCo3-和tandem中的Cl- /hCo3-和Cl- /h+交换来增加CCD Cl-摘要，则2）如果醛固酮通过NEDD4-2通过NEDD4-2提高Cl- cl-cl-cl-cl-cl-cl-by-cl-bycd bycd的临界细胞的作用，并增加3）。 CCD中的NEDD4-2。为了实现这些目标，我们将使用定量的实时PCR，免疫组织化学和免疫荧光，免疫细胞学，免疫造影剂，电脑灌注和灌注型，将检查醛固酮和NEDD4-2对体内和体外小鼠中插入的细胞转运蛋白抽象和功能的影响。

项目成果

Renal intercalated cells and blood pressure regulation.

• DOI: 10.23876/j.krcp.2017.36.4.305
• 发表时间: 2017-12
• 期刊: Kidney research and clinical practice
• 影响因子: 3
• 作者: Wall SM

Reply to Edemir: Physiological regulation and single-cell RNA sequencing.

回复Edemir：生理调节和单细胞RNA测序。

• DOI: 10.1073/pnas.1720333115
• 发表时间: 2018
• 期刊: Proceedings of the National Academy of Sciences of the United States of America
• 影响因子: 11.1
• 作者: Chen,Lihe;Lee,JaeWook;Chou,Chung-Lin;Nair,AnilV;Battistone,MariaA;Păunescu,TeodorG;Merkulova,Maria;Breton,Sylvie;Verlander,JillW;Wall,SusanM;Brown,Dennis;Burg,MauriceB;Knepper,MarkA
• 通讯作者: Knepper,MarkA