Cell biology of vasopressin-induced water channels

加压素诱导的水通道的细胞生物学

基本信息

批准号：
8386101
负责人：
Dennis Brown
金额：
$ 49.35万
依托单位：
MASSACHUSETTS GENERAL HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-09-20 至 2016-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8386101
关键词：
AVPR2 gene Actin-Binding Protein Actins Animals Back Binding Biological Biological Assay Biology Brattleboro Rats Bypass Calcitonin Cell Culture Techniques Cell surface Cells Cellular biology Chemicals Congestive Heart Failure Dehydration Disease Dose Drug Combinations Effectiveness Endocytosis Equilibrium Exocytosis FDA approved Fluid Balance Fluorescence Fluorescence Resonance Energy Transfer Goals Hypertension Hyponatremia Image Analysis In Vitro Intervention Kidney Kidney Diseases Lead Liquid substance Membrane Microscopy Modeling National Center for Research Resources Output Pathway interactions Pharmaceutical Preparations Process Production Proteins Proteus Recycling Regulation Route Screening procedure Signal Transduction Simvastatin Sodium Specificity Systems Biology Testing Time Urine Vasopressins Viagra Water Water consumption Work base catalyst cellular imaging depolymerization design equilibration disorder ezrin high throughput screening in vivo inhibitor/antagonist interdisciplinary approach novel novel therapeutic intervention phosphoric diester hydrolase programs trafficking urinary water channel

AVPR2 gene Actin-Binding Protein Actins Animals Back Binding Biological Biological Assay Biology Brattleboro Rats Bypass Calcitonin Cell Culture Techniques Cell surface Cells Cellular biology Chemicals Congestive Heart Failure Dehydration Disease Dose Drug Combinations Effectiveness Endocytosis Equilibrium Exocytosis FDA approved Fluid Balance Fluorescence Fluorescence Resonance Energy Transfer Goals Hypertension Hyponatremia Image Analysis In Vitro Intervention Kidney Kidney Diseases Lead Liquid substance Membrane Microscopy Modeling National Center for Research Resources Output Pathway interactions Pharmaceutical Preparations Process Production Proteins Proteus Recycling Regulation Route Screening procedure Signal Transduction Simvastatin Sodium Specificity Systems Biology Testing Time Urine Vasopressins Viagra Water Water consumption Work base catalyst cellular imaging depolymerization design equilibration disorder ezrin high throughput screening in vivo inhibitor/antagonist interdisciplinary approach novel novel therapeutic intervention phosphoric diester hydrolase programs trafficking urinary water channel

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项目摘要

DESCRIPTION (provided by applicant): Our finding that AQP2 constitutively recycles in the absence of the antidiuretic hormone, vasopressin (VP), has led to a rethinking of how VP causes AQP2 membrane accumulation and urinary concentration. Indeed, we recently found that an FDA approved drug, simvastatin, blocks AQP2 endocytosis and reduces urine output in VP- deficient Brattleboro rats. Our proposed studies continue the strategy of combining cell biological interrogation of AQP2 trafficking with functional studies in vivo to generate novel therapeutic approaches for disorders of fluid balance including NDI, as well as congestive heart failure and hyponatremia. In Aim 1, we will explore the mechanism(s) underlying our recent discoveries that AQP2 itself is a catalyst for VP-induced actin depolymerization and that the actin-binding protein ezrin is an AQP2 associated protein involved in AQP2 endocytosis. Agents that target the ezrin binding domain on AQP2 would provide increased specificity of action for NDI treatment. Aim 2 will first expand our promising work on phosphodiesterase (PDE5) inhibitors, calcitonin and statins, which promote AQP2 membrane accumulation and increase urine concentration. We will optimize treatment doses, times and delivery routes, and assess the effectiveness of drug combinations. Then, we will further develop our new chemical screening program to identify and characterize compounds that modulate exocytosis and endocytosis in AQP2- expressing cells using high throughput, fluorescence-based assays in cell cultures (we have already identified 40 chemicals that need further characterization). Compounds that modify (increase or decrease) AQP2 membrane accumulation in vitro will ultimately be tested in vivo. We will use a multidisciplinary approach ranging from in vitro protei association assays, novel cell culture models, cellular imaging and analysis (using the PMB Microscopy Core), high throughout chemical screening (via the Center for Systems Biology Chemical Biology COre) and whole animal studies. Our overall goal is to design novel therapies for water balance disorders, including NDI and hyponatremia, using a combination of directed and discovery approaches that bypass the VP/V2R signaling cascade to modulate cell surface expression of AQP2. PUBLIC HEALTH RELEVANCE: The kidney controls how much of our daily water intake is reabsorbed back into the body, and how much is excreted in the urine. Sometimes this process is not balanced, resulting in diseases including hypertension (too much fluid and sodium reabsorption) or dehydration (too much urine production). Our work is aimed at finding the mechanisms by which the kidney controls these processes in order to cure kidney disease.

描述（由申请人提供）：我们发现，在没有抗利尿激素加压素（VP）的情况下，AQP2组成性回收，导致VP引起AQP2膜积累和尿液浓度的重新思考。实际上，我们最近发现，FDA批准的辛伐他汀，阻断了AQP2内吞作用，并减少了VP缺乏的Brattleboro大鼠中的尿量。我们提出的研究继续采用将AQP2运输的细胞生物学询问与体内功能研究结合起来的策略，以生成新的治疗方法，以用于包括NDI，包括NDI，充血性心力衰竭和低钠血症的流体平衡疾病。在AIM 1中，我们将探讨我们最近发现AQP2本身是VP诱导肌动蛋白解聚的催化剂的基础机制，并且肌动蛋白结合蛋白EZRIN是参与AQP2内吞作用的AQP2相关蛋白。靶向Ezrin结合结构域在AQP2上的药物将为NDI治疗提供更高的作用特异性。 AIM 2首先将扩大我们在磷酸二酯酶（PDE5）抑制剂，降钙素和他汀类药物上的有前途的工作，这些抑制剂和他汀类药物促进了AQP2膜的积累并增加尿液浓度。我们将优化治疗剂量，时间和输送路线，并评估药物组合的有效性。然后，我们将进一步开发我们的新化学筛查程序，以识别和表征化合物，这些化合物使用高吞吐量，基于荧光的测定在细胞培养物中调节AQP2表达细胞中的胞吐和内吞作用（我们已经确定了40种需要进一步表征的化学物质）。在体内修饰（增加或减少）AQP2膜积累的化合物最终将在体内进行测试。我们将使用一种多学科方法，从体外蛋白关联分析，新型细胞培养模型，细胞成像和分析（使用PMB显微镜核心），在整个化学筛查中（通过系统生物学生物学中心）和整个动物研究。我们的总体目标是使用定向和发现方法绕过VP/V2R信号级联级联以调节AQP2的细胞表面表达的组合，为水平疾病设计新的水平疗法，包括NDI和低钠血症。公共卫生相关性：肾脏控制着我们每天的水摄入量中的多少被重新吸收到体内，尿液中有多少排泄。有时，此过程无法平衡，导致疾病包括高血压（过多的液体和钠吸收）或脱水（尿液产生过多）。我们的工作旨在找到肾脏控制这些过程以治愈肾脏疾病的机制。