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：
8548321
负责人：
Dennis Brown
金额：
$ 56.91万
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/8548321
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 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 public health relevance screening trafficking urinary water channel

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.

描述（由申请人提供）：我们发现 AQP2 在没有抗利尿激素、加压素 (VP) 的情况下会持续循环，这导致人们重新思考 VP 如何导致 AQP2 膜积聚和尿液浓缩。事实上，我们最近发现 FDA 批准的药物辛伐他汀可以阻断 AQP2 内吞作用并减少 VP 缺陷的 Brattleboro 大鼠的尿量。我们提出的研究继续采用将 AQP2 运输的细胞生物学研究与体内功能研究相结合的策略，以产生针对液体平衡障碍（包括 NDI）以及充血性心力衰竭和低钠血症的新治疗方法。在目标 1 中，我们将探讨我们最近发现的机制，即 AQP2 本身是 VP 诱导的肌动蛋白解聚的催化剂，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 AQP2 上的埃兹蛋白结合域的药物将为 NDI 治疗提供更高的作用特异性。目标 2 将首先扩展我们在磷酸二酯酶 (PDE5) 抑制剂、降钙素和他汀类药物方面的有前景的工作，这些抑制剂可促进 AQP2 膜积聚并增加尿液浓度。我们将优化治疗剂量、时间和给药途径，并评估药物组合的有效性。然后，我们将进一步开发新的化学筛选计划，以在细胞培养物中使用高通量、基于荧光的测定来识别和表征调节 AQP2 表达细胞中的胞吐作用和内吞作用的化合物（我们已经识别了 40 种需要进一步表征的化学物质）。在体外修饰（增加或减少）AQP2 膜积累的化合物最终将在体内进行测试。我们将使用多学科方法，包括体外蛋白质关联测定、新型细胞培养模型、细胞成像和分析（使用 PMB 显微镜核心）、高通量化学筛选（通过系统生物学中心化学生物学 COre）和整体动物研究。我们的总体目标是设计针对水平衡紊乱（包括 NDI 和低钠血症）的新疗法，结合使用定向和发现方法，绕过 VP/V2R 信号级联来调节 AQP2 的细胞表面表达。