Cell biology of vasopressin-induced water channels

描述（由申请人提供）：我们发现AQP 2在不存在抗利尿激素血管加压素（VP）的情况下组成性聚集，这导致对VP如何引起AQP 2膜积聚和尿浓度的重新思考。事实上，我们最近发现FDA批准的药物辛伐他汀阻断了VP缺乏的Brattleboro大鼠的AQP 2内吞作用并减少了尿量。我们提出的研究继续将AQP 2运输的细胞生物学询问与体内功能研究相结合的策略，以产生用于液体平衡障碍（包括NDI）以及充血性心力衰竭和低钠血症的新治疗方法。在目的1中，我们将探讨我们最近发现的AQP 2本身是VP诱导的肌动蛋白解聚的催化剂，肌动蛋白结合蛋白ezrin是参与AQP 2内吞作用的AQP 2相关蛋白的机制。靶向AQP 2上的埃兹蛋白结合结构域的药剂将为NDI治疗提供增加的作用特异性。目的2将首先扩展我们在磷酸二酯酶（PDE 5）抑制剂，降钙素和他汀类药物，促进AQP 2膜积累和增加尿液浓度有前途的工作。我们将优化治疗剂量、时间和递送途径，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程序，以确定和表征在细胞培养物中使用高通量，基于荧光的测定来调节AQP 2表达细胞中的胞吐和胞吞作用的化合物（我们已经确定了40种需要进一步表征的化学物质）。在体外改变（增加或减少）AQP 2膜蓄积的化合物最终将在体内进行测试。我们将使用多学科的方法，包括体外蛋白质缔合测定，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（通过系统生物学化学生物学核心中心）和整个动物研究。我们的总体目标是设计新的治疗水平衡障碍，包括NDI和低钠血症，使用定向和发现方法的组合，绕过VP/V2 R信号级联调节细胞表面AQP 2的表达。

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.