Cell biology of vasopressin-induced water channels

描述(由申请人提供)：我们发现AQP2在缺乏抗利尿激素血管加压素(VP)的情况下进行结构性循环，这导致了对VP如何导致AQP2膜堆积和尿液浓度的重新思考。事实上，我们最近发现FDA批准的药物辛伐他汀可以阻断Vp缺乏的Brattleboro大鼠的AQP2内吞作用并减少尿量。我们建议的研究继续将AQP2转运的细胞生物学询问与体内功能研究相结合的策略，以产生治疗液体平衡障碍的新方法，包括NDI，以及充血性心力衰竭和低钠血症。在目标1中，我们将探索我们最近的发现背后的机制(S)，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内吞的AQP2相关蛋白。靶向AQP2上的Ezrin结合域的药物将为NDI的治疗提供更高的特异性。目的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工作，它们可以促进AQP2膜积聚和增加尿液浓度。优化治疗剂量、次数和给药途径，评估联合用药效果。然后，我们将进一步开发我们的新的化学筛选程序，使用细胞培养中的高通量、基于荧光的分析方法来识别和表征调节AQP2表达细胞的胞吐和内吞作用的化合物(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)AQP2膜蓄积的化合物最终将在体内进行测试。我们将使用多学科方法，从体外蛋白质结合分析、新的细胞培养模型、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核心)和整个动物研究。我们的总体目标是设计治疗水平衡障碍的新疗法，包括NDI和低钠血症，使用定向和发现相结合的方法绕过VP/V2R信号级联来调节AQP2的细胞表面表达。

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.