Novel role of AQP2 in cell migration and kidney tubular injury and repair

描述（由申请方提供）：水通道蛋白2（AQP 2）是一种水通道，在肾脏集合管（CD）中表达。它对哺乳动物体内的水平衡有重要贡献。我们实验室和其他实验室在理解“常规”AQP 2水通道运输的分子基础方面取得了相当大的进展。来自AQP 2敲除动物的有趣观察结果显示，除了预期的多尿/尿崩症（DI）之外，还存在严重的肾小管缺陷、肾衰竭和新生儿死亡。然而，在其他AQP基因敲除动物中没有观察到肾小管异常和早期死亡，这些动物也有类似程度的多尿。此外，在各种转基因动物模型和伴有多尿和肾小管结构和功能缺陷的多囊肾患者中，经常报告AQP 2的异常表达。因此，我们推测AQP 2除了调节和维持水平衡外，对维持肾小管结构和功能的完整性也很重要。我们已经产生了令人信服的证据，出乎意料的是，AQP 2不仅是一个水通道，而且是一个整合素结合膜蛋白，促进细胞迁移。我们已经确定了一个保守的细胞外Arg-Gly-Asp（RGD）整合素结合基序在AQP 2，通过它与整合素1相互作用，调节运输和营业额的整合素1在粘着斑（FA）。AQP 2/整联蛋白相互作用的解偶联导致胞吞作用减少，整联蛋白1的表面保留以及体外细胞迁移和小管形成缺陷。我们将进一步研究AQP 2和整合素在囊泡运输过程中的相互作用及其对迁移细胞中粘着斑周转的作用（目的I）;此外，我们将研究介导AQP 2向前沿极化运输的调节信号，为AQP 2的促迁移功能提供生化基础（目的2）。这些研究结合了广泛的方法，包括亚细胞分级分离，FRAP（光漂白后的荧光恢复），TIRF（全内反射荧光）和活细胞共聚焦荧光成像，以检查整合素1和AQP 2的动态运输。最后，我们将应用多种模型系统，包括胚胎肾器官培养、转基因斑马鱼模型和AQP 2基因敲除动物，研究AQP 2/整合素相互作用的功能意义（目的3）。我们的假设是，AQP 2/整合素相互作用在介导细胞迁移中起作用，并反过来有助于损伤后肾小管的形成和肾小管重塑。因此，我们的研究旨在探索和表征由AQP 2介导的一种意想不到的新机制，该机制有助于肾上皮细胞迁移和肾小管修复，并了解AQP 2在维持肾小管结构和功能方面的关键作用。

DESCRIPTION (provided by applicant): Aquaporin 2 (AQP2) is a water channel, expressed in kidney collecting ducts (CD). It contributes importantly to water homeostasis in mammals. Considerable progress from our lab and others has been made in understanding the molecular basis of "conventional" AQP2 water channel trafficking. Interesting observations from AQP2 knock out animals have revealed the presence of severe tubular defects, renal failure and neonatal mortality in addition to the expected polyuria/diabetes insipidus (DI). Tubular abnormalities and early mortality have, however, not been observed in other AQP knockout animals that also have polyuria even to a similar degree. In addition, aberrant expression of AQP2 has been frequently reported in a variety of transgenic animal models and in patients with polycystic kidney diseases with polyuria and coexisting structural and functional defects of the kidney tubules. Therefore, we hypothesize that AQP2 is important for maintaining the structural and functional integrity of kidney tubules besides regulating and maintaining water balance. We have generated compelling evidence that AQP2 is, unexpectedly, not just a water channel, but is also an integrin binding membrane protein that promotes cell migration. We have identified a conserved extracellular Arg-Gly-Asp (RGD) integrin-binding motif in AQP2, through which it interacts with integrin ¿1, modulates the trafficking and turnover of integrin ¿1 at focal adhesions (FAs). Uncoupling of the AQP2/integrin interaction leads to reduced endocytosis, surface retention of integrin ¿1 and defective cell migration and tubule formation in vitro. We wil further characterize the interaction of AQP2 and integrin during vesicular trafficking and its contribution to the turnover of focal adhesions in migrating cells (Aim I); In addition, we will examine regulatory signal(s) that mediates polarized trafficking of AQP2 to the leading edge, providing a biochemical basis of AQP2's promigratory function (Aim 2). These studies incorporate a wide battery of approaches including subcellular fractionation, FRAP (fluorescence recovery after photo bleaching), TIRF (total internal reflection fluorescence), and live cell confocal fluorescence imaging to examine dynamic trafficking of integrin ¿1 and AQP2. Finally, we will apply multiple model systems including embryonic kidney organ culture, transgenic zebrafish model and AQP2 knockout animals to investigate the functional significance of the AQP2/integrin interaction (Aim 3). Our hypothesis is that the AQP2/integrin interaction plays a role in mediating cell migration, and in turn contributes to kidney tubulogenesis and tubular remodeling after injury. Our studies, therefore, are designed to explore and characterize an unexpected novel mechanism mediated by AQP2 that contributes to renal epithelial cell migration and tubular repair, and to understand the critical role of AQP2 n maintaining kidney tubular structure and function.