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

描述(申请人提供)：水通道蛋白2(AQP2)是一种水通道，表达于肾脏集合管(CD)。它对哺乳动物体内的水分动态平衡有重要贡献。我们实验室和其他实验室在理解“常规”AQP2水通道运输的分子基础方面已经取得了相当大的进展。来自AQP2基因敲除动物的有趣观察显示，除了预期的多尿/尿崩症(DI)外，还存在严重的肾小管缺陷、肾功能衰竭和新生儿死亡。然而，在其他也有类似程度的多尿的AQP基因敲除动物中，还没有观察到肾小管异常和早期死亡率。此外，AQP2的异常表达在各种转基因动物模型中以及多囊肾病伴多尿症和肾小管结构和功能缺陷并存的患者中也经常被报道。因此，我们推测AQP2除了调节和维持水分平衡外，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，证明AQP2出人意料地不仅是一个水通道，而且还是一种促进细胞迁移的整合素结合膜蛋白。我们在AQP2中发现了一个保守的细胞外精氨酸-甘氨酸-天冬氨酸整合素结合基序，通过它与整合素�1相互作用，调节整合素�1在焦点粘连(FA)处的运输和周转。AQP2/整合素相互作用的解偶联导致细胞内吞作用减弱，整合素�1的表面滞留，以及体外细胞迁移和小管形成的缺陷。我们将进一步研究AQP2和整合素在囊泡运输过程中的相互作用及其对迁移细胞内焦点粘连翻转的贡献(AIM I)；此外，我们还将研究介导AQP2极化运输到前沿的调控信号(S)，为AQP2的S迁移功能(AIM 2)提供生化基础。这些研究结合了一系列的方法，包括亚细胞分级、光漂白后的荧光恢复、全内反射荧光和活细胞共聚焦荧光成像，以检测整合素�1和AQP2的动态运输。最后，我们将应用包括胚胎肾脏器官培养、转基因斑马鱼模型和AQP2基因敲除动物在内的多种模型系统来研究AQP2/整合素相互作用的功能意义(目标3)。我们的假设是，AQP2/整合素相互作用在介导细胞迁移中发挥作用，进而促进肾小管的形成和损伤后的肾小管重塑。因此，我们的研究旨在探索和表征AQP2介导的有助于肾上皮细胞迁移和肾小管修复的意想不到的新机制，并了解AQP2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.