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

描述（由申请人提供）：水通道蛋白2 （AQP2）是一种水通道，在肾收集管（CD）中表达。它对哺乳动物体内的水分平衡起着重要作用。我们的实验室和其他实验室在理解“常规”AQP2水渠运输的分子基础方面取得了相当大的进展。从AQP2敲除动物的有趣观察中发现，除了预期的多尿/尿崩症（DI）外，还存在严重的小管缺陷、肾功能衰竭和新生儿死亡率。然而，在其他AQP基因敲除的多尿动物中，没有观察到肾小管异常和早期死亡，即使是类似程度的多尿。此外，在多种转基因动物模型和多囊肾病伴多尿、肾小管结构和功能缺陷共存的患者中，AQP2的异常表达也经常被报道。因此，我们假设AQP2除了调节和维持水平衡外，对维持肾小管的结构和功能完整性也很重要。我们已经获得了令人信服的证据，出乎意料的是，AQP2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胞外arg - gy - asp （RGD）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在局灶粘连（FAs）的转运和转换。在体外，AQP2/整合素相互作用的解耦导致胞吞作用减少，整合素¿1的表面保留以及细胞迁移和小管形成缺陷。我们将进一步表征AQP2和整合素在囊泡运输过程中的相互作用，以及它对迁移细胞局灶黏附转换的贡献（Aim I）；此外，我们将研究介导AQP2向前沿极化运输的调控信号，为AQP2的促迁移功能提供生化基础（Aim 2）。这些研究结合了广泛的方法，包括亚细胞分离，FRAP（光漂白后荧光恢复），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AQP2的动态运输。最后，我们将应用包括胚胎肾器官培养、转基因斑马鱼模型和AQP2敲除动物在内的多个模型系统来研究AQP2/整合素相互作用的功能意义（Aim 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小管重塑。因此，我们的研究旨在探索和表征一个意想不到的由AQP2介导的促进肾上皮细胞迁移和肾小管修复的新机制，并了解AQP2在维持肾小管结构和功能中的关键作用。

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.