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

AQP2 在细胞迁移和肾小管损伤与修复中的新作用

• 批准号: 8505607
• 负责人: HUA A LU
• 金额: $ 36.21万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-20 至 2018-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8505607
• 关键词: Animal Model; Animals; Binding; Biochemical; Biological Assay; Biological Models; Cell Fractionation; Cells; Co-Immunoprecipitations; Complex; Cyclic AMP; Defect; Diabetes Insipidus; Disease; Embryo; Endocytosis; Endometrial Carcinoma; Epithelial; Epithelial Cells; Equilibrium; Event; Fluorescence; Fluorescence Recovery After Photobleaching; Focal Adhesions; Functional disorder; Gentamicins; Homeostasis; Image; In Vitro; Injury; Integrin Binding; Integrins; Kidney; Kidney Failure; Knock-out; Life; Mammals; Mediating; Membrane; Membrane Proteins; Modeling; Molecular; Morphogenesis; Mus; Neonatal Mortality; Organ Culture Techniques; Patients; Phosphorylation; Play; Polycystic Kidney Diseases; Polyuria; Primary Cell Cultures; Process; Proteins; RGD (sequence); Recovery; Recycling; Regulation; Renal tubule structure; Reporting; Retrieval; Role; Signal Pathway; Signal Transduction; Structure; Surface; Surface Plasmon Resonance; Time; Transgenic Animals; Transgenic Organisms; Tubular formation; Vasopressins; Water; Zebrafish; aquaporin-2; base; cancer cell; cell motility; collecting tubule structure; design; extracellular; fluorescence imaging; in vivo; injury and repair; insight; interdisciplinary approach; interest; knockout animal; mortality; mutant; novel; public health relevance; repaired; spine bone structure; trafficking; water channel

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.

描述（由申请人提供）：水通道蛋白2（AQP2）是一种水通道，在肾集合管（CD）中表达。它对哺乳动物的水稳态有重要贡献。我们的实验室和其他实验室在理解“传统”AQP2 水通道运输的分子基础方面取得了相当大的进展。来自 AQP2 敲除动物的有趣观察结果表明，除了预期的多尿症/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率。然而，在其他 AQP 敲除动物中尚未观察到肾小管异常和早期死亡，这些动物也患有类似程度的多尿症。此外，在多种转基因动物模型和患有多尿症并同时存在肾小管结构和功能缺陷的多囊肾病患者中，经常报道AQP2的异常表达。因此，我们假设 AQP2 除了调节和维持水平衡外，对于维持肾小管的结构和功能完整性也很重要。我们已经获得令人信服的证据，表明 AQP2 出乎意料地不仅是水通道，而且还是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 Arg-Gly-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 在粘着斑 (FA) 处的运输和周转。 AQP2/整合素相互作用的解偶联导致胞吞作用减少、整合素 ¿1 的表面保留以及体外细胞迁移和肾小管形成缺陷。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及其对迁移细胞中粘着斑周转的贡献（目标 I）；此外，我们将检查介导 AQP2 极化运输至前缘的调节信号，为 AQP2 的前移功能提供生化基础（目标 2）。这些研究采用了多种方法，包括亚细胞分级、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 AQP2 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AQP2敲除动物来研究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，进而有助于肾小管生成和损伤后肾小管重塑。因此，我们的研究旨在探索和表征 AQP2 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 AQP2 在维持肾小管结构和功能中的关键作用。