Molecular Mechanisms of PTH-Mediated Trafficking in Renal Tubular Cells

描述（由申请人提供）： 甲状旁腺激素紊乱是慢性和终末期肾病的常见特征，可导致显著的发病率和死亡率。甲状旁腺激素（Parathyroid hormone，PTH）是钙、磷稳态的关键调节因子，控制肾小管上皮细胞顶膜磷酸钠共转运蛋白NaPi 2a和NaPi 2c以及钙通道TRPV 5的水平。这些共转运蛋白/通道往返于顶端细胞膜的运输代表了PTH介导其生理作用的最终共同途径。然而，PTH调节顶端运输的精确分子机制仍然未知。本项目的目的是确定PTH调节肾小管细胞顶膜NaPi转运蛋白和TRPV 5通道的运输。迄今为止获得的初步数据表明，有差异的NaPi 2a，NaPi 2c和TRPV 5的贩运，以响应PTH。我的假设是，这些转运蛋白和通道与细胞成分的相互作用不同，这些细胞成分对运输至关重要：支架（PDZ）蛋白，肌动蛋白细胞骨架蛋白和马达蛋白。将使用各种各样的技术来研究贩运问题。将使用全内反射荧光显微镜和光漂白后的荧光恢复来检查活细胞中贩运的动态调节。这些成像技术将通过研究PTH对天然组织中这些转运蛋白/通道的细胞定位的影响来补充。将使用免疫共沉淀研究转运蛋白与PDZ蛋白的相互作用。肌动蛋白细胞骨架的作用将使用修改细胞骨架的试剂进行解剖。将使用电生理学研究PTH对TRPV 5通道的功能作用。这个项目将使我能够将我在研究生阶段获得的电生理学技能与我在赞助商实验室学到的高分辨率动态成像方法结合起来。此外，我将学习新的生物化学技术和先进的生物物理方法。最终，我打算获得必要的技能，使自己成为一名独立调查员。 公共卫生相关性：这项工作将提供对PTH调节钙和磷稳态的见解，这对正常细胞功能至关重要。这一知识可能最终导致开发新的疗法来治疗钙和磷代谢的改变。

DESCRIPTION (provided by applicant): Disorders of parathyroid hormone which result in significant morbidity and mortality are a common feature of chronic and end-stage renal disease. Parathyroid hormone (PTH), a key regulator of calcium and phosphorous homeostasis, controls the levels of sodium-phosphate cotransporters NaPi2a and NaPi2c and the calcium channel TRPV5 in the apical menbrane of renal tubular cells. The trafficking of these cotransporters/channels to and from the apical cell membrane represents the final common pathway through which PTH mediates its physiological action. The precise molecular mechanisms whereby PTH regulates apical trafficking, however, remain unknown. The goal of this project is to determine ho PTH regulates trafficking of NaPi transporters and the TRPV5 channel at the apical membrane of renal tubular cells. Preliminary data obtained thus far indicate that there is differential trafficking of NaPi2a, NaPi2c and TRPV5 in response to PTH. My hypothesis is that these transporters and channels interact differently with the cellular components crucial for trafficking: scaffolding (PDZ) proteins, the actin cytoskeletonand motor proteins. Trafficking will be studied using a wide variety of techniques. Dynamic regulation of trafficking in living cells will be examined using total internal reflection fluorescence microscopy and fluorescence recovery after photobleaching. These imaging techniques will be complemented by investigation of the effects of PTH on the cellular localization of these transporters/channels in native tissue. Interactions of the transporters with PDZ proteins will be studied using co-immunoprecipitation. The role of the actin cytoskeleton will be dissected using agents that modify the cytoskeleton. The functional effects of PTH on TRPV5 channel s will be investigated using electrophysiology. This project will allow me to couple the elctrophysiology skills I acquired as a graduate student with the high-resolution, dynamic imaging methods I have learned in my sponsor's laboratory. In addition, I will learn new biochemical techniques and advanced biophysical methods. Ultimately, I intend to acquire the skills necessary to establish myself as an independent investigator. PUBLIC HEALTH RELEVANCE: This work will provide insights into PTH regulation of calcium and phosphorous homeostasis which is critical for normal cellular function. This knowledge may ultimately lead to the development of novel therapies to treat altered calcium and phosphorous metabolism.