Molecular Mechanisms of PTH-Mediated Trafficking in Renal Tubular Cells

描述(由申请人提供)： 甲状旁腺激素紊乱会导致严重的发病率和死亡率，是慢性和终末期肾脏疾病的共同特征。甲状旁腺激素(PTH)是钙磷稳态的关键调节剂，控制着肾小管上皮细胞膜上钠-磷共转运体NaPi2a和NaPi2c以及钙通道TRPV5的水平。这些共转运体/通道往返于顶端细胞膜，是甲状旁腺素调节其生理作用的最终共同途径。然而，甲状旁腺激素调节心尖部运输的确切分子机制仍不清楚。本项目的目标是确定hO-PTH调节肾小管上皮细胞顶膜上的napi转运体和TRPV5通道的转运。到目前为止获得的初步数据表明，对PTH的反应存在NaPi2a、NaPi2c和TRPV5的不同转运。我的假设是，这些转运蛋白和通道与运输过程中至关重要的细胞成分--支架(PDZ)蛋白、肌动蛋白细胞骨架和马达蛋白--的相互作用是不同的。将使用多种技术研究人口贩运问题。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运输的动态调节。这些成像技术将通过研究甲状旁腺激素对这些转运体/通道在天然组织中的细胞定位的影响来补充。将使用免疫共沉淀法研究转运蛋白与PDZ蛋白的相互作用。肌动蛋白细胞骨架的作用将使用改变细胞骨架的试剂来剖析。用电生理学方法研究甲状旁腺素对TRPV5通道S的功能影响。这个项目将使我能够将研究生时学到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，我还将学习新的生化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的调查员。 公共卫生相关性：这项工作将为甲状旁腺素调节钙和磷的动态平衡提供见解，这对正常的细胞功能至关重要。这一知识可能最终导致治疗钙磷代谢改变的新疗法的开发。

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.