Molecular Mechanisms of PTH-Mediated Trafficking in Renal Tubular Cells

PTH 介导的肾小管细胞贩运的分子机制

• 批准号: 7589936
• 负责人: JUDITH T., BLAINE
• 金额: $ 15.34万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-01 至 2014-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7589936
• 关键词: Actins; Acute; Affect; Aftercare; Apical; Binding; Biochemical; Biotinylation; Brush Border; Calcium; Calcium Channel; Cell Culture Techniques; Cell membrane; Cell physiology; Cell surface; Cells; Chronic; Clathrin; Co-Immunoprecipitations; Complement; Cytoskeleton; Data; Development; Disease; Distal; Distal convoluted renal tubule structure; Dominant-Negative Mutation; Dynamin; Electron Microscopy; Electrophysiology (science); Embryo; End stage renal failure; Endocytosis; Fluorescence Microscopy; Fluorescence Recovery After Photobleaching; Goals; Homeostasis; Hormones; Human; Image; Imaging Techniques; Immunofluorescence Immunologic; In Vitro; Investigation; Kidney; Kinetics; Knockout Mice; Knowledge; Label; Laboratories; Laboratory Study; Lead; Learning; Life; Link; Mediating; Membrane; Membrane Proteins; Metabolism; Methods; Modeling; Molecular; Morbidity - disease rate; Motor; Movement; Mus; Myosin ATPase; PDZ protein; Parathyroid Hormone Receptor; Parathyroid Hormone Receptors; Parathyroid Hormones; Pathway interactions; Phase; Phosphorous; Physiological; Preparation; Principal Investigator; Process; Property; Proteins; Proximal Kidney Tubules; Regulation; Research Personnel; Resolution; Retrieval; Role; Serum; Small Interfering RNA; Surface; System; Techniques; Time; Tissues; Tubular formation; Western Blotting; Work; apical membrane; brush border membrane; calcium disorder; cellular microvillus; graduate student; hormone regulation; human PTH protein; imaging modality; in vivo; insight; jasplakinolide; kidney cell; mortality; mutant; myosin VI; novel; public health relevance; research study; response; scaffold; skills; sodium-phosphate cotransporter proteins; trafficking

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.

描述（由申请人提供）： 导致显着发病率和死亡率的甲状旁腺激素紊乱是慢性和终末期肾病的共同特征。甲状旁腺激素 (PTH) 是钙和磷稳态的关键调节剂，控制肾小管细胞顶膜中钠磷酸盐协同转运蛋白 NaPi2a 和 NaPi2c 以及钙通道 TRPV5 的水平。这些协同转运蛋白/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。该项目的目标是确定 PTH 如何调节肾小管细胞顶膜的 NaPi 转运蛋白和 TRPV5 通道的运输。迄今为止获得的初步数据表明，NaPi2a、NaPi2c 和 TRPV5 响应 PTH 的运输存在差异。我的假设是，这些转运蛋白和通道与对运输至关重要的细胞成分以不同的方式相互作用：支架 (PDZ) 蛋白、肌动蛋白细胞骨架和运动蛋白。将使用多种技术对人口贩运进行研究。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 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。将使用修饰细胞骨架的试剂来剖析肌动蛋白细胞骨架的作用。将使用电生理学研究 PTH 对 TRPV5 通道的功能影响。这个项目将使我能够将我作为研究生获得的电生理学技能与我在资助者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，我打算获得成为一名独立调查员所需的技能。 公共健康相关性：这项工作将深入了解 PTH 对钙和磷稳态的调节，这对正常细胞功能至关重要。这些知识可能最终导致治疗钙磷代谢改变的新疗法的开发。