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: 8535726
• 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 2015-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8535726
• 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 of parathyroid glands; 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; Health; 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 gland; Pathway interactions; Phase; Phosphorus; 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; imaging modality; in vivo; insight; jasplakinolide; kidney cell; mortality; mutant; myosin VI; novel; research study; response; scaffold; skills; sodium-hydrogen exchanger regulatory factor; 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）： 甲状旁腺激素的疾病导致显着发病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）是钙和磷稳态的关键调节剂，控制肾小管细胞顶端磷酸钠磷酸钠共转运蛋白NAPI2A和NAPI2C的水平以及钙通道TRPV5。这些共转运蛋白/通道往返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。但是，PTH调节顶端贩运的精确分子机制仍然未知。该项目的目的是确定HO PTH调节NAPI转运蛋白的运输和肾小管细胞顶膜处的TRPV5通道。到目前为止获得的初步数据表明，响应于PTH的NAPI2A，NAPI2C和TRPV5的运输差异。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的相互作用不同：脚手架（PDZ）蛋白，肌动蛋白细胞骨架和运动蛋白。将使用多种技术研究贩运。在光漂白后，将使用总内反射荧光显微镜和荧光恢复来检查活细胞中运输的动态调节。这些成像技术将通过研究PTH对这些转运蛋白/通道在天然组织中的细胞定位的影响来补充。将使用共免疫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饰细胞骨架的药物进行解剖。 PTH对TRPV5通道S的功能效应将使用电生理学研究。这个项目将使我能够与我在赞助商的实验室中学到的高分辨率，动态成像方法相结合，以使用高分辨率，动态成像方法。此外，我将学习新的生化技术和先进的生物物理方法。最终，我打算获得确立自己为独立调查员的必要技能。 公共卫生相关性：这项工作将为钙和磷稳态的PTH调节提供见解，这对于正常的细胞功能至关重要。这些知识最终可能导致新疗法的发展，以治疗改变的钙和磷代谢。