Protein Handling By Renal Proximal Tubule Epithelial Cells

肾近端小管上皮细胞的蛋白质处理

基本信息

批准号：
8508259
负责人：
Nicholas Ferrell
金额：
$ 12.16万
依托单位：
VANDERBILT UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-08-03 至 2017-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8508259
关键词：
Actins Affect Albumins Apical Appointment Base Composition Biomedical Engineering Bioreactors Blood Cell physiology Cells Cellular biology Chemicals Chronic Kidney Failure Clinic Confocal Microscopy Cytoskeletal Modeling DNA Sequence Rearrangement Development Devices Disease Disease Progression Dolastatin Compound End stage renal failure Endocytosis Epithelial Cells Epithelium Epothilones Equipment Evaluation Excretory function F-Actin Fluorescence Microscopy Funding Goals Growth Health Human Image Immunoglobulins In Vitro Indiana Individual Infiltration Injury Institutes Institution Investigation Kidney Kidney Failure Kinetics Lead Liquid substance Measures Mentors Mentorship Methods Microfluidics Microscopy Microtubule stabilizing agent Microtubules Modeling Molecular Sieve Chromatography Molecular Weight Monitor Nature Nephrology Outcome Paclitaxel Pathology Pathway interactions Perfusion Phenotype Physiological Physiology Plasma Proteins Process Prognostic Marker Proteins Proteinuria Proximal Kidney Tubules Public Health Publications Qualifying Relative (related person)Renal function Research Research Activity Research Institute Research Personnel Research Training Risk Factors Role Severities Simulate Stabilizing Agents System Training Transferrin Tubular formation United States United States National Institutes of Health Universities Urine Workplace base career development combat experience glomerulosclerosis in vitro Model jasplakinolide medical schools meetings monolayer nephrogenesis novel novel strategies prevent programs protein distribution protein transport research and development response shear stress solute therapeutic target trafficking uptake urinary tract obstruction

项目摘要

DESCRIPTION (provided by applicant): The primary goal of this project is to provide me with the mentored career development and research training needed to transition into a role as a productive independent researcher in the field of nephrology. An experienced team of researchers and mentors has been assembled to provide the necessary guidance, monitoring, and evaluation of my research and career development progress. This team is lead by the primary mentor, Dr. William Fissell, a certified nephrologist and associate staff in the Cleveland Clinic's Department of Biomedical Engineering. Dr. Fissell has a strong research program in bioartificial kidney development. Dr. Tyler Miller, a nephrologist with appointment at Case Western Reserve University's Department of Physiology and an expert in epithelial cell biology, will serve as co-mentor. The remainder of the mentoring committee consists of Dr. John Sedor and Dr. Leslie Bruggeman, both of whom have extensive mentorship experience and serve as mentors for my T32 training. The committee will meet quarterly to discuss progress related to career development activities, research progress, and publications. Additional career development activities will consist of a combination of formal coursework, career development seminars, and continued attendance and presentations at national meetings. The project will primarily be carried out at the Cleveland Clinic's Lerner Research Institute. The Institute is a highly collaborative and productive work environment with available expertise in a wide range of health related fields. I will be able to take advantage of the state-of-the-art facilities and equipment available at The Cleveland Clinic. Portions of the career development activities, including training in responsible conduct in research and formal coursework, will be carried out at Case Western Reserve University (the co-mentor's institution). Interaction with Case Western Reserve will also facilitate access to additional expertise through Case's strong research program in epithelial cell biology. A portion of the project will be carried out at Indiana University Medical School at the O'Brien Center for Advanced Renal Microscopy. This is an NIH funded imaging facility that provides access to highly specialized equipment that is critical to the proposed research. For the research portion of the project, we hypothesize that two factors: (1) changes in shear stress resulting from altered tubular flow rates and (2) infiltration of large molecular weight proteins into the proximal tubule, both of which may be present in disease, affect the kinetics and outcome of protein handling by renal proximal tubular epithelial cells. We have developed a novel microfluidic bioreactor system for perfusion culture of renal epithelial cells with controlled shear stress and simulated filtrate composition. This experimental system allows us to approach this topic for a new angle, making us uniquely qualified for this investigation. We will combine this system with state-of-the-art imaging to investigate these phenomena. For Aim 1, cells will be perfused at flow rates ranging from sub-physiological to super- physiological and cytoskeletal reorganization will be characterized by fluorescence microscopy. Cellular uptake and intracellular trafficking of albumin will be characterized under flow and static conditions using spinning disk confocal microscopy and size exclusion chromatography to determine if flow induced cytoskeletal remodeling alters the capacity for protein uptake, intracellular processing, or intracellular composition of intact and degraded protein. For Aim 2, we will investigate the affects of other high molecular weight proteins (transferrin and immunoglobulins) that enter the tubule in disease to evaluate if these proteins compete with albumin in the endocytic processing pathway, thus affecting intracellular composition and cellular response. Finally, we will determine the fate of various proteins following cellular processing at physiological and super-physiological protein concentrations to determine: (1) if degraded proteins are excreted into the apical (urine) or basolateral (blood) compartments and (2) if saturation of the endocytic capacity of the cells results in intracellular accumulation of intact proteins, which may elicit specific cellular responses based on nature and concentration of these proteins. The effects of these two distinct, but potentially interrelated factors, on tubular protein handling may elucidate some of the mechanisms that affect progression of chronic kidney disease and eventual loss of renal function and may offer targeted therapeutic approaches at the tubular level to combat disease progression.

描述（由申请人提供）：该项目的主要目标是为我提供指导的职业发展和研究培训，以帮助我转变为肾脏病学领域富有成效的独立研究员。我们组建了一支经验丰富的研究人员和导师团队，为我的研究和职业发展进展提供必要的指导、监控和评估。该团队由主要导师 William Fissell 博士领导，他是一名经过认证的肾病专家，也是克利夫兰诊所生物医学工程系的助理人员。菲塞尔博士在生物人工肾开发方面拥有强大的研究计划。凯斯西储大学生理学系的肾脏病专家、上皮细胞生物学专家泰勒·米勒博士将担任联合导师。指导委员会的其余成员由 John Sedor 博士和 Leslie Bruggeman 博士组成，他们都拥有丰富的指导经验，并担任我的 T32 培训的导师。该委员会将每季度举行一次会议，讨论与职业发展活动、研究进展和出版物相关的进展。其他职业发展活动将包括正式课程、职业发展研讨会以及继续出席全国会议并进行演讲。该项目将主要在克利夫兰诊所的勒纳研究所进行。该研究所拥有高度协作和高效的工作环境，拥有广泛的健康相关领域的专业知识。我将能够利用克利夫兰诊所最先进的设施和设备。部分职业发展活动，包括负责任的研究行为培训和正式课程，将在凯斯西储大学（共同导师所在机构）进行。与凯斯西储大学的互动还将有助于通过凯斯在上皮细胞生物学方面强大的研究项目获得更多专业知识。该项目的一部分将在印第安纳大学医学院的奥布莱恩高级肾脏显微镜中心进行。这是美国国立卫生研究院 (NIH) 资助的成像设施，可提供对拟议研究至关重要的高度专业化设备。对于该项目的研究部分，我们假设两个因素：（1）由于肾小管流速改变而导致剪切应力的变化，以及（2）大分子量蛋白质渗透到近端肾小管中，这两者都可能存在于疾病中，影响肾近端肾小管上皮细胞处理蛋白质的动力学和结果。我们开发了一种新型微流体生物反应器系统，用于具有受控剪切应力和模拟滤液组成的肾上皮细胞灌注培养。这个实验系统使我们能够从一个新的角度探讨这个主题，使我们具有独特的资格进行这项研究。我们将将该系统与最先进的成像相结合来研究这些现象。对于目标 1，将以亚生理到超生理的流速灌注细胞，并通过荧光显微镜表征细胞骨架重组。将使用转盘共聚焦显微镜和尺寸排阻色谱法在流动和静态条件下表征白蛋白的细胞摄取和细胞内运输，以确定流动诱导的细胞骨架重塑是否改变蛋白质摄取、细胞内加工或完整和降解蛋白质的细胞内组成的能力。对于目标 2，我们将研究疾病中进入肾小管的其他高分子量蛋白质（转铁蛋白和免疫球蛋白）的影响，以评估这些蛋白质是否在内吞加工途径中与白蛋白竞争，从而影响细胞内组成和细胞反应。最后，我们将确定各种蛋白质在生理和超生理蛋白质浓度下细胞处理后的命运，以确定：（1）降解的蛋白质是否排泄到顶端（尿液）或基底外侧（血液）隔室中，以及（2）是否饱和细胞内吞能力的降低导致完整蛋白质在细胞内积累，这可能会根据这些蛋白质的性质和浓度引发特定的细胞反应。这两个不同但可能相互关联的因素对肾小管蛋白处理的影响可能会阐明影响慢性肾病进展和最终肾功能丧失的一些机制，并可能在肾小管水平上提供有针对性的治疗方法以对抗疾病进展。