Protein Handling By Renal Proximal Tubule Epithelial Cells

描述（由申请人提供）：这个项目的主要目标是为我提供指导的职业发展和研究培训，使我成为肾病学领域富有成效的独立研究人员。一个由经验丰富的研究人员和导师组成的团队为我的研究和职业发展进程提供必要的指导、监督和评估。该团队由主要导师威廉·费塞尔（William Fissell）博士领导，他是克利夫兰诊所生物医学工程系的认证肾病专家和助理员工。费塞尔博士在生物人工肾脏发育方面有很强的研究计划。凯斯西储大学（Case Western Reserve University）生理学系肾病专家、上皮细胞生物学专家泰勒·米勒（Tyler Miller）博士将担任联合导师。指导委员会的其他成员包括John Sedor博士和Leslie Bruggeman博士，他们都有丰富的指导经验，并担任我T32培训的导师。委员会将每季度召开一次会议，讨论与职业发展活动、研究进展和出版物有关的进展。其他职业发展活动将包括正式课程、职业发展讨论会和继续出席国家会议并在会议上发言。该项目将主要在克利夫兰诊所的勒纳研究所进行。该研究所是一个高度协作和富有成效的工作环境，在广泛的卫生相关领域拥有专门知识。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训练和正式课程，将在凯斯西储大学（共同导师的机构）进行。与凯斯西储的合作也将有助于通过凯斯在上皮细胞生物学方面的强大研究项目获得更多的专业知识。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。这是一个由美国国立卫生研究院资助的成像设施，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)小管流速改变导致的剪切应力变化和(2)大分子质量蛋白向近端小管浸润，这两者都可能存在于疾病中，影响肾近端小管上皮细胞处理蛋白质的动力学和结果。我们开发了一种新的微流控生物反应器系统，用于肾上皮细胞的灌注培养，控制剪切应力和模拟滤液组成。这个实验系统使我们能够从一个新的角度来研究这个问题，使我们有资格进行这项研究。我们将把这个系统与最先进的成像技术结合起来研究这些现象。对于Aim 1，细胞将以从亚生理到超生理的流速灌注，细胞骨架重组将通过荧光显微镜进行表征。细胞摄取和细胞内白蛋白运输将在流动和静态条件下进行表征，使用旋转盘共聚焦显微镜和尺寸排除色谱，以确定流动诱导的细胞骨架重塑是否改变了蛋白质摄取、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Aim 2，我们将研究在疾病中进入小管的其他高分子量蛋白（转铁蛋白和免疫球蛋白）的影响，以评估这些蛋白是否在内吞加工途径中与白蛋白竞争，从而影响细胞内成分和细胞反应。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加工后的命运，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）室；(2)细胞内吞噬能力的饱和是否导致细胞内完整蛋白质的积累，这可能引发基于这些蛋白质的性质和浓度的特定细胞反应。这两种不同但潜在相关的因素对肾小管蛋白处理的影响可能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靶向治疗方法来对抗疾病进展。

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.