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，需要过渡到一个角色，作为一个富有成效的独立研究人员在肾脏病学领域。一个经验丰富的研究人员和导师团队已经组装，以提供必要的指导，监测和评估我的研究和职业发展的进展。该团队由主要导师William Fissell博士领导，William Fissell博士是克利夫兰诊所生物医学工程部的认证肾病学家和助理人员。Fissell博士在生物人工肾开发方面拥有强大的研究计划。泰勒米勒博士是凯斯西储大学生理学系的肾脏病学家，也是上皮细胞生物学专家，他将担任共同导师。指导委员会的其余成员包括John Sedor博士和Leslie Bruggeman博士，他们都有丰富的指导经验，并担任我的T32培训的导师。委员会将每季度举行一次会议，讨论与职业发展活动、研究进展和出版物有关的进展。其他职业发展活动将包括正式课程、职业发展研讨会和继续出席国家会议并在会上发言。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，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。职业发展活动的一部分，包括负责任的研究行为和正式课程的培训，将在凯斯西储大学（共同导师的机构）进行。与凯斯西储的互动也将有助于通过凯斯在上皮细胞生物学方面的强大研究计划获得更多的专业知识。该项目的一部分将在印第安纳州大学医学院的奥布莱恩高级肾显微镜中心进行。这是一个由NIH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）由肾小管流速改变引起的切应力变化和（2）大分子量蛋白质浸润到近端小管中，这两个因素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，用于在可控剪切力和模拟滤液组成的条件下灌注培养肾上皮细胞。这个实验系统使我们能够从一个新的角度来探讨这个问题，使我们成为这项研究的唯一合格者。我们将联合收割机与国家的最先进的成像系统，以调查这些现象。对于目标1，将以亚生理至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。将在流动和静态条件下使用旋转圆盘共聚焦显微镜和尺寸排阻色谱法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摄取、细胞内加工或完整和降解蛋白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，我们将确定在生理和超生理蛋白质浓度下细胞加工后各种蛋白质的命运，以确定：（1）如果降解的蛋白质被排泄到顶端（尿）或基底外侧（血液）隔室中，和（2）如果细胞的内吞能力饱和导致完整蛋白质的细胞内积累，其可基于这些蛋白质的性质和浓度引发特异性细胞应答。这两种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阐明影响慢性肾脏疾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提供靶向治疗方法以对抗疾病进展。

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.