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.
Chronic kidney disease (CKD) is a significant public health issue in the United States. A clearer understanding of the physiology underlying CKD will aid in development of novel approaches to slow its progression to end stage renal disease. Our study will investigate several factors that may influence the rate of CKD progression in order to guide new therapies to treat kidney failure.