Metabolism Core

Project Summary/Abstract: Metabolism Core The Metabolism Core (Meta-Core) is a Biomedical Resource Core within the Washington University Chronic Kidney Disease National Resource Center. Chronically injured kidneys have altered metabolism, and recent data suggests that these metabolic changes play a causative role in progressive decline in kidney function. The ability to interrogate metabolism is crucial to understanding chronic kidney disease pathophysiology, but there are a number of barriers for kidney researchers to do so. First, many investigators interested in chronic kidney disease do not have expertise in metabolism and metabolic assays. Second, many of these assays require expensive equipment not readily available to many labs. To address these issues, the Meta-Core faculty consisting of Leslie Gewin, M.D., Core Director, Brian Finck, Ph.D., Associate Core Director, Gary Patti, Ph.D., Core Collaborator, and Leah Shriver, Ph.D., Core Collaborator, will perform consultations with Meta- Core users to guide and design the best metabolic assay to answer the research question. Several assays will be available to the Meta-Core users to interrogate metabolism at the cellular or whole kidney level. These assays include Seahorse bioflux analyses with the use of primary cells or freshly isolated tubules ex vivo, both more representative of the highly oxidative proximal tubules than commercially available primary human tubule cells or conditionally immortalized tubule cells. We can measure oxidation of fatty acids or glucose/pyruvate in kidney tissue ex vivo using radioactive substrate oxidation assays. The 3H-palmitate assay, not often performed on kidney tissue, has advantages over the more commonly used 14C-palmitate assay as it detects complete oxidation of long chain fatty acids through the electron transport chain and is easier to perform (collect 3H2O rather than 14CO2). We can also refer users to the Nutrition Obesity Research Center’s high- resolution respirometer (Oroboros Oxygraph 2k) to detect respiration in isolated mitochondria. In addition, untargeted metabolomics and stable isotope tracer studies will be performed on primary cells in vitro and in vivo. The Meta-Core faculty are all committed to sharing validated protocols and methods with the O’Brien Consortium as well as larger scientific community. In addition, we will help train the kidney scientific community how to generate primary proximal tubule cells and tubules and use the Seahorse bioflux analysis in a way that yields consistent, reproducible results. In particular, the importance of validating cell number to use, post- analysis correction for number of cells, and proper concentrations of reagents will be emphasized. The Meta- Core will leverage the expertise and equipment of other Cores at Washington University to produce complementary services for a reduced rate.