Core B: Single Nephron & Metabolomics Core
The Single Nephron and Metabolomics Core provides an important regional, national and international resource for investigators who wish to define the expression, localization and functional characteristics of transport and other relevant proteins in single nephron segments studied in vitro or in defined renal epithelial cells. Moreover, targeted metabolomics services will be available to measure changes in levels of interrelated networks of small molecules involved in regulating nephron function. It is expected that the data generated from the specialized techniques, tools and expertise available through this Core will be complemented by analyses performed in other Center Cores, including the Cellular Physiology, Kidney Imaging, and the Model Organisms Cores. The Single Nephron and Metabolomics Core aims to offer an integrated approach including functional (in vitro microperfusion of isolated segments, measurements of transepithelial ion/solute fluxes, fluorescence functional imaging of single tubular cells), biochemical (microassays of enzyme/transporter activity), molecular (quantitation and analysis of RNA and protein), and analytical (targeted renal metabolomics of interrelated networks of small molecules) strategies applied to microdissected tubules, single cells, and urinary exosomes to address relevant questions proposed by users. The Core will also provide analytical services for determining concentrations of exogenous pharmacological agents and molecular probes achieved within the tissue of interest (for example, levels of xenobiotics in kidneys of zebrafish). The specific objectives of the core are:

• To provide microdissected tubules for quantitation of RNA expression and/or abundance (for real time PCR, microarray profiling), protein expression (immunoblotting), immunolocalization (in collaboration with the Kidney Imaging Core), and enzyme/transporter microassays.

• To perform functional fluorescence assays of channel/transporter function in isolated tubules microperfused in vitro and in cultured renal cells.

• To perform measurements of transepithelial ion/solute fluxes across isolated tubules microperfused in vitro.

• To isolate urinary exosomes from human subjects for total RNA and protein analyses and to correlate the findings with the results of objectives 1-3 above.

• To perform targeted metabolomic analyses of interrelated networks of small molecules in microdissected tubules, cultured renal cells, kidney/tubule perfusate, urinary exosomes and renal tissues and to determine achieved concentrations of exogenous pharmacological agents and molecular probes in renal tissues of interest.

• To provide training in all of the above.