As part of the applicant's ongoing training program, this research project's objective is to characterize acid-base transport in the rabbit cortical collecting duct (CCD) by examining the specific transporters in each cell type (alpha and beta intercalated cells, alpha IC's, beta IC's, and principal cells, PC's) and their roles in intracellular pH (pHi) regulation. By studying primary kidney cell cultures incubated under varying conditions, the mechanisms by which the CCD responds to systemic acid-base conditions will be elucidated. The cultured cells, grown on transparent, permeable membranes, will be mounted on an inverted epi-florescence microscope in a chamber designed to permit separate luminal and bath perfusion. They will be loaded with pH-sensitive dye and excited with alternating beams of 490 and 440 nm light. Paired florescent images, along with a transmitted light DIC image, will be intensified and transferred through a video camera to an image analysis system every several seconds. A computer in communication with the imaging system will divide the 490 and 440 nm images and compute mean two dimensional pHi values for all cells in the field. Experiments will be designed to search for the presence of acid-base transporters felt to be present on the IC's and PC's, to quantify their activity and to localize them to apical or basolateral membranes. Other known and novel transporters, if present, will also be identified. A second group of studies will be designed to evaluate net transepithelial acid-base transport in the same cell preparation. H+ fluxes will be calculated based upon the pH gradients established by confluent cultures in response to experimental conditions. It is known that systemic acid-base status determines whether perfused rabbit CCD's will secrete net acid or net HCO3-secretors. An attempt will be made to mimic the in vivo affect with in vitro changes in culture conditions. An analysis of the mechanisms underlying such an effect will then be done by performing long-term experiments in which individual cells are monitored for a day or more (using the image analysis techniques described) while being perfused with sterile solutions. Acid-base transporters of individual cell will be examined following changes in incubation conditions so that insight may be gained into the mechanisms involved in the known intriguing response. Acid-base transport in cultured medullary collecting duct cells will subsequently be studied in an analogous manner. This research ultimately will provide insight into clinically important disturbances of acid-base regulation.
