The long-term objective of the proposed research is to elucidate the mechanisms of H+ and HCO3- transport in the various cell types of the mammalian nephron, and to evaluate the likely importance of these transport mechanisms for the transepithelial transport of acid/base as well as for the regulation of intracellular pH (pHi). Specifically, the aims are to examine H+ and HCO3 transport in four nephron segments of the rabbit (proximal convoluted tubule (PCT), proximal straight tubule (PST), cortical collecting tubule (CCT), and collecting duct of the inner stripe of the outer medulla (CD/ISOM) and in single cultured cells (PK-1 cell line) derived from pig kidney. The experimental approach is centered on the use of pH-sensitive dyes to continuously monitor pHi. The dyes are available as membrane-permeable ester derivatives. Once inside the cell, the derivatives are cleaved by native esterases, releasing the relatively impermeant dye. Single nephron segments will be isolated and perfused in vitro. Because the PCT, PST and CD/ISOM are of a single cell type at any fixed distance from the glomerulus, the pHi measurements on these segments can be made on several neighboring cells. The optimal technique for making such a pHi determination is the measurement of intracellular dye absorbance, a technique developed during the previous grant period. Because the CCT is made up of two distinct cell types, however, it is best to make separate measurements of pHi for each cell type. The best technique for this purpose, and also for the measurement of pHi on single cultured cells, is a measurement of fluorescence intensity during alternate excitation at two wavelengths. Regardless of the preparation, the approach will be to acutely load the cell with acid (i.e., lower pHi) and monitor the subsequent recovery of pHi. This pHi recovery is due to the extrusion of H+ from the cell and/or the uptake of HCO3-. The mechanism of transport can be inferred from how the pHi recovery rate is affected by variations in the extracellular ionic composition, by pharmacologic agents, and by hormones. This research is important for understanding, at the cellular level, how the kidney regulates the pH of the blood. This impacts directly on several clinical areas, including renal and cardiovascular disease, and the effects of several drugs and hormones on the body's acid/base status.
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