The overall objective of the experiments described in the present proposal is to characterize the mechanism of basal and parathyroid hormone(PTH)-stimulated calcium absorption in the mouse renal cortical thick ascending limb(cTALH). This nephron segment, along with the distal tubule, represents the principal site at which renal calcium absorption is regulated. Previous studies have led to the following dilemma in the understanding of calcium absorption in the isolated cTALH: 1) basal calcium transport is voltage dependent when examined over a range of imposed voltages; 2) PTH and cyclic AMP increase the rate of net calcium absorption; 3) neither PTH nor cyclic AMP increases the transepithelial voltage in the cTALH. A hypothetical model to rationalize these results has been developed and will be evaluated in the proposed studies. According to this model, basal calcium absorption proceeds through the paracellular pathway driven by the spontaneous transepithelial voltage. PTH, cting via cyclic AMP, activates a transcellular, active transport pathway for calcium absorption. To test this model, experiments will be performed on isolated microperfused segments of cTALH. Calcium will be measured with a graphite furnace atomic absorption spectrometer. Preliminary observations are consistent with the aforementioned model since PTH and dibutyryl cyclic AMP stimulated net calcium absorption in the cTALH, but not medullary thick limb, after all transepithelial electromotive forces for calcium movement were abolished. Further, prostaglandin E2 inhibited the PTH-stimulated component of net calcium absorption in the cTALH and thus may be an important regulator of PTH action in vivo.
Four specific aims of the research program have been established: 1) characterization of basal and PTH-stimulated calcium absorption in the cTALH; 2) identification of the mechanism of apical membrane calcium influx and basolateral calcium efflux; 3) characterization of the mechanism of drug-induced dissociation of calcium and sodium transport in the thick limb; and 4) the evaluation of certain electrophysiological correlates of PTH and diuretic action in the cTALH using intracellular voltage recording techniques. The information gained in these studies will aid in elucidating the mechanisms of basal and hormone-stimulated calcium transport and may have practical implications for understanding various complications of advanced renal disease, diuretic therapy, renal stone formation and, in general, disorders of calcium metabolism.
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