The thick ascending limb of Henle's loop has been recognized only recently to play an important role in the renal regulation of systemic acid-base balance. The thick ascending limb influences urinary net acid excretion through a unique capacity to reabsorb actively both bicarbonate and ammonium. Both absorptive processes are regulated homeostatically and are influenced by factors such as potassium concentration, sodium balance, and peptide hormones that commonly are associated with clinical acid-base disorders. The transport events involved in regulation of bicarbonate and ammonium absorption in the mammalian thick ascending limb are largely unknown. Therefore, the goals of this project are to study the cellular mechanisms of bicarbonate and ammonium absorption, and to integrate this information into a comprehensive understanding of the regulation of thick ascending limb acid-base transport. Thick ascending limbs from rats will be studied in vitro using isolated tubule perfusion, epifluorescence, and patch clamp techniques to investigate four specific issues: I.) the functional roles of apical and basolateral membrane H+/HCO3- transporters in transcellular bicarbonate absorption and regulation of intracellular pH, II.) the effects on individual membrane transporters of factors such as vasopressin, dietary sodium intake, and chronic metabolic acidosis that regulate transcellular bicarbonate absorption, III.) the cellular mechanisms of active NH4+ absorption, and the regulation of specific NH4+ transport pathways by factors such as chronic metabolic acidosis that control transcellular NH4+ absorption, and IV.) the role of NH4+ and H+ in regulation of apical membrane K+ channels. The proposed studies also will provide information on issues fundamental to understanding the physiology and pathophysiology of epithelial acid-base transport, including how cells regulate intracellular pH during changes in the rate of transcellular bicarbonate or ammonium transport, the differential function and regulation of apical and basolateral membrane Na+/H+ exchangers, and how transport of H+/HCO3- and ammonium may influence the transport of other ions such as sodium and potassium. These studies will be the first to examine in detail the cellular mechanisms of regulation of bicarbonate and ammonium absorption in the mammalian thick ascending limb, and to correlate the regulation of individual membrane transporters with changes in both transcellular transport rate and intracellular pH. The results will provide information essential for understanding the role of the thick ascending limb in the physiologic and pathophysiologic control of systemic acid-base balance.
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