The thick ascending limb of the loop of Henle contributes to the renal regulation of systemic acid-base balance by reabsorbing both bicarbonate and ammonium. the continuing goal of this project is to determine the cellular and molecular mechanisms of these absorptive processes. Absorption of HCO3 by the medullary thick ascending limb (MTAL) is regulated by several factors (peptide hormones, growth factors, prostaglandins, catecholamines) associated with clinical acid-base disorders and renal disease states, but the mechanisms of this regulation are not understood. the overall goal of the current project is to identify the signal transduction pathways that regulate acide-base transport in the MTAL and to provide an integrated picture of the physiologic regulation ofHCO3 absorption. this requires analytical studies of diverse signaling pathways, focused on understanding how they function and interact in the MTAL. MTALs from rates will be studies in vitro to investigate four specific issues: I. Regulation of Na+/H+ exchange by protein kinase C, II. Regulation of Na+/H+ exchange and HC03 absorption by growth factors, III. Regulation of signaling via protein kinase C, and IV. Signaling via tyrosine kinawse pathways and mitogen activated protein kinases. A basic strategy of the project is that transepithelial transport rates, activities of apical and basolateral membrane transporters, effects of protein kinase inhibitors, and activities of individual signaling proteins will be measured coordinately in the MTAL to provide a detailed and integrative understanding of the regulation of HCO3 absorption. This approach will include newly developed methods for the measurement of protein kinase activities using immune complex assays in microdissected renal tubules. The proposed studies will provide new information on issues fundamental to understanding the physiology and pathophysiology of epithelial acid-base transport, including 1) the signaling proteins that couple extracellular stimuli to the regulation of apical and basolateral membrane acide-base transporters, 2) how different signaling pathways interact to regulate acide-base transport, and 3) several unusual aspects of the regulation of Na+/H+ exchange, including inhibition by hyperosmolatity and growth factors. These studies will be the first to examine in detail the signal transduction pathways that regulate acide-base transport in the mammalian thick ascending limb. 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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