The collecting duct system of the mammalian nephron plays a critical role in regulating water and electrolyte balance. Its cellular heterogeneity, however, has greatly hampered attempts to characterize transport processes and their regulation at the cellular level. Cell specific monoclonal antibodies recently developed by the P.I. have now made it possible to isolate and cluture individual cell types of the collecting duct by immunoadsorption and fluorescence-activated cell sorting and study transport properties of these cells. Primary cultures of intercalated cells (ICC) and principal cells (PC), displaying differentiated functions, such as secretion of H+ and K+ and reabsorption of Na+, PO4, and Ca2+, can be maintained on permeable supports. Such cultures also retain their original hormonal responsiveness and express many of the antigens they posses in situ. Preliminary results obtained by these techniques provide the basis for the long-term objectives of this project, which are to elucidate (1) the mechanism of K+ secretion by PC; and (2) the role of ICC in acid-base homeostasis, Aim (1) proposes a detailed analysis of the cellular mechanism of the action of AVP on K+ secretion in PC monolayers. These studies will address the possibility that AVP exerts a dual action on PC, through the cAMP and intracellular Ca2+-messengers systems and will examine the contribution of alterations in K+ permeability and changes in driving forces for K+ in the mechanism of action of AVP.
Aim (2) will also test for the possibility that K+ itself can modulate K+ secretion by causing adaptive changes, and that aldosterone modulates this response.
Aim (2) involves a study of the mechanism of H+ secretion by cultured ICC. Specifically, we will examine the contribution of an apical H+-ATP-ase to acid secretion and will determine the mechanisms involved in base-disposal through the basolateral membrane. Finally, we will test the effect of aldosterone and changes in ion composition resembling those seen during metabolic/respiratory alkalosis and acidosis. The new information acquired by these studies should help to clarify the mechanisms controlling renal K+ secretion and acidification by the distal nephron, and consequently, lead to a better understanding of the etiology of the disturbances of acid-base and potassium homeostasis.
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