BICARBONATE TRANSPORT BY THE MATURING RENAL TUBULE

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The main objective is to examine the cellular and molecular mechanisms responsible for the regulation of acid-base transport by the renal cortical collecting duct (CCD). Studies performed in our laboratory have shown that there are at least two functionally different types of intercalated cells in the CCD of mature rabbits: one secretes H+ via a luminal H+ATPase (type A) and one secretes HCO3 via an apical Cl/HCO3 exchanger (type B). Type A cells show rapid apical endocytotic activity, whereas type B cells bind peanut agglutinin (PNA). These properties facilitate their identification by functional fluorescent dyes. Exposure to acidic media results in alteration of intercalated cell physiology, characterized by the loss of apical Cl/HCO3 exchangers by some type B cells and conversion of net HCO3 transport by CCDs from secretion to absorption. Although type A cells can be stimulated to increase H+ secretion in response to acid treatment, the cellular mechanisms underlying the response are not well understood; little is known how the type B cells respond. We have suggested that they might be able to reverse functional polarity and secrete H+, but alternative explanations are possible, To address this polarity issue directly we plan to perfuse CCDs at low pH in vitro, using a fluorescent pH sensitive dye (BCECF) to estimate fluxes of H+/HCO3 in individual intercalated cells before and after the incubation. The molecular mechanisms underlying changes in physiology of both subtypes of intercalated cells will be examined by determining the duration of the acid stimulus needed to set off these changes, as well as any requirement for protein synthesis, signal transduction or microfilament-microtubular function. Functional changes will be correlated with fluorescent immunocytochemical studies using antibodies to specific apical and basolateral Cl/HCO3 exchangers, and H+ATPases, as well as Western blots and 2-D protein gels of acid-incubated CCDs. To determine when intercalated cells become competent to transport H+/HCO3 and respond to acid loading, CCDs from mesonephric, neonatal, and maturing kidneys will be incubated in acidic media and examined as described above. Isolated CCDs, as well as a subconfluent ("immature") collecting duct cell line, will be examined by in situ hybridization for the disappearance of proliferative and pattern-formation genes and appearance of acid-base related (differentiated) genes. Finally, since developing intercalated cells might not express mature phenotypes, their lineage will be determined in the neonatal rat kidney using retrovirus-mediated gene transfer. The results of these studies should help us to better understand at the cellular level how the intercalated cell develops and differentiates and how the kidney responds to metabolic acidosis. They also should help to explain why the newborn is less able to regulate acid-base homeostasis than is the adult.