The mammalian renal proximal tubule reabsorbs 80% of the bicarbonate which is filtered by the glomerulus. This biocarbonate moves through the proximal tubule epithelial cell and therefore must be transported across both the apical and basolateral plasma membranes. In the apical membrane, a Na+/H+ exchanger secretes H+ into the tubule lumen which, through the action of carbonic anhydrase, results in the entry of bicarbonate into the cell. The mechanism whereby this bicarbonate exits the cell at the basolateral membrane is still obscure. The rate of bicarbonate reabsorption by the proximal tubule is regulated by factors such as systemic pH and parathyroid hormone. Regulation of bicarbonate reabsorption presumably occurs through an alteration in the activity of the apical and basolateral membrane bicarbonate transporters. As an example of this regulation, apical Na+/H+ exchange activity is increased in chronic metabolic acidosis caused by feeding animals larger amounts of acid than they can excrete. In this grant request, we have proposed experiments to determine the ionic mechanism of bicarbonate transport at the basolateral membrane (Specific Aim 1) and to find out whether this transport mechanism is altered by systemic acidosis (Specific Aim 2). Another series of experiments is aimed at obtaining information about the function and regulation of the apical membrane Na+/H+ exchanger. Specifically we proposed to determine if the increase in exchanger activity observed in acidosis is due to an increase in the number of exchangers or in the intrinsic properties of the exchanger. (Specific Aim 3). Lastly, we have proposed experiments to learn more about the physical properties of both the apical Na/H+ exchanger and the basolateral bicarbonate transporter, including measurements of the activation energies and pH dependencies of these two ion translocating systems (Specific Aims 4,5). The main methods to be used are 1) separation of apical and basolateral plasma membranes by sucrose density gradient centrifugation and 2) measurement of H+ transport across these membranes by various mechanisms using pH sensitive fluorescent probes. We believe that these studies will yield important information on the mechanisms of H+ (and bicarbonate) transport in the kidney and on the cellular mechanisms available for regulation of H+ transport. Because H+ transport is involved in many aspects of cell function other than transepithelial transport (e.g., cell division, response to certain hormones and mitogens), this information will also be of general biological importance.
