RENAL NET ACID SECRETION AND THE NA/K/2C1 CONTRANSPORTER

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Two distinct genes (BSC-1 and BSC-2) encode the NA/+-K/+-2CI- co- transporter. BSC-1, or the "absorptive isoform", is kidney-specific and localizes to the apical membrane of the thick ascending limb. BSC-2, the "secretory" isoform, is widely distributed BSC-1 is responsible for the "single effect" of the countercurrent multiplier, and thereby generates an axial gradient in the medullary interstitium for ammonium and other solutes, facilitating net acid secretion and urinary concentration. In the thick ascending limb, NH/4 uptake by the Na/+-K/+-2CI-co-transporter (BSC-1) is highly regulated. Since BSC-2 localizes to the basolateral membrane of the rat alpha intercalated cell and since it is both an NH/4 amd a CO/transporter, it likely mediates net acid secretion. However, regulation of BSC-2 in the kidney and its role in net acid secretion are untested. Regulation of net CI- and H/+ secretion by the collecting duct requires transport of these ions to be regulated in parallel across the apical and the basolateral membrane of the collecting duct cell. With perturbations in acid-base balance such as chronic metabolic acidosis an increase in apical proton secretion is observed which results from up- regulation of the apical H/+-ATPase in parallel with the basolateral CI- HCO/3-exchanger. However, in the rat contribution of CI-/HCO/3-exchange to transepithelial net acid and CI-secretion has not been established. Thus, another mechanism for net H/+ and/or CI-uptake across the basolateral membrane may be important in mediating or modulating transepithelial net acid secretion. We will determine the contribution of BSC-2 to transepithelial net acid and net CI-secretion. Moreover, we will determine if activity of the co-transporter is modulated in a fashion appropriate for correction of perturbations in acid-base balance. With the recent cloning of both isoforms of the co-transporter, the published structure has been exploited to raise antibodies against co-transporter peptides. These antibodies can be used to study the long term regulation of the co-transporter. Changes in protein immunoreactivity and mRNA can be correlated with changes in transport activity to determine the role of the co-transporter in acid-base homeostasis. Specific Aims are to determine the following in the rat OMCD: 1. If BSC-2 modulates net acid secretion through direct NH/4+ uptake. 2. If BSC-2 is a major contributor to transepithelial net CI- secretion. 4. To determine the mechanism of BSC-2 transport regulation. To answer these questions, isolated renal tubules perfused in vitro will be studied. Transport will be studied with microfluorimetry as well as pH sensitive fluorescent probes. Transporter immunoreactivity and message abundance will be studied using immunoblots, immunocytochemistry and RT PCR.