REGULATION OF RENAL BICARBONATE AND CHLORIDE ABSORPTION

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Chronic acidosis, glomerular hyperfiltration, and K depletion all lead to increased activities of the proximal tubular Na/H antiporter and Na/3HCO3 cotransporter, effects that persist when the transporters are studied outside the altered environment (memory effect). The intracellular signals mediating these adaptations are presently unknown. We postulate that a number of different extracellular signals occurring acidosis, hyperfiltration, and K deficiency, all converge on a common cellular pathway that effects increased H/HCO3 transporter activities at the transcriptional and translational levels. We have established a model for chronic acidosis in cultured proximal tubule cells, where chronic incubation in acid medium leads to a tissue-specific, protein synthesis-dependent increase in Na/H antiporter activity. One aim of this grant is to define the cell signalling mechanisms that chronically regulate H/HCO3 transporter activities; and to examine the role of these pathways in mediating the effect of chronic acid incubation on Na/H antiporter activity. Cell signals that will be examined include: 1) phospholipase C/PKC/cell Ca; 2) adenyl cyclase/CAMP/PKA; and 3) cell [Na] and Ph. We will also attempt to establish cell culture models for K depletion and hyperfiltration. The second aim of this proposal is to study the molecular mechanisms by which the Na/H antiporter cloned by Sardet et al, is chronically regulated by the above determinants. Specifically, we will examine protein abundance, relative distribution of protein between microsomal and plasma membrane fractions, rates of synthesis/degradation, degree of phosphorylation, abundance of MRNA, and rates of MRNA transcription. Lastly, we have a 1400 bp fragment from the promoter/enhancer region of the human Na/H antiporter gene. We will use this to study how the above regulators modify interactions between nuclear proteins and the Na/H antiporter/enhancer region by CAT assay, mobility shift, and Dnase protection assays.