REGULATION OF SODIUM PUMPS IN THE KIDNEY

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Na,K-ATPase, the sodium pump, is an integral membrane protein found in all animal cells which exchanges intracellular Na+ for extracellular K+ coupled to ATP hydrolysis. Regulation of Na,K- ATPase is necessary for cell volume, fluid and electrolyte homeostasis, electrical excitability, and to drive Na+ coupled co- transport of nutrients and countertransport of cations. The overall aim of this proposal is to understand how sodium pump metabolism is regulated in the kidney using a kidney cell line (MDCK) and primary cultures of proximal tubule. We have shown that low K+ treatment and activation of Na+/H+ exchange increases Na,K-ATPase synthesis pretranslationally (at transcription) in MDCK cells; and that thyroid hormone (T3) regulates Na,K-ATPase synthesis in proximal tubule cells. The first aim is to determine if T3 and ionic stimuli act directly or indirectly: determine if T3 induction of Na,K-ATPase synthesis is secondary to changes in ion fluxes by comparing the timecourse of change in ion fluxes to changes in alpha and beta mRNAs following T3; and determine if the ions and T3 increase mRNAs directly or secondary to an intermediary by employing translation inhibitors. The second aim is to identify steps in Na,K-ATPase metabolism regulated by ions and T3: compare changes in subunit mRNA levels to changes in peptide synthesis rates, peptide abundance and Na,K-ATPase activity to determine if additional pre-, co-, or post-translational regulatory steps are involved. The third aim is to identify the ion/T3 sensitive regulatory regions of the Na,K-ATPase subunit genes: determine if regions upstream from the alpha and beta structural genes are capable of conferring ionic/T3 regulation when placed upstream from a heterologous gene not normally regulated by the signals; and determine if the regulatory sequences contain binding sites for putative trans-acting regulatory factors. The fourth aim is to determine if (Na+) or (H+) are signals that regulate Na,K-ATPase expression by examining if changing intracellular Na+ and H+ independently increases synthesis of Na,K-ATPase subunits. Accomplishing these aims will contribute to an explanation of the mechanisms regulating Na,K-ATPase expression in the kidney and may impact the understanding of fluid and electrolyte imbalance in disease states.