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Extracellular Hypotonicity Increases Na,K-ATPase Cell Surface Expression via Enhanced Na⁺ Influx in Cultured Renal Collecting Duct Cells

Manlio Vinciguerra^*, Serge Arnaudeau, David Mordasini^*, Martine Rousselot^*, Marcelle Bens, Alain Vandewalle, Pierre-Yves Martin^*, Udo Hasler^* and Eric Feraille^*

*Service de Néphrologie, Fondation pour Recherches Médicales, Geneva, Switzerland; Department of Physiology, University of Geneva Medical Center, Geneva, Switzerland; and INSERM U478, Faculté de Médecine Xavier Bichat, Paris, France

Correspondence to Dr. Eric Feraille, Service de Néphrologie, Fondation pour Recherches Médicales, 64 rue de la Roseraie, CH-1211 Geneva 4, Switzerland. Phone: 41-22-382-38-37; Fax: 41-22-347-59-79; E-mail: Eric.Feraille{at}medecine.unige.ch

In the renal collecting duct (CD), the Na,K-ATPase, which provides the driving force for Na⁺ absorption, is under tight multifactorial control. Because CD cells are physiologically exposed to variations of interstitial and tubular fluid osmolarities, the effects of extracellular anisotonicity on Na,K-ATPase cell surface expression were studied. Results show that hypotonic conditions increased, whereas hypertonic conditions had no effect on Na,K-ATPase cell surface expression in confluent mpkCCD_cl4 cells. Incubating cells with amphotericin B, which increases [Na⁺]_i, under isotonic or anisotonic conditions, revealed that Na,K-ATPase recruitment to the cell surface was not directly related to variations of cell volume and osmolarity. The effects of amphotericin B and extracellular hypotonicity were not additive, and both were prevented by protein kinase A and proteasome inhibitors, suggesting a common mechanism of action. In line with this hypothesis, extracellular hypotonicity induced a sustained stimulation of the amiloride-sensitive short-circuit current, indicating increased Na⁺ influx through the apical epithelial Na⁺ channel. Moreover, inhibiting apical Na⁺ entry by amiloride, a blocker of epithelial Na⁺ channel, or incubating cells in Na⁺-free medium prevented the cell surface recruitment of Na,K-ATPase in response to extracellular hypotonicity. Altogether, these findings strongly suggest that extracellular hypotonicity stimulates apical Na⁺ influx leading to increased [Na⁺]_i, protein kinase A activation, and recruitment of Na,K-ATPase units to the cell surface of mpkCCD_cl4 cells.

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Copyright © 2008 by the American Society of Nephrology. Online ISSN: 1533-3450 Print ISSN: 1046-6673