Hyperosmolality stimulates Na-K-ATPase gene expression in inner medullary collecting duct cells

Primary cultures of inner medullary collecting duct (IMCD) cells of rats were incubated in hyperosmotic media to determine the effects on Na-K-ATPase alpha(1)- and beta(1)-subunit mRNA expression. Osmolality of the incubation media was raised from 300 up to 500 mosmol/kgH(2)O by adding NaCl, mannitol, raffinose, or urea. Hyperosmotic media supplemented with NaCl, mannitol, or raffinose caused two- to fourfold increases in the alpha(1)-subunit mRNA accumulation and five- to eightfold increases in the beta(1)-subunit mRNA accumulation, with peak elevations of both subunits at 12 h after addition. In sharp contrast, hyperosmolar urea medium had no effect at any time. When NaCl or mannitol was added to the media in amounts ranging from 300 to 600 mosmol/kgH(2)O, the maximal effects on both alpha(1)- and beta(1)-subunit mRNA accumulation occurred at 500 mosmol/kgH(2)O. In urea-supplemented medium, however, there was no significant change at any level of osmolality. The upregulation of alpha(1)- and beta(1)-subunit mRNA induced by hyperosmotic mannitol- or raffinose-supplemented media was markedly inhibited by removal of Na from the culture medium. Furthermore, pretreatment with a protein synthesis inhibitor cycloheximide partially inhibited the upregulation of alpha(1)- and beta(1)-subunit mRNA in IMCD cells exposed to hyperosmotic media treated with NaCl or mannitol. When IMCD cells were incubated with hyperosmotic media (500 mosmol/kgH(2)O) supplemented with NaCl or mannitol for 24 h, Na-K-ATPase activity increased by 78.6 and 82.8%, respectively. In contrast, hyperosmolar urea medium had no significant effect on Na-K-ATPase activity. These results demonstrate that 1) hyperosmolality induced by the poorly permeating solutes (NaCl, mannitol, and raffinose) but not the rapidly permeating solute (urea) stimulates both alpha(1)- and beta(1)-subunit mRNA accumulations in IMCD cells in a time- and an osmolality-dependent manner, 2) the hyperosmolality-induced upregulation of alpha(1) and beta(1)-subunit mRNA leads to an increase in Na-K-ATPase activity; and 3) the above upregulation of alpha(1)- and beta(1)-subunit mRNA in response to hyperosmotic media requires, at least in part, the presence of Na in the extracellular medium and the de novo synthesis of intermediate proteins.