Intracellular Na+ directly modulates Na+,K+-ATPase gene expression in normal rat kidney epithelial cells

Abstract

Background. In a wide variety of cell systems, increases in cell Na+ ([Na+](i)) lead to an induction of N+,K+-ATPase mRNA expression. On the other hand, the increase in [Na+](i) can also induce a rise in cell Ca2+ ([Ca2+](i)) through a secondary inhibition of Na+/Ca2+ exchange and a decrease in cell pH (pH(i)) through a secondary inhibition of Na+/H+ exchange. It is not known whether [Na+](i), [Ca2+](i), and/or pH(i) directly modulate N+,K+-ATPase mRNA expression. Methods. We used normal rat kidney epithelial cells (NRK) to examine the effects of ouabain on N+,K+- ATPase α1- and β1-mRNA accumulation by Northern blot analysis and the relationship between the mRNA accumulation and [Na+](i), [Ca2+](i), or pH(i). [Na+](i), [Ca2+](i), and pH(i) were measured using a Na+-sensitive fluorescent dye (SBFI), a Ca2+ -sensitive fluorescent dye (Fura-2), and a pH-sensitive fluorescent dye (BCECF), respectively. Results. Ouabain (1 mmol/L) significantly increased [Na+](i). Upon addition of ouabain, α1-mRNA levels increased to 2.3 times the control level at three hours, with maximum 3.3-fold elevations at 12 hours. β1-mRNA levels also increased to 2.4 times the control level at 3 hours, with a maximum 3.3-fold increase at 12 hours. The ouabain-mediated α1- and β1-mRNA induction was inhibited by both the RNA transcription inhibitor (actinomycin D) and the protein synthesis inhibitor (cycloheximide). Ouabain at three hours caused an increase in [Ca2+](i). Similar increases in [Ca2+](i), which were elicited by the Ca2+ ionophore (ionomycin) in the presence of extracellular Ca2+, had no effect on α1- or β1-mRNA levels. In Ca2+-free medium treated with EGTA, ouabain at three hours caused a significant increase in [Na+](i) without any changes in [Ca2+](i), and also increased α1- and β1-mRNA levels. Ouabain at three hours caused a significant decrease in pH(i). Similar decreases in pH(i), which were elicited by the specific inhibitor of Na+/H+ exchange (ethylisopropylamiloride), caused no effect on α1- or β1-mRNA levels. Exposure of NRK to the Na+ ionophore (monensin) in the absence of extracellular Ca2+ increased [Na+](i) and α1- and β1-mRNA levels. The increases in α1- and β1-mRNA levels upon addition of ouabain were associated with significant increases in α1- and β1-subunit proteins. Conclusions. In NRK, ouabain causes an increase in [Na+](i), which directly modulates Na+,K+-ATPase α1- and β1-mRNA accumulation.