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Up‐regulation of apical NHE3 in renal OK cells overexpressing the rodent alpha1‐subunit of the Na+ pump

Vectorial Na+ reabsorption across the proximal tubule is mediated by apical entry of Na+, primarily via Na+/H+ exchanger isoform 3 (NHE3), and basolateral extrusion via the Na+ pump (Na+‐K+‐ATPase). We hypothesized that regulation of Na+ reabsorption should involve not only the activity of the basolateral Na+‐K+‐ATPase but also the apical NHE3, in a concerted manner. In order to generate a cell line that overexpresses Na+‐K+‐ATPase, OK cells were transfected with the rodent Na+‐K+‐ATPase alpha1‐subunit (pCMV ouabain vector), and native cells used as a control. The existence of distinct functional classes of Na+‐K+‐ATPase in wild type and transfected cells was confirmed by the inhibition profile of Na+‐K+‐ATPase activity by ouabain. In contrast to wild type cells, transfected cells exhibited two IC50s for ouabain. The first was similar to the IC50 of control cells; the second IC50 was 2 logs greater than the first, consistent with the presence of both rat and opossum alpha1 isozymes. It is shown that transfection of OK cells with Na+‐K+‐ATPase increased both Na+‐K+‐ATPase and NHE3 activities. This was associated with overexpression of the Na+‐K+‐ATPase alpha1‐subunit and NHE3 in transfected OK cells. The abundance of the Na+‐K+‐ATPase beta1‐subunit was slightly lower in transfected OK cells. In conclusion, the increase in both expression and function of Na+‐K+‐ATPase found in cells transfected with the rodent Na+ pump alpha1 cDNA is expected to stimulate apical Na+ influx into the cells, thereby accounting for the observed stimulation of the apical NHE3 activity.