13 Effect of Erythropoietin on Fluid Movement Across the Respiratory Epithelium.

Abstract

Erythropoietin has a wide range of nonhematopoietic effects in the body, including the regulation of certain enzymes in the lung. Clinically, its administration has been associated with a decrease in the number of days in oxygen for sick premature infants. If erythropoietin has a favorable effect on respiratory illness, one mechanism could be a reduction in pulmonary edema. In previous studies, we demonstrated that erythropoietin increases cellular uptake of sodium, a necessary first step in ion transport-linked fluid clearance (Pediatr Res 58:817A, 2005). To further characterize the effect on ion transport, we studied the effect of erythropoietin on the Na-K-ATPase, an enzyme important in sodium translocation. To accomplish this goal, we treated A549 cells, an immortal adenocarcinoma cell line with features of Type II cells, with erythropoietin and measured the uptake of 86Rb+ (a mimic of K+) in the presence and absence of ouabain, an inhibitor of Na-K-ATPase. We found that after 24h, erythropoietin (35 U/ml) increased ouabain-sensitive Rb uptake on average by 25% (n=8, control: 1.6 ± 0.6 vs erythropoietin: 2.0 ± 0.8 nmol Rb/million cells/min, p<0.05), indicating an increase in Na-K-ATPase activity. To confirm this finding, we disrupted the cell membrane and assessed Na-K-ATPase activity by an alternative method in which we measured the release of inorganic phosphate in the presence and absence of ouabain. Similar to our results in intact cells, erythropoietin (24h, 35 U/ml) increased the activity of the Na-K-ATPase (n=7, control: 0.9 ± 0.4 vs erythropoietin: 2.1 ± 1.3 mg phosphate/mg protein, p<0.05). In additional studies, we grew A549 cells on a semipermeable membrane and measured fluid movement across the monolayer by measuring the change in protein concentration in the supernatant as a reflection of fluid loss. After 24 h, erythropoietin increased by nearly 25% the volume of fluid loss from the supernatant (n=6, control: 132 ± 26 vs erythropoietin: 164 ± 34 uL/24 h, p<0.05). We then added amiloride (1mM), an inhibitor of Na channels important in the transepithelial movement of liquid, to monolayers treated with erythropoietin and found that fluid loss from the supernatant decreased to that of control cells (n=6, control: 126 ± 26 uL/24 h). Our findings show that erythropoietin increases Na-K-ATPase activity and transepithelial liquid movement in vitro. We speculate that if erythropoietin were to increase sodium transport and Na-K-ATPase activity in vivo, then lung water balance and respiratory difficulty might improve as well.