Differentiation of proton-pumping activity in cultured renal inner medullary collecting duct cells

Cultured inner medullary collecting duct (IMCD) cells have been shown to secrete protons (H⁺) by two mechanisms: an N-ethylmaleimide-and dicyclohexyl-carbodiimide-sensitive electrogenic H⁺-ATPase or H⁺ pump, and an amiloride-sensitive, secondary active Na⁺/H⁺ exchanger. These cells also express Cl^-/HCO₃^- exchange and carbonic anhydrase activity in common with other renal epithelial cells involved in acid-base transport. Video fluorescence microscopy of individual cells using 2′, 7′-biscarboxyethyl-5(6)-carboxyfluorescein has demonstrated that adjacent-cultured IMCD cells show substantial functional intercellular heterogeneity. The development of H⁺-pumping activity is associated with high-baseline intracellular pH and peanut agglutinin (PNA) affinity, and loss of mitotic activity and of Na⁺/H⁺ exchange. The H⁺-pumping activity may be further enhanced by removal of fetal calf serum for 6-54 h or by selecting cells with high PNA affinity. IMCD cells in their most differentiated state form domes, which consistently showed the highest rates of H⁺-pumping activity, as well as high affinity for peanut lectin. When IMCD were plated at low density, domes developed relatively late (2-4 weeks), at which time cells located in the center of nests of contiguously growing cells were quiescent and showed H⁺-pumping activity but no Na⁺/H⁺ exchange. On the other hand, dense plating was associated with early development of domes (end of 1st week), at which time adjacent cells showed a high mitotic activity and Na⁺/H⁺ exchange, but no H⁺-pumping activity. We speculate that differentiation of IMCD cells results in the development of cell polarity. This could include either loss of the apical Na⁺/H⁺-exchange activity, or localization of this exchanger only to the basolateral membrane, while the H⁺ pump differentiates at the apical membrane.