Development of H⁺ secretion by cultured renal inner medullary collecting duct cells

The purpose of these studies was to characterize the development of H⁺ secretion by cultured inner medullary collecting duct (IMCD) cells. Differentiated IMCD cells derived from rat papillae develop in culture N-ethylmaleimide (NEM)- and dicyclohexylcarbodiimide (DCCD)-sensitive Na⁺-independent H⁺ pump, but showed substantial heterogeneity. Cells with pH(i) ≥ 7.1, measured by 2',7'-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF), showed more H⁺ pumping than did those with pH(i) < 7.1. Quiescent cells in domes and in the center of growing nests often showed active H⁺ pumping activity, but rarely exhibited Na⁺-H⁺ exchange, whereas dividing cells at the periphery of the nests showed Na⁺-independent 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS)-sensitive Cl^--HCO₃^- exchange that functioned best when pH(i) was ≥ 7.1, and homogenates of IMCD cells expressed carbonic anhydrase (CA) activity and CA II mRNA. A subgroup of IMCD cells avidly bound fluorescent peanut agglutinin (PNA); this binding became more extensive with time in culture. The PNA-labeled cells showed increased BCECF uptake, higher pH(i), and more H⁺ pumping activity than did cells not binding PNA. They could be isolated by gel-immobilized PNA and in culture showed more CA activity and H⁺ pumping activity than unselected monolayers. The percentage of cells binding PNA correlated well with CA activity, indicating that this subgroup is better specialized to secrete H⁺. The appearance of PNA binding sites and the development of high CA activity may be characteristic of IMCD cell differentiation in culture.