Differentiated transport functions in primary cultures of rabbit collecting ducts

Fragments of rabbit cortical collecting tubule (CCT) were isolated by solid-phase immunoadsorption with a monoclonal antibody against an ectoantigen on principal cells. Primary cultures, initiated from the "immunodissected" cells, were then grown to confluency in porous bottom dishes. The purity of the freshly isolated and cultured cells was verified by flow cytometry and immunocytochemistry, using a panel of cell-specific monoclonal antibodies and lectins. As a result of continued vectorial transport, the cultured epithelial monolayers generated pronounced transepithelial solute gradients. The composition of the luminal (L) and basolateral (B) media after 48 h of incubation was as follows: [Na], L 119 +/- 1.1, B 145 +/- 3.0; [K], L 11.9 +/- 0.5, B 2.0 +/- 0.3; [PO4], L 2.8 +/- 0.2, B 7.3 +/- 0.3; [Cl], L 134.1 +/- 2.7 B 117.8 +/- 2.2, [mM]; [Ca], L 42.1 +/- 1.2, B 812 +/- 26, [microM]; pH, L 5.52 +/- 0.12, B 7.15 +/- 0.08. The transepithelial osmotic concentration difference was 14.3 +/- 3.5 mosmol/l. The amount of glucose at the luminal side remained unaltered, whereas glucose concentration at the basolateral side decreased from 21 to 7.7 +/- 1.0 mM. Lactic acid accumulated predominantly at the basolateral side (L 1.0 +/- 0.15, B 23.5 +/- 1.7 mM). These data indicate that primary cultures of immunodissected CCT cells retain many of their differentiated transport functions and thus should prove useful for studying the mechanisms of solute transport in the collecting duct system.