Characterization of a mouse cortical collecting duct cell line

Abstract

A cortical collecting duct (CCD) cell line has been developed from a mouse transgenic for the early region of simian virus 40, Tg(SV40E)Bri/7. CCDs were microdissected and placed on collagen gels. Monolayers were subsequently subcultured onto permeable collagen membranes and maintained in serum-supplemented medium. One line, designated M-1, retained many characteristics of the CCD, including a typical epithelial appearance and CCD-specific antigens. M-1 cells, when grown in monolayers on permeable supports, exhibited a high transepithelial resistance (885.7 ± 109.6 ohms/cm2) and developed a lumen negative transepithelial potential difference (PD) of -45.7 ± 3.5 mV. The associated short-circuit current (SCC) averaged 71.8 ± 10.3 μA/ cm2, and was reduced by 95% by luminal application of amiloride. The cultured cells responded to arginine vasopressin (AVP) with a significant increase in SCC. M-1 cells generated significant transepithelial solute gradients. After 24 hours incubation, the composition of the luminal (L) and basolateral (B) media (in mM) was: [Na+], L = 106.7 ± 0.9 and B = 127.4 ± 0.4; [K+], L = 8.6 ± 0.6 and B = 2.1 ± 0.3; [Cl], L = 68.6 ± 5.8 and B = 101.8 ± 6.6; [HCO3], L = 15.5 ± 1.5 and B = 8.6 ± 1.2; while pH was 7.16 ± 0.03 at the luminal and 6.94 ± 0.03 at the basolateral side. The formation of these concentration gradients indicates that the CCD cultures absorb Na+ and Cl- and secrete K+. Lactate accumulated predominantly at the basolateral side (L = 7.1 ± 0.44 and B = 17.5 ± 0.52 mM); osmotic concentration was 272 ± 1.4 at the luminal and 290 ± 3.0 mOsm/kg at the basolateral side. These data demonstrate that the M-1 cell line retains many phenotypic properties of the CCD epithelium and thus should prove useful as a model in studying mechanisms of ion transport in this segment.