Sodium-bicarbonate cotransporter NBCn1 in the kidney medullary thick ascending limb cell line is upregulated under acidic conditions and enhances ammonium transport

Abstract

In this study, we examined the effect of bicarbonate transporters on ammoniumammonia uptake in the medullary thick ascending limb cell line ST-1. Cells were treated with 1 mm ouabain and 0.2 mm bumetanide to minimize carrier-mediated NH4+ transport, and the intracellular accumulation of 14C-methylammoniummethylammonia (14C-MA) was determined. In CO2HCO3--free solution, cells at normal pH briefly accumulated 14C-MA over 7 min and reached a plateau. In CO2HCO3- solution, however, cells markedly accumulated 14C-MA over the experimental period of 30 min. This CO2HCO3--dependent accumulation was reduced by the bicarbonate transporter blocker, 4,4′- diisothiocyanatostilbene-2,2′-disulfonate (DIDS; 0.5 mm). Replacing Cl- with gluconate reduced the accumulation, but the reduction was more substantial in the presence of DIDS. Incubation of cells at pH 6.8 (adjusted with NaHCO3 in 5% CO2) for 24 h lowered the mean steady-state intracellular pH to 6.96, significantly lower than 7.28 for control cells. The presence of DIDS reduced 14C-MA accumulation in control conditions but had no effect after acidic incubation. Immunoblotting showed that NBCn1 was upregulated after acidic incubation and in NH 4Cl-containing media. The Cl--HCO3- exchanger AE2 was present, but its expression remained unaffected by acidic incubation. Expressed in Xenopus oocytes, NBCn1 increased carrier-mediated 14C-MA transport, which was abolished by replacing Na+. Two-electrode voltage clamp of oocytes exhibited negligible current after NH4Cl application. These results suggest that DIDS-sensitive HCO 3- extrusion normally governs NH4+NH3 uptake in the medullary thick ascending limb cells. We propose that, in acidic conditions, DIDS-sensitive HCO3- extrusion is inactivated, while NBCn1 is upregulated to stimulate NH4+ transport. © 2010 The Physiological Society.