Basolateral Na+-dependent HCO3- transporter NBCn1-mediated HCO3- influx in rat medullary thick ascending limb

Abstract

The electroneutral Na+-dependent HCO3- transporter NBCn1 is strongly expressed in the basolateral membrane of rat medullary thick ascending limb cells (mTAL) and is up-regulated during NH4+-induced metabolic acidosis. Here we used in vitro perfusion and BCECF video-imaging of mTAL tubules to investigate functional localization and regulation of Na+-dependent HCO3- influx during NH4+-induced metabolic acidosis. Tubule acidification was induced by removing luminal Na+ (ΔpHi: 0.88 ± 0.11 pH units, n = 10). Subsequently the basolateral perfusion solution was changed to CO2/HCO3- buffer with and without Na+. Basolateral Na+-H+ exchange function was inhibited with amiloride. Na+-dependent HCO3- influx was determined by calculating initial base flux of Na+-mediated re-alkalinization. In untreated animals base flux was 8.4 ± 0.9 pmol min-1 mm-1. A 2.4-fold increase of base flux to 21.8 ± 3.2 pmol min-1 mm-1 was measured in NH4+-treated animals (11 days, n = 11). Na+-dependent re-alkalinization was significantly larger when compared to control animals (0.38 ± 0.03 versus 0.22 ± 0.02 pH units, n = 10). In addition, Na+-dependent HCO3- influx was of similar magnitude in chloride-free medium and also up-regulated after NH4+ loading. Na+-dependent HCO3- influx was not inhibited by 400 μm DIDS. A strong up-regulation of NBCn1 staining was confirmed in immunolabelling experiments. RT-PCR analysis revealed no evidence for the Na+-dependent HCO3- transporter NBC4 or the two Na+-dependent CI-/HCO3- exchangers NCBE and NDCBE. These data strongly indicate that rat mTAL tubules functionally express basolateral DIDS-insensitive NBCn1. Function and protein are strongly up-regulated during NH4+-induced metabolic acidosis. We suggest that NBCn1-mediated basolateral HCO3- influx is important for basolateral NH3 exit and thus NH4+ excretion by means of setting pHi to a more alkaline value. © The Physiological Society 2003.