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 ammonium/ammonia 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 NH(4)(+) transport, and the intracellular accumulation of (14)C-methylammonium/methylammonia ((14)C-MA) was determined. In CO(2)/HCO(3)(-)-free solution, cells at normal pH briefly accumulated (14)C-MA over 7 min and reached a plateau. In CO(2)/HCO(3)(-) solution, however, cells markedly accumulated (14)C-MA over the experimental period of 30 min. This CO(2)/HCO(3)(-)-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 NaHCO(3) in 5% CO(2)) 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 (14)C-MA accumulation in control conditions but had no effect after acidic incubation. Immunoblotting showed that NBCn1 was upregulated after acidic incubation and in NH(4)Cl-containing media. The Cl(-)-HCO(3)(-) exchanger AE2 was present, but its expression remained unaffected by acidic incubation. Expressed in Xenopus oocytes, NBCn1 increased carrier-mediated (14)C-MA transport, which was abolished by replacing Na(+). Two-electrode voltage clamp of oocytes exhibited negligible current after NH(4)Cl application. These results suggest that DIDS-sensitive HCO(3)(-) extrusion normally governs NH(4)(+)/NH(3) uptake in the medullary thick ascending limb cells. We propose that, in acidic conditions, DIDS-sensitive HCO(3)(-) extrusion is inactivated, while NBCn1 is upregulated to stimulate NH(4)(+) transport.

Fig. 1

¹⁴C-MA accumulation in cells. A) ¹⁴C-MA accumulation in ${\mathrm{CO}}_2∕{\mathrm{HCO}}_3^{-}\text{-free}$ solution (pH 7.4). Cells grown on a 24-well plate were treated with ${\mathrm{CO}}_2∕{\mathrm{HCO}}_3^{-}\text{-free}$ ¹⁴C-MA assay solution. Cells were then rapidly rinsed with ice-cold wash solution containing unlabeled MA at different time points. The ¹⁴C-MA assay solution in this and subsequent experiments with ST-1 cells contained 0.2 mM bumetanide and 1 mM ouabain to minimize carrier-mediated CH₃-NH₃⁺ transport. Experiments were done in the presence or absence of 0.5 mM 4,4’-diisothiocyanatostilbene-2,2’-disulfonate (DIDS). The accumulation was presented as pmole/μg total protein. Data were averaged from 3 experiments. B) ¹⁴C-MA accumulation in ${\mathrm{CO}}_2∕{\mathrm{HCO}}_3^{-}$ solution. The experimental procedure was identical to that in A except that the assay solution contained 5% CO₂, 33 mM ${\mathrm{HCO}}_3^{-}$ (pH 7.4). The dotted line was derived from the line in A. Data were averaged from 3 experiments.

Fig. 2

Effects of Cl^– & Na⁺ replacement and K⁺ concentration on ¹⁴C-MA accumulation. All assay solutions contained 5% CO₂, 33 mM ${\mathrm{HCO}}_3^{-}$ (pH 7.4). A) Effect of Cl^– replacement. Cl^– in the ¹⁴C-MA assay solution was replaced by gluconate in the absence or presence of 0.5 mM DIDS (n = 4 for each). B) Effect of raising K⁺ concentration. The K⁺ concentration in the assay solution was raised from 2 mM to 20 mM (n = 4). C) Effect of Na⁺ replacement. Na⁺ in the assay solution was replaced by NMDG⁺ (n = 4).

Fig. 3

${\mathrm{CO}}_2∕{\mathrm{HCO}}_3^{-}\text{-induced}$ stimulation of the pH_i recovery from an alkali load. A) pH_i recovery from an alkali load in the absence of ${\mathrm{CO}}_2∕{\mathrm{HCO}}_3^{-}$. Cells were treated with 20 mM NH₄Cl, and 0.5 mM DIDS was applied during the pH_i recovery from an alkali load (n =5). B) pH_i recovery from an alkali load in the presence of 5%CO₂, 33 mM ${\mathrm{HCO}}_3^{-}$. Cells were subjected to the protocol as described in A except that the experiment was done in the presence of ${\mathrm{CO}}_2∕{\mathrm{HCO}}_3^{-}$ (n =5). C) Summary of the pH_i recovery rate. Data were obtained from the experiments in A and B.

Fig. 4

¹⁴C-MA accumulation in cells after acidic incubation. A) Effect of DIDS on ¹⁴C-MA accumulation. Cells were treated with ¹⁴C-MA assay solution in the absence and presence of 5% CO₂, 33 mM ${\mathrm{HCO}}_3^{-}$ (pH 7.4) for 30 min. The concentration of DIDS was 0.5 mM. The p values for comparison (a–d) are described in the text. Data were averaged from 3 experiments. B) Effect of acidic pH_i on ¹⁴C-MA accumulation. Cells were incubated in the medium of pH 7.4 or 6.8 for 24 hours. Experiments were done by treating cells with ${\mathrm{CO}}_2∕{\mathrm{HCO}}_3^{-}\text{-buffered}$ ¹⁴C-MA assay solution (pH 7.4) for 20 min. Data were averaged from 5 experiments. C) Steady-state pH_i. Cells were incubated in the medium of pH 7.4 or 6.8 for 24 h, and their pH_i was measured in ${\mathrm{CO}}_2∕{\mathrm{HCO}}_3^{-}$ at the corresponding pH (n = 4 for each). The measurements were done using the ratiometric pH-sensitive dye BCECF-AM.

Fig. 5

Effect of acidic incubation on NBCn1 expression. A) Immunoblot for NBCn1. Cells were incubated in the medium of pH 7.4 versus 6.8 for 0 or 24 h. Plasma membranes were isolated and subjected to immunoblot with the anti-NBCn1 antibody. The blots were striped and reprobed with the β-actin antibody. One of three experiments is shown. B) Quantitative measurements of NBCn1. The pixel intensity of NBCn1 was measured and normalized to β-actin (n = 3). The p value less than 0.05 was considered significant. C) Immunoblot for NBCn1 at normal and acidic incubation for 24, 48, and 72 hours. One of three experiments is shown. D) Quantitative measurements of NBCn1. The value of NBCn1/β-actin at pH 6.8 was presented relative to the value at pH 7.4 at each time point. The data were analyzed by one-way ANOVA with Bonferroni post-test. *p value of less than 0.05. E) NBCn1 expression in cells treated with NH₄Cl. Cells were incubated in the medium (pH 7.4) containing 0 or 10 mM NH₄Cl for 24 h. NH₄Cl replaced NaCl. One of three experiments is shown. F) Quantitative measurements of NBCn1.

Fig. 6

Effect of acidic incubation on AE2 expression. A) Immunoblot for AE2. The experimental procedure was similar to that in Fig. 5 except the blot was probed with the anti-AE2 antibody. One of four experiments is shown. B) Quantitative measurements of AE2. The pixel intensity of AE2 was measured and normalized to β-actin.

Fig. 7

Time course of ¹⁴C-MA accumulation in Xenopus oocytes expressing NBCn1. A & B) ¹⁴C-MA accumulation in oocytes injected with water or NBCn1-E cRNA. Oocytes were incubated with the ¹⁴C-MA assay solution (no bumetanide and ouabain) in the absence (A) or presence (B) of ${\mathrm{CO}}_2∕{\mathrm{HCO}}_3^{-}$ (pH 7.4), and then rapidly washed with ice-cold unlabeled MA solution at different time points. The accumulation was expressed as nmole/oocyte. Data were averaged from 4 independent experiments with total 16 oocytes per group. C) ${\mathrm{CO}}_2∕{\mathrm{HCO}}_3^{-}\text{-dependent}$ ¹⁴C-MA accumulation. The accumulation was calculated by subtracting values in ${\mathrm{CO}}_2∕{\mathrm{HCO}}_3^{-}\text{-free}$ ND96 solution from values in ${\mathrm{CO}}_2∕{\mathrm{HCO}}_3^{-}\text{-containing}$ solution in A & B. D) Effect of Na⁺ replacement on ¹⁴C-MA accumulation. Na⁺ was replaced with NMDG⁺ (n = 3 for each).

Fig. 8

Two-electrode voltage clamp of oocytes. A & B) Current-voltage relationships for currents before and after NH₄Cl application. Oocytes were clamped at -60 mV and voltage commands from -120 to +40 mV were applied. Recordings were made in ${\mathrm{CO}}_2∕{{\mathrm{HCO}}^{-}}_3\text{-free}$ ND96 solution and 1 min after switching to a solution containing 20 mM NH₄Cl. Data were averaged from 6 controls and 8 NBCn1-E-expressing oocytes. C) Slope conductance. The conductance was computed near the zero-current potential in A & B.