NHE2-mediated bicarbonate reabsorption in the distal tubule of NHE3 null mice

Abstract

NHE3(-/-) mice display a profound defect in proximal tubule bicarbonate reabsorption but are only mildly acidotic owing to reduced glomerular filtration rate and enhanced H(+) secretion in distal nephron segments. In vivo microperfusion of rat distal tubules suggests that a significant fraction of bicarbonate reabsorption in this nephron segment is mediated by NHE2. Two approaches were used to evaluate the role of distal tubule NHE2 in compensating for the proximal defect of H(+) secretion in NHE3(-/-) mice. First, renal clearance experiments were used to assess the impact of HOE694, an inhibitor with significant affinity for NHE2, on excretion of bicarbonate in NHE3(-/-) and NHE2(-/-) mice. Second, in vivo micropuncture and microperfusion were employed to measure the concentration of bicarbonate in early distal tubule fluid and to measure distal bicarbonate reabsorption during a constant bicarbonate load. Our data show that HOE694 had no effect on urinary bicarbonate excretion in NHE3(+/+) mice, whereas bicarbonate excretion was higher in NHE3(-/-) mice receiving HOE694. HOE694 induced a significant increase in bicarbonate excretion in mice given an acute bicarbonate load, but there was no effect during metabolic acidosis. Bicarbonate excretion was not affected by HOE694 in bicarbonate-loaded NHE2(-/-) mice. In vivo micropuncture revealed that early distal bicarbonate concentration was elevated in both bicarbonate-loaded and NHE3(-/-) mice. Further, microperfusion experiments showed that HOE694-sensitive bicarbonate reabsorption capacity was higher in acidotic and NHE3 null animals. We conclude that NHE2 contributes importantly to acidification in the distal tubule, and that it plays a major role in limiting urinary bicarbonate losses in states in which a high luminal bicarbonate load is presented to the distal tubule, such as in NHE3 null mice.

Figure 1. Infusion protocols for renal clearance studies

All concentrations expressed in mmol l⁻¹. Infusions were maintained for 3 h. After a 60 min equilibration period, baseline measurements were made during the control period followed by infusion of either the NHE2 inhibitor HOE694 or the time control vehicle (1% DMSO) during the experimental period.

Figure 2. Urinary sodium excretion (A) and bicarbonate excretion (B) in NHE3^−/− (squares, n = 13) and NHE3^+/+ mice (circles, n = 12) during infusion of either the NHE2 inhibitor HOE694 (filled symbols) or vehicle (open symbols)

The drug had no significant effect in NHE3^+/+ mice. Two-way ANOVA indicated a significant effect of HOE694 for both sodium and bicarbonate excretion in NHE3^−/− mice (see text). Point-to-point comparisons were made using Bonferroni's post hoc test; *P < 0.05, **P < 0.01.

Figure 3. Sodium and bicarbonate excretion in bicarbonate-loaded mice (A) (triangles; n = 7 in each group) and acidotic mice (B) (diamonds; n = 5 for HOE694 and 4 for time controls) during infusion of either the NHE2 inhibitor HOE694 (filled symbols) or vehicle (open symbols)

Two-way ANOVA indicated a significant effect of HOE694 on both variables in bicarbonate-loaded mice only (see text). Point-to-point comparisons were made using Bonferroni's post hoc test; **P < 0.01.

Figure 4. Sodium and bicarbonate excretion in bicarbonate-loaded NHE2^+/+ mice (open squares, n = 5) and NHE2^−/− mice (filled squares, n = 6) during infusion of the NHE2 inhibitor HOE694

Point-to-point comparisons between groups were made using Bonferroni's post hoc test; *P < 0.05, **P < 0.01.

Figure 5. The effect of HOE694 on renal potassium excretion in four groups of mice. Infusion of the NHE2 inhibitor (black symbols) increased potassium excretion compared to time controls (open symbols) in NHE3^−/− mice and bicarbonate-loaded animals

Point-to-point comparisons were made using Bonferroni's post hoc test; *P < 0.05. Data are means ± s.e.m.

Figure 6. Bicarbonate concentration in the early distal tubule of control (n = 18 tubules/5 mice), chronically acidotic mice (n = 14/7), bicarbonate loaded (n = 8/3) or NHE3^−/− (n = 9/2) mice

The Kruskal–Wallis test indicated a significant difference between the 4 groups (P < 0.001). Point-to-point comparisons were made using Dunn's post hoc test; *P < 0.05, **P < 0.01 versus control. Data are means ± s.e.m.

Figure 7. Bicarbonate reabsorption during perfusion of the early distal tubule with a control perfusate (25 mm HCO₃) in either control (n = 11 tubules/5 mice), chronically acidotic (n = 14 /7) or NHE3^−/− (n = 6/2) mice

The Kruskal–Wallis test indicated a significant difference between the 3 groups (P < 0.01). Point-to-point comparisons were made using Dunn's post hoc test; *P < 0.05, **P < 0.01versus control. Data are means ± s.e.m.

Figure 8. Bicarbonate reabsorption during perfusion of the early distal tubule with a control perfusate (25 mm HCO₃⁻) and the same solution to which HOE694 (100 μmol l⁻¹) had been added in either control (n = 11 tubules/4 mice), chronically acidotic (n = 14/7) or NHE3^−/− (n = 6/2) mice

The drug evoked a significant inhibition of bicarbonate reabsorption in each of the 3 groups, as indicated. Comparisons were made using Student's paired t test.

Figure 9. The net effect of HOE694 (100 μmol l⁻¹) on early distal bicarbonate reabsorption in either control (n = 11 tubules/5 mice), chronically acidotic (n = 14/7) or NHE3^−/− (n = 6/2) mice

The Kruskal–Wallis test indicated a significant difference between the 3 groups (P < 0.01). Point-to-point comparisons were made using Dunn's post hoc test; *P < 0.05, **P < 0.01 versus control. Data are means ± s.e.m.