Salt restriction induces pseudohypoaldosteronism type 1 in mice expressing low levels of the beta-subunit of the amiloride-sensitive epithelial sodium channel

Abstract

The amiloride-sensitive epithelial sodium channel (ENaC) is a heteromultimer of three homologous subunits (alpha-, beta-, and gamma-subunits). To study the role of the beta-subunit in vivo, we analyzed mice in which the betaENaC gene locus was disrupted. These mice showed low levels of betaENaC mRNA expression in kidney (approximately 1%), lung (approximately 1%), and colon (approximately 4%). In homozygous mutant betaENaC mice, no betaENaC protein could be detected with immunofluorescent staining. At birth, there was a small delay in lung-liquid clearance that paralleled diminished amiloride-sensitive Na+ absorption in tracheal explants. With normal salt intake, these mice showed a normal growth rate. However, in vivo, adult betaENaC m/m mice exhibited a significantly reduced ENaC activity in colon and elevated plasma aldosterone levels, suggesting hypovolemia and pseudohypoaldosteronism type 1. This phenotype was clinically silent, as betaENaC m/m mice showed no weight loss, normal plasma Na+ and K+ concentrations, normal blood pressure, and a compensated metabolic acidosis. On low-salt diets, betaENaC-mutant mice developed clinical symptoms of an acute pseudohypoaldosteronism type 1 (weight loss, hyperkalemia, and decreased blood pressure), indicating that betaENaC is required for Na+ conservation during salt deprivation.

Figure 1

Gene targeting strategy for the disruption of the mouse βENaC gene locus. (A) Structure of the wild-type βENaC gene, the targeting vector, and the predicted targeted locus. The “R566Stop” mutation is indicated (asterisk). Expected fragment sizes of the wild-type and mutated βENaC allele after digestion with HincII and hybridization with probe A are indicated. H2 = HincII; H3 = HindIII; K1 = KpnI. The precise exon–intron structure has not been determined. Identified exons are indicated (shaded box). (B) Southern blot analysis of offspring from the breeding of chimeras. Tail DNA was digested with HincII and hybridized with a 5′ flanking probe (probe A). The βENaC genotype is indicated above each lane. Expected fragment sizes of wild-type (5-kb) and mutant (7-kb) allele are indicated. (C) Northern blot analysis of βENaC RNA transcripts from βENaC +/+, m/+, and m/m mice kept on low-salt diets (1 g/kg Na⁺). Total RNA (15 μg) from colon, kidney, and lung of each four independent animals was hybridized with βENaC-specific ³²P-labeled probes.

Figure 2

Immunolocalization of βENaC protein in kidney (A–F) and lung (G–J) of βENaC +/+ and βENaC m/m mice by using C-terminal antibody (A and B) and ectodomain antibody (C–J). (Left) Immunofluorescence. (Right) Corresponding Normarski images. In cortical collecting ducts (CD) from wild-type mice (A–D), immunofluorescence was present in the cytoplasm of principal cells, whereas intercalated cells (arrowheads) were unreactive. Cells of proximal convoluted tubules and glomeruli (G) were unstained. In contrast, no staining was detected in kidney of βENaC m/m mice (E and F). In lung of wild-type mice (G and H), signal was present in bronchioles (B) and terminal bronchioles (TB). No specific immunostaining was present in lung from the βENaC m/m mice (I and J). The faint, nonspecific fluorescence in I is nonepithelial and associated with basement membrane. (Scale bar = 25 μm.)

Figure 3

Liquid absorption in lung explants. (A) Mean whole-lung water content (wet/dry weight ratio) in βENaC m/m mice (closed triangles) and βENaC +/+ mice (open circles). ∗, P < 0.05. (B) Liquid absorptive capacity measured as percentage of cysts that remained inflated after stimulation by 10⁻⁵ M cAMP in explants from βENaC m/m (black bars) and +/+ mice (white bars) at E16–E18. (C and D) Short-circuit currents (ΔI_sc) in tracheal explants from newborn (C) and adult (D) mice. βENaC +/+ (white bars); βENaC m/m (black bars). Treatment with 10⁻⁴ M amiloride (amil), 3 × 10⁻⁴ M terbutaline (terb), and 10⁻⁴ M UTP, all of which stimulate Cl⁻ secretion, confirms the viability of the βENaC m/m explants.

Figure 4

Physiological measurement in adult βENaC +/+ (white bars) and m/m (dark bars) mice on normal-salt (3 g/kg Na⁺) diets. (A) In vivo measurements of ΔPD_amil (βENaC +/+, n = 16; βENaC m/m, n = 17). ∗∗, P < 0.01. (B) Relative values of urinary Na⁺ and K⁺ normalized with creatinine (Creat; for each group, n = 17). ∗, P < 0.05; ∗∗, P < 0.01. (C) Arterial blood pH and HCO₃⁻ (βENaC +/+, n = 5; βENaC m/m, n = 7). ∗, P < 0.05. (D) Plasma aldosterone concentrations (nM; for each group, n = 7). ∗, P < 0.05.

Figure 5

Body-weight measurements in βENaC +/+ (n = 12; open squares) and m/m (n = 8; closed squares) mice. Animals were kept 6 days (days 0–6) on normal-salt diets (3 g/kg Na⁺), which then were replaced by low-salt diets (0.1 g/kg Na⁺) for 8 more days (days 6–14). Body weights of each group are indicated as percentage of reference weight (100% at day 6). Mice were weighed daily at the same time.

Figure 6

Physiologic measurements in adult βENaC m/m (black bars) and +/+ (white bars) mice on low-salt diets (0.1 g/kg Na⁺; A–D). (A) Plasma K⁺. For each group, n = 7. ∗∗, P < 0.01. (B) Relative values of urinary Na⁺ and K⁺ normalized with creatinine (Creat). For βENaC +/+, n = 7; for βENaC m/m, n = 6. (C) Plasma aldosterone levels (nM). For each group, n = 6. ∗∗, P < 0.01. (D) In vivo measurements of ΔPD_amil. For βENaC +/+, n = 6; for βENaC m/m, n = 7. ∗∗, P < 0.01. (E) Mean blood-pressure measurements in βENaC +/+ (n = 7) and m/m (n = 8) mice on normal-salt diets (NS; 3 g/kg Na⁺) and low-salt diets (LS; 0.1 g/kg Na⁺). ∗, P < 0.05.