Airway surface liquid volume regulation determines different airway phenotypes in liddle compared with betaENaC-overexpressing mice

Abstract

Studies in cystic fibrosis patients and mice overexpressing the epithelial Na(+) channel beta-subunit (betaENaC-Tg) suggest that raised airway Na(+) transport and airway surface liquid (ASL) depletion are central to the pathogenesis of cystic fibrosis lung disease. However, patients or mice with Liddle gain-of-function betaENaC mutations exhibit hypertension but no lung disease. To investigate this apparent paradox, we compared the airway phenotype (nasal versus tracheal) of Liddle with CFTR-null, betaENaC-Tg, and double mutant mice. In mouse nasal epithelium, the region that functionally mimics human airways, high levels of CFTR expression inhibited Liddle epithelial Nat channel (ENaC) hyperfunction. Conversely, in mouse trachea, low levels of CFTR failed to suppress Liddle ENaC hyperfunction. Indeed, Na(+) transport measured in Ussing chambers ("flooded" conditions) was raised in both Liddle and betaENaC-Tg mice. Because enhanced Na(+) transport did not correlate with lung disease in these mutant mice, measurements in tracheal cultures under physiologic "thin film" conditions and in vivo were performed. Regulation of ASL volume and ENaC-mediated Na(+) absorption were intact in Liddle but defective in betaENaC-Tg mice. We conclude that the capacity to regulate Na(+) transport and ASL volume, not absolute Na(+) transport rates in Ussing chambers, is the key physiologic function protecting airways from dehydration-induced lung disease.

FIGURE 1.

Effect of the Liddle mutation on airway Na⁺ absorption. A–C, in vivo measurements of basal and amiloride-sensitive nasal PD (A) and ex vivo measurements of basal and amiloride-sensitive I_sc in nasal tissues (B) and tracheal tissues (C) from Liddle (L/L) and WT mice. n = 5–17 mice/group. *, p < 0.05; **, p < 0.001 compared with WT. Error bars, S.E.

FIGURE 2.

Expression of ENaC subunits and ENaC regulators in nasal and tracheal epithelia. A–C, transcript levels of αENaC, βENaC, γENaC, CFTR, Nedd4-2, and Sgk1 in freshly excised nasal and tracheal tissues from L/L and WT mice. A and B, comparison of transcript expression levels in nasal (A) and tracheal (B) tissues from L/L versus WT mice. Data are expressed as -fold changes from WT. n = 5–6 mice/group; *, p < 0.05; **, p < 0.01. C, comparison of expression levels in tracheal tissues (open bars) versus nasal tissues (closed bars) from WT mice. Data are expressed as -fold changes from tracheal tissues. n = 5–6 mice/group; **, p < 0.01; ***, p < 0.001. Error bars, S.E.

FIGURE 3.

Effect of the Liddle mutation on airway Na⁺ absorption in CFTR-deficient mice. A–C, in vivo measurements of basal and amiloride-sensitive nasal PD (A) and ex vivo measurements of basal and amiloride-sensitive I_sc in nasal tissues (B) and tracheal tissues (C) from WT mice, L/L mice, single mutant CFTR-deficient (CF) mice, and double mutant CFTR-deficient Liddle (CF + L/L) mice. A, nasal PD measurements. n = 8–12 mice/group. *, p < 0.001 compared with WT and L/L mice; †, p < 0.001 compared with WT, L/L, and CF mice. B, nasal I_sc measurements. n = 5–7 mice/group. *, p < 0.05 compared with WT and L/L mice; †, p < 0.02 compared with L/L mice; ‡, p < 0.01 compared with WT and L/L mice; §, p < 0.05 compared with CF mice. C, tracheal I_sc measurements. n = 6–9 mice/group. *, p < 0.05 compared with WT mice; †, p < 0.01 compared with L/L mice. ‡, p ≤ 0.02 compared with CF mice; §, p < 0.001 compared with WT and CF mice. Error bars, S.E.

FIGURE 4.

Effect of the Liddle mutation on airway morphology. Lung histology from adult WT mice, L/L mice, CF mice, double mutant CFTR-deficient Liddle (CF + L/L) mice, and βENaC-Tg mice stained with H&E and Alcian blue periodic acid-Schiff (AB-PAS). Scale bars, 100 μm. Results shown are representative for n = 6–14 mice/group.

FIGURE 5.

ASL volume regulation in airway epithelia from Liddle mice and βENaC-Tg mice. A–C, Confocal images (A) and summary of measurements of ASL height (B and C) at 0, 2, 4, 8, and 24 h after the mucosal addition of 20 μl of PBS containing Texas Red dextran to primary tracheal epithelial cultures from βENaC-Tg (A and B), L/L mice (A and C), and the respective WT littermates. Scale bars, 7 μm. n = 3–6 mice/group. *, p < 0.05 compared with WT. **, p < 0.01 compared with WT. Error bars, S.E.

FIGURE 6.

Regulation of ENaC in airway epithelia is abnormal in βENaC-Tg mice but preserved in Liddle mice. A, airway histology from neonatal (3-day-old) βENaC-Tg mice, L/L mice, and their respective WT littermates. Sections were stained with H&E and evaluated for degenerative airway epithelial cells (arrows). Scale bars, 20 μm (upper panels) and 10 μm (lower panels). B, summary of airway epithelial necrosis as determined from the number of degenerative epithelial cells per mm of the basement membrane. n = 3–5 mice for each group. *, p ≤ 0.01 compared with WT. C–E, amiloride dose-response curves (C and D) and summary of IC₅₀ values (E) obtained from tracheal tissues of βENaC-Tg mice, L/L mice, and respective WT littermates. n = 6–12 mice/group. *, p < 0.01 compared with WT mice; †, p = 0.001 compared with L/L mice. Error bars, S.E.

FIGURE 7.

Regulation of ENaC by extracellular proteases is abnormal in airway epithelia from βENaC-Tg mice. Freshly excised tracheal tissues from WT and βENaC-Tg mice were pretreated with luminal aprotinin, and amiloride-sensitive I_sc was measured either in the presence of aprotinin alone (Aprotinin) or after the addition of luminal trypsin (+Trypsin). n = 9–14 mice/group. *, p < 0.05; **, p < 0.001 compared with mice of same genotype; †, p < 0.001 compared with WT mice; §, p < 0.001 compared with βENaC-Tg mice. Error bars, S.E.