Lack of Kcnn4 improves mucociliary clearance in muco-obstructive lung disease

Abstract

Airway mucociliary clearance (MCC) is the main mechanism of lung defense keeping airways free of infection and mucus obstruction. Airway surface liquid volume, ciliary beating, and mucus are central for proper MCC and critically regulated by sodium absorption and anion secretion. Impaired MCC is a key feature of muco-obstructive diseases. The calcium-activated potassium channel KCa.3.1, encoded by Kcnn4, participates in ion secretion, and studies showed that its activation increases Na+ absorption in airway epithelia, suggesting that KCa3.1-induced hyperpolarization was sufficient to drive Na+ absorption. However, its role in airway epithelium is not fully understood. We aimed to elucidate the role of KCa3.1 in MCC using a genetically engineered mouse. KCa3.1 inhibition reduced Na+ absorption in mouse and human airway epithelium. Furthermore, the genetic deletion of Kcnn4 enhanced cilia beating frequency and MCC ex vivo and in vivo. Kcnn4 silencing in the Scnn1b-transgenic mouse (Scnn1btg/+), a model of muco-obstructive lung disease triggered by increased epithelial Na+ absorption, improved MCC, reduced Na+ absorption, and did not change the amount of mucus but did reduce mucus adhesion, neutrophil infiltration, and emphysema. Our data support that KCa3.1 inhibition attenuated muco-obstructive disease in the Scnn1btg/+ mice. K+ channel modulation may be a therapeutic strategy to treat muco-obstructive lung diseases.

Keywords: Epithelial transport of ions and water; Inflammation; Potassium channels; Pulmonology.

Figure 1. KCa3.1 participates in sodium absorption and anion secretion of mouse and human epithelium.

Representative short-circuit current traces of (A) WT and (B) Kcnn4^–/– mouse tracheae used to calculate the following: (C) V_te, (D) R_te, (E) I_SC, (F) Amiloride-sensitive current, (G) amiloride-insensitive current, (H) cAMP-induced anion current and Ca⁺²-activated anion current at (I) peak or (J) plateau phases; n = 9 for each group. (K) Representative Ussing chamber recordings of HBECs incubated with TRAM-34 and controls showing amiloride addition. (L) Summary of TRAM-34 effect on amiloride-sensitive Na⁺ absorption; n = 6 different cell cultures for each condition. Statistical differences were calculated using rank-sum test. Detailed values including data for Kcne3^–/– are included in Table 1.

Figure 2. Mucociliary clearance is enhanced after KCa3.1 inhibition in the mouse.

(A) CBF changes in Kcnn4^–/– or WT cells treated with TRAM-34 (100 nM) after UTP stimulation of tracheal epithelial cell explants. (B) Quantification of the area under the curve of CBF recordings including WT explants treated with amiloride (10 μM) and/or TRAM-34. Differences were calculated using ANOVA on ranks; n = 5, 4, 5, 5 and 6, respectively. Polar plots (75 μm radius) of the trajectory of beads placed in (C) WT (80 beads), (D) Kcnn4^–/– (68 beads), (E) WT plus 10 μM amiloride (84 beads), and (F) Kcnn4^–/– plus 10 μM amiloride (81 beads) tracheae, corresponding to 5 different experiments for each group. Calculated speed of particles (G) and total distance (H) from the experiments shown in C–F. Differences were calculated using ANOVA on ranks. (I) In vivo lung clearance of fluorescently labeled OVA in WT (n = 4) and Kcnn4^–/– (n = 4) mice. Difference was calculated using rank-sum test. CBF, ciliary beating frequency; UTP, uridine-5′-triphosphate.

Figure 3. The genetic silencing of Kcnn4 reduces sodium absorption and increases MCC in the Scnn1b^tg/+ mouse trachea.

(A) Representative I_sc traces showing the extent of amiloride-sensitive currents in the Scnn1b^tg/+ (n = 6) and double mutant (n = 4) mice tracheae. (B) Summary of amiloride-sensitive currents as shown in (A), P value calculated using ANOVA on ranks. Polar plots (75 μm radius) of the trajectory of beads placed in (C) Scnn1b^tg/+ (222 beads) and double mutants (126 beads), from 5 different experiments each. Detailed electrical parameters are given in Table 1. Calculated (D) speed of particles and (E) total distance from the experiments shown in C. Differences were calculated using ANOVA on ranks. MCC, mucociliary clearance.

Figure 4. Genetic silencing of Kcnn4 reduces lung inflammatory disease in mice with muco-obstructive lung disease.

Total cells (A) and macrophages (B) quantification in BALF. *P < 0.05 vs. WT and Kcnn4^–/–. Neutrophils (C) quantification in BALF. *P < 0.05 vs. all other groups and § indicates the difference vs. Scnn1b^tg/+. ANOVA on ranks; n = 13, 13, 13, and 10 for WT, Kcnn4^+/+, Scnn1b^tg/+, and double mutants, respectively. Representative images (n = 5 each group; scale bar: 20 μm) of LY6G/LY6C immunostaining in mucus plugs (D). Quantification of LY6G/LY6C-positive cells (E) in the Scnn1b^tg/+ (n = 5) and double mutants (n = 5). *P < 0.05 calculated by rank-sum test. Mouse lung volume (F); *P < 0.05 vs. all other groups and § indicates the difference vs. Scnn1b^tg/+; ANOVA on ranks; n = 13, 13, 13, and 10 for WT, Kcnn4^+/+, Scnn1b^tg/+, and double mutants, respectively. Mean linear intercept (G) calculated from images as shown in H (scale bar: 200 μm); *P < 0.05 vs. all other groups and § indicates the difference vs. Scnn1b^tg/+; ANOVA on ranks; n = 13, 13, 13, and 10 for WT, Kcnn4^+/+, Scnn1b^tg/+, and double mutants, respectively.

Figure 5. Genetic silencing of Kcnn4 improved mucus clearance in mouse airways.

Intraluminal (A) and intracellular (B) mucus volume was determined in the proximal and distal airways. Differences were calculated with ANOVA on ranks; n = 12, 12, 12 and 9 animals for WT, Kcnn4^+/+, Scnn1b^tg/+, and double mutants, respectively. Representative images of mucus attachment to the epithelial surface for Scnn1b^tg/+ (n = 15) and double mutants (n = 13) (C). Selected areas of main images (scale bar: 50 μm) are noted as 1–3 in red letters, and are shown amplified separately (scale bar: 20 μm). Summary of the percentage of epithelium surface covered my mucus in proximal and distal airways (D). Only paired samples from the same animal were included; n = 13, 16, 15, and 13 for WT, Kcnn4^+/+, Scnn1b^tg/+, and double mutants, respectively. *P < 0.05 vs. WT and Kcnn4^–/– ANOVA on ranks. The P values for each proximal vs. distal airways comparison were calculated by rank-sum test.