Impact of KvLQT1 potassium channel modulation on alveolar fluid homeostasis in an animal model of thiourea-induced lung edema

Abstract

Alveolar ion and fluid absorption is essential for lung homeostasis in healthy conditions as well as for the resorption of lung edema, a key feature of acute respiratory distress syndrome. Liquid absorption is driven by active transepithelial sodium transport, through apical ENaC Na⁺ channels and basolateral Na⁺/K⁺-ATPase. Our previous work unveiled that KvLQT1 K⁺ channels also participate in the control of Na⁺/liquid absorption in alveolar epithelial cells. Our aim was to further investigate the function of KvLQT1 channels and their interplay with other channels/transporters involved in ion/liquid transport in vivo using adult wild-type (WT) and KvLQT1 knock-out (KO) mice under physiological conditions and after thiourea-induced lung edema. A slight but significant increase in water lung content (WLC) was observed in naïve KvLQT1-KO mice, relative to WT littermates, whereas lung function was generally preserved and histological structure unaltered. Following thiourea-induced lung edema, KvLQT1-KO did not worsen WLC or lung function. Similarly, lung edema was not aggravated by the administration of a KvLQT1 inhibitor (chromanol). However, KvLQT1 activation (R-L3) significantly reduced WLC in thiourea-challenged WT mice. The benefits of R-L3 were prevented in KO or chromanol-treated WT mice. Furthermore, R-L3 treatment had no effect on thiourea-induced endothelial barrier alteration but restored or enhanced the levels of epithelial alveolar AQP5, Na⁺/K⁺-ATPase, and ENaC expressions. Altogether, the results indicate the benefits of KvLQT1 activation in the resolution of lung edema, probably through the observed up-regulation of epithelial alveolar channels/transporters involved in ion/water transport.

Keywords: AQP5; ENaC; Na+/K+-ATPase; animal model; ion/liquid transport; lung homeostasis; potassium channels; pulmonary edema.

FIGURE 1

Validation of KvLQT1 extinction in mouse lungs. (A) Detection of the 240 and 370-pb products, specific to the WT and mutant alleles, amplified by PCR from genomic DNA of ear punches and lung tissues from WT and KO mice. Representative immunofluorescence images of KvLQT1 channel protein staining performed on freshly isolated mouse airway epithelial cells (B), n = 4, Magnification: × 200 Scale: 100 µm). (C) Representative short-circuit current traces, measured in Ussing chamber, of primary cultures of airway epithelial cells, isolated from WT (left panel) and KO mice (right panel). After current stabilization, 7.5 µM amphotericin B was added and an apical-to-basolateral K⁺ gradient was established, before addition of 20 µM chromanol (at the basolateral side). Total basolateral (D) and chromanol-sensitive (E) K⁺ currents (I _sc μA/cm²) as well as transepithelial resistance F (Ωxcm²) of WT and KvLQT1-KO cell cultures (n = 10–17). Values are means ± SEM. Non-parametric Mann-Whitney t-test (Agostino/Pearson normality test: negative, panels (D, E) and unpaired t-test (Agostino/Pearson normality test: positive, panel (F). **p < .01, ***p < .001 vs. WT mice.

FIGURE 2

Lung phenotype assessment of the KvLQT1-KO mouse model. (A) Water lung content (mg/g) from control (non-treated) WT and KO mice (n = 16–27). Mean pressure-volume loops, (B) and C_st (quasi-static compliance at 5 cmH₂O, (C) were measured with flexiVent among control (non-treated) WT and KO mice (n = 11–13). Representative images of histological sections from WT and KO mouse lungs stained with hematoxylin-eosin (D), Scale: 200 μm). Values are presented as means ± SEM. Unpaired t-test (Agostino/Pearson normality test: positive) was practiced for panel (A-C). **p < .01 vs. WT mice.

FIGURE 3

Impact of KvLQT1 extinction and pharmacological inhibition on thiourea-induced acute lung edema and function in mice. (A) WT mice were pretreated with PBS (i.n.) or with the KvLQT1 inhibitor chromanol (Chrom, 20 μM, i.n.) 1 h before i.p. injection (150 μl) of PBS (control condition) or thiourea (TU, 5 mg/kg). KO mice also received PBS (i.n) prior to PBS or thiourea injection. 4 h later, lungs were collected for measurement of the water lung content within the six experimental groups in WT mice (i.n/i.p): PBS/PBS, Chrom/PBS, PBS/TU, and Chrom/TU and KO mice: PBS/PBS and PBS/TU, n = 6–27. Mean pressure-volume loops and Cst were also measured in separate WT control (PBS/PBS) and thiourea-treated (PBS/TU) mice (B), n = 9–13 with the flexiVent system as well as in WT and KO thiourea-treated mice (C), WT/TU vs. KO/TU, n = 9–10. Representative images of histological sections from WT and KO thiourea-treated mouse lungs stained with hematoxylin-eosin (D), Scale: 200 μm. Values are presented as means ± SEM. One-way ANOVA and Bonferroni’s multiple comparisons test were practiced (normality Agostino/Pearson test: positive) and One-way ANOVA non-parametric comparison test (normality Agostino/Pearson test: negative) were applied to panel (A). Non-parametric Mann-Whitney t-test (Agostino/Pearson normality test: negative) was practiced for panel (B, C). *p < .05, **p < .01, ****p < .0001 vs. PBS/PBS, ^ΔΔΔ p < .001 vs. Chrom/PBS.

FIGURE 4

Beneficial impact of KvLQT1 activation on thiourea-induced acute lung edema in WT mice. (A) After treatment with the pharmacological KvLQT1 activator R-L3 (4 μM, i.n.) or PBS (i.n.), a group of WT (left panel, n = 6–20) or KO (middle panel, n = 6–18) mice were challenged with thiourea (TU, 5 mg/kg, i.p.) or PBS (i.p.). 4 h after, lungs were collected for measurement of the water lung content within the four experimental groups (i.n/i.p: PBS/PBS, R-L3/PBS, PBS/TU, and R-L3/TU). Also, WT mice were treated with a combination of R-L3+chromanol (R + C, 4 μM and 20 µM respectively, i.n) or PBS before the challenge with thiourea (TU, 5 mg/kg, i.p.) or PBS (i.p.) and the water lung content was compared among the three experimental groups (i.n/i.p: PBS/PBS, PBS/TU, and R + C/TU, n = 12–15) (right panel). Endothelial permeability (B), n = 8, assessed by Evans blue (20 mg/kg, 100 μL, i.v.) extravasation from the circulation to the lungs, was measured in the control condition (i.n./i.p.: PBS/PBS) and after the thiourea challenge (i.p.: TU, 5 mg/kg) in WT mice treated with the KvLQT1 activator R-L3 (i.n/i.p.: R-L3/TU) or not (PBS/TU). Mean pressure-volume loops (C), middle panel and Cst (C), right panel (n = 8–13) in WT control animals (PBS/PBS) and mice treated with R-L3 (i.n/i.p.: R-L3/TU) or not (PBS/TU) were measured with the flexiVent system. Representative images of histological sections from WT thiourea mouse lungs treated or not with the KvLQT1 activator stained with hematoxylin-eosin (D), Scale: 200 μm). Values are presented as means ± SEM. One-way ANOVA and Bonferroni’s multiple comparisons test were practiced (normality Agostino/Pearson test: positive) for panels (A–C). **p < .01 ***p < .001 ****p < .0001 vs. PBS/PBS and ^# p < .05 vs. PBS/TU.

FIGURE 5

Impact of KvLQT1 activation on alveolar type I (AT1) and II (ATII) epithelial cell markers in thiourea-challenged WT mice. Representative immunofluorescence images (left panels) of lung sections (5 μm, Scale: 100 μm) from WT control mice (PBS/PBS), and WT mice challenged with thiourea (TU, 5 mg/kg i.p., PBS/TU) and treated or not (PBS) with the KvLQT1 activator R-L3 (R-L3/TU) stained with the ATI markers podoplanin (A), n = 50 images, AQP5 (B), n = 59–60 or ATII marker pro-SPC (C), n = 48–51). Nuclei were stained by DAPI. Quantification (right panels) of podoplanin, AQP5, and pro-SPC marker intensities was made with a protocol exploited by ICY software. Values are presented as means ± SEM. Non-parametric Mann-Whitney t-test (Agostino/Pearson normality test: negative) for panel (A). One-way ANOVA non-parametric comparison test (normality Agostino/Pearson test: negative) for panel (B, C). *p < .05, ^** p < .01 or ****p < .0001 vs. PBS/PBS, ^## p < .01 vs. PBS/TU.

FIGURE 6

Impact of KvLQT1 activation on α-ENaC and Na⁺/K⁺-ATPase protein expression in alveolar epithelial cells after thiourea-induced lung edema in WT mice. Representative immunofluorescence images (left panels, Scale: 100 μm) of the α-ENaC subunit (A), n = 3 experiments, including a pool of 14 mice) and Na⁺/K⁺-ATPase (B), n = 3 experiments, including a pool of 14 mice staining performed on ATII cells freshly isolated from lungs from WT control mice (i.n/i.p.: PBS/PBS) and WT mice challenged with thiourea (i.p.: TU, 5 mg/kg) and treated or not (PBS) with the KvLQT1 activator (i.n./i.p.: R-L3/TU and PBS/TU, respectively). Nuclei were stained by DAPI. Quantification (right panels) of marker intensity was made with a protocol exploited by ICY software. Values are presented as means ± SEM. One-way ANOVA non-parametric comparison test (normality Agostino/Pearson test: negative) for panel (A). One-way ANOVA and Bonferroni’s multiple comparisons test were practiced (normality Agostino/Pearson test: positive) for panel (B). **p < .01, vs. PBS/PBS and ^# p < .05 vs. PBS/TU for Na⁺/K⁺-ATPase.

FIGURE 7

Schematic model describing the beneficial effect of KvLQT1 activation, by R-L3, on lung edema resorption after thiourea challenge, through the observed up-regulation of ion/water channels/transporter involved in sodium and fluid absorption by the alveolar epithelium. Created by Biorender.com.