Function of KvLQT1 potassium channels in a mouse model of bleomycin-induced acute lung injury

Abstract

Acute respiratory distress syndrome (ARDS) is characterized by an exacerbated inflammatory response, severe damage to the alveolar-capillary barrier and a secondary infiltration of protein-rich fluid into the airspaces, ultimately leading to respiratory failure. Resolution of ARDS depends on the ability of the alveolar epithelium to reabsorb lung fluid through active transepithelial ion transport, to control the inflammatory response, and to restore a cohesive and functional epithelium through effective repair processes. Interestingly, several lines of evidence have demonstrated the important role of potassium (K⁺) channels in the regulation of epithelial repair processes. Furthermore, these channels have previously been shown to be involved in sodium/fluid absorption across alveolar epithelial cells, and we have recently demonstrated the contribution of KvLQT1 channels to the resolution of thiourea-induced pulmonary edema in vivo. The aim of our study was to investigate the role of the KCNQ1 pore-forming subunit of KvLQT1 channels in the outcome of ARDS parameters in a model of acute lung injury (ALI). We used a molecular approach with KvLQT1-KO mice challenged with bleomycin, a well-established ALI model that mimics the key features of the exudative phase of ARDS on day 7. Our data showed that KvLQT1 deletion exacerbated the negative outcome of bleomycin on lung function (resistance, elastance and compliance). An alteration in the profile of infiltrating immune cells was also observed in KvLQT1-KO mice while histological analysis showed less interstitial and/or alveolar inflammatory response induced by bleomycin in KvLQT1-KO mice. Finally, a reduced repair rate of KvLQT1-KO alveolar cells after injury was observed. This work highlights the complex contribution of KvLQT1 in the development and resolution of ARDS parameters in a model of ALI.

Keywords: acute lung injury; alveolar-capillary barrier; animal model; injury and repair; potassium channels; pulmonary inflammation; respiratory function.

FIGURE 1

Assessment of the pulmonary and systemic dysfunction after the bleomycin challenge. Wild-type (WT, n = 8-9, shown on the left side of vertical dotted line) and KvLQT1-KO (KO, n = 10–11, right side) mice were challenged or not (PBS) with bleomycin (3 U/kg, 50 μL, (I) n. on day 0). On day 7, blood gas (A), electrolyte (B), and metabolite (C) levels were measured using the epoc^® system. Each dot represents 1 mouse and values are mean ± SEM. One-way ANOVA test (Agostino/Pearson normality test: positive) was performed for all parameters. **p < 0.01, ***p < 0.005, ****p < 0.001.

FIGURE 2

Evaluation of the lung function in vivo (resistance and elastance). Wild-type (WT, n = 8–10, left side of vertical dotted line) and KvLQT1-KO (KO, n = 6-8, right side) mice were challenged or not (PBS) with bleomycin (3 U/kg, 50 μL, i. n. on day 0). On day 7, several respiratory mechanics parameters reflecting lung resistance (R_rs, R_N and G, panel (A) and elastance E_rs and H, panel (B)) were measured using the flexiVent system (SCIREQ Inc., Montreal, QC, Canada). Each dot represents 1 mouse and values are mean ± SEM. One-way ANOVA test (Agostino/Pearson normality test: positive) was performed for all parameters. *p < 0.05, **p < 0.01.

FIGURE 3

Evaluation of the lung function in vivo (compliance). Wild-type (WT, n = 8–10, left side of vertical dotted line) and KvLQT1-KO (KO, n = 6-8, right side) mice were challenged or not (PBS) with bleomycin (3 U/kg, 50 μL, i. n. on day 0). On day 7, several respiratory mechanic parameters reflecting lung compliance (PV loop, C_rs and C_st) were measured using the flexiVent system. Each dot represents 1 mouse and values are mean ± SEM. One-way ANOVA test (Agostino/Pearson normality test: positive) was performed for all parameters. *p < 0.05, **p < 0.01.

FIGURE 4

Alteration of the alveolar-capillary barrier by bleomycin in WT and KvLQT1-KO mice. Wild-type (WT, left side of vertical dotted line) and KvLQT1-KO (KO, right side) mice were challenged or not (PBS) with bleomycin (3 U/kg, 50 μL, i. n. on day 0). On day 7, water content of the lungs (A), (n = 9–13), protein content in broncho-alveolar lavages (BALs) (B), (n = 10–20) and Evans Blue concentration (C), (n = 8–10) were measured. Each dot represents 1 mouse and values are mean ± SEM. One-way ANOVA test (Agostino/Pearson normality test: positive) for all parameters was performed for (A–C). *p < 0.05, ***p < 0.005, ****p < 0.0001.

FIGURE 5

Effect of KvLQT1 modulation on bleomycin-induced inflammatory response. Wild-type (WT, left side of vertical dotted line) and KvLQT1-KO (KO, right side) mice were challenged or not (PBS) with bleomycin (3 U/kg, 50 μL, i. n. on day 0). On day 7, BAL (n = 9–14) was collected and total immune cell count was performed (A). The cell pellet was then cytocentrifuged and stained with hematoxylin-eosin to obtain differential cell counts (in %) of neutrophils (B), macrophages (C) and lymphocytes (D). Pro-inflammatory cytokine (E): TNF-α, (F) IL-1β, and (G) IL-6 and chemokine (H): KC and (I) MCP-1 mRNA fold change expression (normalized to β-actin housekeeping gene) was detected from lung tissue of WT (n = 10) and KO (n = 7) mice exposed or not to bleomycin on day 7 and presented as log₂ (∆∆Ct). Each dot represents 1 mouse and values are mean ± SEM. One-way ANOVA test (Agostino/Pearson normality test: positive) was performed for (A–C and F). Kruskal–Wallis nonparametric test (Agostino/Pearson normality test: negative) was performed for (D,E,G and H). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

FIGURE 6

Histological evidence of bleomycin-induced inflammatory tissue damage. On day 7, fixed lung tissues from wild-type (WT, n = 7) and KvLQT1-KO (KO, n = 7) mice challenged or not (PBS) with bleomycin (3 U/kg, 50 μL, i. n. on day 0) were collected for further hematoxylin-eosin staining. Three representative zones (scale: 300 µm) from each experimental group are shown in (A). A lung injury score representing the proportion of septa responsible for gas exchange (B) was defined using the Visiomorph^® software. The percentage (%) of injured/inflamed lung area (C) with inflammatory damage was also evaluated on the whole histological sections. The score of inflammatory infiltration intensity was also defined by pathologists from the CRCHUM molecular platform (D). Each dot represents 1 mouse and values are mean ± SEM. One-way ANOVA test (Agostino/Pearson normality test: positive) was performed for (B,C, and D) **p < 0.01, ***p < 0.001, ****p < 0.0001.

FIGURE 7

Assessment of alveolar epithelial markers after bleomycin challenge. Representative immunofluorescence images of lung sections (scale: 100 µm) from WT and KvLQT1-KO mice challenged or not (PBS) with bleomycin (3 U/kg, 50 μL, (I) n. on day 0). On day 7, lung tissues were fixed and immunostained for pro-SPC (A), n = 50 fields, ATII marker and AQP5 (B), n = 50 fields, ATI marker. Cell nuclei were stained with DAPI. Quantification of marker intensity was performed using a protocol exploited by ICY software. Values are mean ± SEM. One-way ANOVA test (Agostino/Pearson normality test: positive) was performed for (A,B) *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

FIGURE 8

Repair rates of alveolar cell monolayers from WT and KvLQT1-KO mice. Wound-healing rates after mechanical injury of primary cultures (day 4) of alveolar cells isolated from the lungs of WT (n = 7) and KvLQT1-KO mice (n = 6) were monitored over a period of 6 h and plotted individually (each dot represents one alveolar cell culture). Images were taken at t = 0 and t = 6 h to evaluate the area of repair and then to calculate the wound-healing rates. Values are mean ± SEM. Unpaired t-test was performed. ***p < 0.001.