Inhibition of NKCC1 Modulates Alveolar Fluid Clearance and Inflammation in Ischemia-Reperfusion Lung Injury via TRAF6-Mediated Pathways

Abstract

Background: The expression of Na-K-2Cl cotransporter 1 (NKCC1) in the alveolar epithelium is responsible for fluid homeostasis in acute lung injury (ALI). Increasing evidence suggests that NKCC1 is associated with inflammation in ALI. We hypothesized that inhibiting NKCC1 would attenuate ALI after ischemia-reperfusion (IR) by modulating pathways that are mediated by tumor necrosis-associated factor 6 (TRAF6). Methods: IR-ALI was induced by producing 30 min of ischemia followed by 90 min of reperfusion in situ in an isolated and perfused rat lung model. The rats were randomly allotted into four groups comprising two control groups and two IR groups with and without bumetanide. Alveolar fluid clearance (AFC) was measured for each group. Mouse alveolar MLE-12 cells were cultured in control and hypoxia-reoxygenation (HR) conditions with or without bumetanide. Flow cytometry and transwell monolayer permeability assay were carried out for each group. Results: Bumetanide attenuated the activation of p-NKCC1 and lung edema after IR. In the HR model, bumetanide decreased the cellular volume and increased the transwell permeability. In contrast, bumetanide increased the expression of epithelial sodium channel (ENaC) via p38 mitogen-activated protein kinase (p38 MAPK), which attenuated the reduction of AFC after IR. Bumetanide also modulated lung inflammation via nuclear factor-κB (NF-κB). TRAF6, which is upstream of p38 MAPK and NF-κB, was attenuated by bumetanide after IR and HR. Conclusions: Inhibition of NKCC1 by bumetanide reciprocally modulated epithelial p38 MAPK and NF-κB via TRAF6 in IR-ALI. This interaction attenuated the reduction of AFC via upregulating ENaC expression and reduced lung inflammation.

Keywords: Na-K-2Cl cotransporter 1; acute lung injury; alveolar fluid clearance; bumetanide; epithelial sodium channel; ischemia-reperfusion; p38 mitogen-activated protein kinase; tumor necrosis-associated factor 6.

Figure 1

Effects of BMT on lung edema. (A) Lung weight gain, (B) pulmonary microvascular permeability (Kf), (C) lung wet/dry (W/D) weight ratios, (D) lung weight/body weight (LW/BW), and (E) protein concentration in bronchoalveolar lavage fluid (BALF). The increase of these parameters in the ischemia–reperfusion (IR) group was significantly attenuated by treatment with BMT. BMT, bumetanide 70 μg/kg; CTRL, control. Data are expressed as the mean ± SD (n = 6 per group). *P < 0.05 compared with the control group; ^#P < 0.05 compared with the IR group.

Figure 2

Expressions of p-NKCC1 in rat lung tissues and MLE-12 cells. (A) Representative images of p-NKCC1 immunofluorescence staining (FITC-labeled green; original magnification ×400) of rat lung. Nuclei were counterstained with DAPI (blue). (B) T-NKCC1 and p-NKCC1 expressions in MLE-12 cells determined by western blot analysis (n = 5 per group). (C) Histogram of p-NKCC1 determined by flow cytometry in MLE-12 cells (n = 3 per group). BMT attenuated the activation of epithelial p-NKCC1 in IR-ALI. BMT, bumetanide 20-μM in HR model and 70 μg/kg in IR model; CTRL, control. Data are expressed as the means ± SD. *P < 0.05 compared with the control group; ^#P < 0.05 compared with the HR group.

Figure 3

Cell size and paracellular permeability in MLE-12 cells. (A) Representative flow cytometry dot plots showing the gating strategy for identification of side scatter (SSC) and forward scatter (FSC) in MLE-12 cells, followed by hierarchical subgating according to negative 7-Amino-Actinomycin D (7-AAD) staining (P1). (B) Histograms of cell sizes measured by forward scatter (FSC) after 1 h of reoxygenation. Data are expressed as the means ± SD from independent experiments (n = 3 per group). (C) Paracellular permeability assay using MLE-12 cells cultured with FITC-dextran (4 kDa, 2 mg/ml). Data are expressed as the mean ± SD (n = 9 per group). BMT, bumetanide 20-μM; CTRL, control. *P < 0.05 compared with the control group; ^#P < 0.05 compared with the HR group.

Figure 4

α-ENaC expression and alveolar fluid clearance (AFC). (A) Histogram show the expression of α-ENaC on the MLE-12 cells (P2). Cells were gated according to negative 7-Amino-Actinomycin D (7-AAD) staining (P1; n = 3 per group). (B) α-ENaC levels in MLE-12 cells determined by western blot analysis (n = 5 per group). (C) Effects of AFC in response to BMT treatment (n = 5 per group). BMT, bumetanide 20-μM in HR model and 70 μg/kg in IR model; CTRL, control. Data are expressed as the means ± SD. *P < 0.05 compared with the control group; ^#P < 0.05 compared with the HR group.

Figure 5

Expressions of MAPKs and α-ENaC in MLE-12 cells. (A) Total Erk MAPK (T-Erk) and phosphorylated Erk MAPK (p-Erk), (B) total JNK MAPK (T-JNK) and phosphorylated JNK MAPK (p-JNK), (C) total p38 MAPK (T-p38) and phosphorylated p38 MAPK (p-p38) after HR treated with BMT. (D) α-ENaC expression after HR treated by p38 MAPK inhibitor, BIRB-796 10 μM. BMT, bumetanide 20-μM; CTRL, control. Data are expressed as the mean ± SD (n = 5 per group). *P < 0.05 compared with the control group; ^#P < 0.05 compared with the HR group.

Figure 6

Expressions of epithelial NF-κB and inflammatory parameters in IR-ALI. (A) IκB-α and nuclear phosphorylated NF-κB p65 levels in MLE-12 cells treated with BMT. (B) Hematoxylin and eosin staining for lung tissue (200× magnification). (C) Lung injury score in IR model. (D) TNF-α level in the perfusate in IR model. TATA and β-actin served as loading controls for nuclear and cytoplasmic proteins, respectively. BMT, bumetanide 20-μM in HR model and 70 μg/kg in IR model; CTRL, control. Data are expressed as the means ± SD (n = 5 per group). *P < 0.05 compared with the control group; ^#P < 0.05 compared with the HR group.

Figure 7

TRAF6 expression in IR and HR models. (A) TRAF6 mRNA levels in MLE-12 cells. (B) Protein levels in MLE-12 cells determined by western blot analysis. (C) Immunohistochemical staining for TRAF6 in lung tissue (200× magnification). In the IR and HR groups, TRAF6 expressions were significantly increased and decreased after BMT treatment, respectively. BMT, bumetanide 20-μM in HR model and 70 μg/kg in IR model. All values are expressed as the means ± SD (n = 5 per group). *p < 0.05 compared with the control group; ^#P < 0.05 compared with the HR group.

Figure 8

The mechanisms of NKCC1 modulating alveolar fluid clearance and inflammation in IR-ALI. IR stress causes phosphorylation of NKCC1 and activation of TRAF6, which result in cell swelling and inflammation of alveolar epithelium. Inhibition of NKCC1 by bumetanide reciprocally modulates epithelial TRAF6 expression. This interaction suppresses downstream p38 MAPK and NF-κB pathways, which attenuates the reduction of AFC via upregulating α-ENaC expression and reduces the alveolar inflammation.