Correlation of apical fluid-regulating channel proteins with lung function in human COPD lungs

Abstract

Links between epithelial ion channels and chronic obstructive pulmonary diseases (COPD) are emerging through animal model and in vitro studies. However, clinical correlations between fluid-regulating channel proteins and lung function in COPD remain to be elucidated. To quantitatively measure epithelial sodium channels (ENaC), cystic fibrosis transmembrane conductance regulator (CFTR), and aquaporin 5 (AQP5) proteins in human COPD lungs and to analyze the correlation with declining lung function, quantitative western blots were used. Spearman tests were performed to identify correlations between channel proteins and lung function. The expression of α and β ENaC subunits was augmented and inversely associated with lung function. In contrast, both total and alveolar type I (ATI) and II (ATII)-specific CFTR proteins were reduced. The expression level of CFTR proteins was associated with FEV1 positively. Abundance of AQP5 proteins and extracellular superoxide dismutase (SOD3) was decreased and correlated with spirometry test results and gas exchange positively. Furthermore, these channel proteins were significantly associated with severity of disease. Our study demonstrates that expression of ENaC, AQP5, and CFTR proteins in human COPD lungs is quantitatively associated with lung function and severity of COPD. These apically located fluid-regulating channels may thereby serve as biomarkers and potent druggable targets of COPD.

Figure 1. SOD3, AQP5, and pro-SPC proteins in control, moderate, and severe COPD lungs.

A. Two bands (32 and 30 kDa) are recognized by a specific polyclonal antibody against human SOD3 (Santa Cruz, sc-32219) via Western blot. The blots were stripped and reprobed with a monoclonal anti-β actin antibody. B. Quantitative densitometry of SOD3 relative to corresponding β actin. One-way ANOVA. *P<0.05 and ***P<0.001 vs control or moderate group. N = 17. C. Representative images of control, moderate, and severe groups. H & E stain. 10×. D. AQP5 proteins are detected by a specific antibody against human AQP5 (Santa Cruz, sc-28628) via Western blot. Pro-SPC is detected (21 kDa) using a specific antibody (Millipore, AB3786). E. Densitometric measurements of AQP5 over β actin. N = 17. *P<0.05 versus controls. F. Relative pro-SPC protein expression. One-way ANOVA. *P<0.05 versus controls and **P<0.01 versus moderate group.

Figure 2. Expression of ENaC proteins.

A. Representative Western blots. α (90 kDa), β (95 kDa) and γ ENaC protein signals are found in blots probed with specific antibodies against α (Santa Cruz, sc-22239), β (Santa Cruz, sc-21013), and γ (Abcam, ab3468) subunits. B. Total expression levels of ENaC subunits. β actin is used as a loading control. C & D. Expression of ENaC proteins in ATI and ATII cells. The expression levels of ENaC proteins related to AQP5 (C) and pro-SPC (D) were adjusted to compensate for the alterations in AQP5 and pro-SPC (dotted bars). One-way ANOVA. *P<0.05 and ***P<0.001 versus control groups.

Figure 3. CFTR expression in COPD lungs.

A. Western blots of CFTR expression. A predominant band at 165 kDa is detected by a specific anti-CFTR antibody (R&D Systems, MAB25031) in human lungs. β actin serves as a loading control. B–D. Densitometry of total CFTR expression (B), relative expression in ATI (C), and ATII (D) cells. Dotted bars show the values due to reduced expression of AQP5 and pro-SPC. One-way ANOVA. *P<0.05 and **P<0.01 versus control groups.

Figure 4. Association of spirometry and expression of SOD3 and ENaC in COPD lungs.

A. Total SOD3 and perdlco. B. α ENaC in ATII cells and spirometry. C. α ENaC expressed in ATI cells and fev1pd1a. D. β ENaC in ATI cells and lung function test. E. Total γ ENaC level and fev1prd1.

Figure 5. Correlation of aquaporin 5 with spirometry test.

Dotted lines were created by linear regression analysis of the paired experimental data.

Figure 6. Positive correlation of CFTR with spirometry test in COPD lungs.

A. Total CFTR expression and fev1prd2. B. Expression of CFTR in ATI cells and three spirometric parameters. C. CFTR proteins in ATII cells and spirometry test.

Figure 7. Expression of protein levels and COPD severity.

A. SOD3, total CFTR (tCFTR), and CFTR in ATI cells (CFTR1) are associated with GOLD classification of COPD (see Methods for details). B. CFTR expressed in ATII cells correlates with GOLD1. C. β ENaC in ATI cells is positively associated with GOLD1.