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

Zhao R, Liang X, Zhao M, Liu SL, Huang Y, Idell S, Li X, Ji HL - PLoS ONE (2014)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Molecular Biology, University of Texas Health Science Center at Tyler, Tyler, Texas, United States of America; Texas Lung Injury Institute, University of Texas Health Science Center at Tyler, Tyler, Texas, United States of America.

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.

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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.
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pone-0109725-g001: 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.

Mentions: Extracellular superoxide dismutase (SOD3) is a major superoxide scavenger in the respiratory system. Antioxidants may protect the airways and lungs against excessive superoxide free radicals from tobacco and inflammatory cells. SOD3 proteins are reduced in COPD lung tissues [25], most likely from attenuating oxidative fragmentation of the extracellular matrix (i.g., collagen I, hyaluronan, and heparin sulfate) to protect against alveolar enlargement [26], [27]. SOD3 was associated with FEV1 in COPD patients [28]. This was supported by the association of inherited SOD3 mutations and FVC% in two population studies [29]. To evaluate the reliability of our samples, we set out to analyze SOD3 expression in lung tissues. Two bands (32 and 31 kDa) were recognized by a SOD3-specific antibody (Figure 1A). SOD3 proteins showed a significant decrease in both moderate and severe COPD lungs (Figure 1A & B). These observations were consistent with a recent report in the LTRC-provided samples [25], indicating that the tissues were suitable for protein assays. We therefore used SOD3 as a control in subsequent experiments and statistical analyses. SOD3 proteins were reduced to a lesser extent in moderate and severe COPD lung tissues compared with controls. The inflammatory responses may affect the translation and posttranslational modifications of SOD3 and ion channel proteins. Indeed, pulmonary inflammation is more severe in moderate versus severe COPD lungs, as determined by leukocyte infiltration, airspace flooding, and hemorrhage (Figure 1C).


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

Zhao R, Liang X, Zhao M, Liu SL, Huang Y, Idell S, Li X, Ji HL - PLoS ONE (2014)

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.
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pone-0109725-g001: 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.
Mentions: Extracellular superoxide dismutase (SOD3) is a major superoxide scavenger in the respiratory system. Antioxidants may protect the airways and lungs against excessive superoxide free radicals from tobacco and inflammatory cells. SOD3 proteins are reduced in COPD lung tissues [25], most likely from attenuating oxidative fragmentation of the extracellular matrix (i.g., collagen I, hyaluronan, and heparin sulfate) to protect against alveolar enlargement [26], [27]. SOD3 was associated with FEV1 in COPD patients [28]. This was supported by the association of inherited SOD3 mutations and FVC% in two population studies [29]. To evaluate the reliability of our samples, we set out to analyze SOD3 expression in lung tissues. Two bands (32 and 31 kDa) were recognized by a SOD3-specific antibody (Figure 1A). SOD3 proteins showed a significant decrease in both moderate and severe COPD lungs (Figure 1A & B). These observations were consistent with a recent report in the LTRC-provided samples [25], indicating that the tissues were suitable for protein assays. We therefore used SOD3 as a control in subsequent experiments and statistical analyses. SOD3 proteins were reduced to a lesser extent in moderate and severe COPD lung tissues compared with controls. The inflammatory responses may affect the translation and posttranslational modifications of SOD3 and ion channel proteins. Indeed, pulmonary inflammation is more severe in moderate versus severe COPD lungs, as determined by leukocyte infiltration, airspace flooding, and hemorrhage (Figure 1C).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Molecular Biology, University of Texas Health Science Center at Tyler, Tyler, Texas, United States of America; Texas Lung Injury Institute, University of Texas Health Science Center at Tyler, Tyler, Texas, United States of America.

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.

Show MeSH
Related in: MedlinePlus