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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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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.
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pone-0109725-g002: 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.

Mentions: Transgenic mice over-expressing ENaC had COPD-like phenotype [14]. We hypothesized that ENaC translation in COPD lungs is increased. Expression levels of the four ENaC subunits are not identical and are non-coordinately regulated by hormones [30]. The well-characterized anti-α and anti-β ENaC antibodies recognized a 90 kDa and a 95 kDa single band, respectively, by Western blot. By comparison, a specific anti-γ ENaC antibody identified two forms of proteins: full-length parental (95 kDa) and proteolytically cleaved protein (85 kDa) (Figure 2A). Average α ENaC level relative to corresponding β actin was significantly elevated, while γ ENaC level was reduced, in severe COPD lungs as determined by densitometry, (Figure 2B–D). ENaC proteins are expressed in both alveolar type I (ATI) and II (ATII) cells. Squamous ATI cells cover approximately 93% of airspace surface area (140 m2), while ATI cells account for up to 35% of the cell population [31]. In COPD/emphysema lungs, the airspace surface area was reduced by up to 75% due to alveolar enlargement [32], [33]. To examine whether the changes in ENaC expression in subpopulations of alveolar cells varied, we analyzed ENaC expression in ATI and ATII cells separately. Our data showed an increase in α ENaC protein in both ATI and ATII cells (Figure 2C & D). Expression of β ENaC was enhanced significantly in COPD lungs too (Figure 2C). In contrast, neither full-length nor trimmed peptide γ ENaC proteins underwent a significant change in both cell types (Figure 2C & D). Our results for the first time suggest that the expression of α and β ENaC proteins is augmented in human COPD lung tissues.


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)

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.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4201481&req=5

pone-0109725-g002: 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.
Mentions: Transgenic mice over-expressing ENaC had COPD-like phenotype [14]. We hypothesized that ENaC translation in COPD lungs is increased. Expression levels of the four ENaC subunits are not identical and are non-coordinately regulated by hormones [30]. The well-characterized anti-α and anti-β ENaC antibodies recognized a 90 kDa and a 95 kDa single band, respectively, by Western blot. By comparison, a specific anti-γ ENaC antibody identified two forms of proteins: full-length parental (95 kDa) and proteolytically cleaved protein (85 kDa) (Figure 2A). Average α ENaC level relative to corresponding β actin was significantly elevated, while γ ENaC level was reduced, in severe COPD lungs as determined by densitometry, (Figure 2B–D). ENaC proteins are expressed in both alveolar type I (ATI) and II (ATII) cells. Squamous ATI cells cover approximately 93% of airspace surface area (140 m2), while ATI cells account for up to 35% of the cell population [31]. In COPD/emphysema lungs, the airspace surface area was reduced by up to 75% due to alveolar enlargement [32], [33]. To examine whether the changes in ENaC expression in subpopulations of alveolar cells varied, we analyzed ENaC expression in ATI and ATII cells separately. Our data showed an increase in α ENaC protein in both ATI and ATII cells (Figure 2C & D). Expression of β ENaC was enhanced significantly in COPD lungs too (Figure 2C). In contrast, neither full-length nor trimmed peptide γ ENaC proteins underwent a significant change in both cell types (Figure 2C & D). Our results for the first time suggest that the expression of α and β ENaC proteins is augmented in human COPD lung tissues.

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