The Effects of the Anti-aging Protein Klotho on Mucociliary Clearance

Abstract

α-klotho (KL) is an anti-aging protein and has been shown to exert anti-inflammatory and anti-oxidative effects in the lung and pulmonary diseases such as chronic obstructive pulmonary disease (COPD) and cystic fibrosis. The current study investigated the direct effect of KL on the bronchial epithelium in regards to mucociliary clearance parameters. Primary human bronchial and murine tracheal epithelial cells, cultured, and differentiated at the air liquid interface (ALI), were treated with recombinant KL or infected with a lentiviral vector expressing KL. Airway surface liquid (ASL) volume, airway ion channel activities, and expression levels were analyzed. These experiments were paired with ex vivo analyses of mucociliary clearance in murine tracheas from klotho deficient mice and their wild type littermates. Our results showed that klotho deficiency led to impaired mucociliary clearance with a reduction in ASL volume in vitro and ex vivo. Overexpression or exogenous KL increased ASL volume, which was paralleled by increased activation of the large-conductance, Ca²⁺-activated, voltage-dependent potassium channel (BK) without effect on the cystic fibrosis transmembrane conductance regulator (CFTR). Furthermore, KL overexpression downregulated IL-8 levels and attenuated TGF-β-mediated downregulation of LRRC26, the γ subunit of BK, necessary for its function in non-excitable cells. In summary, we show that KL regulates mucociliary function by increasing ASL volume in the airways possibly due to underlying BK activation. The KL mediated BK channel activation may be a potentially important target to design therapeutic strategies in inflammatory airway diseases when ASL volume is decreased.

Keywords: TGF-β; airway surface liquid volume; calcium activated potassium channels; klotho; mucociliary clearance.

Figure 1

(A) Representative images showing hematoxylin staining of whole lung sections from kl^−/− (KO) and kl^+/+ (WT) mice (4X magnification, scale bar = 100 μm) and representative images of μOCT recordings from kl^−/− (KO) and kl^+/+ (WT) tracheas. (B) Comparison of fold changes in airway surface liquid (ASL) depth, (C) ciliary beat frequency (CBF), and (D) mucociliary transport ex vivo from excised tracheas of kl^−/− (KO) and kl^+/+ (WT) mice using μOCT. (E) Bronchoalveolar lavage fluid (BALF) analysis showing a trend increase of total neutrophil cell count in three representative WT and KO mice. Statistics: Student's t-test showing mean ± S.D. with ^**p < 0.01, as indicated in appropriate graphs (n = 7–11- animals per group).

Figure 2

(A) Dot plot showing a significant decrease of ASL volume in murine tracheal epithelial cells (MTECs), isolated from kl^−/− mice and their wild type littermates and using meniscus scanning. (B) Bar graphs indicating ASL volume change in human bronchial epithelial cells (HBEC), differentiated at the ALI interface and treated with TGF-β (2.5 ng/ml) ± recombinant KL (100 ng/ml) for 24 and 48 h. (C) ASL volume change in HBEC, infected with either control or klotho and differentiated at the ALI interface and effect of TGF-β (2.5 ng/ml) for 24 and 48 h. (n = 3 independent experiments showing mean ± S.E. with ^*P < 0.05 and ^**P < 0.01).

Figure 3

(A) Effect of treatment with recombinant KL on the activation of CFTR, ENaC, and BK channels, assessed in human ALI cultures using Ussing chambers. (B) Effect of klotho overexpression on basolateral IL-8 secretion in HBECs. (C) Bar graphs indicating changes in relative mRNA expression of CFTR and BK channels subunits after treatment with KL, (D) TGF-β (2.5 ng/mL) after 24 h and (E) 48 h. (F) Effect of TGF-β ± KL on BK channel conductance in HBEC. (G) 10 nM GSK1016790A, a TRPV4-selective agonist, transiently increases intracellular calcium in GCaMP6s expressing HBECs. This response is partially blocked in the presence of TRPV4 antagonist HC-067047 (10 μM, n = 1). (H) Acute KL exposure (0.01 μg/ml, 5 min) transiently increases intracellular calcium (n = 3). (I) Pre-exposure to HC-067047 amplified KL-mediated calcium effects. 100 μM UTP served as a positive control for calcium influx (n = 3 independent experiments from 3 to 5 different lungs showing mean ± S.E. with ^*P < 0.05, ^**P < 0.01, and ^***P < 0.005).

Figure 4

Diagram summarizing working hypothesis. Klotho exerts an activating effect on the apical BK channel through a direct action but also indirectly by attenuating (1) a TGF-β-mediated decrease in LRRC26, the regulatory subunit of the BK channel and (2) a TGF-β-mediated inhibition of BK. In addition, klotho decreases basolateral IL-8 secretion, which is pro-inflammatory thereby contributing to ASL volume dysregulation.