TRPV4 channels are essential for alveolar epithelial barrier function as protection from lung edema
Jonas Weber, Suhasini Rajan, Christian Schremmer, Yu-Kai Chao, Gabriela Krasteva-Christ, Martina Kannler, Ali Önder Yildirim, Monika Brosien, Johann Schredelseker, Norbert Weissmann, Christian Grimm, Thomas Gudermann, Alexander Dietrich
Jonas Weber, Suhasini Rajan, Christian Schremmer, Yu-Kai Chao, Gabriela Krasteva-Christ, Martina Kannler, Ali Önder Yildirim, Monika Brosien, Johann Schredelseker, Norbert Weissmann, Christian Grimm, Thomas Gudermann, Alexander Dietrich
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Research Article Cell biology Pulmonology

TRPV4 channels are essential for alveolar epithelial barrier function as protection from lung edema

Abstract

Ischemia/reperfusion-induced edema (IRE), one of the most significant causes of mortality after lung transplantation, can be mimicked ex vivo in isolated perfused mouse lungs (IPL). Transient receptor potential vanilloid 4 (TRPV4) is a nonselective cation channel studied in endothelium; however, its role in the lung epithelium remains elusive. Here, we show enhanced IRE in TRPV4-deficient (TRPV4–/–) IPL compared with that of WT controls, indicating a protective role of TRPV4 in maintenance of the alveolar epithelial barrier. By immunohistochemistry, mRNA profiling, and electrophysiological characterization, we detected TRPV4 in bronchial epithelium, alveolar epithelial type I (ATI), and alveolar epithelial type II (ATII) cells. Genetic ablation of TRPV4 resulted in reduced expression of the water-conducting aquaporin-5 (AQP-5) channel in ATI cells. Migration of TRPV4–/– ATI cells was reduced, and cell barrier function was impaired. Analysis of isolated primary TRPV4–/– ATII cells revealed a reduced expression of surfactant protein C, and the TRPV4 activator GSK1016790A induced increases in current densities only in WT ATII cells. Moreover, TRPV4–/– lungs of adult mice developed significantly larger mean chord lengths and altered lung function compared with WT lungs. Therefore, our data illustrate essential functions of TRPV4 channels in alveolar epithelial cells and in protection from edema formation.

Authors

Jonas Weber, Suhasini Rajan, Christian Schremmer, Yu-Kai Chao, Gabriela Krasteva-Christ, Martina Kannler, Ali Önder Yildirim, Monika Brosien, Johann Schredelseker, Norbert Weissmann, Christian Grimm, Thomas Gudermann, Alexander Dietrich

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Figure 4

Identification of alveolar epithelial type II cells and differentiation to alveolar epithelial type I cells.

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Identification of alveolar epithelial type II cells and differentiation ...
Representative cell cluster 1 day after isolation in a phase-contrast image (scale bar: 100 μm) (A) and stained with a fluorescence-coupled specific pro-surfactant protein-C (pSP-C) antibody (scale bar: 50 μm) (B). Nuclei staining was performed with Hoechst dye (blue). Electrophysiological whole-cell measurements of basal and GSK-induced (GSK-induced) current densities in WT and TRPV4–/– primary alveolar epithelial type II (ATII) cells (C and D). Representative current density–voltage curves of WT (gray, blue traces) and TRPV4–/– (red trace) ATII cells before (gray trace) and during application of GSK (blue and red traces) (C). Summary of current densities at ±100 mV before (white bars) and after application of GSK analyzed in WT (black bars) and TRPV4–/– (gray bars) ATII cells (D). Representative Western blot analysis of pSP-C expression in WT and TRPV4–/– ATII cells (E) and summary of pSP-C expression in TRPV4–/– and WT ATII cells (F). Image of confluent cells on day 6 after ATII cell isolation (scale bar: 100 μm) (G) and analysis of AQP-5 expression in cells grown for 3, 4, and 6 days in plastic cell culture dishes by Western blotting (H). β-Actin was used as loading control in each blot. Data represent mean ± SEM from at least 3 independent cell preparations of 5 mice each. Significance between means was analyzed using 1-way ANOVA (C) or 2-tailed unpaired Student’s t test (F); *P < 0.05, **P < 0.01.