Transient receptor vanilloid 4 (TRPV4) channels are essential for alveolar epithelial cell function

Ischemia-reperfusion(IR)-induced edema formation can be mimicked ex-vivo in isolated perfused mouse lungs (IPL). Here we show enhanced edema formation in transient receptor potential vanilloid 4 (TRPV4)-deficient (TRPV4-/-) IPL compared to wild-type (WT) controls in response to IR, indicating a protective role of TRPV4 to maintain the alveolar epithelial barrier. By immunohistochemistry, mRNA profiling or electrophysiological analysis we detected TRPV4 in bronchial epithelium, alveolar type I (ATI) and alveolar type II (ATII) cells. Genetic ablation of TRPV4 resulted in reduced expression of aquaporin-5 (AQP-5) channels in ATI as well as decreased production of pro surfactant protein C (pSP-C) in ATII cells. Migration of TRPV4-deficient ATI cells was reduced and cell barrier function was impaired. Moreover, adult TRPV4−/− lungs developed emphysema-like changes and altered lung parameters compared to WT lungs. Therefore, our data highlight novel essential functions of TRPV4 channels in alveolar epithelial cells and in the protection from edema formation. eLife digest Transient receptor potential vanilloid 4 (TRPV4) is a non-selective Ca2+ permeable cation channel expressed in lung endothelium where increased channel activity has been shown to compromise endothelial barrier function. In other tissues however, the channel maintains physiological cell barriers, e.g. in skin, the urogenital tract and the corneal epithelium. In tracheal epithelial cells TRPV4 channels regulate ciliar beat frequency and in alveolar epithelial cells TRPV4 activation by 4α-phorbol esters produced blebs and breaks in lung septa by unknown molecular mechanisms. To understand the channels role in lung function Weber et al. employed ex-vivo isolated perfused mouse lungs (IPL) to mimic ischemia-reperfusion-induced edema as one of the most common and significant causes of morbidity and mortality after lung transplantation in human patients. TRPV4-deficient (TRPV4−/−) IPL developed enhanced edema formation compared to wild-type (WT) controls in response to ischemia and reperfusion, indicating a protective role of TRPV4 to maintain the alveolar epithelial barrier. TRPV4 was detected in bronchial epithelium, alveolar type I (ATI) and alveolar type II (ATII) cells by immunohistochemistry or mRNA profiling. Genetic ablation of TRPV4 resulted in reduced expression and plasma membrane insertion of water conducting aquaporin-5 (AQP-5) channels in ATI cells compared to WT mice. Analysis of isolated primary TRPV4−/− ATII cells revealed a reduced expression of pro surfactant protein-C (pSP-C) a precursor of a protein important for decreasing surface tension and for alveolar fluid homeostasis. Moreover, the TRPV4 activator GSK1016790A induced increases in current densities only in WT but not in TRPV4−/− ATII cells. On a molecular level ablation of TRPV4 induced less Ca2+-mediated nuclear translocation of nuclear factor of activated T-cells (NFAT) to the nucleus, which may be responsible for reduced expression of the identified proteins. Although the ability of TRPV4−/− ATII to differentiate to ATI cells was unchanged, migration of TRPV4-deficient ATI cells was reduced and cell barrier function was impaired. Moreover, TRPV4−/− lungs of adult mice developed significantly larger mean chord lengths and altered lung function compared to WT lungs. The findings of Weber et al. highlights novel essential functions of TRPV4 channels in alveolar epithelial cells and in the protection from edema formation.

Abstract Ischemia-reperfusion(IR)-induced edema formation can be mimicked ex-vivo in isolated perfused mouse lungs (IPL). Here we show enhanced edema formation in transient receptor potential vanilloid 4 (TRPV4)-deficient (TRPV4-/-) IPL compared to wild-type (WT) controls in response to IR, indicating a protective role of TRPV4 to maintain the alveolar epithelial barrier. By immunohistochemistry, mRNA profiling or electrophysiological analysis we detected TRPV4 in bronchial epithelium, alveolar type I (ATI) and alveolar type II (ATII) cells. Genetic ablation of TRPV4 resulted in reduced expression of aquaporin-5 (AQP-5) channels in ATI as well as decreased production of pro surfactant protein C (pSP-C) in ATII cells. Migration of TRPV4-deficient ATI cells was reduced and cell barrier function was impaired. Moreover, adult TRPV4-/-lungs developed emphysema-like changes and altered lung parameters compared to WT lungs. Therefore, our data highlight novel essential functions of TRPV4 channels in alveolar epithelial cells and in the protection from edema formation. eLife digest Transient receptor potential vanilloid 4 (TRPV4) is a non-selective Ca 2+ permeable cation channel expressed in lung endothelium where increased channel activity has been shown to compromise endothelial barrier function. In other tissues however, the channel maintains physiological cell barriers, e.g. in skin, the urogenital tract and the corneal epithelium. In tracheal epithelial cells TRPV4 channels regulate ciliar beat frequency and in alveolar epithelial cells TRPV4 activation by 4α-phorbol esters produced blebs and breaks in lung septa by unknown molecular mechanisms.
To understand the channels role in lung function Weber et al. employed ex-vivo isolated perfused mouse lungs (IPL) to mimic ischemia-reperfusion-induced edema as one of the most common and significant causes of morbidity and mortality after lung transplantation in human patients. TRPV4-deficient (TRPV4-/-) IPL developed enhanced edema formation compared to wild-type (WT) controls in response to

Introduction
The alveolar epithelium serves multiple functions in the lung. On the one hand, the epithelial layer forms a natural barrier to the external environment protecting the body from invading microorganisms and toxicants, while, on the other hand, alveolar epithelial cells facilitate gas exchange. In the adult lung, the alveolar epithelium consists of two epithelial cell types, which are crucial to maintain lung homeostasis and tissue repair (Mutze et al., 2015). Alveolar epithelial type I (ATI) cells are elongated cells with a large cell surface and high barrier function in close proximity to endothelial cells of the alveolar capillaries facilitating gas exchange (Mutze et al., 2015). ATI cells are also highly water permeable, allowing for ion transport and maintenance of lung fluid balance (Dobbs et al., 2010). Although the latter cells cover the largest surface area of the lung (Weibel, 2015), alveolar epithelial type II (ATII) cells, which exhibit a cubic morphology, by far outnumber ATI cells (Stone et al., 1992). ATII cells are also involved in ion transport and liquid homeostasis (Fehrenbach, 2001) and aremost importantly -responsible for the production, storage, secretion and recycling of pulmonary surfactant. Surfactant lowers the surface tension at the tissue-air barrier to allow proper inflation and deflation of the lung during breathing (Halliday, 2008).
Moreover, ATII cells also serve as progenitors for ATI cells and are capable of long- Transient receptor potential vanilloid 4 (TRPV4) is the fourth cloned member of the vanilloid family of TRP channels (Nilius and Szallasi, 2014). Like most TRP channels TRPV4 harbors an invariant sequence, the TRP box (containing the amino acid sequence: EWKFAR), in its intracellular C-terminal tail as well as ankyrin repeats in the intracellular N-terminus. The protein is composed of six membrane-spanning helices (S1-6), and a presumed pore-forming loop between S5 and S6 (Dietrich et al., 2017;Nilius and Szallasi, 2014). Four of these monomers of the same type preferentially assemble in a functional homotetrameric complex (Hellwig et al., 2005), Moreover, TRPV4-deficient mice were protected from bleomycin-induced pulmonary fibrosis, due to the channel's constitutive expression and function in lung fibroblasts (Rahaman et al., 2014). In lung endothelium, where its role was most extensively  (Balakrishna et al., 2014). Therefore, TRPV4 channels may function as chemosensors of toxicants in the lung epithelium (reviewed in (Steinritz et al., 2018)).
In here, we set out to study pulmonary functions of TRPV4 channels capitalizing on a TRPV4-deficient mouse model. Triggered by ischemia-reperfusion, we observed enhanced lung edema formation, probably due to down-regulation of aquaporine-5 channels in alveolar type I (ATI) cells, reduced pro surfactant protein-C (pSP-C) production in ATII cells and/or emphysema-like changes in the overall lung architecture. Our data suggest an essential role of TRPV4 channels in the alveolar epithelium.

Ablation of TRPV4 increases ischemia-reperfusion (IR)-induced edema formation in isolated perfused mouse lungs
To investigate the role of TRPV4 in IR-induced edema formation, we isolated lungs from wild-type (WT) and TRPV4-deficient (TRPV4-/-) mice. Initial characterization of these mice revealed impaired pressure sensation in dorsal root ganglia (Suzuki et al., 2003) and impaired osmotic sensation by exaggerated arginine vasopressin (AVP) secretion in the brain (Mizuno et al., 2003). Loss of TRPV4-protein was confirmed in lung lysates. While in WT controls a protein of the appropriate size of 100 kDa was detected by Western blotting with TRPV4 specific antibodies, TRPV4-/-lungs did not express any TRPV4 protein ( Figure 1A). Murine embryonic fibroblasts (MEF) (Kalwa et al., 2015) like pulmonary fibroblasts express TRPV4 protein (Rahaman et al., 2014) and served as an additional positive control. After initial perfusion for 15 min followed by 90 min ischemia and 120 min reperfusion, TRPV4-deficient lungs show enhanced lung edema formation as evidenced by a considerable gain in lung weight as opposed to WT lungs ( Figure 1B). Weight increased to a similar extent as already described by us previously (Weissmann et al., 2012). These results clearly contrast with observations on TRPC6-deficient lungs, which are protected from IR-induced edema due to reduced endothelial permeability (Weissmann et al., 2012). Therefore, we generated a TRPV4/TRPC6 double deficient (TRPV4/TRPC6-/-) mouse model, whose lungs lack the increase in IR-induced edema formation, but developed edema similar to WT mice ( Figure 1B). Moreover, lung edema formation in TRPV4-/-lungs was clearly visible ( Figure 1C) and consistently wet to dry weight ratio gain doubled in TRPV4-/-, but only slightly increased in TRPV4/TRPC6-/-lungs ( Figure 1D).

TRPV4 is expressed in alveolar epithelial type I (ATI) and type II (ATII) cells
As TRPV4 is highly expressed in lung endothelium, and its activation results in an increase of endothelial permeability (reviewed in (Simmons et al., 2018)), we focused on its possible function in epithelial cells representing the second natural barrier regulating edema formation. Analysis of mice carrying an eGFP-reporter protein under the control of the TRPV4 promoter/enhancer region revealed expression of TRPV4 protein in endothelium, bronchial as well as alveolar epithelium (Figure 2A). In the bronchial epithelium we detected TRPV4 in ciliated cells by co-staining with a β-tubulin IV antibody (Figure 2-

Loss of TRPV4 resulted in decreased aquaporin-5 expression in TRPV4-/-lungs
Staining of lung slices with fluorescence-coupled antibodies specific for the water conducting channel AQP-5 revealed lower total expression levels in septa-forming ATI cells and reduced plasma membrane localization in TRPV4-/-lungs compared to WT lungs ( Figure 3A-E). These results were confirmed by Western blotting analysis of lung lysates probed with an AQP-5-specific antibody (Figure 3F-G). In clear contrast to these results, protein levels of aquaporin-1 (AQP-1) -the major aquaporin channel in the microvascular endothelium -were not significant different in TRPV4-/endothelial compared to WT cells (Figure 3-figure supplement 1A-E).
Identification of currents induced by the TRPV4 activator GSK1016790A only in primary ATII cells from WT mice producing more pro surfactant protein-C (pSP-

C) than TRPV4-/-cells
To investigate the role of TRPV4 on a cellular level, we first isolated ATII epithelial cells ( Figure 4A) from WT and TRPV4-/-mice, which showed no morphological differences. ATII cells were identified by staining with fluorophore-coupled antibodies directed against pro surfactant protein-C (pSP-C) (Figure 4B), which is produced by ATII cells (reviewed in (Fehrenbach, 2001)). Patch clamp analysis of primary ATII cells revealed significantly larger currents induced by the selective TRPV4 activator GSK1016790A (reviewed in (Dietrich, 2019)) only in WT cells. Moreover, GSK1016790A-induced currents in TRPV4-/-cells were not significantly from the basal currents in WT cells ( Figure 4C-D). Western blotting analysis of protein lysates from ATII cells revealed lower pSP-C levels in TRPV4-/-ATII cells compared to WT cells (Figure 4E-F). We then differentiated ATII to ATI cells by growing them to confluency in plastic cell culture dishes for at least 6 days as described (Mutze et al., 2015) ( Figure 4G). After 6 days WT cells expressed AQP-5 protein as an ATI cell marker ( Figure 4H).

TRPV4-/-mice expose emphysema-like lung structures and altered lung function
To analyze differences in lung anatomy as a consequence of altered ATI cell function, we quantified mean chord lengths in histological lung sections ( Figure 6A). TRPV4ablation significantly increased mean chord length of the alveolar lumen in adult (three to twelve months old ( Figure 6C-D)) mice compared to WT lungs, while young mice (three weeks old) showed no differences ( Figure 6B). Moreover, lung function was altered ( Figure 6E-H): TRPV4-/-lungs showed increased inspiratory capacity and compliance ( Figure 6E, G, H) as well as decreased elastance ( Figure 6F) significantly different from WT mice of the same age.

Discussion
Although TRPV4 is highly expressed in lungs, its exact function is still elusive (reviewed in (Dietrich, 2019)). Activation of TRPV4 in endothelial cells by mechanical stress e.g.  Figure 1B) and a higher wet-to-dry weight ratio gain ( Figure 1D) when compared to control wild-type (WT) mice. Barrier function was rescued by consecutive breeding TRPV4-/-mice with TRPC6-/-mice, because lung edema formation in double deficient mice was similar to WT animals ( Figure 1B).  (Figure 3A-G). To analyze TRPV4 function on a cellular level, we isolated ATII cells identified by their expression of pro surfactant protein-C (pSP-C) (Figure 4A-B). We were able to identify significantly larger currents induced by the TRPV4 activator GSK1016790A in WT but not in TRPV4-/-ATII cells (Figure 4C-D). To our knowledge these data show for the first time that TRPV4 channels are not only expressed, but are also functional in ATII cells. Quantifying pSP-C levels by Western

As TRPV4 activation in endothelial cells has been shown to result in higher
Blotting revealed a reduced expression in TRPV4-/-cells compared to WT cells ( Figure 4E-F). The role of surfactant proteins in the prevention of alveolar edema by reducing surface tension as a driving force for fluid flow across the air blood barrier, is still a matter of debate (Hills, 1999), but might also explain exaggerated edema formation in TRPV4-/-mice.
Next, we differentiated ATII to ATI cells (Mutze et al., 2015), monitored by the expression of two ATI cell markers: AQP-5 and podoplanin. As AQP-5 protein expression was reduced in TRPV4-/-ATI cells (Figure 5A-B) while podoplanin levels were not altered ( Figure 5C-D), it seems rather unlikely that TRPV4-deficency and/or a reduction of pSP-C expression results in reduced ATII to ATI differentiation in general. Plasma membrane translocation of AQP-5 as well as AQP-5 expression may depend on nuclear localization of the transcription factor nuclear factor of activated T cells (NFAT) by a rise of intracellular Ca 2+ via TRPV4 similar to TRPC channels (Curcic et al., 2019). Therefore, we quantified nuclear NFAT levels and detected significantly lower levels in TRPV4-/-cells in comparison to WT control cells ( Figure   5E-F). A major breakthrough in our understanding of AQP-5 function for water transport across apical membranes of ATI cells, was the analysis of AQP-5-deficient mice (Ma et al., 2000). Although lack of AQP-5 entailed a 10-fold decrease in alveolar permeability in response to an osmotic gradient, AQP-5-/-mice are indistinguishable from WT mice with regard to hydrostatic pulmonary edema as well as isosmolar fluid transport from the alveolar space (King et al., 2000; Ma et al., 2000). Cognizant of this scenario, a role for AQP-5 in the clearance of fluid from the alveolar space after IR-induced lung edema cannot entirely be ruled out, but appears to be unlikely, and we tried to dissect other additional mechanisms for the vulnerability of TRPV4-/-lungs to edema formation.
As two reports demonstrated decreased migration of human epithelial ovarian cancer Thus, we analyzed lung alveolar histology in WT and TRPV4-/-lungs in young and adult mice. Mean chord length as a measure of alveolar size was increased in adult (3 months -1 year old) but not in young (3 weeks old) TRPV4-/-mice compared to WT mice of the same age (Figure 6A-D). We concluded that differences were not caused by defects in embryonic lung development, but were due to ongoing growth and repair processes in adult animals. Most interestingly, the emphysema-like changes in lung morphology were also detected in SP-C-deficient mice (Glasser et al., 2003), raising the possibility that reduced pSP-C levels in TRPV4-/-ATII cells may also contribute to the phenotype. In the same vein, adult TRPV4-/-mice showed altered lung function with increased inspiratory capacity and compliance as well as decreased elastance ( Figure 6E-H) compared to WT mice of the same age. Loss of septa formation because of reduced pSP-C levels in adult TRPV4-/-mice may be responsible for decreased clearance of fluid from the alveolar space and may therefore explain higher edema formation in TRPV4-/-lungs.
In summary, loss of TRPV4 channels in alveolar epithelial cells results in decreased production of pSP-C production in ATII and lower AQP-5 expression and membrane localization in ATI cells. The latter proteins are likely to be involved in continuously ongoing repair processes in adult mice, resulting in emphysema-like changes in TRPV4-/-mice. These molecular events may define a protective function of TRPV4 channels against lung edema formation, in clear contrast to their detrimental role in endothelial cells.

Isolated, perfused mouse lung (IPL)
Quantification of edema formation in isolated perfused mouse lungs (IPL) were done as described (Weissmann et al., 2012). In brief, mice were anesthetized by intraperitoneal injection of ketamine (100 mg/kg body weight (bw)), xylazine (0,7 mg/kg bw) and anticoagulated with heparin (500 iU./kg bw). Animals were intubated via a small incision in the trachea, ligated and ventilated with room air using the VCM type 681 (positive end−expiratory pressure, 3 cmH2O; positive end-inspiratory pressure 3 cmH2O; respiratory rate was 90 breaths/min). The sternum was opened, the ribs were spread and the right ventricle was incised to place the air-free perfusion catheter into the pulmonary artery. After ligation the perfusion was started with 0.5 ml/min perfusion solution (7.19 g sodium chloride, 0.33 g potassium chloride, 0.27 g magnesium hexahydrate, 0.36 g calcium chloride dihydrate, 0.15 g potassium dihydrogen orthophosphate, 2.67 g glucose monohydrate, 51.28 g hydroxyethyl starch

Analysis of functional parameters of the respiratory tract
Mice were anesthetized with ketamine (270 mg/kg bw) and xylazin (11 mg/kg bw), intratracheally intubated through a small incision of the trachea and connected to the flexiVent system (Scireq, Montreal, Canada).

Primary murine alveolar epithelial cells
Isolation of alveolar epithelial cells type 2 (ATII) was done as described (Corti et al., 1996;Dobbs, 1990;Mutze et al., 2015). In brief, lungs were flushed via a catheter through the pulmonary artery with 0.9% NaCl solution (B. Braun Melsungen AG, Melsungen, Germany), inflated with 1 ml dispase (BD Bioscience, San Jose, CA) followed by 500 µl 1% low-melting-point agarose (Sigma-Aldrich, St Louis, MO) and incubated for 1 h at RT. Subsequently, lung lobes were separated and dissected using two forceps, filtered through 100 µm, 20 µm and 10 µm nylon filters (Sefar, Heiden, Switzerland) and centrifuged for 10 min at 200 x g. Cell pellets were resuspended in DMEM (Sigma-Aldrich) and plated on CD45 and CD16/32 (BD Bioscience) coated culture dishes for a negative selection of macrophages and lymphocytes and incubated for 30 min at 37 °C. Non-adherent cells were collected and seeded on uncoated dishes to negatively select fibroblasts at 37 °C for 25 min. Cells were collected and live cells were counted by trypan blue staining in a Neubauer counting chamber. Two x 10 6 cells/well of a 6-well plate were seeded in DMEM containing 10% FCS (Invitrogen, Carlsbad; USA), 1% HEPES (Carl Roth, Karlsruhe, Germany) and 1% penicillin/streptomycin (Lonza, Basel, Switzerland) and used for analysis or grown for at least 6 d for ATI cell differentiation. ATII cells were transfected with 1 μM Accell SMARTpool siRNA for TRPV4 (in starving medium, 0.1% FCS) two days after isolation.
On day 6, the cells were washed once and kept in starving medium. A non-coding pool of the Accell siRNA in starving medium served as control (see Table 1 for siRNA sequences).

Patch Clamp recordings of ATII cells
Conventional whole-cell recordings were carried out at RT 24 hours after isolation of ATII cells from WT and TRPV4-/-mice. The following bath solution containing 140 mM NaCl, 1.3 mM MgCl2, 2.4 mM CaCl2, 10 mM glucose, 10 mM HEPES (pH 7.4 with NaOH) and resulting in an osmolality of 310 mOsm kg-1 was used for patch-clamp recordings. The pipette solution contained 135 mM CsCl, 2 mM Na-ATP, 1 mM MgCl2, 5 mM EGTA and 10 mM HEPES (pH 7.2 with CsOH), resulting in an osmolality of 296 mOsm kg 1. Patch pipettes made of borosilicate glass (Science Products, Hofheim, Germany) had resistances of 2.2-3.5 MΩ for whole-cell measurements. Data were collected with an EPC10 patch clamp amplifier (HEKA, Lambrecht, Germany) using the Patchmaster software. Current density-voltage relations were obtained before and after application of the TRPV4 activator GSK1016790A (1mM) to the bath solution using voltage ramps from -100 to +100 mV, each lasting 5 s. Data were acquired at a frequency of 40 kHz after filtering at 2.8 kHz. The current density-voltage curves and the current density amplitudes at ±100 mV were extracted at minimal or maximal currents, respectively.

Western blot analysis
Western Blotting was done as previously described (Hofmann et al., 2017).

Nanostring® nCounter expression analysis
Direct quantification of TRPV4 mRNA in murine lung cells was done as described (Kannler et al., 2018). In brief: total RNA from pulmonary murine cells was isolated using the Qia RNeasy Mini Kit (Qiagen, Hilden, Germany). Quantity, purity, and integrity of the RNA samples were controlled by spectrophotometry (NanoQuant, Tecan, Männedorf, Switzerland). Two probes (the reporter and the capture probe) were hybridized to their specific target mRNAs. Then, the target-probe complexes were immobilized in the imaging surface of the nCounter Cartridge by binding of the capture probe. Finally, the sample cartridges were scanned by an automated fluorescence microscope and molecular barcodes (fluorophores contained in the reporter probe) for each specific target were counted. For expression analysis by nCounter NanoString technology, 200 ng total RNA was hybridized with a Nanostring Gene Expression CodeSet and analysed using the nCounter Digital Analyzer.

Migration assay
Around 4.4 x 10 6 ATII cells/well were seeded on a 2 well silicone insert with a 500 µm cell-free gap (ibidi GmbH, Martinsried, Germany) and grown in DMEM (10% FCS, 1% HEPES and 1% penicillin/streptomycin) for 5 days to obtain ATI like cells.
Subsequently cells were starved in serum reduced medium (0.1% FCS) for 24 h before insert detachment to create a defined cell-free gap. Images were taken 0, 1, 3, 5, 8, 12 and 24 h after gap creation. Migration was analyzed by measuring the remaining gap width with ImageJ software in 3 pictures per time point and replicate.

Isolation of nuclear fractions
Isolation of nuclear protein extracts from ATI-like cells after 6 days of culture was performed with a Nuclear Extract Kit according to the manufacturer's instructions (Active Motif, 40010, La Hulpe, Belgium) as described (Hofmann et al., 2017). In brief, cells were first washed with PBS containing phosphatase inhibitors. Cytoplasmic protein fractions were collected by adding hypotonic lysis buffer and detergent, causing leakage of cytoplasmic proteins into the supernatant. After centrifugation (14.000 × g for 30 s) nuclear protein fractions were obtained by resuspending pellets in detergentfree lysis buffer containing protease inhibitors. NFAT proteins were analyzed by Western blotting as described below using an NFATc1 specific (mouse, SantaCruz Biotechnology, sc-7294, 1:600) antiserum and lamin B1 (rabbit, Thermo Fisher Scientific, PA5-19468, 1:5000) antibodies as loading control. Protein bands were normalized to loading controls and quantified by an Odyssey® Fc unit (Licor, Lincoln, USA).

Quantification of cell resistance by electrical cell impedance sensing (ECIS)
Resistance changes of ATII to ATI cells was analyzed using an electric cell impedance sensing (ECIS) device (Applied Biophysics, Troy, NY, USA). Freshly isolated epithelial cells were seeded on ECIS culture ware (8W10E+; Applied Biophysics, Troy, NY, USA), which was preincubated with FCS for three hours and connected to the ECIS device. A total of 1 x 10 4 cells were seeded per chamber and grown at 37°C and 5% CO2 in an incubator. Resistance (Ω) was analyzed at 2000 Hz over 160 h.

Statistics
All our data are based on experiments with mice. According to the 3R (for reduce, refine, replace) rules German and European guidelines aim to reduce animal experiments. Therefore, we stopped all experiments with animals as soon as a clear trend for significance or no significance was observed. Experimental groups were defined by genotype of mice or treatment (ischemia or no ischemia; GSK activator or solvent). All statistical test were performed using GraphPad Prism 7 (GraphPad Software, San Diego, USA).  The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Author contributions
JW, AD Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article; MB, NW, AÖY, JS Contributed essential unpublished data or reagents; Y-KC, MK, GK-C, SR, CG Acquisition of data, Analysis and interpretation of data; TG Drafting or revising the article

Ethics
Animal experimentation: Experiments involving animals were done in accordance with the EU Animal Welfare Act and were approved by the local councils on animal care (permit No. 55.2.-1-54-2532(permit No. 55.2.-1-54- -7-2015 from Government of Oberbayern, Germany.  Significance between means was analyzed using two way ANOVA (B) or two tailed unpaired Student's t-test (D) and indicated as *** for p<0.001, ** for p<0.01 and * for p<0.05.  Significance between means was analyzed using two tailed unpaired Student's t-test and indicated as *** for p<0.001, ** for p<0.01 and * for p<0.05.  Electrical cell resistance was quantified with an ECIS device for WT and TRPV4-/-ATI cells (cell preparation from 4 mice) for 160 h (I). Data represent means ± SEM from at least 3 independent cell preparations of 5 mice each. Significance between means was analyzed using two tailed unpaired Student's t-test and indicated as *** for p<0.001 and * for p<0.05. (D) WT and TRPV4-/-mice. Inspiratory capacity (IC (E)), elastance of the respiratory system (Ers (F)), static compliance (Cst (G)) and compliance of the respiratory system (Crs (H)) of 6 months old WT and TRPV4-/-mice. Data represent means ± SEM from at least 3 mice. Significance between means was analyzed using two tailed unpaired Student's t-test and indicated as *** for p<0.001, ** for p<0.01 and * for p<0.05.  and TRPV4-deficient endothelial cells (B). Summaries of AQP-1 protein expression in plasma membranes (% AQP-1 in membranes (C)) and in relation to the cytosol (% AQP-1 membrane/cytosol (D)). Data represent means ± SEM from 9 lungs. No significance between means was identified using two tailed unpaired Student's t-test.