Biokinetic modeling of nanoparticle interactions with lung alveolar epithelial cells: uptake, intracellular processing, and egress.

Studies on health effects of engineered nanomaterials (ENMs) in the lung have provided information on ENM toxicity and translocation across airway and alveolar epithelial barriers. Various inhaled ENMs (e.g., gold and iridium nanoparticles) have been reported to partially cross the air-blood barrier in the lung, enter the vasculature, and distribute in several end organs, including the heart, liver, spleen, and kidney. Using an in vitro primary rat alveolar epithelial cell (AEC) monolayer model, we reported transport rates of relatively nontoxic polystyrene nanoparticles (PNPs), which appear to be taken up via nonendocytic processes into AECs. PNPs internalized into cytoplasm then trigger autophagy, followed by delivery of PNPs from autophagosomes into lysosomes, from where PNPs are exocytosed. We used the data from these experiments to perform biokinetic modeling that incorporates the processes associated with internalization and intracellular distribution of PNPs, autophagy, lysosomal exocytosis of PNPs, and several putative mechanisms of action that extend our previous understanding of AEC processing of PNPs. Results suggest that entry of PNPs into AECs, subsequent activation of autophagy by cytosolic PNPs, accumulation of PNPs in lysosomes, and lysosomal exocytosis are interwoven by proposed regulatory mechanisms.

Austalide K from the Fungus Penicillium rudallense Prevents LPS-Induced Bone Loss in Mice by Inhibiting Osteoclast Differentiation and Promoting Osteoblast Differentiation.

Osteoporosis is a chronic disease that has become a serious public health problem due to the associated reduction in quality of life and its increasing financial burden. It is known that inhibiting osteoclast differentiation and promoting osteoblast formation prevents osteoporosis. As there is no drug with this dual activity without clinical side effects, new alternatives are needed. Here, we demonstrate that austalide K, isolated from the marine fungus Penicillium rudallenes, has dual activities in bone remodeling. Austalide K inhibits the receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast differentiation and improves bone morphogenetic protein (BMP)-2-mediated osteoblast differentiation in vitro without cytotoxicity. The nuclear factor of activated T cells c1 (NFATc1), tartrate-resistant acid phosphatase (TRAP), dendritic cell-specific transmembrane protein (DC-STAMP), and cathepsin K (CTSK) osteoclast-formation-related genes were reduced and alkaline phosphatase (ALP), runt-related transcription factor 2 (Runx2), osteocalcin (OCN), and osteopontin (OPN) (osteoblast activation-related genes) were simultaneously upregulated by treatment with austalide K. Furthermore, austalide K showed good efficacy in an LPS-induced bone loss in vivo model. Bone volume, trabecular separation, trabecular thickness, and bone mineral density were recovered by austalide K. On the basis of these results, austalide K may lead to new drug treatments for bone diseases such as osteoporosis.

Wavelet scattering networks for atomistic systems with extrapolation of material properties.

The dream of machine learning in materials science is for a model to learn the underlying physics of an atomic system, allowing it to move beyond the interpolation of the training set to the prediction of properties that were not present in the original training data. In addition to advances in machine learning architectures and training techniques, achieving this ambitious goal requires a method to convert a 3D atomic system into a feature representation that preserves rotational and translational symmetries, smoothness under small perturbations, and invariance under re-ordering. The atomic orbital wavelet scattering transform preserves these symmetries by construction and has achieved great success as a featurization method for machine learning energy prediction. Both in small molecules and in the bulk amorphous LiαSi system, machine learning models using wavelet scattering coefficients as features have demonstrated a comparable accuracy to density functional theory at a small fraction of the computational cost. In this work, we test the generalizability of our LiαSi energy predictor to properties that were not included in the training set, such as elastic constants and migration barriers. We demonstrate that statistical feature selection methods can reduce over-fitting and lead to remarkable accuracy in these extrapolation tasks.

Multidirectional Cylindrical Piezoelectric Force Sensor: Design and Experimental Validation.

A common design concept of the piezoelectric force sensor, which is to assemble a bump structure from a flat or fine columnar piezoelectric structure or to use a specific type of electrode, is quite limited. In this paper, we propose a new design of cylindrical piezoelectric sensors that can detect multidirectional forces. The proposed sensor consists of four row and four column sensors. The design of the sensor was investigated by the finite element method. The response of the sensor to various force directions was observed, and it was demonstrated that the direction of the force applied to the sensor could be derived from the signals of one row sensor and three column sensors. As a result, this sensor proved to be able to detect forces in the area of 225° about the central axis of the sensor. In addition, a cylindrical sensor was fabricated to verify the proposed sensor and a series of experiments were performed. The simulation and experimental results were compared, and the actual sensor response tended to be similar to the simulation.

Austalides, Osteoclast Differentiation Inhibitors from a Marine-Derived Strain of the Fungus Penicillium rudallense.

Four new meroterpenoids, austalides V-X (1-3) and a farnesylated phthalide derivative (4), were isolated from the culture of the marine fungus Penicillium rudallense, together with eight known meroterpenoids derivatives (5-12). Their structures, including absolute configurations, were determined by spectroscopic methods. All of the isolated compounds were evaluated for their inhibitory activities on the receptor activator of nuclear factor κB ligand (RANKL)-induced osteoclast differentiation. Compounds 1, 2, 5-7, and 10 exhibited potent osteoclast differentiation inhibitory activity with ED50 values of 1.9-2.8 µM.

Evidence for Nanoparticle-Induced Lysosomal Dysfunction in Lung Adenocarcinoma (A549) Cells.

BACKGROUND: Polystyrene nanoparticles (PNP) are taken up by primary rat alveolar epithelial cell monolayers (RAECM) in a time-, dose-, and size-dependent manner without involving endocytosis. Internalized PNP in RAECM activate autophagy, are delivered to lysosomes, and undergo [Ca2+]-dependent exocytosis. In this study, we explored nanoparticle (NP) interactions with A549 cells. METHODS: After exposure to PNP or ambient pollution particles (PM0.2), live single A549 cells were studied using confocal laser scanning microscopy. PNP uptake and egress were investigated and activation of autophagy was confirmed by immunolabeling with LC3-II and LC3-GFP transduction/colocalization with PNP. Mitochondrial membrane potential, mitophagy, and lysosomal membrane permeability (LMP) were assessed in the presence/absence of apical nanoparticle (NP) exposure. RESULTS: PNP uptake into A549 cells decreased in the presence of cytochalasin D, an inhibitor of macropinocytosis. PNP egress was not affected by increased cytosolic [Ca2+]. Autophagy activation was indicated by increased LC3 expression and LC3-GFP colocalization with PNP. Increased LMP was observed following PNP or PM0.2 exposure. Mitochondrial membrane potential was unchanged and mitophagy was not detected after NP exposure. CONCLUSIONS: Interactions between NP and A549 cells involve complex cellular processes leading to lysosomal dysfunction, which may provide opportunities for improved nanoparticle-based therapeutic approaches to lung cancer management.

Water Extracts of Hull-less Waxy Barley (Hordeum vulgare L.) Cultivar 'Boseokchal' Inhibit RANKL-induced Osteoclastogenesis.

Osteoporosis is a disease that leads to reduced bone mineral density. The increase in patient and medical costs because of global aging is recognized as a problem. Decreased bone mass is a common symptom of bone diseases such as Paget's disease, rheumatoid arthritis, and multiple myeloma. Osteoclasts, which directly affect bone mass, show a marked increase in differentiation and activation in the aforementioned diseases. Moreover, these multinucleated cells made from monocytes/macrophages under the influence of RANKL and M-CSF, are the only cells capable of resorbing bones. In this study, we found that the water extracts of Boseokchal (BSC-W) inhibited osteoclast differentiation in vitro and investigated its inhibitory mechanism. BSC-W was obtained by extracting flour of Boseokchal using hexane and water. To osteoclast differentiation, bone marrow-derived macrophage cells (BMMs) were cultured with the vehicle (0.1% DMSO) or BSC-W in the presence of M-CSF and RANKL for 4 days. Cytotoxicity was measured by CCK-8. Gene expression of cells was confirmed by real-time PCR. Protein expression of cells was observed by western blot assay. Bone resorption activity of osteoclast evaluated by bone pit formation assay using an Osteo Assay Plate. BSC-W inhibited RANKL-induced osteoclastogenesis in a dose-dependent manner without exerting a cytotoxic effect on BMMs. BSC-W decreased the transcriptional and translational expression of c-Fos and NFATc1, which are regulators of osteoclastogenesis and reduced the mRNA expression level of TRAP, DC-STAMP, and cathepsin K, which are osteoclast differentiation marker. Furthermore, BSC-W reduced the resorption activity of osteoclasts. Taken together, our results indicate that BSC-W is a useful candidate for health functional foods or therapeutic agents that can help treat bone diseases such as osteoporosis.

Alveolar epithelial cell processing of nanoparticles activates autophagy and lysosomal exocytosis.

Using confocal microscopy, we quantitatively assessed uptake, processing, and egress of near-infrared (NIR)-labeled carboxylated polystyrene nanoparticles (PNP) in live alveolar epithelial cells (AEC) during interactions with primary rat AEC monolayers (RAECM). PNP fluorescence intensity (content) and colocalization with intracellular vesicles in a cell were determined over the entire cell volume via z stacking. Isotropic cuvette-based microfluorimetry was used to determine PNP concentration ([PNP]) from anisotropic measurements of PNP content assessed by confocal microscopy. Results showed that PNP uptake kinetics and steady-state intracellular content decreased as diameter increased from 20 to 200 nm. For 20-nm PNP, uptake rate and steady-state intracellular content increased with increased apical [PNP] but were unaffected by inhibition of endocytic pathways. Intracellular PNP increasingly colocalized with autophagosomes and/or lysosomes over time. PNP egress exhibited fast Ca2+ concentration-dependent release and a slower diffusion-like process. Inhibition of microtubule polymerization curtailed rapid PNP egress, resulting in elevated vesicular and intracellular PNP content. Interference with autophagosome formation led to slower PNP uptake and markedly decreased steady-state intracellular content. At steady state, cytosolic [PNP] was higher than apical [PNP], and vesicular [PNP] (~80% of intracellular PNP content) exceeded both cytosolic and intracellular [PNP]. These data are consistent with the following hypotheses: 1) autophagic processing of nanoparticles is essential for maintenance of AEC integrity; 2) altered autophagy and/or lysosomal exocytosis may lead to AEC injury; and 3) intracellular [PNP] in AEC can be regulated, suggesting strategies for enhancement of nanoparticle-driven AEC gene/drug delivery and/or amelioration of AEC nanoparticle-related cellular toxicity.

Atomistic Simulation Derived Insight on the Irreversible Structural Changes of Si Electrode during Fast and Slow Delithiation.

Quantifying the irreversible chemical and structural changes of Si during cycling remains challenging. In this study, a continuous reactive molecular dynamics delithiation algorithm, with well-controlled potential gradient and delithiation rate, was developed and used to investigate the "natural" delithiation responses of an aluminum-oxide coated silicon thin-film. Fast delithiation led to the formation of dense Si network near the surface and nanoporosity inside the a-LixSi, resulting in 141% volume dilation and significant amount of Li trapped inside (a-Li1.2Si) at the end of delithiation process. In contrast, slow delithiation allowed the a-LixSi to shrink by near-equilibrium condition, demonstrating no permanent inner pore with nearly Li-free structure (a-Li0.2Si) and minimal volume dilation (44%). However, even without trapped Li, the delithiated a-LixSi still exhibited higher volume (lower density) than the equilibrium structure with the same Li concentration, despite delithiation rate. The origin of this excess volume is the loss of directly bonded Si-Si pairs, which made the subsequent relithiation faster. On the basis of the atomistic modeling and the quantified degradation mechanism, battery operating guidelines, including the delithiation rate and the depth of charge to avoid trapped Li and coating delamination, were suggested to improve the durability Si electrodes.

Suppression Effect of Astaxanthin on Osteoclast Formation In Vitro and Bone Loss In Vivo.

Osteoporosis is characterized by a reduction of the bone mineral density (BMD) and microarchitectural deterioration of the bone, which lead to bone fragility and susceptibility to fracture. Astaxanthin (AST) has a variety of biological activities, such as a protective effect against asthma or neuroinflammation, antioxidant effect, and decrease of the osteoclast number in the right mandibles in the periodontitis model. Although treatment with AST is known to have an effect on inflammation, no studies on the effect of AST exposure on bone loss have been performed. Thus, in the present study, we examined the antiosteoporotic effect of AST on bone mass in ovariectomized (OVX) mice and its possible mechanism of action. The administration of AST (5, 10 mg/kg) for 6 weeks suppressed the enhancement of serum calcium, inorganic phosphorus, alkaline phosphatase, total cholesterol, and tartrate-resistant acid phosphatase (TRAP) activity. The bone mineral density (BMD) and bone microarchitecture of the trabecular bone in the tibia and femur were recovered by AST exposure. Moreover, in the in vitro experiment, we demonstrated that AST inhibits osteoclast formation through the expression of the nuclear factor of activated T cells (NFAT) c1, dendritic cell-specific transmembrane protein (DC-STAMP), TRAP, and cathepsin K without any cytotoxic effects on bone marrow-derived macrophages (BMMs). Therefore, we suggest that AST may have therapeutic potential for the treatment of postmenopausal osteoporosis.

Protective Effects of 2,3,5,4'-Tetrahydroxystilbene-2-O-ß-d-glucoside on Ovariectomy Induced Osteoporosis Mouse Model.

2,3,5,4'-Tetrahydroxystilbene-2-O-ß-d-glucoside (TSG), an active polyphenolic component of Polygonum multiflorum, exhibits many pharmacological activities including antioxidant, anti-inflammation, and anti-aging effects. A previous study demonstrated that TSG protected MC3T3-E1 cells from hydrogen peroxide (H2O2) induced cell damage and the inhibition of osteoblastic differentiation. However, no studies have investigated the prevention of ovariectomy-induced bone loss in mice. Therefore, we investigated the effects of TSG on bone loss in ovariectomized mice (OVX). Treatment with TSG (1 and 3 µg/g; i.p.) for six weeks positively affected body weight, uterine weight, organ weight, bone length, and weight change because of estrogen deficiency. The levels of the serum biochemical markers of calcium (Ca), inorganic phosphorus (IP), alkaline phosphatase (ALP), and total cholesterol (TCHO) decreased in the TSG-treated mice when compared with the OVX mice. Additionally, the serum bone alkaline phosphatase (BALP) levels in the TSG-treated OVX mice were significantly increased compared with the OVX mice, while the tartrate-resistant acid phosphatase (TRAP) activity was significantly reduced. Furthermore, the OVX mice treated with TSG showed a significantly reduced bone loss compared to the untreated OVX mice upon micro-computed tomography (CT) analysis. Consequently, bone destruction in osteoporotic mice as a result of ovariectomy was inhibited by the administration of TSG. These findings indicate that TSG effectively prevents bone loss in OVX mice; therefore, it can be considered as a potential therapeutic for the treatment of postmenopausal osteoporosis.

The Dual Role of Oat Bran Water Extract in Bone Homeostasis Through the Regulation of Osteoclastogenesis and Osteoblast Differentiation.

The number of patients with bone metabolic disorders including osteoporosis is increasing worldwide. These disorders often facilitate bone fractures, which seriously impact the patient's quality of life and could lead to further health complications. Bone homeostasis is tightly regulated to balance bone resorption and formation. However, many anti-osteoporotic agents are broadly categorized as either bone forming or anti-resorptive, and their therapeutic use is often limited due to unwanted side effects. Therefore, safe and effective therapeutic agents are needed for osteoporosis. This study aims to clarify the bone protecting effects of oat bran water extract (OBWE) and its mode of action. OBWE inhibited RANKL (receptor activator of nuclear factor-κB ligand)-induced osteoclast differentiation by blocking c-Fos/NFATc1 through the alteration of I-κB. Furthermore, we found that OBWE enhanced BMP-2-stimulated osteoblast differentiation by the induction of Runx2 via Smad signaling molecules. In addition, the anti-osteoporotic activity of OBWE was also evaluated using an in vivo model. OBWE significantly restored ovariectomy-induced bone loss. These in vitro and in vivo results showed that OBWE has the potential to prevent and treat bone metabolic disorders including osteoporosis.

KCNK1 inhibits osteoclastogenesis by blocking the Ca2+ oscillation and JNK-NFATc1 signaling axis.

KCNK1 (K(+) channel, subfamily K, member 1) is a member of the inwardly rectifying K(+) channel family, which drives the membrane potential towards the K(+) balance potential. Here, we investigated its functional relevance during osteoclast differentiation. KCNK1 was significantly induced during osteoclast differentiation, but its functional overexpression significantly inhibited osteoclast differentiation induced by RANKL (also known as TNFSF11), which was accompanied by the attenuation of the RANKL-induced Ca(2+) oscillation, JNK activation and NFATc1 expression. In contrast, KCNK1 knockdown enhanced the RANKL-induced osteoclast differentiation, JNK activation and NFATc1 expression. In conclusion, we suggest that KCNK1 is a negative regulator of osteoclast differentiation; the increase of K(+) influx by its functional blockade might inhibit osteoclast differentiation by inhibiting Ca(2+) oscillation and the JNK-NFATc1 signaling axis. Together with the increased attention on the pharmacological possibilities of using channel inhibition in the treatment of osteoclast-related disorders, further understanding of the functional roles and mechanisms of K(+) channels underlying osteoclast-related diseases could be helpful in developing relevant therapeutic strategies.

Oligopeptide Transport in Rat Lung Alveolar Epithelial Cells is Mediated by Pept2.

PURPOSE: Studies were conducted in primary cultured rat alveolar epithelial cell monolayers to characterize peptide transporter expression and function. METHODS: Freshly isolated rat lung alveolar epithelial cells were purified and cultured on permeable support with and without keratinocyte growth factor (KGF). Messenger RNA and protein expression of Pept1 and Pept2 in alveolar epithelial type I- and type II-like cell monolayers (±KGF, resp.) were examined by RT-PCR and Western blotting. 3H-Glycyl-sarcosine (3H-gly-sar) transmonolayer flux and intracellular accumulation were evaluated in both cell types. RESULTS: RT-PCR showed expression of Pept2, but not Pept1, mRNA in both cell types. Western blot analysis revealed presence of Pept2 protein in type II-like cells, and less in type I-like cells. Bi-directional transmonolayer 3H-gly-sar flux lacked asymmetry in transport in both types of cells. Uptake of 3H-gly-sar from apical fluid of type II-like cells was 7-fold greater than that from basolateral fluid, while no significant differences were observed from apical vs. basolateral fluid of type I-like cells. CONCLUSIONS: This study confirms the absence of Pept1 from rat lung alveolar epithelium in vitro. Functional Pept2 expression in type II-like cell monolayers suggests its involvement in oligopeptide lung disposition, and offers rationale for therapeutic development of di/tripeptides, peptidomimetics employing pulmonary drug delivery.

Knockout Mice Reveal a Major Role for Alveolar Epithelial Type I Cells in Alveolar Fluid Clearance.

Active ion transport by basolateral Na-K-ATPase (Na pump) creates an Na(+) gradient that drives fluid absorption across lung alveolar epithelium. The α1 and ß1 subunits are the most highly expressed Na pump subunits in alveolar epithelial cells (AEC). The specific contribution of the ß1 subunit and the relative contributions of alveolar epithelial type II (AT2) versus type I (AT1) cells to alveolar fluid clearance (AFC) were investigated using two cell type-specific mouse knockout lines in which the ß1 subunit was knocked out in either AT1 cells or both AT1 and AT2 cells. AFC was markedly decreased in both knockout lines, revealing, we believe for the first time, that AT1 cells play a major role in AFC and providing insights into AEC-specific roles in alveolar homeostasis. AEC monolayers derived from knockout mice demonstrated decreased short-circuit current and active Na(+) absorption, consistent with in vivo observations. Neither hyperoxia nor ventilator-induced lung injury increased wet-to-dry lung weight ratios in knockout lungs relative to control lungs. Knockout mice showed increases in Na pump ß3 subunit expression and ß2-adrenergic receptor expression. These results demonstrate a crucial role for the Na pump ß1 subunit in alveolar ion and fluid transport and indicate that both AT1 and AT2 cells make major contributions to these processes and to AFC. Furthermore, they support the feasibility of a general approach to altering alveolar epithelial function in a cell-specific manner that allows direct insights into AT1 versus AT2 cell-specific roles in the lung.

Acredinone C and the Effect of Acredinones on Osteoclastogenic and Osteoblastogenic Activity.

A new inhibitor, acredinone C (1), of receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast differentiation was isolated from the culture broth of the fungus Acremonium sp. (F9A015) along with acredinones A (2) and B (3). The structure of acredinone C (1), which incorporates benzophenone and xanthone moieties, was established by the analyses of combined spectroscopic data including 1D and 2D NMR and MS. All of the acredinones studied efficiently inhibited the RANKL-induced formation of TRAP(+)-MNCs in a dose-dependent manner without any cytotoxicity up to 10 µM. Acredinone A showed dual activity in both osteoclast and osteoblast differentiation in vitro and good efficacy in an animal disease model of bone formation.

Cytotoxic Effects of ZnO Nanoparticles on the Expression of ROS-Responsive Genes in the Human Cell Lines.

In the present investigation, engineered ZnO nanoparticles were tested for their induced oxidative stress in T47D tumor cell lines. The expressions of reactive oxygen species (ROS) related genes, glutathione S-transferase (GST) and catalase were quantified by real time-polymerase chain reaction (RT-PCR). In addition, qualitative analysis of GST was also performed at the cell level using molecular beacon (MB) technology. The tested nanoparticles were 20 nm in size, water-dispersible and treated on human breast tumor epithelial cell lines at 20, 40, 80 µg/ml concentration with 14, 28, 48 h incubation times. Nanoparticles induced expressions of ROS responsive genes at molecular and cellular level, produces consistent results with respect to different dosage and incubation time. The experiment showed that the expression of both GST and catalase genes were maximized at 28 h with 80 µg/ml concentration. However, the toxic effect of the monodisperse ZnO nanoparticles was not significant compared with control experiments, demonstrating its high potential in the applications of nanomedicines for a diagnostic and therapeutic tool.

Praeruptorin A inhibits in vitro migration of preosteoclasts and in vivo bone erosion, possibly due to its potential to target calmodulin.

Excessive activity and/or increased number of osteoclasts lead to bone resorption-related disorders. Here, we investigated the potential of praeruptorin A to inhibit migration/fusion of preosteoclasts in vitro and bone erosion in vivo. Praeruptorin A inhibited the RANKL-induced migration/fusion of preosteoclasts accompanied by the nuclear translocation of NFATc1, a master regulator of osteoclast differentiation. Antimigration/fusion activity of praeruptorin A was also confirmed by evaluating the mRNA expression of fusion-mediating molecules. In silico binding studies and several biochemical assays further revealed the potential of praeruptorin A to bind with Ca(2+)/calmodulin and inhibit its downstream signaling pathways, including the Ca(2+)/calmodulin-CaMKIV-CREB and Ca(2+)/calmodulin-calcineurin signaling axis responsible for controlling NFATc1. In vivo application of praeruptorin A significantly reduced lipopolysaccharide-induced bone erosion, indicating its possible use to treat bone resorption-related disorders. In conclusion, praeruptorin A has the potential to inhibit migration/fusion of preosteoclasts in vitro and bone erosion in vivo by targeting calmodulin and inhibiting the Ca(2+)/calmodulin-CaMKIV-CREB-NFATc1 and/or Ca(2+)/calmodulin-calcineurin-NFATc1 signaling axis.

In vitro cytotoxic evaluation of MgO nanoparticles and their effect on the expression of ROS genes.

Water-dispersible MgO nanoparticles were tested to investigate their cytotoxic effects on oxidative stress gene expression. In this in vitro study, genes related to reactive oxygen species (ROS), glutathione S-transferase (GST) and catalase, were quantified using real-time polymerase chain reactions (molecular level) and molecular beacon technologies (cellular level). The monodispersed MgO nanoparticles, 20 nm in size, were used to treat human cancer cell lines (liver cancer epithelial cells) at different concentrations (25, 75 and 150 µg/mL) and incubation times (24, 48 and 72 h). Both the genetic and cellular cytotoxic screening methods produced consistent results, showing that GST and catalase ROS gene expression was maximized at 150 µg/mL nanoparticle treatment with 48 h incubation. However, the genotoxic effect of MgO nanoparticles was not significant compared with control experiments, which indicates its significant potential applications in nanomedicine as a diagnostic and therapeutic tool.

Knockout mice reveal key roles for claudin 18 in alveolar barrier properties and fluid homeostasis.

Claudin proteins are major constituents of epithelial and endothelial tight junctions (TJs) that regulate paracellular permeability to ions and solutes. Claudin 18, a member of the large claudin family, is highly expressed in lung alveolar epithelium. To elucidate the role of claudin 18 in alveolar epithelial barrier function, we generated claudin 18 knockout (C18 KO) mice. C18 KO mice exhibited increased solute permeability and alveolar fluid clearance (AFC) compared with wild-type control mice. Increased AFC in C18 KO mice was associated with increased ß-adrenergic receptor signaling together with activation of cystic fibrosis transmembrane conductance regulator, higher epithelial sodium channel, and Na-K-ATPase (Na pump) activity and increased Na-K-ATPase ß1 subunit expression. Consistent with in vivo findings, C18 KO alveolar epithelial cell (AEC) monolayers exhibited lower transepithelial electrical resistance and increased solute and ion permeability with unchanged ion selectivity. Claudin 3 and claudin 4 expression was markedly increased in C18 KO mice, whereas claudin 5 expression was unchanged and occludin significantly decreased. Microarray analysis revealed changes in cytoskeleton-associated gene expression in C18 KO mice, consistent with observed F-actin cytoskeletal rearrangement in AEC monolayers. These findings demonstrate a crucial nonredundant role for claudin 18 in the regulation of alveolar epithelial TJ composition and permeability properties. Increased AFC in C18 KO mice identifies a role for claudin 18 in alveolar fluid homeostasis beyond its direct contributions to barrier properties that may, at least in part, compensate for increased permeability.