RESUMO
BACKGROUND: High altitude pulmonary edema (HAPE) affects individuals and is characterized by alveolar flooding with protein-rich edema as a consequence of blood-gas barrier disruption. In this study, we hypothesized that aquaporin 5 (AQP5) which is one kind of water channels may play a role in preservation of alveolar epithelial barrier integrity in high altitude pulmonary edema (HAPE). METHODS: Therefore, we established a model in Wildtype mice and AQP5 -/- mice were assingned to normoxic rest (NR), hypoxic rest (HR) and hypoxic exercise (HE) group. Mice were produced by training to walk at treadmill for exercising and chamber pressure was reduced to simulate climbing an altitude of 5000 m for 48 hours. Studies using BAL in HAPE mice to demonstrated that edema is caused leakage of albumin proteins and red cells across the alveolarcapillary barrier in the absence of any evidence of inflammation. RESULTS: In this study, the Lung wet/dry weight ratio and broncholalveolar lavage protein concentrations were slightly increased in HE AQP5 -/- mice compared to wildtype mice. And histologic evidence of hemorrhagic pulmonary edema was distinctly shown in HE group. The lung Evan's blue permeability of HE group was showed slightly increased compare to the wildtype groups, and HR group was showed a medium situation from normal to HAPE development compared with NR and HE group. CONCLUSIONS: Deletion of AQP5 slightly increased lung edema and lung injury compared to wildtype mice during HAPE development, which suggested that the AQP5 plays an important role in HAPE formation induced by high altitude simulation.
Assuntos
Doença da Altitude/metabolismo , Altitude , Aquaporina 5/metabolismo , Barreira Alveolocapilar/metabolismo , Hipertensão Pulmonar/metabolismo , Alvéolos Pulmonares/metabolismo , Albuminas/metabolismo , Doença da Altitude/genética , Doença da Altitude/patologia , Animais , Aquaporina 5/deficiência , Aquaporina 5/genética , Barreira Alveolocapilar/patologia , Líquido da Lavagem Broncoalveolar/química , Permeabilidade Capilar , Modelos Animais de Doenças , Hipertensão Pulmonar/genética , Hipertensão Pulmonar/patologia , Hipóxia/complicações , Masculino , Camundongos , Camundongos Knockout , Esforço Físico , Alvéolos Pulmonares/patologia , Fatores de TempoRESUMO
BACKGROUND: Aquaporin (AQP) is a membrane protein that facilitates osmotic water transport. Aquaporin 5 (AQP5) expresses at type I alveolar epithelia of apical membrane that confers high osmotic water permeability. Osmosis or stretch challenge in alveoli significantly up-regulates AQP5 expression, which suggests that AQP5 may play a role in the maintenance of epithelia barrier function. Pseudomonas aeruginosa (PA), a leading gram-negative bacterial frequently isolated from ventilation-associated pneumonia patients, disrupts alveolar and airway epithelial cells and subsequently leads to blood dissemination. In this study, we hypothesized that AQP5 might be protective in acute lung injury induced by PA, and deletion of AQP5 might lead to aggravated lung injury. METHODS: Lung injury model was induced by intratracheal instillation of PA (1 × 10(6) colony-forming unit) in wild-type and AQP5 knockout mice, 2 hours and 6 hours later, blood and lung lysate were cultured to detect blood dissemination, bronchoalveolar lavage fluid and lung tissue were collected for histology analysis. Lung injury assessment, wet/dry weight ratio, protein leakage, and Evan's blue dye extravasation were evaluated for pulmonary barrier function. RESULTS: AQP5 deficiency led to increased bacterial blood dissemination and aggravated lung injury during PA infection, and AQP5 deletion also reduced mucin production in lung. Moreover, AQP5 deficiency showed declined activation of mitogen-activated protein kinase and nuclear factor-kappa B pathways in lungs before and after PA infection. CONCLUSION: Our data demonstrated that AQP5 plays a protective role in the maintenance of pulmonary barrier function against PA infection.
Assuntos
Lesão Pulmonar Aguda/microbiologia , Lesão Pulmonar Aguda/prevenção & controle , Aquaporina 5/fisiologia , Infecções por Pseudomonas/prevenção & controle , Análise de Variância , Animais , Aquaporina 5/deficiência , Western Blotting , Lavagem Broncoalveolar , Modelos Animais de Doenças , Extravasamento de Materiais Terapêuticos e Diagnósticos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NF-kappa B/metabolismo , Tamanho do Órgão , Pseudomonas aeruginosa , Reação em Cadeia da Polimerase em Tempo Real , Células-TroncoRESUMO
BACKGROUND: Aquaporin 5 (AQP5) is widely expressed in various organ and tissues. In light of the novel oncogenic properties of aquaporins (AQPs), here we investigated the effect of AQP5 knockdown by RNAi on transmembrane osmotic water permeability, cell migration potential and cell volume regulation ability. METHODS: AQP5 expression was inhibited by short hairpin RNA in SPC-A1 cells, a lung adenocarcinoma cell line. Cells loaded with a fluoroprobe (calcein-AM) were immersed in either isosmotic, hyperosmotic or hyposmotic solutions, and fluorescence intensity was recorded using confocal microscopy. These measurements were used to calculate osmotic water permeability coefficients (Pf) and to monitor regulated volume decrease (RVD). Tumor cell migration and invasion assays were performed in a modified Boyden chamber. Wound healing and colony forming ability were also tested. RESULTS: Although self-quenching was not found in SPC-A1 cells, we observed a linear relationship between fluorescence intensity and cell water volume, suggesting that this method is a sensitive and reproducible way to measure single-cell transmembrane water permeability. Cells in which the AQP5 gene was silenced showed a 49.4% decrease in osmotic water permeability, a 55.3% decrease in migration and a 28.4% decrease in invasion potential. In addition, RVD decreased remarkably with reduced osmotic water permeability. CONCLUSION: Our results suggest that AQP5, which mediates water permeability and thus regulates cell shape and volume, is a potentially important determinant in cell migration.
Assuntos
Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Aquaporina 5/deficiência , Movimento Celular/fisiologia , Tamanho Celular , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Adenocarcinoma de Pulmão , Aquaporina 5/metabolismo , Transporte Biológico/fisiologia , Linhagem Celular Tumoral , Técnicas de Silenciamento de Genes/métodos , Humanos , Água/metabolismoRESUMO
Airway inflammation and mucus hyperproduction play the central role in the development of asthma, although the mechanisms remain unclear. The aquaporin (AQP)-5 may be involved in the process due to its contribution to the volume of liquid secreted from the airways. The present study firstly found the overexpression of AQP5 in the airway epithelium and submucosal glands of asthmatics. Furthermore, we aimed at evaluating the role of AQP5 in airway inflammation and mucous hyperproductions during chronic allergic responses to house dust mite (HDM). Bronchoalveolar lavage levels of interleukin (IL)-2, IL-4, IL-10, interferon-γ and Mucin 5AC (MUC5AC), and number of peribronchial and perivascular cells were measured in AQP5 wild-type and AQP5 knockout (KO) mice. We found that HDM induced airway inflammation, lung Th2 cell accumulation and mucin hypersecretion in C57BL/6 mice rather than AQP5 KO mice. Expression of MUC5AC and MUC5B proteins and genes in the lung tissue was significantly lower in AQP5 KO mice. Thus, our results implicate involvement of AQP5 in the development of airway inflammation and mucous hyperproduction during chronic asthma.
Assuntos
Aquaporina 5 , Asma/metabolismo , Líquido da Lavagem Broncoalveolar/química , Mucina-5AC/biossíntese , Mucina-5B/biossíntese , Muco/imunologia , Pyroglyphidae/imunologia , Sistema Respiratório/metabolismo , Animais , Antígenos/imunologia , Antígenos/metabolismo , Aquaporina 5/deficiência , Aquaporina 5/genética , Asma/imunologia , Líquido da Lavagem Broncoalveolar/imunologia , Citocinas/imunologia , Citocinas/metabolismo , Deleção de Genes , Hipersensibilidade/imunologia , Hipersensibilidade/metabolismo , Inflamação/imunologia , Inflamação/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Muco/metabolismo , Pyroglyphidae/metabolismo , Sistema Respiratório/imunologia , Sistema Respiratório/patologia , Células Th2/imunologia , Células Th2/metabolismoRESUMO
PURPOSE: Tear film composition depends on water and ion transport across ocular surface epithelia and on fluid secretion by lacrimal glands. The purpose of this study was to establish in situ fluorescence methods to measure tear film ionic concentrations and pH in mice and to determine whether tear film composition is sensitive to deficiency of the major ocular surface aquaporin water channels. METHODS: Tear film ionic concentrations and pH were measured in anesthetized mice by ratio imaging fluorescence microscopy after topical application of ion/pH-sensing, dual-wavelength fluorescent indicators. [Na(+)], [K(+)], and [Cl(-)] were measured with membrane-impermeant indicators developed by our laboratory, and pH was measured with bis(carboxyethyl)-carboxyfluorescein fluorescence-conjugated dextran. Measurements were performed on wild-type mice and on knockout mice lacking aquaporins AQP1, AQP3, and AQP5. RESULTS: In wild-type mice, tear film [Na(+)] was 139 +/- 8 mM, [K(+)] was 48 +/- 1 mM, [Cl(-)] was 127 +/- 4 mM, and pH was 7.59 +/- 0.2 (SE; n = 5-8). pH did not differ significantly in the AQP knockout mice. [Na(+)] was increased by approximately twofold in AQP5 null mice (230 +/- 20 mM) and was greatly reduced after exposure of the ocular surface to a humidified atmosphere. [K(+)] was mildly reduced in AQP1 null mice. CONCLUSIONS: These results establish an in situ optical methodology to measure tear film [Na(+)], [K(+)], [Cl(-)], and pH in living mice, without the need for fluid sampling. Tear film hypertonicity in AQP5 deficiency is likely caused by reduced transcorneal water secretion in response to evaporative water loss.
Assuntos
Aquaporina 5/fisiologia , Cloretos/metabolismo , Córnea/metabolismo , Potássio/metabolismo , Sódio/metabolismo , Lágrimas/metabolismo , Animais , Aquaporina 1/deficiência , Aquaporina 1/fisiologia , Aquaporina 3/deficiência , Aquaporina 3/fisiologia , Aquaporina 5/deficiência , Água Corporal/metabolismo , Corantes Fluorescentes , Concentração de Íons de Hidrogênio , Hibridização in Situ Fluorescente , Transporte de Íons/fisiologia , Camundongos , Camundongos Knockout , Microscopia de Fluorescência , Concentração OsmolarRESUMO
Regulation of cell volume in response to changes in osmolarity is critical for cell function and survival. However, the molecular basis of osmosensation and regulation of cell volume are not clearly understood. We have examined the mechanism of regulatory volume decrease (RVD) in salivary gland cells and report a novel association between osmosensing TRPV4 (transient receptor potential vanalloid 4) and AQP5 (aquaporin 5), which is required for regulating water permeability and cell volume. Exposure of salivary gland cells and acini to hypotonicity elicited an increase in cell volume and activation of RVD. Hypotonicity also activated Ca2+ entry, which was required for subsequent RVD. Ca2+ entry was associated with a distinct nonselective cation current that was activated by 4alphaPDD and inhibited by ruthenium red, suggesting involvement of TRPV4. Consistent with this, endogenous TRPV4 was detected in cells and in the apical region of acini along AQP5. Importantly, acinar cells from mice lacking either TRPV4 or AQP5 displayed greatly reduced Ca2+ entry and loss of RVD in response to hypotonicity, although the extent of cell swelling was similar. Expression of N terminus-deleted AQP5 suppressed TRPV4 activation and RVD but not cell swelling. Furthermore, hypotonicity increased the association and surface expression of AQP5 and TRPV4. Both these effects and RVD were reduced by actin depolymerization. These data demonstrate that (i) activation of TRPV4 by hypotonicity depends on AQP5, not on cell swelling per se, and (ii) TRPV4 and AQP5 concertedly control regulatory volume decrease. These data suggest a potentially important role for TRPV4 in salivary gland function.
Assuntos
Aquaporina 5/metabolismo , Tamanho Celular , Glândulas Salivares/citologia , Glândulas Salivares/metabolismo , Canais de Cátion TRPV/metabolismo , Animais , Aquaporina 5/química , Aquaporina 5/deficiência , Aquaporina 5/genética , Cálcio/metabolismo , Linhagem Celular , Humanos , Soluções Hipotônicas , Camundongos , Camundongos Knockout , Mutagênese , Concentração Osmolar , Ratos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Deleção de Sequência , Canais de Cátion TRPV/deficiência , Canais de Cátion TRPV/genéticaRESUMO
Airway mucus is regulated by many inflammatory mediators such as ILs, TNF-alpha, EGF, PGF2alpha, LT, and so on. Recently, the relationship between membrane ion channel and mucus production has been under investigation. The present study aimed to examine whether AQP5 was involved in modulation of mucin expression and secretion in airway submucosal gland cells (SPC-A1). A recombinant plasmid (pShAQP5) containing small hairpin RNA expression cassette targeting AQP5 sequence was constructed. In pShAQP5 transiently transfected cells, ELISA showed MUC5AC synthesis and secretion were increased by 57.9% and 85.3%, respectively, on day 5 after pShAQP5 transfection. While in five stably transfected clones (shAQP5-G1, G2, G3, A2, and A5), the upregulated levels of MUC5AC mRNA were 118%, 165%, 65%, 123%, and 38%, respectively. The elevated levels of MUC5AC synthesis and secretion varied from 59-156% and 33-166%, respectively. This is the first reliable investigation of the regulation of MUC5AC mucin secretion by silencing AQP5. Further study of the regulatory mechanism between AQPs and mucins may provide new strategies for development of novel antihypersecretory drugs in airway diseases.