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Knee cartilage is in an aqueous environment filled with synovial fluid consisting of water, various nutrients, and ions to maintain chondrocyte homeostasis. Aquaporins (AQPs) are water channel proteins that play an important role in water exchange in cells, and AQP1, -3, and -4 are known to be expressed predominantly in cartilage. We evaluated the changes in AQP expression in chondrocytes from human knee articular cartilage in patients of different ages and identified the key factor(s) that mediate age-induced alteration in AQP expression. The mRNA and protein expression of AQP1, -3 and -4 were significantly decreased in fibrocartilage compared to hyaline cartilage and in articular cartilage from older osteoarthritis patients compared to that from young patients. Gene and protein expression of AQP1, -3 and -4 were altered during the chondrogenic differentiation of C3H10T1/2 cells. The causative factors for age-associated decrease in AQP included H 2 O 2 , TNFα, and HMGB1 for AQP1, -3, and -4, respectively. In particular, the protective effect of AQP4 reduction following HMGB1 neutralization was noteworthy. The identification of other potent molecules that regulate AQP expression represents a promising therapeutic approach to suppress cartilage degeneration during aging., (© 2021. The Author(s).)

(1) Background: The unusual accumulation of Na,K-ATPase complexes in the brush border membrane of choroid plexus epithelial cells have intrigued researchers for decades. However, the full range of the expressed Na,K-ATPase subunits and their relation to the microvillus cytoskeleton remains unknown. (2) Methods: RT-PCR analysis, co-immunoprecipitation, native PAGE, mass spectrometry, and differential centrifugation were combined with high-resolution immunofluorescence histochemistry, proximity ligase assays, and stimulated emission depletion (STED) microscopy on mouse choroid plexus cells or tissues in order to resolve these issues. (3) Results: The choroid plexus epithelium expresses Na,K-ATPase subunits α1, α2, β1, β2, β3, and phospholemman. The α1, α2, β1, and β2, subunits are all localized to the brush border membrane, where they appear to form a complex. The ATPase complexes may stabilize in the brush border membrane via anchoring to microvillar actin indirectly through ankyrin-3 or directly via other co-precipitated proteins. Aquaporin 1 (AQP1) may form part of the proposed multi-protein complexes in contrast to another membrane protein, the Na-K-2Cl cotransporter 1 (NKCC1). NKCC1 expression seems necessary for full brush border membrane accumulation of the Na,K-ATPase in the choroid plexus. (4) Conclusion: A multitude of Na,K-ATPase subunits form molecular complexes in the choroid plexus brush border, which may bind to the cytoskeleton by various alternative actin binding proteins.

The accumulation of amyloid-β (Aβ) is a characteristic event in the pathogenesis of Alzheimer's disease (AD). Aquaporin 1 (AQP1) is a membrane water channel protein belonging to the AQP family. AQP1 levels are elevated in the cerebral cortex during the early stages of AD, but the role of AQP1 in AD pathogenesis is unclear. We first determined the expression and distribution of AQP1 in brain tissue samples of AD patients and two AD mouse models (3xTg-AD and 5xFAD). AQP1 accumulation was observed in vulnerable neurons in the cerebral cortex of AD patients, and in neurons affected by the Aβ or tau pathology in the 3xTg-AD and 5xFAD mice. AQP1 levels increased in neurons as aging progressed in the AD mouse models. Stress stimuli increased AQP1 in primary cortical neurons. In response to cellular stress, AQP1 appeared to translocate to endocytic compartments of β- and γ-secretase activities. Ectopic expression of AQP1 in human neuroblastoma cells overexpressing amyloid precussir protein (APP) with the Swedish mutations reduced β-secretase (BACE1)-mediated cleavage of APP and reduced Aβ production without altering the nonamyloidogenic pathway. Conversely, knockdown of AQP1 enhanced BACE1 activity and Aβ production. Immunoprecipitation experiments showed that AQP1 decreased the association of BACE1 with APP. Analysis of a human database showed that the amount of Aβ decreases as the expression of AQP1 increases. These results suggest that the upregulation of AQP1 is an adaptive response of neurons to stress that reduces Aβ production by inhibiting the binding between BACE1 and APP., (© The Author(s) 2020. Published by Oxford University Press on behalf of The Gerontological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: Drought stress can severely affect plant growth and crop yield. The cloning and identification of drought-inducible promoters would be of value for genetically-based strategies to improve resistance of crops to drought., Results: Previous studies showed that the MaPIP1;1 gene encoding an aquaporin is involved in the plant drought stress response. In this study, the promoter pMaPIP1;1, which lies 1362 bp upstream of the MaPIP1;1 transcriptional initiation site, was isolated from the banana genome..And the transcription start site(A) is 47 bp before the ATG. To functionally validate the promoter, various lengths of pMaPIP1;1 were deleted and fused to GUS to generate pMaPIP1;1::GUS fusion constructs that were then transformed into Arabidopsis to generate four transformants termed M-P1, M-P2, M-P3 and M-P4.Mannitol treatment was used to simulate drought conditions. All four transformants reacted well to mannitol treatment. M-P2 (- 1274 bp to - 1) showed the highest transcriptional activity among all transgenic Arabidopsis tissues, indicating that M-P2 was the core region of pMaPIP1;1. This region of the promoter also confers high levels of gene expression in response to mannitol treatment. Using M-P2 as a yeast one-hybrid bait, 23 different transcription factors or genes that interacted with MaPIP1;1 were screened. In an dual luciferase assay for complementarity verification, the transcription factor MADS3 positively regulated MaPIP1;1 transcription when combined with the banana promoter. qRT-PCR showed that MADS3 expression was similar in banana leaves and roots under drought stress. In banana plants grown in 45% soil moisture to mimic drought stress, MaPIP1;1 expression was maximized, which further demonstrated that the MADS3 transcription factor can synergize with MaPIP1;1., Conclusions: Together our results revealed that MaPIP1;1 mediates molecular mechanisms associated with drought responses in banana, and will expand our understanding of how AQP gene expression is regulated. The findings lay a foundation for genetic improvement of banana drought resistance.

Neural crest migration requires cells to move through an environment filled with dense extracellular matrix and mesoderm to reach targets throughout the vertebrate embryo. Here, we use high-resolution microscopy, computational modeling, and in vitro and in vivo cell invasion assays to investigate the function of Aquaporin 1 (AQP-1) signaling. We find that migrating lead cranial neural crest cells express AQP-1 mRNA and protein, implicating a biological role for water channel protein function during invasion. Differential AQP-1 levels affect neural crest cell speed and direction, as well as the length and stability of cell filopodia. Furthermore, AQP-1 enhances matrix metalloprotease activity and colocalizes with phosphorylated focal adhesion kinases. Colocalization of AQP-1 with EphB guidance receptors in the same migrating neural crest cells has novel implications for the concept of guided bulldozing by lead cells during migration., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)

Water influx through aquaporin-1 (AQP-1) has been linked to the ability of different cell types to migrate, and therefore plays an important part in processes like metastasis and angiogenesis. Since the erythroid growth factor erythropoietin (Epo) is now recognized as an angiogenesis promoter, we investigated the participation of AQP-1 as a downstream effector of this cytokine in the migration of endothelial cells. Inhibition of AQP-1 with either mercury ions (Hg 2+ ) or a specific siRNA led to an impaired migration of EA.hy926 endothelial cells exposed to Epo (wound-healing assays). Epo also induced the expression of AQP-1 at mRNA and protein levels, an effect which was dependent on the influx of extracellular calcium through L-type calcium channels as well as TRPC3 channels. The relationship between Epo and AQP-1 was further confirmed at shorter exposure times, as the cytokine was unable to trigger calcium influxes in cells where AQP-1 had previously been knocked down. Moreover, Epo promoted changes in the subcellular localization of AQP-1 as well as rearrangements in the actin cytoskeleton, which are consistent with a migratory phenotype. Worthy of note, carbamylated erythropoietin (cEpo), the non-erythropoietic and non-promigratory derivative of Epo, was incapable of AQP-1 modulation. The therapeutical implications of aquaporin targeting in angiogenesis-related diseases highlight the importance of the present results in the context of the relationship between AQP-1 and Epo., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Multidrug resistance-associated protein 2 (MRP2/ABCC2), a hepatocyte canalicular transporter involved in bile secretion, is downregulated in cholestasis triggered by lipopolysaccharide. The human aquaporin-1 (hAQP1) adenovirus-mediated gene transfer to liver improves cholestasis by incompletely defined mechanisms. Here we found that hAQP1 did not affect MRP2/ABCC2 expression, but significantly increased its transport activity assessed in situ with endogenous and exogenous substrates, likely by a hAQP1-induced increase in canalicular membrane cholesterol amount. Our results suggest that hAQP1-induced MRP2/ABCC2 activation contributes to the cholestasis improvement., (Copyright © 2019 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

Background: Inflammatory cytokines enhanced the progress of the pathogenesis of osteoarthritis, however the mechanisms remain unclear. The objective is to determine aquaporins (AQPs) in the pathogenesis of osteoarthritis., Methods and Findings: Primary rat articular chondrocytes were treated with IL-1β to mimic the early stage of osteoarthritis in vitro. Early osteoarthritis animal model was established by intra-articular injection of 4% papain. Micro- or ultra-structure histopathologic changes, cell viability, apoptosis cells and cell membrane permeability, locations and expressions of AQP1 and AQP3 and matrix were detected in the cartilage or in the chondrocytes of knee. IL-1β could reduce the chondrocytes viability, increase the apoptosis cells, and also impair the cell membrane and organelles. IL-1β significantly induced the up-regulation of AQP1 and AQP3 in the chondrocytes. In the chondrocytes, AQPs were mainly clustered in both membrane and perinuclear region of cytoplasm, while higher AQPs were detected in the superficial and middle layers of the cartilage. With the up-regulation of AQPs, the cartilage matrix was considerably decreased in both the chondrocytes and in the osteoarthritis cartilage. In the early osteoarthritis rat model, serum and synovial fluid confirmed that higher IL-1β could increase the expressions of AQPs, and decrease the cartilage matrix in both the chondrocytes and the cartilage., Conclusions: Inflammatory cytokine IL-1β via up-regulation of AQPs caused the abnormal metabolism of water transport and loss of the cartilage matrix in the chondrocytes, and ultimately exacerbated the pathogenesis of early osteoarthritis. Therefore, AQPs may be a candidate therapeutic target for prevention and treatment of osteoarthritis., Competing Interests: The authors have declared that no competing interests exist.

Objective- Hypoxic pulmonary hypertension (HPH) is characterized by proliferative vascular remodeling. Abnormal pulmonary artery smooth muscle cells proliferation and endothelial dysfunction are the primary cellular bases of vascular remodeling. AQP1 (aquaporin-1) is regulated by oxygen level and has been observed to play a role in the proliferation and migration of pulmonary artery smooth muscle cells. The role of AQP1 in HPH pathogenesis has not been directly determined to date. To determine the possible roles of AQP1 in the pathogenesis of HPH and explore its possible mechanisms. Approach and Results- Aqp1 knockout mice were used, and HPH model was established in this study. Primary pulmonary artery smooth muscle cells, primary mouse lung endothelial cells, and lung tissue sections from HPH model were used. Immunohistochemistry, immunofluorescence and Western blot, cell cycle, apoptosis, and migration analysis were performed in this study. AQP1 expression was upregulated by chronic hypoxia exposure, both in pulmonary artery endothelia and medial smooth muscle layer of mice. Aqp1 deficiency attenuated the elevation of right ventricular systolic pressures and mitigated pulmonary vascular structure remodeling. AQP1 deletion reduced abnormal cell proliferation in pulmonary artery and accompanied with accumulation of HIF (hypoxia-inducible factor). In vitro, Aqp1 deletion reduced hypoxia-induced proliferation, apoptosis resistance, and migration ability of primary cultured pulmonary artery smooth muscle cells and repressed HIF-1α protein stability. Furthermore, Aqp1 deficiency protected lung endothelial cells from apoptosis in response to hypoxic injury. Conclusions- Our data showed that Aqp1 deficiency could attenuate hypoxia-induced vascular remodeling in the development of HPH. AQP1 may be a potential target for pulmonary hypertension treatment.

The Ascomycete fungus Fusarium graminearum, the causal agent of Fusarium head blight of wheat and barley, has become a predominant model organism for the study of fungal phytopathogens. Aquaporins (AQPs) have been implicated in the transport of water, glycerol, and a variety of other small molecules in yeast, plants and animals. However, the role of these proteins in phytopathogenic fungi is not well understood. Here, we identified and attempted to elucidate the function of the five aquaporin genes in F. graminearum. The phylogenetic analysis revealed that FgAQPs are divided into two clades, with FgAQP1 in the first clade. The ∆AQP1 mutant formed whitish colonies with longer aerial hyphae and reduced conidiation and perithecium formation. The ∆AQP1 mutant conidia were morphologically abnormal and appeared to undergo abnormal germination. The ∆AQP1 mutant and the wild type strain were equally pathogenic, while the mutant produced significantly higher quantities of deoxynivalenol (DON). The ∆AQP1 mutant also exhibited increased resistance to osmotic and oxidative stress as well as cell-wall perturbing agents. Using FgAQP1-GFP and DAPI staining, we found that FgAQP1 is localized to the nuclear membrane in conidia. Importantly, deletion of FgAQP1 increased the severity of conidium autophagy. Taken together, these results suggest that FgAQP1 is involved in hyphal development, stress responses, secondary metabolism, and sexual and asexual reproduction in F. graminearum. Unlike the ∆AQP1 mutant, the ∆AQP2, ∆AQP3, ∆AQP4 and ∆AQP5 mutants had no variable phenotypes.

In response to collagen stimulation, platelets use a coordinated system of fluid entry to undergo membrane ballooning, procoagulant spreading, and microvesiculation. We hypothesized that water entry was mediated by the water channel aquaporin-1 (AQP1) and aimed to determine its role in the platelet procoagulant response and thrombosis. We established that human and mouse platelets express AQP1 and localize to internal tubular membrane structures. However, deletion of AQP1 had minimal effects on collagen-induced platelet granule secretion, aggregation, or membrane ballooning. Conversely, procoagulant spreading, microvesiculation, phosphatidylserine exposure, and clot formation time were significantly diminished. Furthermore, in vivo thrombus formation after FeCl3 injury to carotid arteries was also markedly suppressed in AQP1-null mice, but hemostasis after tail bleeding remained normal. The mechanism involves an AQP1-mediated rapid membrane stretching during procoagulant spreading but not ballooning, leading to calcium entry through mechanosensitive cation channels and a full procoagulant response. We conclude that AQP1 is a major regulator of the platelet procoagulant response, able to modulate coagulation after injury or pathologic stimuli without affecting other platelet functional responses or normal hemostasis. Clinically effective AQP1 inhibitors may therefore represent a novel class of antiprocoagulant antithrombotics.

Background: Peritoneal dialysis (PD) contributes to restore acid-base homeostasis in patients with end-stage renal disease. The transport pathways for buffers and carbon dioxide (CO2) across the peritoneal membrane remain poorly understood., Methods: Combining well-established PD protocols, whole-body plethysmography and renal function studies in mice, we investigated molecular mechanisms of acid-base regulation in PD, including the potential role of the water channel aquaporin-1 (AQP1)., Results: After instillation in peritoneal cavity, the pH of acidic dialysis solutions increased within minutes to rapidly equilibrate with blood pH, whereas the neutral pH of biocompatible solutions remained constant. Predictions from the three-pore model of peritoneal transport suggested that local production of HCO3- accounts at least in part for the changes in intraperitoneal pH observed with acidic solutions. Carbonic anhydrase (CA) isoforms were evidenced in the peritoneal membrane and their inhibition with acetazolamide significantly decreased local production of HCO3- and delayed changes in intraperitoneal pH. On the contrary, genetic deletion of AQP1 had no effect on peritoneal transport of buffers and diffusion of CO2. Besides intraperitoneal modifications, the use of acidic dialysis solutions enhanced acid excretion both at pulmonary and renal levels., Conclusions: These findings suggest that changes in intraperitoneal pH during PD are mediated by bidirectional buffer transport and by CA-mediated production of HCO3- in the membrane. The use of acidic solutions enhances acid excretion through respiratory and renal responses, which should be considered in patients with renal failure.

Antiangiogenic therapies have failed to confer survival benefits in patients with metastatic breast cancer (mBC). However, to date, there has not been an inquiry into the roles for acquired versus innate drug resistance in this setting. In this study, we report roles for these distinct phenotypes in determining therapeutic response in a murine model of mBC resistance to the antiangiogenic tyrosine kinase inhibitor sunitinib. Using tumor measurement and vascular patterning approaches, we differentiated tumors displaying innate versus acquired resistance. Bioluminescent imaging of tumor metastases to the liver, lungs, and spleen revealed that sunitinib administration enhances metastasis, but only in tumors displaying innate resistance to therapy. Transcriptomic analysis of tumors displaying acquired versus innate resistance allowed the identification of specific biomarkers, many of which have a role in angiogenesis. In particular, aquaporin-1 upregulation occurred in acquired resistance, mTOR in innate resistance, and pleiotrophin in both settings, suggesting their utility as candidate diagnostics to predict drug response or to design tactics to circumvent resistance. Our results unravel specific features of antiangiogenic resistance, with potential therapeutic implications. Cancer Res; 77(4); 1008-20. ©2016 AACR ., (©2016 American Association for Cancer Research.)

Malignant pleural mesothelioma (MPM) is an aggressive cancer. MPM cells express aquaporin-1 (AQP1) that in other cancers has been shown to participate in the tumor metastasis processes. However, in MPM patients AQP1 overexpression is an independent prognostic factor favoring survival. In this study we aimed at evaluating the role of AQP1 in cell adhesion, migration, and tumor sphere formation in nonmalignant mesothelial cells (MeT-5A) and in epithelioid (M14K) and sarcomatoid (ZL34) MPM cell lines. We used fibronectin (FN) or homologous cell-derived extracellular martrix (ECM) substratum to investigate the role of AQP1 in these experimental phenotypes, inhibiting AQP1 by 10(-5) M mercury chloride (MC). Deposited ECM during cell culture exhibited significant concentration differences among cell types. ZL34 cell adhesion was significantly higher than MeT-5A or M14K cells on FN and ECM. MeT-5A and M14K cell adhesion on FN was sensitive to AQP1 inhibition, whereas AQP1 inhibition on ECM was limited to M14K cells. Wound healing in ZL34 cells was significantly higher than MeT-5A and M14K cells on FN and ECM. AQP1 inhibition significantly lowered cell migration in ZL34 cells on FN and ECM. Sphere formation was not dependent on FN or ECM in the media. AQP1 inhibition in FN media reduced sphere formation in M14K cells, whereas, in ECM, all three cell types were sensitive to AQP1 inhibition., (Copyright © 2016 the American Physiological Society.)

Objective: To study the change of airway mucus secretion under a high temperature and humidity environment, and explore the effects of hot-humid stress and acclimation on the morbidity and mortality of respiratory disease., Methods: Forty-five BABL/c mice were randomly divided into five groups:normal group, hot-humid group I, hot-humid group Ⅱ, hot-humid group Ⅲ, hot-humid group IV, with 9 mice in each group. Mice in normal group were continuously placed in the common environment and sacrificed after 7 days. Mice in other groups were housed in a temperature-and-humidity-controlled environment (33℃±0.5℃, 95%±5%). Mice in hot-humid group I, hot-humid group Ⅱ, hot-humid group Ⅲ and hot-humid group IV were sacrificed after 12 hours, 24 hours, 4 days, 7 days respectively. The protein expression of mucin 5AC(MUC5AC)、epidermal growth factor receptor (EGFR)、aquaporin 1(AQP1) and aquaporin 5(AQP5) in lung were tested by immunohistochemisty., Results: After housed in a high temperature and humidity environment, immunohistochemisty revealed a significant increase of AQP5 12 h later, MUC5AC and EGFR 24 h later, compared with normal group( P <0.05). There was a significant decrease of MUC5AC 7 d later, compared with normal group( P <0.05). There was no significant difference in MUC5AC, EGFR and AQP5 expression among all groups at other time points. There was no difference of AQP1 in humid heat groups, compared with normal group, but a significant decrease in humid heat Ⅲ and IV groups, compared with humid heat I and Ⅱ groups., Conclusions: These findings indicate that hot-humid stress induces mucus hypersecretion in airways, which may be related to the up-regulation of EGFR and down-regulation of AQP5 in MUC5AC. Although acclimation mitigates above-mentioned response, a series of more complex responses may be induced if still in the hot-humid environment.

In marine teleosts, such as the gilthead seabream, several aquaporin paralogs are known to be expressed during the hyperosmotic induction of spermatozoon motility in seawater. Here, we used immunological inhibition of channel function to investigate the physiological roles of Aqp1aa, Aqp1ab, and Aqp7 during seabream sperm activation. Double immunofluorescence microscopy of SW-activated sperm showed that Aqp1aa and Aqp7 were respectively distributed along the flagellum and the head, whereas Aqp1ab accumulated in the head and in discrete areas toward the anterior tail. Inhibition of Aqp1aa reduced the rise of intracellular Ca(2+), which is independent of external Ca(2+) and normally occurs upon activation, and strongly inhibited sperm motility. Impaired Aqp1aa function also prevented the intracellular trafficking of Aqp8b to the mitochondrion, where it acts as a peroxiporin allowing H2O2 efflux and ATP production during activation. However, restoring the Ca(2+) levels with a Ca(2+) ionophore in spermatozoa with immunosuppressed Aqp1aa function fully rescued mitochondrial Aqp8b accumulation and sperm motility. In contrast, exposure of sperm to Aqp1ab and Aqp7 antibodies did not affect motility during the initial phase of activation, but latently compromised the trajectory and the pattern of movement. These data reveal the coordinated action of spatially segregated aquaporins during sperm motility activation in a marine teleost, where flagellar-localized Aqp1aa plays a dual Ca(2+)-dependent role controlling the initiation of sperm motility and the activation of mitochondrial detoxification mechanisms, while Aqp1ab and Aqp7 in the head and anterior tail direct the motion pattern., (© 2015 by the Society for the Study of Reproduction, Inc.)

Background: Marine mammals are well adapted to their hyperosmotic environment. Several morphological and physiological adaptations for water conservation and salt excretion are known to be present in cetaceans, being responsible for regulating salt balance. However, most previous studies have focused on the unique renal physiology of marine mammals, but the molecular bases of these mechanisms remain poorly explored. Many genes have been identified to be involved in osmotic regulation, including the aquaporins. Considering that aquaporin genes were potentially subject to strong selective pressure, the aim of this study was to analyze the molecular evolution of seven aquaporin genes (AQP1, AQP2, AQP3, AQP4, AQP6, AQP7, and AQP9) comparing the lineages of cetaceans and terrestrial mammals., Results: Our results demonstrated strong positive selection in cetacean-specific lineages acting only in the gene for AQP2 (amino acids 23, 83, 107,179, 180, 181, 182), whereas no selection was observed in terrestrial mammalian lineages. We also analyzed the changes in the 3D structure of the aquaporin 2 protein. Signs of strong positive selection in AQP2 sites 179, 180, 181, and 182 were unexpectedly identified only in the baiji lineage, which was the only river dolphin examined in this study. Positive selection in aquaporins AQP1 (45), AQP4 (74), AQP7 (342, 343, 356) was detected in cetaceans and artiodactyls, suggesting that these events are not related to maintaining water and electrolyte homeostasis in seawater., Conclusions: Our results suggest that the AQP2 gene might reflect different selective pressures in maintaining water balance in cetaceans, contributing to the passage from the terrestrial environment to the aquatic. Further studies are necessary, especially those including other freshwater dolphins, who exhibit osmoregulatory mechanisms different from those of marine cetaceans for the same essential task of maintaining serum electrolyte balance.

Background: Aquaporin-1 (AQP-1), found in the early 1990s, a water channel protein in the cell membranes of mammals, has been reported to play an important role in water balance of the respiratory system. However, there are a few studies about the role of AQP in occupational pulmonary disease such as silicosis. This study is to explore the information of aquaporin-1 (AQP-1) in the pathogenesis of silicosis by examining AQP expression, distribution, and location in the lung tissue of a silicotic rat model., Methods: Male Wistar SPF rats were divided randomly into the following 8 groups (n = 8 per group): (1) saline control group: instillation of 1 mL sterile physiological saline; (2) silica groups (ld, 7d, 14d, 28d, 42d, 56d): instillation of a suspension of 50 mg silica dust in a total volume of 1 mL sterile physiological saline; (3) the normal control group without treatment. Immunohistochemistry, immunofluorescence, and western blot were used to detect distribution and expression of AQP-1 in the lung tissue of rats exposed to silica., Results: The expression of AQP-1 between normal and the saline control rats showed no significant difference, but was decreased in the silicotic model rats' lung., Conclusions: The expression of AQP-1 decreased in silicotic rats, which suggests that AQP-1 may play an important role in the formation of silicosis.

The nasal respiratory mucosa is the primary site for evaporative water loss in panting species, necessitating the movement of water across the nasal epithelium. Aquaporins (AQP) are protein channels that facilitate water movement in various fluid transporting tissues of non-panting species. Whether the requirement for enhanced capacity for transepithelial water movement in the nasal respiratory mucosa of panting species has led to differences in AQP localization is unknown. Using immunohistochemistry, we report the localization of AQP1, 3, and 5 in the nasal respiratory mucosa of sheep being exposed to ambient temperatures of ~21 °C or ~38 °C for 4.5 h before death (n=3/treatment). Exposure to either treatment resulted in panting. While exposure to ~38 °C resulted in a higher respiratory frequency (mean difference: 82 breaths min(-1); P<0.001) than exposure to ~21 °C, there was no difference in the localization of AQPs. Connective tissue and vascular endothelial cells expressed AQP1. Glandular acini expressed AQP1 and apically localized AQP5, which was also present in glandular duct cells. Ciliated columnar epithelial cells expressed AQP5 apically and AQP3 basolaterally. Basal cells expressed AQP3. The distribution and co-localization of AQPs in the ovine nasal respiratory mucosa is different to that reported in non-panting species and may reflect the physiological demands associated with enhanced respiratory evaporation. We propose that AQP1, 3, and 5 may constitute a transepithelial water pathway via glandular secretions and across the surface epithelium, which provides a possible means for rapid and controllable water movement in the nasal respiratory mucosa of a panting species., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Introduction: The homeostasis of maternal-fetal fluid exchange is critically important in pregnancy. We sought to investigate the function of aquaporin 1 (AQP1) during pregnancy by examining fluid compartments of AQP1 knockout (AQP1-KO) mice., Methods: Homozygous pregnant AQP1 knockout (KO) mice and control pregnant wild type CD1 mice were sacrificed. Placenta and fetal membranes were examined by immunohistochemical staining for expression of AQP1. The total number of embryos, atrophic embryos, and fetal and placental weight was recorded in each subgroup. Amniotic fluid amount in each sac was measured and amniotic fluid composition was determined. Analysis of variance of factorial design and one-factor analysis of variance were used for statistics., Results: Immunohistochemistry performed on CD1, though not AQP1-KO, placenta revealed that AQP1 was expressed at the vascular endothelial cell, trophocyte, and amnion epithelial cell. In AQP1-KOs, the number of embryos decreased with advancing gestational age. Although the fetal weight of AQP1-KO mice was significantly lower than wild type, amniotic fluid amount was increased in AQP1-KO mice. The AQP1-KO placenta demonstrated increased degeneration with evidence of altered blood vessel structure and increased syncytiotrophoblast nodules., Conclusions: These findings demonstrate a critical role of AQP1 in placental and fetal growth and maternal-fetal fluid homeostasis.

The water channels, aquaporins (AQPs) are key mediators of transcellular fluid transport. However, their expression and role in cardiac tissue is poorly characterized. Particularly, AQP1 was suggested to transport other molecules (nitric oxide (NO), hydrogen peroxide (H2O2)) with potential major bearing on cardiovascular physiology. We therefore examined the expression of all AQPs and the phenotype of AQP1 knockout mice (vs. wild-type littermates) under implanted telemetry in vivo, as well as endothelium-dependent relaxation in isolated aortas and resistance vessels ex vivo. Four aquaporins were expressed in wild-type heart tissue (AQP1, AQP7, AQP4, AQP8) and two aquaporins in aortic and mesenteric vessels (AQP1-AQP7). AQP1 was expressed in endothelial as well as cardiac and vascular muscle cells and co-segregated with caveolin-1. AQP1 knockout (KO) mice exhibited a prominent microcardia and decreased myocyte transverse dimensions despite no change in capillary density. Both male and female AQP1 KO mice had lower mean BP, which was not attributable to altered water balance or autonomic dysfunction (from baroreflex and frequency analysis of BP and HR variability). NO-dependent BP variability was unperturbed. Accordingly, endothelium-derived hyperpolarizing factor (EDH(F)) or NO-dependent relaxation were unchanged in aorta or resistance vessels ex vivo. However, AQP1 KO mesenteric vessels exhibited an increase in endothelial prostanoids-dependent relaxation, together with increased expression of COX-2. This enhanced relaxation was abrogated by COX inhibition. We conclude that AQP1 does not regulate the endothelial EDH or NO-dependent relaxation ex vivo or in vivo, but its deletion decreases baseline BP together with increased prostanoids-dependent relaxation in resistance vessels. Strikingly, this was associated with microcardia, unrelated to perturbed angiogenesis. This may raise interest for new inhibitors of AQP1 and their use to treat hypertrophic cardiac remodeling.

Glucose-based peritoneal dialysis (PD) solutions dilate the parietal and visceral peritoneal microvasculature by endothelium-dependent mechanisms that primarily involve hyperosmolality. This PD-mediated dilation occurs by active intracellular glucose uptake and adenosine Al receptor activation, and by hyperosmolality-stimulated glibenclamide-sensitive potassium channels. Both pathways invoke NO as a second messenger for vasodilation. We hypothesized that during crystalloid-induced osmosis, the osmotic water flux through the transendothelial water-exclusive aquaporin 1 (AQP1) channels is the primary mechanism whereby the endothelium is being stimulated to instigate hyperosmolality-driven vasodilation. Four microvascular levels (diameters in the range 6 - 100 microm) were visualized by intravital videomicroscopy of the terminal ileum in anesthetized rats. Microvascular diameters and flow were measured after topical exposure to a 5% hypertonic mannitol or 2.5% glucose-based PD solution, at baseline and after brief tissue pre-treatment (with 0.1% glutaraldehyde for 10 seconds) or after combined tissue pre-treatment and pharmacologic blockade of AQP1 with HgCl2 (100 micromol/L). Vascular endothelial integrity was verified by the response to acetylcholine (10(-4) mol/L) and sodium nitroprusside (10(-4) mol/L). The hyperosmolar solutions both caused rapid and sustained vasodilation at all microvascular levels, which was not altered by tissue pre-treatment. Inhibition of AQP1 completely abolished the mannitol-induced vasodilation and markedly attenuated the PD fluid-mediated vasodilation. Neither glutaraldehyde pre-treatment nor HgCl2 affected tissue integrity or endothelial cell function. We conclude that the peritoneal microvascular vasodilation caused by hyperosmolar PD fluid is instigated by the osmotic water flux through AQP1. Clinical PD solutions have components other than hyperosmolality that can induce endothelium-dependent peritoneal microvascular vasodilation independent of the AQP1-mediated osmosis.

Deletions of claudin-2 (Cldn2) and aquaporin1 (AQP1) reduce proximal fluid reabsorption (PFR) by about 30% and 50%, respectively. Experiments were done to replicate these observations and to determine in AQP1/claudin-2 double knockout mice (DKO) if the effects of deletions of these established water pores are additive. PFR was determined in inactin/ketamine-anesthetized mice by free-flow micropuncture using single-nephron I(125)-iothalamate (io) clearance. Animal means of PFR [% of glomerular filtration rate (GFR)] derived from TF/Piothalamate ratios in 12 mice in each of four groups [wild type (WT), Cldn2(-/-), AQP1(-/-), and DKO) were 45.8 ± 0.85 (51 tubules), 35.4 ± 1 (54 tubules; P < 0.01 vs. WT), 36.8 ± 1 (63 tubules; P < 0.05 vs. WT), and 33.9 ± 1.4 (69 tubules; P < 0.01 vs. WT). Kidney and single-nephron GFRs (SNGFR) were significantly reduced in all mutant strains. The direct relationship between PFR and SNGFR was maintained in mutant mice, but the slope of this relationship was reduced in the absence of Cldn2 and/or AQP1. Transtubular osmotic pressure differences were not different between WT and Cldn2(-/-) mice, but markedly increased in DKO. In conclusion, the deletion of Cldn2, AQP1, or of both Cldn2 and AQP1 reduces PFR by 22.7%, 19.6%, and 26%, respectively. Our data are consistent with an up to 25% paracellular contribution to PFR. The reduced osmotic water permeability caused by absence of AQP1 augments luminal hypotonicity. Aided by a fall in filtered load, the capacity of non-AQP1-dependent transcellular reabsorption is sufficient to maintain PFR without AQP1 and claudin-2 at 75% of control.

The pathophysiology of arterial capillary proliferation accompanying fibrosis in human cirrhosis remains unclear. However, evidence regarding the molecules participating in the pathophysiological process has been accumulating. Water channel proteins known as aquaporins (AQP)s, notably AQP-1, appear to be involved in the arterial capillary proliferation in the cirrhotic liver.

Neuromyelitis optica (NMO) is an autoimmune disease targeting aquaporin 4 (AQP4), localized mainly at the astrocytic foot processes. Loss of AQP4 and glial fibrillary acidic protein (GFAP) was reported, but the pathological significance of astrocytopathy is still controversial. Here we show that active lesions in NMO display a wide spectrum of pathology even within a single tissue block of an individual patient. We have distinguished six different lesion types. The first reflects complement deposition at the surface of astrocytes, associated with granulocyte infiltration and astrocyte necrosis and followed by demyelination, global tissue destruction and the formation of cystic, necrotic lesions (lesion type 2). Such destructive lesions lead to Wallerian degeneration in lesion-related tracts (lesion type 3). Around active NMO lesions AQP4 may selectively be lost in the absence of aquaporin 1 (AQP1) loss or other structural damage (lesion type 4). Another pattern is characterized by clasmatodendrosis of astrocytes, defined by cytoplasmic swelling and vacuolation, beading and dissolution of their processes and nuclear alterations resembling apoptosis, which was associated with internalization of AQP4 and AQP1 and astrocyte apoptosis in the absence of complement activation. Such lesions give rise to extensive astrocyte loss, which may occur in part in the absence of any other tissue injury, such as demyelination or axonal degeneration (lesion type 5). Finally, lesions with a variable degree of astrocyte clasmatodendrosis are found, which show plaque-like primary demyelination that is associated with oligodendrocyte apoptosis, but with preservation of axons (lesion type 6). In active multiple sclerosis (MS) lesions astrocytes reveal changes of reactive protoplasmatic or fibrillary gliosis. Only in a subset of lesions, in patients with aggressive disease, loss of AQP4 is observed in the initial stage of their formation, which is associated with retraction of astrocyte processes in the absence of complement deposition, granulocyte infiltration or loss of AQP1 or astrocytes. Our data underline the primary assault of astrocytes in NMO lesions, but also indicate that different mechanisms of tissue injury operate in parallel in the same patient and even within the same lesion.

Context: Aquaporin-1 (AQP1) has been proposed as a mediator of estrogen-induced angiogenesis in human breast cancer and endometrial cancer. Elucidation of the molecular mechanisms governing AQP1-mediated, estrogen-induced angiogenesis may contribute to an improved understanding of tumor development., Objective: Our objective was to identify the estrogen-response element (ERE) in the promoter of the Aqp1 gene and investigate the effects and mechanisms of AQP1 on estrogen-induced tubulogenesis of vascular endothelial cells., Setting: The study was conducted in a university hospital in eastern China., Main Outcome Measures: Immunohistological, real-time PCR and Western blot analyses were used to determine the expression AQP1 mRNA and protein in vascular endothelial cells. Chromatin immunoprecipitation analyses and luciferase reporter assays identified ERE-like motif in the promoter of the Aqp1 gene., Results: Expression of AQP1 in blood vessels of human breast and endometrial carcinoma tissues were significantly higher than controls. Estradiol (E2) dose-dependently increased the expression levels of AQP1 mRNA and protein in human umbilical vein endothelial cells (HUVECs). A functional ERE-like motif was identified in the promoter of the Aqp1 gene. AQP1 colocalized with ezrin, a component of the ezrin/radixin/moesin protein complex, and, ezrin colocalized with filamentous actin in HUVECs. Knockdown of AQP1 or ezrin with specific small interfering RNA significantly attenuated the formation of transcytoplasmic filamentous actin stress fibers induced by E2 and inhibited E2-enhanced cell proliferation, migration, invasion, and tubule formation of HUVECs., Conclusions: Estrogen induces AQP1 expression by activating ERE in the promoter of the Aqp1 gene, resulting in tubulogenesis of vascular endothelial cells. These results provide new insights into the molecular mechanisms underpinning the angiogenic effects of estrogen.

Aquaporin-1 (AQP1) is a member of aquaporin family that was previously proven to be involved in myocardial dysfunction; however, the role of AQP1 in myocardial stunning is less clear. To determine the change of AQP1 expression level in the heart and its effect on oedema after global myocardial ischemia, 40 adult goats underwent cardiopulmonary bypass (CPB) with an aortic cross-clamp time of 2 h and total bypass time of 6, 12, 24, 48 and 72 h followed by subsequent reperfusion. AQP1 function of eight goats was inhibited by HgCl(2) during the 24 h on CPB. All groups were compared with eight sham bypass control goats. Myocardial water content was measured, and the APQ1 mRNA and protein levels were detected by RT-PCR and immunoblotting, respectively. The results showed that the degree of myocardial oedema increased significantly at 6, 12, 24 and 48 h of reperfusion after CPB as compared with the control and recovered at 72 h of subsequent reperfusion. Expression levels of AQP1 mRNA and protein began to increase at 12 h and peaked at 24 h of CPB following reperfusion. Furthermore, myocardial oedema was reduced in the HgCl(2) group compared with the time-matched CPB and control groups. These data suggested that AQP1 expression increases in CPB and AQP1 plays an important role in myocardial oedema during CPB., (Copyright © 2012 John Wiley & Sons, Ltd.)

Background/aims: Most cardiac procedures involve the use of cardiopulmonary bypass (CPB), which pumps oxygenated blood to the body while the heart and lungs are isolated. CPB can cause profound alterations V in the homeostasis of physiological fluids, which often results in myocardial edema. In our study, we used sheep CPB model of in vivo and in vitro to assess the relationship between cGMP and AQP1 during CPB., Methods: ODQ, a specific inhibitor of soluble guanylate cyclase (sGC), was used to treat the CPB animals or cardiomyocytes. Left ventricular function of each group was determined by pressure-volume system. Water content of myocardial tissue was assessed by dry-wet weight, and cardiomyocytes water permeability was also calculated. The concentration of cGMP was determined by Radioimmunoassay (RIA). mRNA and protein expression of AQP1 were detected by real-time PCR and western blot, respectively., Results: The relative expression level of AQP1 mRNA and protein at each time point (0, 6, 12, 24 or 48 h) after CPB was significantly increased (1.18-fold at 12 h, 1.77-fold at 24 h and 2.18-fold at 48h) compared with each sham group, the protein expression of AQP1 also showed a rising trend after CPB. The degree of myocardial edema (75.1% at 12 h, 79.3% at 24 h and 81.0% at 48h) increased following the CPB surgery. The mRNA expression level of AQP1 was significantly decreased by 39.7% (p<0.01) upon treatment with ODQ compared with the CPB-only group, and inhibition of cGMP pathway also can significantly decrease the degree of myocardial edema (84.7% in control group, while 75.2% in ODQ group) and improve cardiac function in sheep after CPB. Results of in vitro experiments showed the same changing trends as in vivo., Conclusion: cGMP pathway controls water channels and then affects water intake during CPB through an AQP1-mediated pathway., (© 2013 S. Karger AG, Basel.)

The cochlear spiral ligament is a connective tissue that plays diverse roles in normal hearing. Spiral ligament fibrocytes are classified into functional sub-types that are proposed to carry out specialized roles in fluid homeostasis, the mediation of inflammatory responses to trauma, and the fine tuning of cochlear mechanics. We derived a secondary sub-culture from guinea pig spiral ligament, in which the cells expressed protein markers of type III or "tension" fibrocytes, including non-muscle myosin II (nmII), α-smooth muscle actin (αsma), vimentin, connexin43 (cx43), and aquaporin-1. The cells formed extensive stress fibers containing αsma, which were also associated intimately with nmII expression, and the cells displayed the mechanically contractile phenotype predicted by earlier modeling studies. cx43 immunofluorescence was evident within intercellular plaques, and the cells were coupled via dye-permeable gap junctions. Coupling was blocked by meclofenamic acid (MFA), an inhibitor of cx43-containing channels. The contraction of collagen lattice gels mediated by the cells could be prevented reversibly by blebbistatin, an inhibitor of nmII function. MFA also reduced the gel contraction, suggesting that intercellular coupling modulates contractility. The results demonstrate that these cells can impart nmII-dependent contractile force on a collagenous substrate, and support the hypothesis that type III fibrocytes regulate tension in the spiral ligament-basilar membrane complex, thereby determining auditory sensitivity.

Urea transport (UT) proteins facilitate the concentration of urine by the kidney, suggesting that inhibition of these proteins could have therapeutic use as a diuretic strategy. We screened 100,000 compounds for UT-B inhibition using an optical assay based on the hypotonic lysis of acetamide-loaded mouse erythrocytes. We identified a class of triazolothienopyrimidine UT-B inhibitors; the most potent compound, UTB(inh)-14, fully and reversibly inhibited urea transport with IC(50) values of 10 nM and 25 nM for human and mouse UT-B, respectively. UTB(inh)-14 competed with urea binding at an intracellular site on the UT-B protein. UTB(inh)-14 exhibited low toxicity and high selectivity for UT-B over UT-A isoforms. After intraperitoneal administration of UTB(inh)-14 in mice to achieve predicted therapeutic concentrations in the kidney, urine osmolality after administration of 1-deamino-8-D-arginine-vasopressin was approximately 700 mosm/kg H(2)O lower in UTB(inh)-14-treated mice than vehicle-treated mice. UTB(inh)-14 also increased urine output and reduced urine osmolality in mice given free access to water. UTB(inh)-14 did not reduce urine osmolality in UT-B knockout mice. In summary, these data provide proof of concept for the potential utility of UT inhibitors to reduce urinary concentration in high-vasopressin, fluid-retaining conditions. The diuretic mechanism of UT inhibitors may complement the action of conventional diuretics, which target sodium transport.

In addition to a constitutive water channel activity, several studies suggest Aquaporin-1 (AQP1) functions as a nonselective monovalent cation channel activated by intracellular cGMP, although variability in responsiveness between preparations has led to controversy in the field. Data here support the hypothesis that responsiveness of the AQP1 ionic conductance to cGMP is governed by tyrosine phosphorylation. Wild-type and mutant human AQP1 channels expressed in Xenopus laevis oocytes were characterized by two-electrode voltage clamp and optical osmotic swelling analyses. Quadruple mutation by site-directed mutagenesis of barrier hydrophobic residues (Val50, Leu54, Leu170, Leu174) to alanines in the central pore induced inward rectification of the ionic current and shifted reversal potential by approximately +10 mV, indicating increased permeability of tetraethylammonium ion. Introduction of cysteine at lysine 51 in the central pore (K51C) in a cysteine-less template created new sensitivity to block of the conductance by mercuric ion. Mutations of candidate consensus sites and pharmacological manipulation of serine and threonine phosphorylation did not alter cGMP-dependent responses; however, mutation of tyrosine Y253C or pharmacological dephosphorylation prevented ion channel activation. Modification of Y253C by covalent addition of a negatively charged group [2-sulfonatoethyl methanethiosulfonate sodium salt (MTSES)] rescued the cGMP-activated conductance response, an effect reversed by dithiothreitol. Results support the proposal that phosphorylation of tyrosine Tyr253 in the carboxyl terminal domain, confirmed by Western blot, acts as a master switch regulating responsiveness of AQP1 ion channels to cGMP, and the tetrameric central pore is the ion permeation pathway. These findings advance resolution of a standing controversy and expand our understanding of AQP1 as a multifunctional regulated channel.

Background: Aquaporin (AQP)-1 is expressed in most microvasculature endothelial cells forming water channels that play major roles in a variety of physiologic processes. Our aim was to investigate the regulatory functions of AQP1 on trancellular and paracellular permeability., Methods: We designed, synthesized, and used small interfering RNAs (siRNAs) selective for AQP1 and investigated their effectiveness in altering AQP1-mediated permeability in human pulmonary microvascular endothelial cells., Results: Twenty-four hours after transfection of ECs with siRNAs targeting two different regions of the AQP1 transcript, AQP1 protein was inhibited by 47.8% to 74.6%. siRNAs containing the same percent of base pairs as the AQP1-siRNAs but in random sequence (i.e., scrambled siRNAs) had no effect. Suppression of AQP1 expression in ECs resulted in decreases in epithelial Na+ channel (ENaC) and Na-K ATPase of ECs, and the suppression ENaC α, β, γ, and Na-K ATPase were 43.1% to 48.2%,70.0% to 76.0%, 52.6% to 55.0%, and 72.7% to 79.3%, respectively. The reduced AQP1expression also resulted in decreased cell-cell junction protein level of VE-cadherin, which was suppressed by 36.5% to 59.5% but had no effect on occludin protein. Tube formation assay and tranwell assay showed AQP1 siRNAs induced high permeability of human pulmonary microvascular endothelial cells. Rho-kinase (ROCK) I and ROCK II were increased by 46.0% to 50.0% and 59% to 81%, respectively, AQP1 siRNA treatment accelerated the formation of F-actin bundles, demonstrating the activation of Rho/ROCK signaling pathway, and decreased mitochondrial membrane potential after AQP1 siRNA treatment, showing an important event of apoptosis process., Conclusions: The data demonstrate that AQP1 is a critical participate in regulating endothelial permeability and barrier function and provide direct evidence of the contribution of AQP1 to blood vessel formation.

Several studies have implicated the aquaporins (aqp) 1, 4, and 9 in the pathogenesis of malignant brain tumours, suggesting that they contribute to motility, invasiveness, and oedema formation and facilitate metabolism in tumour cells under hypoxic conditions. We have studied the expression of aqp1, 4, and 9 in biopsies from glioblastomas, isolated tumour stem cells grown in a tumoursphere assay and analyzed the progenitor and differentiated cells from these cultures. We have compared these to the situation in normal rat brain, its stem cells, and differentiated cells derived thereof. In short, qPCR in tumour tissue showed presence of aqp1, 4, and 9. In the tumour progenitor population, aqp9 was markedly more highly expressed, whilst in tumour-derived differentiated cells, aqp4 was downregulated. However, immunostaining did not reveal increased protein expression of aqp9 in the tumourspheres containing progenitor cells; in contrast, its expression (both mRNA and protein) was high in differentiated cultures. We, therefore, propose that aquaporin 9 may have a central role in the tumorigenesis of glioblastoma.

The preovulatory hydration of teleost oocytes is a unique process among vertebrates. The hydration mechanism is most pronounced in marine acanthomorph teleosts that spawn pelagic (floating) eggs; however, the molecular pathway for water influx remains poorly understood. Recently, we revealed that whole-genome duplication (WGD) resulted in teleosts harboring the largest repertoire of molecular water channels in the vertebrate lineage and that a duplicated aquaporin-1 paralog is implicated in the oocyte hydration process. However, the origin and function of the aquaporin-1 paralogs remain equivocal. By integrating the molecular phylogeny with synteny and structural analyses, we show here that the teleost aqp1aa and -1ab paralogs (previously annotated as aqp1a and -1b, respectively) arose by tandem duplication rather than WGD and that the Aqp1ab C-terminus is the most rapidly evolving subdomain within the vertebrate aquaporin superfamily. The functional role of Aqp1ab was investigated in Atlantic halibut, a marine acanthomorph teleost that spawns one of the largest pelagic eggs known. We demonstrate that Aqp1ab is required for full hydration of oocytes undergoing meiotic maturation. We further show that the rapid structural divergence of the C-terminal regulatory domain causes ex vivo loss of function of halibut Aqp1ab when expressed in amphibian oocytes but not in zebrafish or native oocytes. However, by using chimeric constructs of halibut Aqp1aa and -1ab and antisera specifically raised against the C-terminus of Aqp1ab, we found that this cytoplasmic domain regulates in vivo trafficking to the microvillar portion of the oocyte plasma membrane when intraoocytic osmotic pressure is at a maximum. Interestingly, by coinjecting polyA(+) mRNA from postvitellogenic halibut follicles, ex vivo intracellular trafficking of Aqp1ab is rescued in amphibian oocytes. These data reveal that the physiological role of Aqp1ab during meiosis resumption is conserved in teleosts, but the remarkable degeneracy of the cytoplasmic domain has resulted in alternative regulation of the trafficking mechanism.

Previous studies using bloodstream form Trypanosoma brucei have shown that glycerol transport in this parasite occurs via specific membrane proteins, namely a glycerol transporter and glycerol channels [1]. Later, we cloned, expressed and characterized the transport properties of all three aquaglyceroporins (AQP1-3) [2], which were found permeable for water, glycerol and other small uncharged solutes like dihydroxyacetone [3]. Here, we report on the cellular localization of TbAQP1 and TbAQP3 in bloodstream form trypanosomes. Indirect immunofluorescence analysis showed that TbAQP1 is exclusively localized in the flagellar membrane, whereas TbAQP3 was found in the plasma membrane.In addition, we analyzed the functions of all 3 AQPs, using an inducible inheritable double-stranded RNA interference methodology. All AQP knockdown cell lines were still able to survive hypo-osmotic stress conditions, except AQP2 knockdown parasites. Depleted TbAQP2 negatively impacted cell growth and the regulatory volume recovery, whereas AQP1 und 3 knockdown trypanosomes displayed phenotypes consistent with their localization in external membranes. A simultaneous knockdown of all 3 AQPs showed that the cells were able to substitute the missing glycerol uptake capability through a putative glycerol transporter., (Copyright © 2011 S. Karger AG, Basel.)

Peritoneal dialysis involves diffusive and convective transports and osmosis through the highly vascularized peritoneal membrane. Several lines of evidence have demonstrated that the water channel aquaporin-1 (AQP1) corresponds to the ultrasmall pore predicted by the modelization of peritoneal transport. Proof-of-principle studies have shown that up-regulation of the expression of AQP1 in peritoneal capillaries is reflected by increased water permeability and ultrafiltration, without affecting the osmotic gradient and the permeability for small solutes. Inversely, studies in Aqp1 mice have shown that haploinsufficiency in AQP1 is reflected by significant attenuation of water transport. Recent studies have identified lead compounds that could act as agonists of aquaporins, as well as putative binding sites and potential mechanisms of gating the water channel. By modulating water transport, these pharmacological agents could have clinically relevant effects in targeting specific tissues or disease states. These studies on the peritoneal membrane also provide an experimental framework to investigate the role of water channels in the endothelium and various cell types.

Activation of V2 receptors (V2R) during antidiuresis increases the permeability of the inner medullary collecting duct to urea and water. Extracellular osmolality is elevated as the concentrating capacity of the kidney increases. Osmolality is known to contribute to the regulation of collecting duct water (aquaporin-2; AQP2) and urea transporter (UT-A1, UT-A3) regulation. AQP1KO mice are a concentrating mechanism knockout, a defect attributed to the loss of high interstitial osmolality. A V2R-specific agonist, deamino-8-D-arginine vasopressin (dDAVP), was infused into wild-type and AQP1KO mice for 7 days. UT-A1 mRNA and protein abundance were significantly increased in the medullas of wild-type and AQP1KO mice following dDAVP infusion. The mRNA and protein abundance of UT-A3, the basolateral urea transporter, was significantly increased by dDAVP in both wild-type and AQP1KO mice. Semiquantitative immunoblots revealed that dDAVP infusion induced a significant increase in the medullary expression of the endoplasmic reticulum (ER) chaperone GRP78. Immunofluorescence studies demonstrated that GRP78 expression colocalized with AQP2 in principal cells of the papillary tip of the renal medulla. Using immunohistochemistry and immunogold electron microscopy, we demonstrate that vasopressin induced a marked apical targeting of GRP78 in medullary principal cells. Urea-sensitive genes, GADD153 and ATF4 (components of the ER stress pathway), were significantly increased in AQP1KO mice by dDAVP infusion. These findings strongly support an important role of vasopressin in the activation of an ER stress response in renal collecting duct cells, in addition to its role in activating an increase in UT-A1 and UT-A3 abundance.

Internalization of the obligate intracellular apicomplexan parasite, Cryptosporidium parvum, results in the formation of a unique intramembranous yet extracytoplasmic niche on the apical surfaces of host epithelial cells, a process that depends on host cell membrane extension. We previously demonstrated that efficient C. parvum invasion of biliary epithelial cells (cholangiocytes) requires host cell actin polymerization and localized membrane translocation/insertion of Na(+)/glucose cotransporter 1 (SGLT1) and of aquaporin 1 (Aqp1), a water channel, at the attachment site. The resultant localized water influx facilitates parasite cellular invasion by promoting host-cell membrane protrusion. However, the molecular mechanisms by which C. parvum induces membrane translocation/insertion of SGLT1/Aqp1 are obscure. We report here that cultured human cholangiocytes express several nonmuscle myosins, including myosins IIA and IIB. Moreover, C. parvum infection of cultured cholangiocytes results in the localized selective aggregation of myosin IIB but not myosin IIA at the region of parasite attachment, as assessed by dual-label immunofluorescence confocal microscopy. Concordantly, treatment of cells with the myosin light chain kinase inhibitor ML-7 or the myosin II-specific inhibitor blebbistatin or selective RNA-mediated repression of myosin IIB significantly inhibits (P < 0.05) C. parvum cellular invasion (by 60 to 80%). Furthermore ML-7 and blebbistatin significantly decrease (P < 0.02) C. parvum-induced accumulation of SGLT1 at infection sites (by approximately 80%). Thus, localized actomyosin-dependent membrane translocation of transporters/channels initiated by C. parvum is essential for membrane extension and parasite internalization, a phenomenon that may also be relevant to the mechanisms of cell membrane protrusion in general.

Peritoneal dialysis involves diffusive and convective transport and osmosis through the highly vascularized peritoneal membrane. Several lines of evidence have demonstrated that the water channel aquaporin-1 (AQP1) corresponds to the ultrasmall pore predicted by the model of peritoneal transport. Proof-of-principle studies have shown that upregulation of the expression of AQP1 in peritoneal capillaries results in increased water permeability and ultrafiltration, without affecting the osmotic gradient or small solute permeability. Conversely, studies in Aqp1 mice have shown that haplo-insufficiency for AQP1 results in significant attenuation of water transport. Recent studies have demonstrated that AQP1 is involved in the migration of different cell types, including endothelial cells. In parallel, chemical screening has identified lead compounds that could act as antagonists or agonists of AQPs, with description of putative binding sites and potential mechanisms of gating the water channel. By modulating water transport, these pharmacological agents could have clinically relevant effects in targeting specific tissues or disease states.

Peritoneal dialysis involves diffusive and convective transports and osmosis through the highly vascularized peritoneal membrane. Several lines of evidence have demonstrated that the water channel aquaporin-1 (AQP1) corresponds to the ultrasmall pore predicted by the modelization of peritoneal transport. Proof-of-principle studies have shown that upregulation of the expression of AQP1 in peritoneal capillaries is reflected by increased water permeability and ultrafiltration, without affecting the osmotic gradient and the permeability for small solutes. Inversely, studies in Aqp1 mice have shown that haplo-insufficiency in AQP1 is reflected by significant attenuation of water transport. Recent studies have identified lead compounds that could act as agonists of aquaporins, as well as putative binding sites and potential mechanisms of gating the water channel. By modulating water transport, these pharmacological agents could have clinically relevant effects in targeting specific tissues or disease states. These studies on the peritoneal membrane also provide an experimental framework to investigate the role of water channels in the endothelium and various cell types.

Recent studies revealed an important role of aquaporins (AQPs) in cell migration and migration-associated cell function such as angiogenesis, wound healing, and neutrophil motility. Migration of tumor cells is a crucial step in tumor invasion and metastasis. In the present study, we investigated the expression of AQP1 in human HT20 colon cancer cells and characterized its function in cell migration. By reverse transcription-polymerase chain reaction (RT-PCR) and immunoblot analysis, expression of AQP1 was identified in HT20 cell lines. Immunofluorescence analysis indicated expression of AQP1 protein in the plasma membrane of HT20 cells. The recombinant adenovirus expressing human AQP1 increased the mRNA and protein expression of AQP1 in HT20 cells. In contrary, the RNA interference vector of AQP1 effectively inhibited the mRNA and protein expression of AQP1 in HT20 cells. Adenovirus-mediated high expression of AQP1 in HT20 cells increased relative plasma membrane water permeability and migration rate in both wound healing and invasive transwell migration assays. In contrary, RNA interference vector-mediated low expression of AQP1 in HT20 cells reduced relative plasma membrane water permeability and migration rate. AQP1 expression induced relocalization of actin protein and activation of RhoA and Rac. In nude mice, AQP1 increased extravasation of HT20 Cells in lung after tail vein injection. The results provided the direct evidence that aquaporin-mediated plasma membrane water permeability plays an important role in colon cancer cell migration and may be associated with colon cancer invasion and metastasis.

The mechanism by which extracellular hypotonicity stimulates release of renin from juxtaglomerular (JG) cells is unknown. We hypothesized that osmotically induced renin release depends on water movement through aquaporin-1 (AQP1) water channels and subsequent prostanoid formation. We recorded membrane capacitance (C(m)) by whole-cell patch clamp in single JG cells as an index of exocytosis. Hypotonicity increased C(m) significantly and enhanced outward current. Indomethacin, PLA(2) inhibition, and an antagonist of prostaglandin transport impaired the C(m) and current responses to hypotonicity. Hypotonicity also increased exocytosis as determined by a decrease in single JG cell quinacrine fluorescence in an indomethacin-sensitive manner. In single JG cells from COX-2(-/ -) and AQP1(-/ -) mice, hypotonicity increased neither C(m) nor outward current, but 0.1-muM PGE(2) increased both in these cells. A reduction in osmolality enhanced cAMP accumulation in JG cells but not in renin-producing As4.1 cells; only the former had detectable AQP1 expression. Inhibition of protein kinase A blocked the hypotonicity-induced C(m) and current response in JG cells. Taken together, our results show that a 5 to 7% decrease in extracellular tonicity leads to AQP1-mediated water influx in JG cells, PLA(2)/COX-2-mediated prostaglandin-dependent formation of cAMP, and activation of PKA, which promotes exocytosis of renin.

The number of experimentally derived structures of cellular components is rapidly expanding, and this phenomenon is accompanied by the development of a new semiotic system for teaching. The infographic approach is shifting from a schematic toward a more realistic representation of cellular components. By realistic we mean artist-prepared or computer graphic images that closely resemble experimentally derived structures and are characterized by a low level of styling and simplification. This change brings about a new challenge for teachers: designing course instructions that allow students to interpret these images in a meaningful way. To determine how students deal with this change, we designed several image-based, in-course assessments. The images were highly relevant for the cell biology course but did not resemble any of the images in the teaching documents. We asked students to label the cellular components, describe their function, or both. What we learned from these tests is that realistic images, with a higher apparent level of complexity, do not deter students from investigating their meaning. When given a choice, the students do not necessarily choose the most simplified representation, and they were sensitive to functional indications embedded in realistic images.

We examined the expression of aquaporin (AQP)-1 in monolayers of the rat gastric epithelial cell line RGM1 and investigated the roles AQP1 plays in the epithelial restitution during wound healing. A round wound of constant size was produced in the center of a confluent cell monolayer using a rotating silicon tip. The RGM1 cells expressed AQP1 mRNA. Hg(2+) (HgCl(2)), an inhibitor of AQPs, suppressed cell migration during wound repair in a concentration-dependent manner. Likewise, cell migration was also impaired in cells in which AQP1 was knocked down by RNA interference, resulting in a marked delay of wound healing. The AQP1 knockdown RGM1 cells showed a decrease in the formation of membrane protrusions (lamellipodia) at the leading edge of the wound as compared with control RGM1 cells. These results suggest for the first time that AQP1 plays a crucial role in gastric epithelial cell migration during wound healing.

Aquaporin-1 (AQP1) water channels are expressed in corneal keratocytes, which become activated and migrate following corneal wounding. The purpose of this study was to investigate the role of AQP1 in keratocyte migration. Keratocyte primary cell cultures from wildtype and AQP1-null mice were compared, as well as keratocyte cultures from pig cornea in which AQP1 expression was modulated by RNAi knockdown and adenovirus-mediated overexpression. AQP1 expression was found in a plasma membrane pattern in corneal stromal and cultured keratocytes. Osmotic water permeability, as measured by calcein fluorescence quenching, was AQP1-dependent in cultured keratocytes, as was keratocyte migration following a scratch wound. Keratocyte migration in vivo was compared in wildtype and AQP1 knockout mice by histology and immunofluorescence of corneal sections at different times after partial-thickness corneal stromal debridement. AQP1 expression in keratocytes was increased by 24h after corneal debridement. Wound healing and keratocyte appearance near the wound margin were significantly reduced in AQP1 knockout mice, and the number of neutrophils was increased. These results implicate AQP1 water permeability as a new determinant of keratocyte migration in cornea.