Differential regulation of the water channel protein aquaporins in chondrocytes of human knee articular cartilage by aging.

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 H2O2, 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.

Multiple Na,K-ATPase Subunits Colocalize in the Brush Border of Mouse Choroid Plexus Epithelial Cells.

(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, ß1, ß2, ß3, and phospholemman. The α1, α2, ß1, and ß2, 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.

Neuronal Aquaporin 1 Inhibits Amyloidogenesis by Suppressing the Interaction Between Beta-Secretase and Amyloid Precursor Protein.

The accumulation of amyloid-ß (Aß) 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 Aß 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 ß- and γ-secretase activities. Ectopic expression of AQP1 in human neuroblastoma cells overexpressing amyloid precussir protein (APP) with the Swedish mutations reduced ß-secretase (BACE1)-mediated cleavage of APP and reduced Aß production without altering the nonamyloidogenic pathway. Conversely, knockdown of AQP1 enhanced BACE1 activity and Aß production. Immunoprecipitation experiments showed that AQP1 decreased the association of BACE1 with APP. Analysis of a human database showed that the amount of Aß 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 Aß production by inhibiting the binding between BACE1 and APP.

Identification of transcription factors interacting with a 1274 bp promoter of MaPIP1;1 which confers high-level gene expression and drought stress Inducibility in transgenic Arabidopsis thaliana.

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.

Aquaporin-1 plays a key role in erythropoietin-induced endothelial cell migration.

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 (Hg2+) 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.

Neural crest cells bulldoze through the microenvironment using Aquaporin 1 to stabilize filopodia.

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.

Inflammatory cytokines via up-regulation of aquaporins deteriorated the pathogenesis of early osteoarthritis.

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-1ß 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-1ß could reduce the chondrocytes viability, increase the apoptosis cells, and also impair the cell membrane and organelles. IL-1ß 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-1ß could increase the expressions of AQPs, and decrease the cartilage matrix in both the chondrocytes and the cartilage. CONCLUSIONS: Inflammatory cytokine IL-1ß 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.

Improved hepatic MRP2/ABCC2 transport activity in LPS-induced cholestasis by aquaporin-1 gene transfer.

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.

Aqp-1 Gene Knockout Attenuates Hypoxic Pulmonary Hypertension of Mice.

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.

Aquaporin-1 regulates platelet procoagulant membrane dynamics and in vivo thrombosis.

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.

Aquaporin1 regulates development, secondary metabolism and stress responses in Fusarium graminearum.

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.

Mechanisms of acid-base regulation in peritoneal dialysis.

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.

Sunitinib Treatment Enhances Metastasis of Innately Drug-Resistant Breast Tumors.

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.

Influence of AQP1 on cell adhesion, migration, and tumor sphere formation in malignant pleural mesothelioma is substratum- and histological-type dependent.

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.

[Influence of hot-humid stress and acclimation on airway mucus production in lungs of mice].

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.

Decreased Expression of Aquaporin-1 in Lung Tissue of Silicotic Rats.

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.

Coordinated Action of Aquaporins Regulates Sperm Motility in a Marine Teleost.

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.

Evidence of Positive Selection of Aquaporins Genes from Pontoporia blainvillei during the Evolutionary Process of Cetaceans.

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.

Hyperosmolality-mediated peritoneal microvascular vasodilation is linked to aquaporin function.

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.