AQP1 Protein Research Articles

Postmenopausal response to angiotensin II‐induced hypertension is blunted during perimenopause: a study in the accelerated ovarian failure (AOF) model of menopause

A female's risk of developing hypertension (HTN) increases dramatically after menopause. This study aimed to investigate the onset of ANG II‐induced HTN in the AOF model of menopause (VCD model). The AOF model preserves a perimenopausal period in mice, which is increasingly recognized as a critical timeframe for the treatment and prevention of diseases. We hypothesized that ANG II‐infusion would elicit a differential response when administered in peri vs postmenopause and examined the cardiovascular and renal impact. Female VCD treated BL/6 mice (peri and postmenopause) received 800 ng/kg/min ANG II via osmotic mini‐pump for 10 days. ANG II elicited a significant increase in MAP in postmenopausal females (ANG/POST) vs cycling females (C 105 vs ANG 128 vs ANG/POST 143 mmHg, P<0.05). In contrast, there was no difference in MAP in the perimenopause study; ANG II increased MAP in cycling and PERI groups (~126 mmHg). AQP2 protein expression significantly decreased in the renal cortex of ANG/POST mice alone (C 100 vs ANG 98 vs ANG/POST 78%). Renal inflammation, measured by ICAM‐1and F4/80 staining, increased in ANG/POST vs all other groups. Cardiac collagen volume fraction significantly increased in ANG/POST vs ANG (C 0.36 vs ANG 1.36 vs ANG/POST 2.94%). Our data suggests that the perimenopause to menopause transition is a key stage in the loss of a female's protection against developing HTN, and renal and CV complications.

935 OVEREXPRESSION OF CREM ALPHA IN CD4+ T CELLS ACCELERATES ACUTE ConA-INDUCED HEPATITIS BUT DOES NOT INFLUENCE FIBROSIS

Introduction: Aquaporins (AQPs) are a family of channel proteins that facilitate the osmotic movement of water and small solutes across biological membranes. In the hepatobiliary tract, AQPs participate in formation and flow of bile. We have previously shown that Liver X Receptor b (LXRb), a nuclear receptor activated by oxysterols, controls the expression of AQP-1 in pancreas and kidney. Aim and Methods: In the present study we aimed to investigate the role of LXRb in controlling water channel homeostasis in the hepatobiliary tract, in-vivo. Both male and female WT and LXRb−/− mice were studied at the age of 12 months. Results: In WT mice, LXRb protein was strongly expressed in the nuclei of intrahepatic cholangiocytes while a weak expression was detected in hepatocytes. Male LXRb−/− mice demonstrated a significant increase of serum levels of both total bilirubin and alkaline phosphatase, indicating the presence of a mild cholestasis. In the whole liver, there was a significant reduction in the levels of both the mRNA of AQP-1 and AQP-4 in male LXRb−/− mice. Interestingly, AQP-4 protein expression was detected on the plasma membrane of male WT hepatocytes while in LXRb−/− mice, the immunoreactivity was identified in the cytosol, suggesting a possible defect in its transport to the membrane. No AQP-4 was detected in female mice either WT or LXRb−/−. In the kidney, AQP-2 trafficking is controlled by caveolin-1, a major component of caveolae, that is involved in transcytosis, endocytosis and signal transduction. In prostate cancer cells, caveolin-1 is upregulated by testosterone and acts as Androgen Receptor (AR) co-regulator being involved in non-genomic effects of AR. In LXRb−/− male mice, strong immunoreactivity of caveolin-1 was detected in the cytosol while in WT mice it was on the cell surface. Thus there is impaired translocation of caveolin-1 in the mutant mice. Interestingly, in LXRb−/− male mice, serum testosterone levels were in normal range. Conclusion: LXRb regulates the level of AQP-1 and AQP-4 mRNA as well as the cellular localization of AQP-4, via caveolin-1, in male mice.

917 EXPRESSION, LOCALISATION AND POTENTIAL CLINICAL SIGNIFICANCE OF AQUAPORINS IN UROTHELIAL BLADDER CANCER

You have accessJournal of UrologyBladder Cancer: Basic Research (II)1 Apr 2013917 EXPRESSION, LOCALISATION AND POTENTIAL CLINICAL SIGNIFICANCE OF AQUAPORINS IN UROTHELIAL BLADDER CANCER Peter Rubenwolf, Jennifer Southgate, Christian Thomas, Joachim Thüroff, Stefan Denzinger, Wolf Wieland, and Wolfgang Otto Peter RubenwolfPeter Rubenwolf Mainz, Germany More articles by this author , Jennifer SouthgateJennifer Southgate York, United Kingdom More articles by this author , Christian ThomasChristian Thomas Mainz, Germany More articles by this author , Joachim ThüroffJoachim Thüroff Mainz, Germany More articles by this author , Stefan DenzingerStefan Denzinger Regensburg, Germany More articles by this author , Wolf WielandWolf Wieland Regensburg, Germany More articles by this author , and Wolfgang OttoWolfgang Otto Regensburg, Germany More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2013.02.494AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Human urothelium has previously been shown to express several aquaporins (AQP). Alongside a fundamental role in numerous physiological processes, there is strong presumptive evidence that AQPs are involved in carcinogenesis, specifically in tumor angiogenesis and cell migration. As yet, expression of aquaporins in bladder cancer cell lines (BCCL) and urothelial bladder carcinoma (UBC) has not been systematically studied. The aim of this study was to investigate the expression of AQPs in BCCL and in human tumor samples of UBC of various stages and grades. METHODS AQP transcript and protein expression by BCCL RT4, RT112 and T24 were investigated by reverse transcriptaseûpolymerase chain reaction (RT-PCR) and immunolabelling. Immunohistochemistry was used to assess AQP 3 protein expression in 95 human specimens of UBC of various grades and stages. RESULTS Transcripts for AQP 3, 4, 7, 9, and 11 were detected by all three BCCL. AQP 0, 1, 2, 5, 6, 8, 10 and 12 were not expressed. Immunofluorescence microscopy confirmed expression of AQP 3, 4, 7 and 9 at the protein level. Overall, AQP 3 was shown to be intensely expressed at cell membranes by 68% of samples of human UBC, whereas 32% of tumors did not express the marker. With regard to stage, 91%, 67% and 36% of stage pTa, pT1 and pT2, respectively, were shown to be AQP3-positive. Relative to grade, 93%, 77% and 60% of G1, G2 and G3 tumors were found to express AQP 3. The observed differences in AQP3 expression between grades and stages were statistically significant (p<0.05). CONCLUSIONS This is the first study to provide prima facie evidence that several AQPs are expressed in bladder cancer cell lines and human UBC. Our results indicate that there is a correlation between loss of AQP3 protein expression and tumor stage and grade. However, whether AQPs play any biological role in the carcinogenesis and progression of UBC and, in particular, whether this could be of prognostic or therapeutic value, has yet to be determined. Our study provides a platform for further investigations into the biological and clinical relevance of AQPs in human UBC. © 2013 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 189Issue 4SApril 2013Page: e377 Peer Review Report Advertisement Copyright & Permissions© 2013 by American Urological Association Education and Research, Inc.MetricsAuthor Information Peter Rubenwolf Mainz, Germany More articles by this author Jennifer Southgate York, United Kingdom More articles by this author Christian Thomas Mainz, Germany More articles by this author Joachim Thüroff Mainz, Germany More articles by this author Stefan Denzinger Regensburg, Germany More articles by this author Wolf Wieland Regensburg, Germany More articles by this author Wolfgang Otto Regensburg, Germany More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Aqp5 is a new transcriptional target of Dot1a and a regulator of Aqp2.

Dot1l encodes histone H3 K79 methyltransferase Dot1a. Mice with Dot1l deficiency in renal Aqp2-expressing cells (Dot1lAC) develop polyuria by unknown mechanisms. Here, we report that Aqp5 links Dot1l deletion to polyuria through Aqp2. cDNA array analysis revealed and real-time RT-qPCR validated Aqp5 as the most upregulated gene in Dot1lAC vs. control mice. Aqp5 protein is barely detectable in controls, but robustly expressed in the Dot1lAC kidneys, where it colocalizes with Aqp2. The upregulation of Aqp5 is coupled with reduced association of Dot1a and H3 dimethyl K79 with specific subregions in Aqp5 5′ flanking region in Dot1lAC vs. control mice. In vitro studies in IMCD3, MLE-15 and 293Tcells using multiple approaches including real-time RT-qPCR, luciferase reporter assay, cell surface biotinylation assay, colocalization, and co-immunoprecipitation uncovered that Dot1a represses Aqp5. Human AQP5 interacts with AQP2 and impairs its cell surface localization. The AQP5/AQP2 complex partially resides in the ER/Golgi. Consistently, AQP5 is expressed in none of 15 normal controls, but in all of 17 kidney biopsies from patients with diabetic nephropathy. In the patients with diabetic nephropathy, AQP5 colocalizes with AQP2 in the perinuclear region and AQP5 expression is associated with impaired cellular H3 dimethyl K79. Taken together, these data for the first time identify Aqp5 as a Dot1a potential transcriptional target, and an Aqp2 binding partner and regulator, and suggest that the upregulated Aqp5 may contribute to polyuria, possibly by impairing Aqp2 membrane localization, in Dot1lAC mice and in patients with diabetic nephropathy.

Verification and spatial localization of aquaporin-5 in the ocular lens

Until recently, the lens was thought to express only two aquaporin (AQP) water channels, AQP1 and AQP0. In this study we confirm lenticular AQP5 protein expression by Western blotting and mass spectrometry in lenses from a variety of species. In addition, confocal microscopy was used to map cellular distributions of AQP5 in mouse, rat and human lenses. Tandem mass spectrometry of a human lens membrane preparation revealed extensive sequence coverage (56.2%) of AQP5. Western blotting performed on total fiber cell membranes from mouse, rat, bovine and human lenses confirmed AQP5 protein expression is conserved amongst species. Western blotting of dissected lens fractions suggests that AQP5 is processed in the lens core by C-terminal truncation. Immunohistochemistry showed that AQP5 signal was most abundant in the lens outer cortex and decreased in intensity in the lens core. Furthermore, AQP5 undergoes differentiation-dependent changes in subcellular location from an intracellular localization in differentiating fiber cells to the plasma membrane of mature fiber cells upon the loss of fiber cell nuclei. Our results show that AQP5 is a significant component of lens fiber cell membranes, representing the second most abundant water channel in these cells. Together, the changes to AQP5 distribution and structure are likely to modulate the functional role of AQP5 in different regions of the lens.

Prognostic Value of Combined Aquaporin 3 and Aquaporin 5 Overexpression in Hepatocellular Carcinoma

Background. Aquaporin (AQP) 3 and AQP 5 are involved in tumorigenesis and tumor progression of several tumor types. Aim. To investigate expression patterns and clinical significance of AQP3 and AQP5 in hepatocellular carcinoma (HCC). Methods. Immunohistochemistry was performed to detect the expression of AQP3 and AQP5 in HCC tissues. Results. Immunohistochemistry analysis showed the increased expression of AQP3 and AQP5 protein levels in HCC tissues compared with their adjacent nonneoplastic tissues (both P < 0.001). In addition, combined AQP3 and AQP5 protein expression was significantly associated with serum AFP (P = 0.008), tumor stage (P = 0.006), and tumor grade (P = 0.006). Moreover, HCC patients highly expressing both AQP3 and AQP5 proteins had worse 5-year disease-free survival and 5-year overall survival (P = 0.002 and 0.005, resp.). Furthermore, the Cox proportional hazards model showed that combined AQP3 and AQP5 protein expression was an independent poor prognostic factor for both 5-year disease-free survival (P = 0.009) and 5-year overall survival (P = 0.01) in HCC. Conclusion. Our data suggest for the first time that the aberrant expression of AQP3 and AQP5 proteins may be strongly related to tumor progression and prognosis in patients with HCC. The overexpression of AQP3 in combination with upregulation of AQP5 may be an unfavorable prognostic factor for HCC.

Time-Dependent Expression Patterns of Cardiac Aquaporins Following Myocardial Infarction

Aquaporins (AQPs) are expressed in myocardium and the implication of AQPs in myocardial water balance has been suggested. We investigated the expression patterns of AQP subtypes in normal myocardium and their changes in the process of edema formation and cardiac dysfunction following myocardial infarction (MI). Immunostaining demonstrated abundant expression of AQP1, AQP4, and AQP6 in normal mouse heart; AQP1 in blood vessels and cardiac myocytes, AQP4 exclusively on the intercalated discs between cardiac myocytes and AQP6 inside the myocytes. However, neither AQP7 nor AQP9 proteins were expressed in CD1 mouse myocardium. Echocardiography revealed that cardiac function was reduced at 1 week and recovered at 4 weeks after MI, whereas myocardial water content determined by wet-to-dry weight ratio increased at 1 week and rather reduced below the normal at 4 weeks. The expression of cardiac AQPs was up-regulated in MI-induced groups compared with sham-operated control group, but their time-dependent patterns were different. The time course of AQP4 expression coincided with that of myocardial edema and cardiac dysfunction following MI. However, expression of both AQP1 and AQP6 increased persistently up to 4 weeks. Our findings suggest a different role for cardiac AQPs in the formation and reabsorption of myocardial edema after MI.

The Role of Aquaporin 1 Activated by cGMP in Myocardial Edema Caused by Cardiopulmonary Bypass in Sheep

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.

Reduced expression of aquaporin 9 in tubal ectopic pregnancy

Previous studies demonstrate significant roles for passive water channels (aquaporins, AQPs) in maintaining water homeostasis in cell membranes of endometrial cells during decidualisation and embryo implantation. However, there is little information regarding the role of AQPs in the human fallopian tube, specifically their role in human tubal ectopic pregnancy. In this study we took tissue samples from the site of implantation of tubal ectopic pregnancy (group 1, N = 30, mean age 32 years, range 23-42) and the corresponding non-implantation site in women undergoing salpingectomy for tubal pregnancy (group 2). Ampullary fallopian tubes during mid-secretory phase were collected as control group (group 3, N = 17, mean age 37 years, range 30-50). Thin sections were prepared and stained with anti-AQP9, and, for estrogen and progesterone receptors in each group. Immunohistochemical studies showed that AQP9 proteins localize in the cytoplasm of epithelial cells of Fallopian tube. Expression of AQP9 was significantly reduced during tubal pregnancy compared to controls (group 1 vs. group 3, P = 0.036; group 2 vs. group 3, P = 0.029), and, this reduced expression was not related to estrogen receptor or progesterone receptor status (group 2, ER vs. AQP9, Pearson r = 0.173, P = 0.361; PR vs. AQP9, Pearson r = 0.124, P = 0.514, respectively). Similarly, there is no correlation between AQP9 and estrogen receptor or progesterone receptor status in the normal group (group 3, ER vs. AQP9, Pearson r = -0.026, P = 0.923; PR vs. AQP9, Pearson r = -0.292, P = 0.255, respectively). Reduced expression of AQP9 in human fallopian tube may contribute to aspects of pathophysiology of tubal ectopic pregnancy.

Marked Depletion of the Water-Channel Protein, AQP5, in the Canine Nictitating Membrane Glands Might Contribute to the Development of KCS

The objectives of this study were to investigate the normal histological localization of aquaporin (AQP) 5 protein in the lacrimal and nictitating membrane glands and to compare this localization in healthy and keratoconjunctivitis sicca (KCS) dogs. Lacrimal and nictitating membrane glands of 5 healthy Beagles and nictitating membrane glands of 5 KCS dogs (3 Beagles and 2 mongrel dogs: 0-13 years) were used for the present study. The owners of the KCS dogs did not consent to perform biopsies of the lacrimal glands. The localization and distribution of AQP5 protein were investigated by an immunohistochemical technique. In immunohistochemical staining, AQP5 was localized in the apical site of acinar epithelial and ductal epithelial cells from both the lacrimal and nictitating membrane glands in healthy dogs. However, AQP5 was not detected in the 5 KCS dogs. These results for immunohistochemical AQP5 localization might correlate with the deficiency in tear secretion found in KCS dogs.

Relaxin Regulates Hyaluronan Synthesis and Aquaporins in the Cervix of Late Pregnant Mice

Cervical ripening is associated with loss of structural integrity and tensile strength, thus enabling the cervix to dilate at term. It is characterized by changes in glycosaminoglycan composition, increased water content, and a progressive reorganization of the collagen network. The peptide hormone relaxin via interaction with its receptor, relaxin family peptide receptor 1 (RXFP1), promotes tissue hydration and increases cervical hyaluronan (HA) concentrations, but the mechanisms that regulate these effects are not known. This study in relaxin mutant (Rln(-/-)) mice tested the hypothesis that relaxin regulates HA synthase and aquaporin (AQP) expression in the cervix. We also assessed expression of the RXFP1 protein by immunohistochemistry. Pregnant Rln(-/-) mice had lower Has2 and Aqp3 expression on d 18.5 of pregnancy and decreased cervical HA compared with wild-type Rln(+/+) mice. Chronic infusion of relaxin for 4 or 6 d in pregnant Rln(-/-) mice reversed these phenotypes and increased Has2 and Aqp3 compared with placebo controls. Relaxin-treated mice also had lower Has1 and Aqp5. Changes in gene expression were paralleled by increases in cervical HA and variations in AQP3 and AQP5 protein localization in epithelial cells of Rln(-/-) cervices. Our findings demonstrate that relaxin alters AQP expression in the cervix and initiates changes in glycosaminoglycan composition through increased HA synthesis. These effects are likely mediated through RXFP1 localized to subepithelial stromal cells and epithelial cells. We suggest these actions of relaxin collectively promote water recruitment into the extracellular matrix to loosen the dense collagen fiber network.

Experimental research Expression of pulmonary aquaporin 1 is dramatically upregulated in mice with pulmonary fibrosis induced by bleomycin

IntroductionPulmonary fibrosis occurs due to fibroblast proliferation and collagen production in the lung and begins with alveolar inflammatory edema. Aquaporins (AQPs) play pivotal roles in lung fluid transport. In this study we establish the experimental model for pulmonary fibrosis in C57BL/6 mice to investigate expressions of AQP1 and AQP5 in lung tissue.Material and methodsMice in model groups were treated intratracheally with bleomycin with the dose of 5 mg/kg body weight. The mice were sacrificed at 1 week, 2 weeks and 3 weeks respectively. The left upper lungs were harvested for histopathologic H-E and Masson's staining. The mRNAs of AQP1 and AQP5 were analyzed by real-time polymerase chain reaction (real-time PCR) and the proteins of AQP1 and AQP5 were analyzed by western blotting.ResultsReal-time PCR showed that AQP1 mRNA in bleomycin 1 w, 2 w, and 3 w groups increased by 377%, 880% and 823% respectively compared to that in the control group (p < 0.01). Western blotting showed that the expression of AQP1 protein in bleomycin 1 w, 2 w, and 3 w groups increased by 53%, 144%, and 141%, respectively (p < 0.05). AQP5 mRNA in bleomycin 1 w and 2 w group decreased by 78% and 66%, respectively (p < 0.05). In bleomycin 2 w and 3 w groups it decreased by 69% and 80% (p < 0.05).ConclusionsThe expression of AQP1 dramatically increased in pulmonary fibrosis. AQP1 plays an important role in the progress of pulmonary fibrosis.

Loss of aquaporin 3 protein expression constitutes an independent prognostic factor for progression-free survival: an immunohistochemical study on stage pT1 urothelial bladder cancer.

BackgroundTreatment of patients with stage pT1 urothelial bladder cancer (UBC) continues to be a challenge due to its unpredictable clinical course. Reliable molecular markers that help to determine appropriate individual treatment are still lacking. Loss of aquaporin (AQP) 3 protein expression has previously been shown in muscle-invasive UBC. The aim of the present study was to investigate the prognostic value of AQP3 protein expression with regard to the prognosis of stage pT1 UBC.MethodAQP 3 protein expression was investigated by immunohistochemistry in specimens of 87 stage T1 UBC patients, who were diagnosed by transurethral resection of the bladder (TURB) and subsequent second resection at a high-volume urological centre between 2002 and 2009. Patients underwent adjuvant instillation therapy with Bacillus Calmette-Guérin (BCG). Loss of AQP3 protein expression was defined as complete absence of the protein within the whole tumour. Expression status was correlated retrospectively with clinicopathological and follow-up data (median: 31 months). Multivariate Cox regression analysis was used to assess the value of AQP3 tumour expression with regard to recurrence-free (RFS), progression-free (PFS) and cancer-specific survival (CSS). RFS, PFS and CSS were calculated by Kaplan-Meier analysis and Log rank test.Results59% of patients were shown to exhibit AQP3-positive tumours, whereas 41% of tumours did not express the marker. Loss of AQP3 protein expression was associated with a statistically significantly worse PFS (20% vs. 72%, p=0.020). This finding was confirmed by multivariate Cox regression analysis (HR 7.58, CI 1.29 – 44.68; p=0.025).ConclusionsLoss of AQP3 protein expression in pT1 UBC appears to play a key role in disease progression and is associated with worse PFS. Considering its potential prognostic value, assessment of AQP3 protein expression could be used to help stratify the behavior of patients with pT1 UBC.

Terlipressin Resolves Ascites of Cirrhotic Rats through Downregulation of Aquaporin 2

To investigate the presence of aquaporin (AQP) 1 and AQP2 in kidneys of cirrhotic rats with ascites, and to determine the effect of terlipressin on AQP1 and AQP2 levels and its therapeutic efficacy in ascites treatment. Eighteen rats were randomly divided into normal noncirrhotic rats treated with saline, cirrhotic rats treated with saline and cirrhotic rats treated with terlipressin (n = 6 per group). In all rats, 24-h net fluid-excretion volume, presence or absence of ascites and portal vein pressure were measured; AQP1 and AQP2 mRNA and protein levels in renal tissue were evaluated. Terlipressin resolved ascites in all animals in the terlipressin-treated group, and significantly increased the 24-h net fluid-excretion volume and decreased portal vein pressure compared with saline treatment. AQP1 and AQP2 were significantly upregulated in cirrhotic rat kidneys compared with normal control rat kidneys. Terlipressin administration significantly down regulated AQP2 in rat kidneys but did not affect AQP1. AQP1 and AQP2 are important factors in ascites induction. Terlipressin appears to be an effective drug for the treatment of ascites due to liver cirrhosis in a rat model, possibly due to AQP2 reduction in kidneys.

Gender-specific effect of physical training on AQP7 protein expression in human adipose tissue

AQP7 is a glycerol channel in adipose tissue with a suggested role in controlling the accumulation of triglycerides and secondly development of obesity and type-2 diabetes. In the present study, we aimed to test the hypotheses that (1) AQP7 is localized to the capillaries within human adipose tissue, (2) genetic predisposition to type-2 diabetes is associated with a low expression of AQP7 in abdominal subcutaneous adipose tissue (SAT) and (3) physical training increases AQP7 expression in SAT. The cellular localization of AQP7 in adipose tissue was investigated by immunohistochemistry. The relative expression of AQP7 protein in abdominal SAT was analysed before and after ending a 10-week exercise training programme in first-degree relatives to type-2 diabetic patients and control individuals. Non-obese first-degree relatives to type-2 diabetic patients (n = 20) and control (n = 11) men and women participated in this study. By this, we find that AQP7 is localized to the capillary endothelial cells within adipose tissue. We were unable to evidence a link between a low AQP7 abundance in SAT and genetic predisposition type-2 diabetes. Instead we demonstrate that physical training influences the expression of AQP7 in SAT in a gender-specific manner. Thus, women responds by increasing the abundance of AQP7 by 2.2-fold (p = 0.03) whereas in men a reduced expression is observed (p = 0.00009), resulting in a more than twofold higher abundance of AQP7 in women as compared with men. In conclusion, the adipose tissue glycerol channel, AQP7, is regulated in response to physical training in a gender-dependent manner in SAT.

Beneficial effects of carbamylated erythropoietin against oxygen–glucose deprivation/reperfusion-induced astrocyte swelling: Proposed molecular mechanisms of action

Carbamylated erythropoietin (C-EPO), one of the erythropoietin derivatives, retains strong anti-edema and neuroprotective properties while lacking the hematopoietic complications of erythropoietin. This study investigated the intracellular and molecular mechanisms underlying the anti-edema property of C-EPO. An in vitro model of astrocyte swelling was created by 5h of oxygen–glucose deprivation and subsequent reperfusion (OGD/Rep). Astrocyte cultures were then treated with C-EPO or left as control cells. Here we show that increases in astrocyte volume, morphological cell swelling, and changes in ultrastructure after OGD/Rep were significantly mitigated by treatment with C-EPO (10ng/ml). The decreases in AQP-4 phosphorylation after OGD/Rep were remarkably recovered by C-EPO treatment. The OGD/Rep-induced upregulations of AQP-4 mRNA and protein were also prevented by C-EPO treatment. Additional treatment with phorbol myristate acetate, an activator of protein kinase C (PKC), enhanced C-EPO-mediated neuroprotective effects, while that of H-7, an inhibitor of PKC, blocked these protections. Our findings establish that C-EPO effectively mitigates astrocyte swelling induced by ischemia and reperfusion-like injury. The modulation of AQP-4 phosphorylation and expression via the PKC pathway is participated in the neuroprotective effects of C-EPO.

Effects of curcumin on levels of nitric oxide synthase and AQP-4 in a rat model of hypoxia–ischemic brain damage

This study examines the preventive and therapeutic effects of curcumin on brain edema after hypoxic–ischemic brain damage (HIBD) in a rat model. Male Sprague-Dawley rats were divided into four groups: a sham group (SH), a hypoxic–ischemic group (HI) without drug treatment, a hypoxic–ischemic group (CU) with curcumin injection, and a hypoxic–ischemic group with DMSO injection (solvent control, SC). HIBD treatment led to edema and ultrastructural changes in the hippocampus, increased the activity levels of nitric oxide synthase (NOS) in the brain (P<0.05), and raised the expression of water channel protein 4 (Aquaporin-4, AQP-4) in the blood–brain barrier (BBB) (P<0.05). Curcumin injection, but not the control DMSO injection, partially reversed HIBD-induced brain edema and morphological changes, as well as HIBD-induced increase in NOS activities and AQP-4 expression (P<0.05). In conclusion, our results showed that BBB ultrastructural changes may play an important role in the formation and development of brain edema after HIBD. Curcumin may protect the BBB ultrastructure and thus decrease brain edema following HIBD by down-regulating HIBD-induced increase in NOS activities and AQP-4 protein expression.

Effects of arteriolar constriction on retinal gene expression and müller cells in an experimental retinal vein occlusion

AbstractPurpose To investigate the effect of the laser‐induced arteriolar constriction (AC) on branch retinal vein occlusion (BRVO) induced Müller cell responses and alterations in gene expression of factors implicated in the development of edema.Methods In Brown‐Norway rats the BRVO was induced by laser photocoagulation of the veins in one half of the retina. AC of the afferent arterioles was performed 30 minutes later. The expression of Vegfa, Vegfb, Pedf, Kir4.1, Aqp4, Aqp1, Il1ß, and Il6 was determined with RT‐PCR in the retina and retinal pigment epithelium (RPE) after 1, 3 and 7 days. Potassium currents were recorded in Müller cells 3 days after BRVO. Immunostaining against GFAP, Aqp4 and Kir4.1 was performed on day 1 and 3.Results BRVO resulted in the neuroretinal transient upregulation of Vegfa on day 1. The expression of the Kir4.1, Aqp4, and Aqp1 channels were downregulated, and Il1ß and Il6 strongly upregulated, on day 1 and 3. The retinal distribution of GFAP and Aqp4 proteins remained unaltered, while the Kir4.1 protein displayed redistribution from a polarized to a uniform retinal distribution. Müller cells showed cellular hypertrophy and a decrease in potassium currents. AC accelerated the restoration of downregulated Kir4.1, Aqp4, and Aqp1 in the RPE, of Kir4.1 in the neuroretina and of upregulated Il6 in the neuroretina. AC did not influence the gliotic alterations of Müller cells and the redistribution of Kir4.1 protein.Conclusion The constriction of the afferent artery in the BRVO region had only marginal effect on the BRVO‐evoked alterations in retinal gene expression.

TNF‐α inhibits aquaporin 5 expression in human salivary gland acinar cells via suppression of histone H4 acetylation

Sjögren's syndrome is a systemic autoimmune disease characterized by reductions in salivary and lacrimal secretions. The mechanisms underlying these reductions remain unclear. We have previously shown that TNF-α plays an important role in the destruction of acinar structures. Here we examined TNF-α's function in the expression of aquaporin (AQP) 5 in human salivary gland acinar cells. Immortalized human salivary gland acinar (NS-SV-AC) cells were treated with TNF-α, and then the expression levels of AQP5 mRNA and protein were analysed. In addition, the mechanisms underlying the reduction of AQP5 expression by TNF-α treatment were investigated. TNF-α-treatment of NS-SV-AC cells significantly suppressed the expression levels of AQP5 mRNA and protein, and reduced the net fluid secretion rate. We examined the expression and activation levels of DNA methyltransferases (Dnmts) in NS-SV-AC cells treated with TNF-α. However, no significant changes were observed in the expression or activation levels of Dnmt1, Dnmt3a or Dnmt3b. Although we also investigated the role of NF-κB activity in the TNF-α-induced suppression of AQP5 expression in NS-SV-AC cells, we detected similar TNF-α suppression of AQP5 expression in non-transfected cells and in a super-repressor form of IκBα cDNA-transfected cell clones. However, interestingly, chromatin immunoprecipitation analysis demonstrated a remarkable decrease in levels of acetylated histone H4 associated with the AQP5 gene promoter after treatment with TNF-α in NS-SV-AC cells. Therefore, our results may indicate that TNF-α inhibition of AQP5 expression in human salivary gland acinar cells is due to the epigenetic mechanism by suppression of acetylation of histone H4.

Transient hyperosmolality modulates expression of cardiac aquaporins

PurposeHyperosmolarity is a common complication in intensive care patients, dysregulating water balance in many organs including brain and heart. The aquaporin (AQP) water channels, in particular AQP1 and −4, have been suggested to play an important role in fluid homeostasis of the myocardium. In many organs AQP expression is regulated by osmolarity, drastically altering water permeability of the cell membranes. The aim of our study was to investigate if plasma hyperosmolality may regulate cardiac expression of AQP1 and −4, and if so, at which magnitude and time frame such regulation takes place. MethodsC57Bl6 mice were injected intraperitoneally with either 1.5ml 0.154Mol (isoosmotic), 0.5ml 1Mol (mild hyperosmotic) or 0.5ml 2Mol (strong hyperosmotic) NaCl. Plasma, hearts, and forebrains were harvested before injection (“time 0”), and after 1, 4, 8 and 24h. AQP1 and −4 expression were analyzed using qPCR and Western blot. ResultsIsoosmotic and mild hyperosmotic injections caused no important changes in cardiac AQP expression. Strong hyperosmotic NaCl injections induced an upregulation of AQP1 mRNA and glycosylated fraction of AQP1 protein in the heart without changes of the total protein. AQP4 mRNA and protein decreased in the heart and increased in the brain after hyperosmotic NaCl. The change in AQP4 protein content in the brain preceded the increase of mRNA. ConclusionAs in the brain, expression of AQP1 and −4 in the heart is influenced by changes in plasma osmolality. Changes in AQP expression may alter cardiac function in hyperosmotic states.