Aquaporin-4 Protein Expression Research Articles

Aquaporin-1 Translocation and Degradation Mediates the Water Transportation Mechanism of Acetazolamide

BackgroundDiuretic agents are widely used on the treatment of water retention related diseases, among which acetazolamide (AZA) acts originally as a carbonic anhydrase (CA) inhibitor. Aquaporin-1 (AQP1) being located in renal proximal tubules is required for urine concentration. Previously our lab has reported AZA putatively modulated AQP1. Aim of this study is to testify our hypothesis that regulating AQP1 may mediate diuretic effect of AZA.Methodology/Principal FindingsFor in vivo study, we utilized Sprague Dawley rats, as well as AQP1 knock-out (AQP1−/−) mice to examine urine volume, and human kidney-2 (HK-2) cell line was used for in vitro mechanism study. In our present study we found that AZA decreased CAs activity initially but the activity gradually recovered. Contrarily, diuretic effect was consistently significant. AQP1 protein expression was significantly decreased on day 7 and 14. By utilizing AQP1−/− mice, we found diuretic effect of AZA was cancelled on day 14, while urine volume continuously increased in wild-type mice. Surface plasmon resonance (SPR) results indicated AQP1 was physiologically bound by myosin heavy chain (MHC), immunoprecipitation and immunofluorescence results confirmed this protein interaction. In vitro study results proved AZA facilitated AQP1 translocation onto cell membrane by promoting interaction with MHC, dependent on ERK/ myosin light chain kinase (MLCK) pathway activation. MHC inhibitor BDM and ERK inhibitor U0126 both abolished above effect of AZA. Eventually AZA induced AQP1 ubiquitination, while proteasome inhibitor MG132 reversed AZA's down-regulating effect upon AQP1.Conclusions/SignificanceOur results identified AZA exerted diuretic effect through an innovative mechanism by regulating AQP1 and verified its inhibitory mechanism was via promoting MHC-dependent translocation onto cell membrane and then ubiquitin mediated degradation, implicating a novel mechanism and target for diuretic agent discovering.

Decreased expression of aquaporin 2 in the collecting duct of mice lacking the vasopressin V1a receptor

Vasopressin V1a receptor null (V1aR(-/-)) mice recently showed incomplete urinary concentration due to higher urine volume during control and water diuresis (euhydration), but showed normal response during dehydration (Aoyagi et al., Am J Physiol 295: F100-7, 2008). Water balance, plasma vasopressin, plasma and urine osmolality, and aquaporin 2 (AQP2) expression in the kidney of wild-type (WT) and V1aR(-/-) mice were therefore further examined using improved methods of urine collection (urinary bladder urine). V1aR(-/-) mice demonstrated a lower urine osmolality (3,360 ± 138 vs. 3,610 ± 47 mOsm/kgH2O) and a higher plasma osmolality (354.3 ± 1.3 vs. 342.5 ± 1.5 mOsm/kgH2O) after dehydration for 24 h compared to WT mice (P < 0.05). In contrast, the plasma vasopressin concentration was significantly (P < 0.001) higher in the V1aR(-/-) mice (48.8 ± 4.8 vs. 22.1 ± 2.4 pg/ml). On the other hand, although the AQP2 protein expression in the kidney was increased after dehydration, the basal (control) and dehydration-induced AQP2 protein levels were significantly lower in V1aR(-/-) mice compared to WT mice (by Western blotting). Staining by an anti-AQP2 antibody in the luminal membrane of the collecting ducts was increased in both V1aR(-/-) and WT mice after dehydration, but was relatively weaker in the V1aR(-/-) mice (by immunohistochemistry). Moreover, urinary excretion of AQP2 protein, an index of the luminal AQP2 expression, was significantly (P < 0.05) lower in the V1aR(-/-) mice. V1aR signaling may be fundamentally important for the expression of AQP2 in the collecting ducts during control conditions and dehydration.

Aquaporin‐1 expression in membranes from proliferative vitreoretinopathy and in epiretinal membranes

AbstractPurpose Aquaporin‐1 (AQP1) is involved in cell migration and proliferation. This study aims to investigate AQP1 expression in membranes from proliferative vitreoretinopathy (PVR) and epiretinal membranes (ERM)Methods 20 Membranes PVR and ERM were collected following eye surgery from 20 eyes from 20 patients. AQP1 mRNA and protein expression were determined by RT‐qPCR and immunofluorescence in both PVR and ERMResults AQP1 mRNA and protein were expressed in both PVR and ERM as shown by both RT‐qPCR and immunofluorescence. Moreover, AQP1 protein expression was heterogeneous among and between PVR and ERM, and was highly colocalized with alpha‐smooth muscle actin (SMA or Acta2) and slightly colocalized with glial fibrillary acidic protein (GFAP).Conclusion AQP1 mRNA and protein were expressed in membranes from PVR and in ERM. Due to the absence of SMA and GFAP colocalization, it is likely that AQP1 is expressed by at least two distinct cells types. AQP1 might play a role in cell migration and proliferation occurring during the formation of PVR and ERM, and could represent a new therapeutic target

Atorvastatin Prevents the Downregulation of Aquaporin-2 Receptor After Bilateral Ureteral Obstruction and Protects Renal Function in a Rat Model

To assess the effects of atorvastatin (ATORV) on renal function after bilateral ureteral obstruction (BUO), measuring inulin clearance and its effect on renal hemodynamic, filtration, and inflammatory response, as well as the expression of Aquaporin-2 (AQP2) in response to BUO and after the release of BUO. Adult Munich-Wistar male rats were subjected to BUO for 24 hours and monitored during the following 48 hours. Rats were divided into 5 groups: sham operated (n = 6); sham + ATORV (n = 6); BUO (n = 6); BUO + ATORV (10 mg/kg in drinking water started 2 days before BUO [n = 5]; and BUO + ATORV (10 mg/kg in drinking water started on the day of the release of BUO [n = 5]). We measured blood pressure (BP, mm Hg); inulin clearance (glomerular filtration rate [GFR]; mL/min/100 g); and renal blood flow (RBF, mL/min, by transient-time flowmeter). Inflammatory response was evaluated by histologic analysis of the interstitial area. AQP2 expression was evaluated by electrophoresis and immunoblotting. Renal function was preserved by ATORV treatment, even if initiated on the day of obstruction release, as expressed by GFR, measured by inulin clearance. Relative interstitial area was decreased in both BUO + ATORV groups. Urine osmolality was improved in the ATORV-treated groups. AQP2 protein expression decreased in BUO animals and was reverted by ATORV treatment. ATORV administration significantly prevented and restored impairment in GFR and renal vascular resistance. Furthermore, ATORV also improved urinary concentration by reversing the BUO-induced downregulation of AQP2. These findings have significant clinical implication in treating obstructive nephropathy.

Effect of remote ischemic postconditioning on an intracerebral hemorrhage stroke model in rats

Background and purpose: While recent studies suggest that remote ischemic postconditioning (RIP) therapy may be of benefit to patients with acute ischemic stroke, RIP’s effects on intracerebral hemorrhage (ICH) still remains unclear. In the present study, the use of RIP in a rat model ICH was investigated to elucidate any potential beneficial or detrimental effects as determined by motor testing, blood brain barrier integrity, and brain water content, as well as aquaporin-4 (AQP-4) and matrix metalloproteinase-9 (MMP-9) expression.Methods: ICH was induced in Sprague–Dawley rats and they were randomized into either a control (n = 24) or RIP treatment (n = 24) group. RIP was performed by repetitive, brief occlusion and release of the bilateral femoral arteries. Functional outcome in each group was assessed by neurologic deficits on vibrissae-elicited forelimb placing test and a 12-point outcome scale. At 72 hours, brain blood volume, water content, blood–brain barrier (BBB) permeability, and protein expression of AQP-4 and MMP-9 were determined.Results: This collagenase model yielded well-defined striatal hematomas. Vibrissae-elicited forelimb placement was significantly (P<0·01) affected by ICH. However, there was no significant difference between the RIP and control groups at either 24 or 72 hours. A 12-point neurological deficit score also failed to differentiate between the RIP and control. There were no significant differences between the two groups in cerebral blood volumes, brain water content, Evans blue extravasations, and expressions of AQP-4 and MMP-9.Conclusions: Although RIP did not show a beneficial effect in our ICH model, treatment with RIP did not exacerbate ICH.

Effects of hyperosmolality on expression of urea transporter A2 and aquaporin 2 in mouse medullary collecting duct cells

In this study, the effects of hyperosmolality on the expression of urea transporter A2 (UTA2) and aquaporin 2 (AQP2) were investigated in transfected immortalized mouse medullary collecting duct (mIMCD3) cell line. AQP2-GFP-pCMV6 and UTA2-GFP-pCMV6 plasmids were stably transfected into mIMCD3 cells respectively. Transfected mIMCD3 and control cells were cultured in different hypertonic media, which were made by NaCl alone, urea alone, or an equiosmolar mixture of NaCl and urea. The mRNA and protein expression of AQP2 was elevated by the stimulation of NaCl alone, urea alone and NaCl plus urea in AQP2-mIMCD3 cells; whereas NaCl alone and NaCl plus urea rather than urea alone increased the mRNA and protein expression of UTA2 in UTA2-mIMCD3 cells, and all the expression presented an osmolality-dependent manner. Moreover, the mRNA and protein expression of UTA2 rather than AQP2 was found to be synergistically up-regulated by a combination of NaCl and urea in mIMCD3 cells. It is concluded that NaCl and urea synergistically induce the expression of UTA2 rather than AQP2 in mIMCD3 cells, and hyperosmolality probably mediates the expression of AQP2 and UTA2 through different mechanisms.

Molecular mechanisms of therapeutic hypothermia on neurological function in a swine model of cardiopulmonary resuscitation

ObjectiveTo explore the molecular mechanisms by which mild hypothermia following resuscitation improves neurological function in a porcine model of cardiac arrest. MethodsThirty-three inbred Chinese Wuzhishan (WZS) minipigs were used. After 8min of untreated ventricular fibrillation (VF), the surviving animals (n=29) were randomly divided into two groups including serum group (n=16) and molecular group (n=13). Serum group animals were used to measure porcine-specific tumour necrosis factor-alpha (TNF-α), interleukin (IL-6, IL-10), matrix metalloproteinase (MMP9), Aquaporin-4 (AQP4), tissue inhibitor to metalloproteinase-1 (TIMP1), neuron-specific enolase (NSE) and S100B at 0.5h, 6h, 12h, 24h and 72h recovery by enzyme-linked immunosorbent assay (ELISA). Molecular group animals were used to measure cerebral cortex messenger RNA (mRNA) and protein expression of nuclear factor-κB (NF-κB), MMP9 and AQP4 by real-time (RT) quantitative polymerase chain reaction (PCR) and Western blotting at 24h and 72h recovery. Animals were further divided into either normothermia or hypothermia groups. Hypothermia (33°C) was maintained for 12h using an endovascular cooling device. Swine neurologic deficit scores (NDS) were used to evaluate neurological function at 24-h and 72-h recovery. ResultsTwenty-nine of the 33 (87.9%) animals were successfully resuscitated. The hypothermia group exhibited higher survival rates at 24h (75%) and 72h (62.5%) compared to the normothermia group (37.5% and 25%, respectively). Hypothermia markedly inhibited expression of NF-κB, TNF-α, MMP9 and NSE, and promoted expression of TIMP1 (P<0.01). The mean NDS at 24-h and 72-h recovery was 112.5 and 61, respectively, in the hypothermic group, and 230 and 207.5, respectively, in the normothermia group. ConclusionBrain protection induced by hypothermia involves inhibition of inflammatory and brain edema pathways.

Hypoxia-induced Up-regulation of Aquaporin-1 Protein in Prostate Cancer Cells in a p38-dependent Manner

Background/Aims: Aquaporin-1 (AQP1) is a glycoprotein that mediates osmotic water transport, its expression has been found to correlate with tumour stage in some tumours. However, the mechanism by which AQP1 protein expression is regulated in tumor cells remains to be fully elucidated. We hypothesized that hypoxia might play an important role in AQP1 induction during tumorigenesis and at the late stages of tumor development. Methods: Isotonic and serum-free hypoxic models were used to investigate AQP1 expression in PC-3M human prostate cancer cells. Results: AQP1 expression was up-regulated by density-induced pericellular hypoxia and cobalt(II) chloride (CoCl₂)-induced hypoxia at the transcriptional level. Moreover, phosphorylation of p38 mitogen-activated protein kinase (MAPK) was induced by density-induced pericellular hypoxia and CoCl₂-induced hypoxia, specific inhibitors of p38 MAPK could concentration-dependently block those effects of hypoxia on AQP1 expression. Intracellular calcium ion (Ca²⁺) and protein kinase C (PKC) were shown to be responsible for the activation of p38 MAPK pathway. In addition, AQP1 induction in dense cultures was dependent on lowered oxygen (O₂) tension. In high cell density culture, certain secretory proteins might induce AQP1 expression indirectly. Conclusion: These findings suggest that AQP1 could be induced by hypoxia at transcription level, and the regulation of AQP1 in PC-3M cells is dependent on calcium, PKC and p38 MAPK, as well as low oxygen tension.

Interleukin-1β Decreases Aquaporin-3 Expression via Trans-Repression by CCAAT Enhancer Binding Protein

Aquaporin-3 (AQP3), which is expressed in the basal layer of keratinocytes and airway epithelial cells, regulates not only water and glycerol permeability at plasma membrane, but also cell migration. We have previously shown that the expression of AQP3 is decreased by inflammatory cytokines, such as TNF-α and IL-1β. However, underlining mechanism for this decrease in AQP3 remains to be clear. In the present study, therefore, we examined the effect of IL-1β on AQP3 mRNA expression and promoter activity in A549 lung epithelial cells. IL-1β decreased both AQP3 protein and mRNA expression, in a concentration- (0.5-50 ng/ml) dependent manner. IL-1β also decreased promoter (−990/+88) activity, suggesting that IL-1β repressed AQP3 gene transcription. The IL-1β-induced decrease in AQP3 was relieved with PD98059, a MEK/ERK inhibitor. We then prepared a series of 5’-deletion constructs of AQP3 promoter. Among these, IL-1β did not decrease the activity of mutant promoters without −374/-272 region. The promoter, which has point mutation at CCAAT enhancer binding protein (C/EBP) binding element located at −276/-262, was also insensitive to IL-1β. Taken together, it was suggested that IL-1β decreases AQP3 expression via MEK/ERK- and C/EBP-dependent signaling.

Gypsum fibrosum and its major component CaSO4 increase cutaneous aquaporin-3 expression levels

Ethnopharmacological relevanceWe have previously reported that Byakkokaninjinto improves cutaneous pruritus by increasing the expression level of aquaporin-3 (AQP3). In this study, we examined the effect of Gypsum fibrosum (main component: CaSO4), which is the main component of Byakkokaninjinto, on the cutaneous AQP3 expression level. Materials and methodsKKAy mice were given a diet containing 0.3% Gypsum fibrosum extract, or a diet containing 0.3% CaSO4 for 4 weeks. The urine volume, plasma glucose levels, cutaneous AQP3 protein expression, and the Ca2+ content were measured. ResultsThe 24-h urine volumes and the plasma glucose levels in the Gypsum fibrosum extract group were not significantly different from those in the control group. In the Gypsum fibrosum extract group, the cutaneous AQP3 protein levels increased significantly, by approximately 3.2-fold, compared to the control group. The cutaneous Ca2+ content in the control group was approximately 35μg/g. In the Gypsum fibrosum extract group, the Ca2+ content increased to approximately 51μg/g, which was significant compared to the control group. In the CaSO4 group, an increase in the AQP3 protein expression levels and Ca2+ content were observed; the extent of these increases were similar to those in the Gypsum fibrosum extract group. ConclusionsThe results of this study suggest that Gypsum fibrosum plays an important role in the increased levels of cutaneous AQP3 expression enhanced by Byakkokaninjinto. The results also indicate that the increase in AQP3 caused by Gypsum fibrosum is attributable to an increase in the cutaneous Ca2+ content from its main component, CaSO4.

Abstract 17216: Aquaporin-1 is Required for Vascular Differentiation of Human Induced Pluripotent Stem Cells Following Exposure to Glucose-Induced Hyperosmolarity

Background and objective: Diabetic hyperglycemia increases plasma osmolarity, leading to adaptive cell reponses. Aquaporin-1 (AQP1) is induced by hyperosmolarity and plays a role in the vascular permeability. Induced pluripotent stem (iPS) cells offer a disease-relevant cellular model for the interplay of risk factors in the vascular complications of diabetes. We tested the hypothesis that glucose-induced hyperosmolarity promotes angiogenesis in human iPS cells through activation of AQP1 and downstream osmosignaling pathway, thus orchestrating cell sprouting and migration. Methods and results: Human iPS cells were generated from skin fibroblasts by lentiviral transduction of four reprogramming factors (Oct4, SOX2, Klf4 and c-Myc). Following 3 weeks compact refractive embryonic stem cell-like colonies emerged. All the iPS cell colonies expressed OCT4, SSEA3, SSEA4 and SOX2, characteristic of successful dedifferentiation. After reprogramming, iPS cells were transfected with siRNA-AQP1 or scrambled controls, and exposed to 5.5 mmol/L glucose (normoglycemia), high glucose (HG) at 12.5, 25 and 45 mmol/L or with high mannitol (HM) at 12.5, 25 and 45 mmol/L for 24-72 hours. Exposure to either HG or HM increased expression of AQP1 and tonicity enhancer binding protein (TonEBP) while decreasing the iPS cell numbers. In iPS cells, AQP1 could be co-immunoprecipitated with β-catenin. HG and HM strongly induced the expression of β-catenin (n=3, p&lt;0.001 vs normal glucose by ANOVA). Under these conditions, proteins co-immunoprecipitated with anti-AQP1 and β-catenin showed increased ratio of F-actin vs G-actin (n=3, p&lt;0.01 vs normal glucose by ANOVA). iPS cells formed bundles in methylcellulose matrix and tubing networks in matrigel, especially when they were exposed to HG and HM (HG 2.8±0.2 fold; HM 3.3±0.5 fold; n=3, p&lt;0.01 by ANOVA. SiRNA to AQP1 reverted the inducing effects of HG and HM on β-catenin expression, actin polymerization and angiogenic activities. Conclusions: The hyperosmolarity serves as a biophysical factor that promotes angiogenesis in human iPS cells. This effect may occur through an AQP1-associated cytoskeleton remodeling. Targeting the osmosignaling pathway offers a novel strategy to reduce vascular complications of diabetes.

AQP2 is necessary for vasopressin- and forskolin-mediated filamentous actin depolymerization in renal epithelial cells

SummaryRemodeling of the actin cytoskeleton is required for vasopressin (VP)-induced aquaporin 2 (AQP2) trafficking. Here, we asked whether VP and forskolin (FK)-mediated F-actin depolymerization depends on AQP2 expression. Using various MDCK and LLC-PK1 cell lines with different AQP2 expression levels, we performed F-actin quantification and immunofluorescence staining after VP/FK treatment. In MDCK cells, in which AQP2 is delivered apically, VP/FK mediated F-actin depolymerization was significantly correlated with AQP2 expression levels. A decrease of apical membrane associated F-actin was observed upon VP/FK treatment in AQP2 transfected, but not in untransfected cells. There was no change in basolateral actin staining under these conditions. In LLC-PK1 cells, which deliver AQP2 basolaterally, a significant VP/FK mediated decrease in F-actin was also detected only in AQP2 transfected cells. This depolymerization response to VP/FK was significantly reduced by siRNA knockdown of AQP2. By immunofluorescence, an inverse relationship between plasma membrane AQP2 and membrane-associated F-actin was observed after VP/FK treatment again only in AQP2 transfected cells. This is the first report showing that VP/FK mediated F-actin depolymerization is dependent on AQP2 protein expression in renal epithelial cells, and that this is not dependent on the polarity of AQP2 membrane insertion.

Aquaporin-2 Promoter Is Synergistically Regulated by Nitric Oxide and Nuclear Factor of Activated T Cells

Background/Aims: We have previously shown that aquaporin-2 (AQP2) is down-regulated in the renal medulla of rats made hypertensive by chronic inhibition of nitric oxide synthase. It has been shown that AQP2 expression is regulated by the calcineurin/nuclear factor of activated T cells (NFATc). Nitric oxide (NO) regulates the activity of NFATc via c-Jun-N-terminal kinase 2 (JNK2). Therefore, we hypothesized that increases in NO enhance NFATc-mediated up-regulation of AQP2 promoter activity. Methods: AQP2 mRNA and protein expression were detected in mouse renal papilla.AQP2 promoter luciferase reporter- and NFAT luciferase reporter-transfected MDCK cells were used to determine AQP2 promoter activity and NFATc activity, respectively. Cells were incubated with classic activators and inhibitors of NFATc and the NO pathway. Results: Our results demonstrate that both Ca²⁺ and NO have a synergistic effect resulting in an increase in AQP2 mRNA and protein in mouse papilla and activation of the AQP2 promoter in kidney-derived cells. In addition, NO enhances Ca²⁺-induced NFATc activation. The underlying mechanism involves increased NFATc nuclear import and decreased export via protein kinase G-mediated inhibition of JNK1/2. Conclusions: This is the first study defining novel regulatory roles for NO and NFATc in the control of AQP2, which is an important renal protein.

Expression of aquaporin-4 during brain edema in rats with thioacetamide-induced acute encephalopathy

To investigate the expression of aquaporin-4 (AQP4) during brain edema in rats with thioacetamide-induced acute liver failure and encephalopathy. The rat model of acute hepatic failure and encephalopathy was induced by intraperitoneal injection of thioacetamide (TAA) at a 24-hour interval for 2 consecutive days. Thirty-two SD rats were randomly divided into the model group (n = 24) and the control group (normal saline, n = 8). And then the model group was further divided into 3 subgroups by the timepoint of decapitation: 24 h (n = 8), 48 h (n = 8) and 60 h (n = 8). Then we observed their clinical symptoms and stages of HE, indices of liver function and ammonia, liver histology and brain water content. The expression of AQP4 protein in brain tissues was measured with Western blot and the expression of AQP4mRNA with RT-PCR (reverse transcription-polymerase chain reaction). Typical clinical manifestations of hepatic encephalopathy occurred in all TAA-administrated rats. The model rats showed the higher indices of ALT (alanine aminotransferase), AST (aspartate aminotransferase), TBIL (total bilirubin) and ammonia than the control rats (P < 0.05). The brain water content was significantly elevated in TAA-administrated rats compared with the control (P < 0.05). The expressions of AQP4 protein and mRNA in brain tissues significantly increased in TAA-administrated rats (P < 0.05). In addition, the expressions of AQP4 protein and mRNA were positively correlated with brain water content (r = 0.536, P < 0.01; r = 0.566, P = 0.01). The high expression of AQP4 in rats with TAA-induced acute liver failure and encephalopathy plays a significant role during brain edema. AQP4 is one of the molecular mechanisms for the occurrence of brain edema in hepatic encephalopathy.

Aquaporin 5 distribution pattern during development of the mouse sublingual salivary gland.

Aquaporin 5 (AQP5) is important in salivary fluid secretion, and has been found in acinar cells of salivary glands in several species. Recently, studies have shown the AQP5 transcript and protein expression patterns as well as the temporal-spatial protein distribution during development of the mouse submandibular salivary gland. The AQP5 distribution pattern of the closely located sublingual gland (SLG) is, however, not well known. Thus, in this study, the Aqp5 RNA expression pattern and the temporal-spatial distribution of AQP5 protein in prenatal development and in adult mouse SLG was investigated. SLGs from embryonic day 14.5 (E14.5) to 18.5 and postnatal days 0 (P0), 25, and 60 were examined using real time PCR and immunohistochemistry. The Aqp5 transcript was detected from E13.5 and was found to increase towards birth and in young adults. The protein was first detected in a scattered pattern in the canalicular stage and became more organized in the luminal membrane of the acinar cells towards birth. During all postnatal developmental stages studied, AQP5 was localized in the luminal and lateral membrane of acinar cells. AQP5 was also detected in the intercalated duct and additional apical membrane staining in the entire intralobular duct was found in the terminal bud stage. These results indicate that AQP5 plays a role during embryonic salivary gland development.

Different effects of CsA and FK506 on aquaporin-2 abundance in rat primary cultured collecting duct cells

Calcineurin (Cn) inhibitors (CnI) such as cyclosporine A (CsA) and FK506 are nephrotoxic immunosuppressant drugs, which decrease tubular function. Here, we examined the direct effect of CnI on aquaporin-2 (AQP2) expression in rat primary cultured inner medullary collecting duct cells. CsA (0.5-5μM) but not FK 506 (0.01-1μM) decreased expression of AQP2 protein and messenger RNA (mRNA) in a concentration and time dependent manner, without affecting mRNA stability. This effect was observed despite similar inhibition of Cn activity by both CnI, thereby suggesting that the CsA-dependent decrease in AQP2 expression was Cn independent. Another inhibitor of cyclophilin A, the primary intracellular target of CsA, had no effect on AQP2 expression. In order to investigate the mechanism of decreased AQP2 transcription, we studied activation status of two suggested transcriptional regulators of AQP2, cAMP-responsive element binding protein (CREB), and tonicity enhancer binding protein (TonEBP). Localization of TonEBP, as well as TonEBP-mediated gene transcription, was not affected by CsA. Phosphorylation of CREB at an activating phosphorylation site (S133) was decreased by CsA, but not by FK506. However, both CnI did not affect cellular cAMP levels. We show that CsA decreases transcription of AQP2, a process that is in part independent of Cn or cyclophilin A and suggests dependence on decreased activity of CREB.

Calcitonin gene-related peptide prevents blood–brain barrier injury and brain edema induced by focal cerebral ischemia reperfusion

Cerebral ischemia is one of the diseases that most compromise the human species. Therapeutic recovery of blood–brain barrier (BBB) disruption represents a novel promising approach to reduce brain injury after stroke. To determine the effects of calcitonin gene-related peptide (CGRP) on the BBB participate in stroke progression, rat cerebral ischemia reperfusion injury was induced by a 2-hour left transient middle cerebral artery occlusion (MCAO) using an intraluminal filament, followed by 46h of reperfusion. CGRP (1μg/ml) at the dose of 3μg/kg (i.p.) was administered at the beginning of reperfusion. Subsequently, 48h after MCAO, arterial blood pressure, infarct volume, water content, BBB permeability, BBB ultrastructure, levels of aquaporin-4 (AQP4) and its mRNA were evaluated. CGRP could reduce arterial blood pressure (P<0.001), infarct volume (P<0.05), cerebral edema (P<0.01), BBB permeability (P<0.05), AQP4 mRNA expression (P<0.05) and AQP4 protein expression (P<0.01). Furthermore, CGRP treatment improved ultrastructural damage of capillary endothelium cells and decreased the loss of the tight junction observed by transmission electronic microscopy (TEM) after 46h of reperfusion. Our findings show that CGRP significantly reduced postischemic increase of brain edema with a 2-hour therapeutic window in the transient model of focal cerebral ischemia. Moreover, it seems that at least part of the anti-edematous effects of CGRP is due to decrease of BBB disruption by improving ultrastructural damage of capillary endothelium cells, enhancing basal membrane, and inhibiting AQP4 and its mRNA over-expression. The data of the present study provide a new possible approach for acute stroke therapy by administration of CGRP.

Effects of Downregulation of Aquaporin1 by Peptidoglycan and Lipopolysaccharide via MAPK Pathways in MeT-5A Cells

This study was designed to investigate the signaling pathway involved in aquaporin1 (AQP1) expression caused by peptidoglycan (PGN) from Staphylococcus aureus and lipopolysaccharide (LPS) in human pleural mesothelial cell lines (MeT-5A) in vitro. RT-PCR, immunoblot analysis, and immunofluorescence assay were used to determine the relative mRNA and protein levels of AQP1 caused by PGN and LPS in MeT-5A cells. Activation of MAPKs by PGN and LPS was reflected by detecting the phosphorylation constituents of extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun N-terminal kinase (JNK), and p38 using immunoblot. MAPKs inhibitors were used to determine the effects of PGN- and LPS-induced AQP1 expression by immunoblot. AQP1 transcription and protein expression were decreased by PGN and LPS in dose- and time-dependent manners in MeT-5A cells. Both PGN and LPS activated p38/ERK/JNK pathways in MeT-5A cells. Furthermore, downregulation of AQP1 expression by LPS was blocked by SB203580, SP600125, and PD98059, which are inhibitors of p38, JNK, and ERK1/2, respectively. In contrast, downregulation of AQP1 expression by PGN was blocked only by SB203580, not by SP600125 or PD98059, underlying the importance of p38 MAPK in the downregulation of AQP1 expression by PGN in MeT-5A cells. AQP1 expression was decreased by both PGN and LPS in dose- and time-dependent manners in MeT-5A cells. AQP1 expression was down-regulated by PGN via p38 MAPK pathway, while AQP1 expression was down-regulated by LPS via p38/JNK/ERK pathways.

Proliferation inhibition of cisplatin and aquaporin 5 expression in human ovarian cancer cell CAOV3

The purpose of this study is to investigate the association between proliferation inhibition of cisplatin and aquaporin 5 (AQP5) expression and its regulation in ovarian carcinoma cell CAOV3. Cell growth rate was measured by MTT after CAOV3 cells were incubated with cisplatin or NF-κB inhibitor PDTC. Western blot and RT-PCR were used to detect the expression of AQP5 and NF-κB p65. Our results showed that expression of AQP5, NF-κB in cytoplasm and karyon and IκBα in cytoplasm protein in CAOV3 cells can be induced to decrease by cisplatin with concentration-dependent manner, and there is a positive correlation between AQP5 protein and cell growth rate (r=0.607, P<0.05). When cells were incubated with 10μg/ml cisplatin, AQP5, NF-κB p65, and IκBα increased rapidly at 6-12h, but decreased at 24h, remain on low level until to 72h. Expression of AQP5 could be induced to decrease by PDTC, and a positive correlation between AQP5 protein expression and NF-κB p65 and IκBα (r=0.894, 0.857; P<0.05). Proliferation inhibition of cisplatin is related with AQP5 expression, and NF-κB may be involved in mechanism of AQP5 regulation. AQP5 will be potential target for therapy of ovarian carcinoma.

Upregulation of Aquaporin-3 Is Involved in Keratinocyte Proliferation and Epidermal Hyperplasia

Aquaporin-3 (AQP3) is a water/glycerol-transporting protein expressed in keratinocytes of the epidermis. We previously showed that AQP3-mediated transport of water and glycerol is involved in keratinocyte migration and proliferation, respectively. However, the involvement of AQP3 in epidermal hyperplasia in skin diseases, such as atopic dermatitis (AD), is unknown. In this study, we found significantly increased AQP3 transcript and protein expression in the epidermis of human AD lesions. The upregulation of AQP3 expression in human keratinocytes by transfection with human AQP3 DNA plasmid was associated with increased cellular glycerol and ATP, as well as increased cell proliferation. Among several cytokines and chemokines produced in the skin, CCL17, which is highly expressed in AD, was found to be a strong inducer of AQP3 expression and enhanced keratinocyte proliferation. In mouse AD models, AQP3 was strongly overexpressed in the epidermis in wild-type mice. Epidermal hyperplasia was reduced in AQP3-deficient mice, with a decreased number of proliferating keratinocytes. These results suggest the involvement of AQP3 in epidermal hyperplasia by a mechanism involving upregulated AQP3 expression and consequent enhancement of keratinocyte proliferation.