Downregulation Of Aquaporin-2 Research Articles

Hyperglycemia Exacerbates Intracerebral Hemorrhage via the Downregulation of Aquaporin-4

Intracerebral hemorrhage (ICH) is associated with high mortality and neurological deficits, and concurrent hyperglycemia usually worsens clinical outcomes. Aquaporin-4 (AQP-4) is important in cerebral water movement. Our aim was to investigate the role of AQP-4 in hyperglycemic ICH. Hyperglycemia was induced by intraperitoneal injection of streptozotocin (STZ; 60 mg/kg) in adult Sprague-Dawley male rats. ICH was induced by stereotaxic infusion of collagenase/heparin into the right striatum. One set of rats was repeatedly monitored by MRI at 1, 4, and 7 days after ICH induction so as to acquire information on the formation of hematoma and edema. Another set of rats was killed and brains were examined for differences in the degree of hemorrhage and edema, water content, blood-brain barrier destruction, and AQP-4 expression. Hyperglycemia ICH rats exhibited increased brain water content, more severe blood-brain barrier destruction, and greater vasogenic edema as seen on diffusion-weighted MRI. Significant downregulation of AQP-4 was observed in STZ-treated rats after ICH as compared with non-STZ-treated rats. Apoptosis was greater on day 1 after ICH in STZ-treated rats. The expression of AQP-4 in the brain is downregulated in hyperglycemic rats as compared with normoglycemic rats after ICH. This change is accompanied by increased vasogenic brain edema and more severe blood-brain barrier destruction.

Effect of erythropoietin on expression of aquaporin-2 in kidney of young rats with partial unilateral ureter obstruction

Objective To investigate the effect of erythropoietin (EPO) on expression of aquaporin-2 (AQP2) in the kidney of young SD rats with partial unilateral ureter obstruction (PUUO). Methods Twenty-four young SD rats were randomly divided into 3 groups (PUUO group, n=8; PUUO+ EPO group, n=8; sham group, n=8). The PUUO model was built by embedding the upper quarter of the left ureter into psoas muscle.EPO(500 U/kg) was given to PUUO+ EPO rats at 1 h after surgery, and then repeated 1, 3, 5 days later after surgery by intraperitoneal injection and the same volume of 9 g/L saline was simultaneously given to PUUO rats.The sham group was prepared in parallel by way of laparotomy and free dissection of the left ureter.In 1 week after surgery, the left kidneys of rats were harvested to examine the effect of EPO on the expression of AQP2 in inner medulla by immunohistochemistry, real-time PCR and Western blot. Results Immunohistochemistry showed that the expression of AQP2 in the collecting duct in PUUO group was significantly downregulated than those in sham group and PUUO+ EPO group, whereas, it was slightly weaker in PUUO+ EPO group than those in sham group.These results were further confirmed by Western blot, and the relative quantity of AQP2 in PUUO group was also lower than those in PUUO+ EPO group and sham group (0.290±0.016 vs 0.640±0.106 vs 0.840±0.134, P<0.05). Real-time PCR showed that the level of AQP2 mRNA in PUUO group was lower than those in PUUO+ EPO group and sham group (ΔCt values of 3 groups were -7.60±0.18 vs -9.70±0.54 vs -10.80±0.31, P<0.05). Conclusions EPO can partially prevent the downregulation of AQP2 in young PUUO rats, and its mechanism needs to be further investigated. Key words: Erythropoietin; Partial unilateral ureter obstruction; Aquaporin-2; Kidney

Abstract TP97: Improved Long-Term Outcome after Transient Cerebral Ischemia in Aquaporin-4 Knockout Mice

Introduction: Aquaporin-4 (AQP4) is an abundant aquaporin in brain that has been hypothesized to play a central role in edema formation. Furthermore, AQP4 downregulation has been correlated with connexin 43 (Cx43) downregulation, a gap junction protein important for ion and water transport through astrocyte networks. AQP4 deletion in mice was shown to be beneficial at 24 hours after permanent occlusion of the middle cerebral artery (MCAO). However, the long-term consequences of AQP4 deletion on behavior, edema, and tissue properties after stroke have not been studied. Objective: Following transient MCAO, behavior, lesion volumes, blood brain barrier (BBB) integrity, neuroinflammation, and astrocyte network properties with Cx43 expressions were assessed for 14 days in wild type (WT) and AQP4 -/- mice. Methods: Transient (30-min) filament MCAO was performed on AQP4 -/- (n=5) and WT (n=8) mice on a CD1 genetic background. Behavioral outcomes were assessed by rotarod, beam balance, and foot fault tests from 1 to 14 days (d) post injury. Temporal magnetic resonance imaging (MRI) was undertaken to assess lesion volume. Mice were transcardially perfused at 15 d and the brains were extracted and frozen to perform immunohistochemical analysis for IgG, AQP4, Cx43, and glial fibrillary acidic protein (GFAP). Results: MRI showed significantly smaller lesion volume in AQP4 -/- at 1, 3, 7, and 14d. AQP4 -/- mice also had improved motor function recovery with 54% and 75% decreased number of foot-faults than WT mice respectively at 1 and 3 d; and 60% increased time spent on the rotarod compared to WT over the 14 days after stroke onset. AQP4 -/- mice had reduced IgG extravasation and Cx43 expression but increased GFAP staining compared to WT at 14d, suggesting, respectively, less BBB disruption and modified astrocyte network properties combined with altered neuroinflammation fates. Conclusion: AQP4 deletion resulted in improved long-term functional recovery associated with decreased lesion volumes and improved BBB integrity. Although more work must be done, the beneficial outcomes in AQP4 -/- may partly be due to decreased Cx43 as well, resulting in not only decreased edema formation through AQP4, but also in decreased spread of edema through the astrocyte network.

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.

Down-Regulation of Aquaporin 1 in Rats With Experimental Acute Necrotizing Pancreatitis

The objectives of this study were to investigate the expression of aquaporin 1 in capillary endothelial cells of rats with experimental acute necrotizing pancreatitis (ANP) and to explore its pathogenic role in capillary leak. Sixty-four male Sprague-Dawley rats were randomly divided into control (n = 32) and ANP groups (n = 32). Eight rats in each group were killed at 3, 6, 12, and 18 hours after induction of experimental models. Quantity of ascites and levels of serum amylases were measured. Capillary permeability in pancreas, lung, and intestinal tissue was detected by Evans blue extravasation method. Aquaporin 1 expression in pancreas, lung, and intestinal tissue was detected by real-time polymerase chain reaction, immunohistochemical staining, and Western blot. Serum amylase level was significantly higher in ANP group than in controls (P < 0.05). Evans blue concentration in tissues in the ANP group was significantly higher than that in controls (P < 0.05). Aquaporin 1 mRNA and protein expressions in tissues were significantly less in the ANP group than in the controls (P < 0.05). The expression of aquaporin 1 was down-regulated in the pancreas, lung, and intestinal tissue of ANP rats, which could play an important role in the pathogenesis of capillary leak syndrome.

Chronic use of chloroquine disrupts the urine concentration mechanism by lowering cAMP levels in the inner medulla

Chloroquine, a widely used anti-malaria drug, has gained popularity for the treatment of rheumatoid arthritis, systemic lupus erythematosus (SLE), and human immunodeficiency virus (HIV). Unfortunately, chloroquine may also negatively impact renal function for patients whose fluid and electrolyte homeostasis is already compromised by diseases. Chronic administration of chloroquine also results in polyuria, which may be explained by suppression of the antidiuretic response of vasopressin. Several of the transporters responsible for concentrating urine are vasopressin-sensitive including the urea transporters UT-A1 and UT-A3, the water channel aquaporin-2 (AQP2), and the Na(+)-K(+)-2Cl(-) cotransporter (NKCC2). To examine the effect of chloroquine on these transporters, Sprague-Dawley rats received daily subcutaneous injections of 80 mg·kg(-1)·day(-1) of chloroquine for 4 days. Twenty-four hour urine output was twofold higher, and urine osmolality was decreased by twofold in chloroquine-treated rats compared with controls. Urine analysis of treated rats detected the presence chloroquine as well as decreased urine urea and cAMP levels compared with control rats. Western blot analysis showed a downregulation of AQP2 and NKCC2 transporters; however, UT-A1 and UT-A3 abundances were unaffected by chloroquine treatment. Immunohistochemistry showed a marked reduction of UT-A1 and AQP2 in the apical membrane in inner medullary collecting ducts of chloroquine-treated rats. In conclusion, chloroquine-induced polyuria likely occurs as a result of lowered cAMP production. These findings suggest that chronic chloroquine treatment would exacerbate the already compromised fluid homeostasis observed in diseases like chronic kidney disease.

Transcriptional Regulation of Aquaporins in the Ischemic Rat Retina: Upregulation of Aquaporin-9

Purpose/aim: To determine the transcriptional regulation of retinal aquaporins (AQPs) in rat models of transient and permanent retinal ischemia, and to prove the effects of chemical hypoxia, oxidative stress, glucose, and osmotic alterations on the expression of AQP9 in cultured human retinal pigment epithelium (RPE) cells.Materials and methods: Transient retinal ischemia-reperfusion in rats was induced by elevation of the intraocular pressure for 1 hour. Permanent retinal ischemia was induced by argon laser-induced retinal vein occlusion. The mRNA levels were determined one day after ischemia.Results: Transient and permanent ischemia of the rat retina resulted in downregulation of AQPs 1, 3, 4, 5, 6, 8, and 11 in the RPE and/or neural retina. Pressure-induced transient retinal ischemia-induced upregulation of AQP9 in the neuroretina and RPE, and of AQ12 in the neuroretina. Retinal vein occlusion induced upregulation of AQP0 in the neuroretina and RPE, and of AQP9 and AQP12 in the neuroretina. In cultured human RPE cells, transcriptional expression of AQP9 was stimulated by chemical hypoxia, oxidative stress, VEGF, and high glucose.Conclusions: The data may suggest that the expression of retinal AQP9 is regulated by metabolic and oxidative stress. Upregulation of AQP9 in RPE cells may prevent lactic acidosis and subretinal edema under ischemic and oxidative stress conditions.

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.

Neuromyelitis Optica Immunoglobulin G present in sera from neuromyelitis optica patients affects aquaporin‐4 expression and water permeability of the astrocyte plasma membrane

NMO-IgG autoantibody selectively binds to aquaporin-4 (AQP4), the most abundant water channel in the central nervous system and is now considered a useful serum biomarker of neuromyelitis optica (NMO). A series of clinical and pathological observations suggests that NMO-IgG may play a central role in NMO physiopathology. The current study evaluated, in well-differentiated astrocytes cultures, the consequences of NMO-IgG binding on the expression pattern of AQP4 and on plasma membrane water permeability. To avoid or to facilitate AQP4 down-regulation, cells were exposed to inactivated sera in two different situations (1 hr at 4°C or 12 hr at 37°C). AQP4 expression was detected by immunofluorescence studies using a polyclonal anti-AQP4 or a human anti-IgG antibody, and the water permeability coefficient was evaluated by a videomicroscopy technique. Our results showed that, at low temperatures, cell exposure to either control or NMO-IgG sera does not affect either AQP4 expression or plasma membrane water permeability, indicating that the simple binding of NMO-IgG does not affect the water channel's activity. However, at 37°C, long-term exposure to NMO-IgG induced a loss of human IgG signal from the plasma membrane along with M1-AQP4 isoform removal and a significant reduction of water permeability. These results suggest that binding of NMO-IgG to cell membranes expressing AQP4 is a specific mechanism that may account for at least part of the pathogenic process.

Down-regulation of aquaporins (AQP3) expression by RNA interference suppresses human lung cancer cell proliferation

Down-regulation of aquaporins (AQP3) expression by RNA interference suppresses human lung cancer cell proliferation

Lithium reduces aquaporin-2 transcription independent of prostaglandins

Vasopressin (AVP)-stimulated translocation and transcription of aquaporin-2 (AQP2) water channels in renal principal cells is essential for urine concentration. Twenty percent of patients treated with lithium develop nephrogenic diabetes insipidus (NDI), a disorder in which the kidney is unable to concentrate urine. In vivo and in mouse collecting duct (mpkCCD) cells, lithium treatment coincides with decreased AQP2 abundance and inactivation of glycogen synthase kinase (Gsk) 3β. This is paralleled in vivo by an increased renal cyclooxygenase 2 (COX-2) expression and urinary prostaglandin PGE(2) excretion. PGE(2) reduces AVP-stimulated water reabsorption, but its precise role in lithium-induced downregulation of AQP2 is unclear. Using mpkCCD cells, we here investigated whether prostaglandins contribute to lithium-induced downregulation of AQP2. In these cells, lithium application reduced AQP2 abundance, which coincided with Gsk3β inactivation and increased COX-2 expression. Inhibition of COX by indomethacin, leading to reduced PGE(2) and PGF(2α) levels, or dexamethasone-induced downregulation of COX-2 both increased AQP2 abundance, while PGE(2) addition reduced AQP2 abundance. However, lithium did not change the prostaglandin levels, and indomethacin and dexamethasone did not prevent lithium-induced AQP2 downregulation. Further analysis revealed that lithium decreased AQP2 protein abundance, mRNA levels and transcription, while PGE(2) reduced AQP2 abundance by increasing its lysosomal degradation, but not by reducing AQP2 gene transcription. In conclusion, our data reveal that in mpkCCD cells, prostaglandins decrease AQP2 protein stability by increasing its lysosomal degradation, indicating that in vivo paracrine-produced prostaglandins might have a role in lithium-induced NDI via this mechanism. However, lithium affects also AQP2 gene transcription, which is prostaglandin independent.

Bilateral ureteral obstruction induces early downregulation and redistribution of AQP2 and phosphorylated AQP2.

Bilateral ureteral obstruction (BUO) is characterized by impairment of urine flow from the kidneys and altered expression of specific membrane proteins in the kidney involved in regulation of renal water and salt transport. Importantly, 24-h BUO reduces the abundance of the collecting duct water channel aquaporin-2 (AQP2) and AQP2 phosphorylated at serine 256 (AQP2pS256). To investigate the mechanism behind downregulation of AQP2 in BUO, rats were subjected to BUO and examined after 2, 6, 12, and 24 h. Q-PCR and immunoblotting showed significantly decreased AQP2 mRNA expression after 2-h BUO and decreased abundance of total AQP2 after 12 and 24 h. In parallel, immunohistochemistry showed weaker labeling of AQP2 at the apical surface of inner medullary collecting ducts (IMCD) compared with controls. The abundance of AQP2pS256 was significantly reduced from 6-h BUO and was confirmed by immunohistochemistry. Importantly, immunoblotting showed reduced abundance of AQP2pS261 after 12- and 24-h BUO mimicking total AQP2. Immunohistochemistry demonstrated early changed intracellular localization of AQP2pS261 in BUO, and colocalization studies showed redistribution from the apical membrane to early endosomes and lysosomes. In conclusion, BUO induces a very early regulation of AQP2 both at the level of abundance and on cellular localization. AQP2 and AQP2 phosphorylated at ser261 redistribute to more intracellular localizations and colocalize with the early endosomal marker EEA1 and the lysosomal marker cathepsin D, suggesting that early downregulation of AQP2 could in part be caused by degradation of AQP2 through a lysosomal degradation pathway.

Disruption of cyclooxygenase‐2 prevents down‐regulation of cortical AQP2 and AQP3 in response to bilateral ureteral obstruction

Previously, we demonstrated that bilateral ureteral obstruction (BUO) in rats was associated with increased cyclooxygenase type 2 (COX-2) expression in renal inner medulla (IM) and that selective COX-2 inhibition prevented downregulation of aquaporin 2 (AQP2) in response to BUO. We hypothesized that mice with disrupted COX-2 are protected from BUO-induced changes in transporters. COX-2 deficient and wild type littermates (COX-2−/− and WT C57BL/6J) were employed to determine aquaporin-2 and -3 and vasopressin V2 receptor protein level in response to BUO. The kidneys were removed and prepared for QPCR and immunoblotting analyses. Plasma osmolality and creatinine increased in both WT and COX-2−/− mice subjected to BUO. Plasma urea was increased in COX-2−/− mice compared to WT at baseline and in response to BUO. COX-2 protein level increased in WT mice in response to BUO and was not detectable in COX-2−/−. V2 receptor protein abundance was lower COX-2−/− mice compared to WT mice after BUO. Downregulation of AQP2 and AQP3 mRNA and protein was prevented in the cortex of COX-2−/− mice subjected to BUO. In IM, AQP2 and AQP3 protein level decreased in response to BUO in both COX-2−/− and WT mice compared to sham. In conclusion, disrupted COX-2 prevents down-regulation of AQP2 and AQP3 in response to BUO in cortex, but not in inner medulla, suggesting a differential regulation of AQP2 and -3 expression in the separate kidney zones.

Hypotonicity-induced Reduction of Aquaporin-2 Transcription in mpkCCD Cells Is Independent of the Tonicity Responsive Element, Vasopressin, and cAMP

The syndrome of inappropriate antidiuretic hormone secretion is characterized by excessive water uptake and hyponatremia. The extent of hyponatremia, however, is less than anticipated, which is ascribed to a defense mechanism, the vasopressin-escape, and is suggested to involve a tonicity-determined down-regulation of the water channel aquaporin-2 (AQP2). The underlying mechanism, however, is poorly understood. To study this, we used the mouse cortical collecting duct (mpkCCD) cell line. MpkCCD cells, transfected with an AQP2-promoter luciferase construct showed a reduced and increased AQP2 abundance and transcription following culture in hypotonic and hypertonic medium, respectively. This depended on tonicity rather than osmolality and occurred independently of the vasopressin analog dDAVP, cAMP levels, or protein kinase A activity. Although prostaglandins and nitric oxide reduced AQP2 abundance, inhibition of their synthesis did not influence tonicity-induced AQP2 transcription. Also, cells in which the cAMP or tonicity-responsive element (CRE/TonE) in the AQP2-promoter were mutated showed a similar response to hypotonicity. Instead, the tonicity-responsive elements were pin-pointed to nucleotides -283 to -252 and -157 to -126 bp. In conclusion, our data indicate that hypotonicity reduces AQP2 abundance and transcription, which occurs independently of vasopressin, cAMP, and the known TonE and CRE in the AQP2-promoter. Increased prostaglandin and nitric oxide, as found in vivo, may contribute to reduced AQP2 in vasopressin-escape, but do not mediate the effect of hypotonicity on AQP2 transcription. Our data suggest that two novel segments (-283 to -252 and -157 to -126 bp) in the AQP2-promoter mediate the hypotonicity-induced AQP2 down-regulation during vasopressin-escape.

Downregulation of Aquaporin-1 in Alveolar Microvessels in Lungs Adapted to Chronic Heart Failure

The threshold pressure for lung edema formation is increased in severe chronic heart failure (CHF) due to reduced microvascular permeability. The water channel aquaporin-1 (AQP1) is present in the pulmonary microvascular endothelium, and a number of studies suggest the importance of AQP1 as a molecular determinant of pulmonary microvascular water transport. The present study examined the abundance and localization of AQP1 in lungs from rats with CHF. We used two different models of CHF: ligation of the left anterior descending coronary artery (LAD ligation) and aorta-banding (AB). Sham-operated rats served as controls. Echocardiographic verification of left ventricular dysfunction, enhanced left ventricular end-diastolic pressure, and right ventricular hypertrophy confirmed the presence of CHF. Western blotting of whole-lung homogenates revealed significant downregulation of AQP1 in LAD-ligated rats (24h: 58±5% of sham; 3weeks: 8±3% of sham; 9weeks: 16±6% of sham) and after AB (30weeks: 37±5% of sham), whereas the protein levels of the specific endothelial cell marker PECAM-1 was increased 3weeks after LAD ligation (229±20% of sham), but unchanged after 9weeks and in the AB rats compared to controls. Immunohistochemical examination 3weeks after LAD ligation showed intact labeling of PECAM-1 but an almost complete absence of AQP1 in the pulmonary alveolar microvessels in the CHF rats. These results suggest that downregulation of AQP1 in the alveolar microvessels may act as a compensatory mechanism to protect against formation of excessive pulmonary edema in CHF.

αENaC-Mediated Lithium Absorption Promotes Nephrogenic Diabetes Insipidus

Lithium-induced nephrogenic diabetes insipidus (NDI) is accompanied by polyuria, downregulation of aquaporin 2 (AQP2), and cellular remodeling of the collecting duct (CD). The amiloride-sensitive epithelial sodium channel (ENaC) is a likely candidate for lithium entry. Here, we subjected transgenic mice lacking αENaC specifically in the CD (knockout [KO] mice) and littermate controls to chronic lithium treatment. In contrast to control mice, KO mice did not markedly increase their water intake. Furthermore, KO mice did not demonstrate the polyuria and reduction in urine osmolality induced by lithium treatment in the control mice. Lithium treatment reduced AQP2 protein levels in the cortex/outer medulla and inner medulla (IM) of control mice but only partially reduced AQP2 levels in the IM of KO mice. Furthermore, lithium induced expression of H(+)-ATPase in the IM of control mice but not KO mice. In conclusion, the absence of functional ENaC in the CD protects mice from lithium-induced NDI. These data support the hypothesis that ENaC-mediated lithium entry into the CD principal cells contributes to the pathogenesis of lithium-induced NDI.

Chemical preconditioning-induced reactive astrocytosis contributes to the reduction of post-ischemic edema through aquaporin-4 downregulation

Aquaporins are a family of membrane proteins that promote the transmembrane diffusion of water. Aquaporin-4 (AQP4) is a predominant water channel protein in the brain and is concentrated in the end-feet of astrocytes. A critical question is what role astrocytic AQP4 plays in pathological conditions. Another matter to be elucidated is the relationship between morphological changes in astrocytes and AQP4 expression in such cases. We investigated the correlation between AQP4 expression and post-ischemic brain edema formation with astrocytic molecular markers after 3-nitropropionic acid (3NP) preconditioning. 3NP is a mitochondrial toxin, which can induce tolerance to ischemia at subtoxic levels. Rats were treated with 3NP at the tolerance-inducible and the non-tolerance-inducible stage (TS or NTS) before focal ischemia. The control group was injected with physiological saline. After ischemia, the hemispheric enlargement (HE) was volumetrically measured. Immunohistochemical and immunofluorescence analyses of AQP4, glial fibrillary acidic protein (GFAP), and glutamine synthetase (GS) were also conducted after the 3NP treatment and a vehicle was applied. HE was found to be significantly smaller in the TS group than in the vehicle group or the NTS group. The immunofluorescence analyses demonstrated that the AQP4 immunoreactivity in the cortex and striatum was significantly reduced in the TS group but not in the NTS group. In contrast, both GFAP expression and GS expression in the TS group were enhanced, with reactive astrocytosis. AQP4 downregulation in reactive astrocytosis may be one of the factors contributing to the role of 3NP preconditioning in attenuating post-ischemic edema.

Effect of salinity on water relations of wild barley plants differing in salt tolerance.

Certain lines of wild barley (Hordeum spontaneum) are more tolerant of salinity than others. The physiological basis of this difference is examined in a comparative study of a saline-tolerant and saline-intolerant line that emphasizes plant water relations. Effects of salt-treatment (75 mM maximum) extending from a few hours to 3 weeks were quantified in 8-day-old seedlings of a saline-sensitive wild barley line ('T-1') and a less saline-sensitive line ('20-45'). Plants were grown in nutrient culture. Levels of mRNA of the HtPIP2;4 aquaporin (AQP) gene were determined together with a range of physiological responses including root hydraulic conductivity, osmotic potential of root xylem sap, transpiration, leaf relative water content, root water content, leaf water potential, leaf sap osmolality, leaf length, leaf area and chlorophyll content. Salt treatment inhibited transpiration and hydraulic conductivity more in salt-tolerant '20-45' plants than in salt-sensitive 'T-1'. In '20-45', the effect was paralleled by a fast (within a few hours) and persistent (3 days) down-regulation of aquaporin. In salt-sensitive 'T-1' plants, aquaporin down-regulation was delayed for up to 24 h. Greater tolerance in '20-45' plants was characterized by less inhibition of leaf area, root fresh weight, leaf water content and chlorophyll concentration. Leaf water potentials were similar in both lines. (i) Decline in hydraulic conductivity in salt-treated barley plants is important for stomatal closure, (ii) lowered transpiration rate is beneficial for salt tolerance, at least at the seedling stage and (iii) changes in AQP expression are implicated in the control of whole plant hydraulic conductivity and the regulation of shoot water relations.

Amiloride blocks lithium entry through the sodium channel thereby attenuating the resultant nephrogenic diabetes insipidus

Lithium therapy frequently induces nephrogenic diabetes insipidus; amiloride appears to prevent its occurrence in some clinical cases. Amiloride blocks the epithelial sodium channel (ENaC) located in the apical membrane of principal cells; hence one possibility is that ENaC is the main entry site for lithium and the beneficial effect of amiloride may be through inhibiting lithium entry. Using a mouse collecting duct cell line, we found that vasopressin caused an increase in Aquaporin 2 (AQP2) expression which was reduced by clinically relevant lithium concentrations similar to what is seen with in vivo models of this disease. Further amiloride or benzamil administration prevented this lithium-induced downregulation of AQP2. Amiloride reduced transcellular lithium transport, intracellular lithium concentration, and lithium-induced inactivation of glycogen synthase kinase 3beta. Treatment of rats with lithium downregulated AQP2 expression, reduced the principal-to-intercalated cell ratio, and caused polyuria, while simultaneous administration of amiloride attenuated all these changes. These results show that ENaC is the major entry site for lithium in principal cells both in vitro and in vivo. Blocking lithium entry with amiloride attenuates lithium-induced diabetes insipidus, thus providing a rationale for its use in treating this disorder.

REDUCED LUNG WATER TRANSPORT RATE ASSOCIATED WITH DOWNREGULATION OF AQUAPORIN‐1 AND AQUAPORIN‐5 IN AGED MICE

1. The purpose of the present study was to examine lung water transport properties and the expression and regulation of the alveolar endothelial water channel aquaporin (AQP)-1 and the epithelial water channel AQP-5 in aged mouse lung using gene expression analysis and water permeability measurements. 2. In aged (20-24-month-old) mice, AQP-1 and AQP-5 mRNA expression decreased by 55.5 and 50.3%, respectively, compared with that in young (8-10-week-old) mice (P < 0.01). In addition, AQP-1 and AQP-5 protein expression decreased in aged mice by 36.9 and 44.6%, respectively, compared with that in young mice (P < 0.01). 3. The osmotically driven water transport rate between the airspace and capillary compartments was reduced by 31.7% in aged mice compared with young mice (2.8 +/- 0.3 vs 4.1 +/- 0.3 mg/s, respectively; P < 0.01). The hydrostatically driven lung water accumulation rate in response to a 10 cmH(2)O increase in pulmonary artery pressure was also reduced in aged mice by 21.9% compared with young mice (0.32 +/- 0.06 vs 0.41 +/- 0.04 mg/s, respectively; P < 0.01). 4. There was a 62.7% decrease in serum glucocorticoids in aged mice compared with young mice (67.6 +/- 26.8 vs 181.3 +/- 44.4 nmol/L, respectively; P < 0.01). In vivo administration of dexamethasone (4 mg/kg) for 5 consecutive days to aged mice increased lung AQP-1 mRNA and protein expression by 2.1 +/- 0.1 fold (P < 0.01) and 1.8 +/- 0.2 fold (P < 0.01), respectively. Accordingly, osmotically and hydrostatically driven water transport rates increased by 35.6% (P < 0.01) and 31.2% (P < 0.01), respectively. 5. The present study provides the first evidence of altered lung water transport associated with downregulation of AQPs in aged lung. Blood glucocorticoid hormone levels are important to maintain normal AQP-1 expression in the lung microvascular endothelium. Corticosteroid-induced AQP-1 upregulation may contribute to the role of corticosteroids in accelerating oedema clearance in aged lung.