Aquaporin-1 Knockout Research Articles

TMIC-27. THE ROLE OF AQUAPORIN-4 IN GLIOBLASTOMA-ASSOCIATED VASOGENIC CEREBRAL EDEMA

Abstract Glioblastoma (GBM) is the most common and aggressive primary brain tumor in adults. Vasogenic cerebral edema is a severe complication that occurs in a majority of GBM patients, leading to a significant increase in intracranial pressure, accompanying neurological deficits, and increased mortality rates. Edema in GBM patients is almost exclusively managed by the corticosteroid dexamethasone (DEX), which has recently been found to have several drawbacks, including immunosuppression and interference with radiation therapy. Thus, there is an urgent need to better understand the mechanisms of edema resolution in GBM in order to identify alternative treatment strategies. The water channel aquaporin-4 (AQP4) is a critical player in the brain's water homeostasis, with increased levels in GBM compared to the normal brain. To gain more insight into the role of AQP4 in GBM-associated edema, we used the RCAS/tv-a system to generate de novo GBM with different driver mutations in Aqp4 knockout (KO), heterozygous (HET), and wild-type (WT) mice. Multiplex IF staining combined with RNAScope of tumors showed that AQP4 expression co-localized with GFAP-positive reactive astrocytes in perivascular areas. Our results indicated that tumor-bearing mice in an Aqp4-deficient background had increased edema content and decreased survival, suggesting that AQP4 is important for edema resolution from tumors. Loss of Aqp4 also significantly decreased astrocyte coverage in the tumor core, but not in the tumor periphery. Moreover, anti-VEGFA treatment only improved vessel leakage and reduced edema in WT tumor-bearing mice. Ongoing studies are focused on characterizing the impact of Aqp4 loss on the gene expression profile of tumor core-specific astrocytes using single-cell RNA sequencing and spatial transcriptomics. We are also studying the cellular distribution of AQP4 in GBM and the effect of DEX treatment on AQP4 levels.

Tanshinone IIA changed the amniotic fluid volume and regulated expression of AQP1 and AQP3 in amniotic epithelium cells: a promising drug treating abnormal amniotic fluid volume

BackgroundMany studies have confirmed the association of aquaporins (AQPs) with abnormal amniotic fluid volume (AFV). In our previous experiments, we found that Tanshinone IIA was able to regulate the expression of AQP1 and AQP3. However, the exact mechanism by which Tanshinone IIA regulates AQPs protein expression and its effect on AFV remains unclear. The purpose of this study was to investigate the effects of Tanshinone IIA on AFV and the possible molecular mechanism of regulation of AQP1 and AQP3.MethodsThe expression of AQPs protein in the amniotic membranes was compared between pregnant women with normal pregnancy and those with isolated oligohydramnios. The AQP1 knockout (AQP1-KO) mice and wild-type (WT) mice were treated with saline or Tanshinone IIA (10 mg/kg) at 13.5GD and 16.5GD. Human amniotic epithelium cells (hAECs) from pregnant women with normal AFV and isolated oligohydramnios were incubated with 35 μmmol/L Tanshinone IIA or 25 mmol/L LiCl [inhibitor of glycogen synthetic kinase 3β (GSK-3β)]. The protein expressions of AQPs, GSK-3β, phospho-GSK-3β (Ser9) in fetal membranes of mice and human amniotic epithelium cells were detected by western blotting.ResultsThe expression of AQP1 protein in the amniotic membrane of isolated oligohydramnios was increased compared with normal pregnancy. The AFV in AQP1-KO mice is higher than that in WT mice. In wild-type mice, AFV in Tanshinone IIA group was significantly higher than that in control group, and AQP1 protein expression was significantly lower than that in control group, but in AQP1 knockout mice, Tanshinone IIA reduced amniotic fluid volume and AQP3 protein expression at 16.5GD. Tanshinone IIA reduced AQP1, AQP3 and p-GSK-3β (Ser9) protein expression in normal hAECs, and this effect was inhibited by LiCl. In hAECs with oligohydramnios, the down-regulation of AQP1 and up-regulation of AQP3 by Tanshinone IIA was independent of GSK-3β signaling pathway.ConclusionsTanshinone IIA may increase AFV in normal pregnancy by downregulating AQP1 protein expression in the fetal membranes, which may be associated with p-GSK-3β signaling pathway. But a larger AFV in AQP1-KO mice was significantly attenuated by Tanshinone IIA, which may be related to AQP3. Tanshinone IIA is a promising drug for the treatment of amniotic fluid abnormality.

AQP1 in the Gastrointestinal Tract of Mice: Expression Pattern and Impact of AQP1 Knockout on Colonic Function.

Aquaporin 1 (AQP1) is one of thirteen known mammalian aquaporins. Its main function is the transport of water across cell membranes. Lately, a role of AQP has been attributed to other physiological and pathological functions including cell migration and peripheral pain perception. AQP1 has been found in several parts of the enteric nervous system, e.g., in the rat ileum and in the ovine duodenum. Its function in the intestine appears to be multifaceted and is still not completely understood. The aim of the study was to analyze the distribution and localization of AQP1 in the entire intestinal tract of mice. AQP1 expression was correlated with the hypoxic expression profile of the various intestinal segments, intestinal wall thickness and edema, as well as other aspects of colon function including the ability of mice to concentrate stools and their microbiome composition. AQP1 was found in a specific pattern in the serosa, the mucosa, and the enteric nervous system throughout the gastrointestinal tract. The highest amount of AQP1 in the gastrointestinal tract was found in the small intestine. AQP1 expression correlated with the expression profiles of hypoxia-dependent proteins such as HIF-1α and PGK1. Loss of AQP1 through knockout of AQP1 in these mice led to a reduced amount of bacteroidetes and firmicutes but an increased amount of the rest of the phyla, especially deferribacteres, proteobacteria, and verrucomicrobia. Although AQP-KO mice retained gastrointestinal function, distinct changes regarding the anatomy of the intestinal wall including intestinal wall thickness and edema were observed. Loss of AQP1 might interfere with the ability of the mice to concentrate their stool and it is associated with a significantly different composition of the of the bacterial stool microbiome.

Assessing the Role of Aquaporin 4 in Skeletal Muscle Function.

Water transport across the biological membranes is mediated by aquaporins (AQPs). AQP4 and AQP1 are the predominantly expressed AQPs in the skeletal muscle. Since the discovery of AQP4, several studies have highlighted reduced AQP4 levels in Duchenne muscular dystrophy (DMD) patients and mouse models, and other neuromuscular disorders (NMDs) such as sarcoglycanopathies and dysferlinopathies. AQP4 loss is attributed to the destabilizing dystrophin-associated protein complex (DAPC) in DMD leading to compromised water permeability in the skeletal muscle fibers. However, AQP4 knockout (KO) mice appear phenotypically normal. AQP4 ablation does not impair physical activity in mice but limits them from achieving the performance demonstrated by wild-type mice. AQP1 levels were found to be upregulated in DMD models and are thought to compensate for AQP4 loss. Several groups investigated the expression of other AQPs in the skeletal muscle; however, these findings remain controversial. In this review, we summarize the role of AQP4 with respect to skeletal muscle function and findings in NMDs as well as the implications from a clinical perspective.

Non-Transport Functions of Aquaporins.

Although it has been more than 20years since the first aquaporin was discovered, the specific functions of many aquaporins are still under investigation, because various mice lacking aquaporins have no significant phenotypes. And in many studies, the function of aquaporin is not directly related to its transport function. Therefore, this chapter will focus on some unexpected functions of aquaporins, such the decreased tumor angiogenesis in AQP1 knockout mice, and AQP1 promotes cell migration, possibly by accelerating the water transport in lamellipodia of migrating cells. AQP transports glycerol, and water regulates glycerol content in epidermis and fat, thereby regulating skin hydration/biosynthesis and fat metabolism. AQPs may also be involved in neural signal transduction, cell volume regulation, and organelle physiology. AQP1, AQP3, and AQP5 are also involved in cell proliferation. In addition, AQPs have also been reported to play roles in inflammation in various tissues and organs. The functions of these AQPs may not depend on the permeability of small molecules such as water and glycerol, suggesting AQPs may play more roles in different biological processes in the body.

Fasting influences aquaporin expression, water transport and adipocyte metabolism in the peritoneal membrane.

The water channels aquaporin-1 (AQP1) and AQP7 are abundantly expressed in the peritoneal membrane. While AQP1 facilitates water transport during peritoneal dialysis (PD), the role of AQP7, which mediates glycerol transport during fasting, remains unknown. We investigated the distribution of AQP7 and AQP1 and used a mouse model of PD to investigate the role of AQP7 in the peritoneal membrane at baseline and after fasting. Single nucleus RNA-sequencing revealed that AQP7 was mostly detected in mature adipocytes, whereas AQP1 was essentially expressed in endothelial cells. Fasting induced significant decreases in whole body fat, plasma glucose, insulin and triglycerides, as well as higher plasma glycerol and corticosterone levels in mice, paralleled by major decreases in adipocyte size and levels of fatty acid synthase and leptin, and increased levels of hormone-sensitive lipase mRNAs in the peritoneum. Mechanistically, fasting upregulated the expression of AQP1 and AQP7 in the peritoneum, with increased ultrafiltration but no change in small solute transport. Studies based on Aqp1 and Aqp7 knockout mice and RU-486 inhibition demonstrated that the glucocorticoid induction of AQP1 mediates the increase in ultrafiltration whereas AQP7 regulates the size of adipocytes in the peritoneum. Fasting induces a coordinated regulation of lipolytic and lipogenic factors and aqua(glycero)porins in the peritoneum, driving structural and functional changes. These data yield novel information on the specific roles of aquaporins in the peritoneal membrane and indicate that fasting improves fluid removal in a mouse model of PD.

The Involvement of Aquaporin-4 in the Interstitial Fluid Drainage Impairment Following Subarachnoid Hemorrhage.

The mechanism of brain injury following subarachnoid hemorrhage (SAH) has not yet been clarified. The glymphatic system (GS), a glia-dependent waste clearance pathway, drains away soluble waste proteins and metabolic products, even some toxic factors from the brain. Aquaporin-4 (Aqp4) is highly expressed on the astrocyte foot processes and facilitates the interstitial fluid (ISF) transportation in the GS system. In this study, the role of Aqp4 in the GS injury after SAH was explored using Aqp4 gene knockout (Aqp4−/−) Sprague Dawley rats. The results of MRI, fluorescent imaging, and transmission electron microscopy (TEM) indicated that, after SAH, the inflow of cerebrospinal fluid (CSF) into the brain and the clearance of ISF from the brain were both significantly decreased. Meanwhile, the expression level of Aqp4 around the artery was markedly higher than that around the vein following SAH. Aqp4 knockout exacerbated the GS damage after SAH. In summary, after SAH, there was an apparent GS impairment, and Aqp4 played key roles in modulating the function of GS in the brain.

Down-regulation of Aquaporin-1 mediates a microglial phenotype switch affecting glioma growth

Aquaporin 1 (AQP1), a transmembrane protein that forms water channels, has previously been shown to facilitate growth and progression of many types of tumors by modulating tumor cell migration, proliferation and angiogenesis. Here, we determined the impact of AQP1 expression in the tumor environment on the progression of brain tumors. Primary microglia from wild type(WT) and AQP1 knockout(KO) mice were used to test AQP1 effect on microglia function by using Western blot, quantative PCR, in an experimental in vivo mouse glioma model and organotypic brain slice culture. Deletion of AQP1 in the host tissue significantly reduced the survival of the mice implanted with GL261 glioma cells. The density of glioma-associated microglia/macrophages was almost doubled in AQP1KO mice. We found that factors secreted from GL261 cells decrease microglial AQP1 expression via the MEK/ERK pathway, and that inhibition of this pathway with Trametinib reduced tumor growth and prolonged the survival of tumor bearing mice, an effect which required the presence of microglia. Deletion of AQP1 in cultured microglia resulted in an increase in migratory activity and a decrease in TLR4-dependent innate immune responses. Our study demonstrates a functional relevance of AQP1 expression in microglia and hints to AQP1 as a potential novel target for glioma therapy.

Effect of aquaporin 1 on mouse peritoneal mesothelial cells after a long-term peritoneal dialysis.

Aquaporin 1 (AQP1) is one member of the aquaporin family, also the deeply studied one. It is widely located on the endothelial cells, but the effect of AQP1 on the peritoneal mesothelial cells (PMCs) after long-term peritoneal dialysis (PD) has not been reported before. We divided normal mice into two groups, control group and dialysis group, to confirm the fibrotic changes and expression of APQ1 on peritoneal mesothelial cells. Then we assigned normal mice and AQP1 knockout mice into four groups: Control group, normal dialysis group, AQP1 knockout control group and AQP1 knockout dialysis group. The two dialysis groups received 4.25% glucose dialysis for 28 days. We found that mice in both dialysis groups showed peritoneal fibrotic changes, which were most severe in the AQP1 knockout dialysis group; the peritoneal thickness in the AQP1 knockout dialysis group was also thicker than that in the dialysis group (P < .05). We used electron microscopy to detect ultrastructural changes and observed changes in microvilli and vacuolar degeneration in mesothelial cells from all groups except the control group. The basement membranes were damaged in the AQP1 knockout dialysis group, and peritoneal mesothelial cells were disrupted and detached in this group. Together our findings indicate that AQP1 plays an important role in maintaining the physiological functions of peritoneal mesothelial cells, and AQP1 can protect mesothelial cells during dialysis.

Increased cerebral vascularization and decreased water exchange across the blood-brain barrier in aquaporin-4 knockout mice.

Aquaporin-4 (AQP4) plays an important role in regulating water exchange across the blood-brain barrier (BBB) and brain-cerebrospinal fluid interface. Studies on AQP-4 knockout mice (AQP4-KO) have reported considerable protection from brain edema induced by acute water intoxication and ischemic stroke, identifying AQP4 as a potential target for therapeutic interventions. However, the long-term effects of chronic AQP4 suppression are yet to be elucidated. In the current study, we evaluated the physiological and structural changes in adult AQP4-KO mice using magnetic resonance imaging (MRI) and immunohistochemical analysis. Water exchange across BBB was assessed by tracking an intravenous bolus injection of oxygen-17 (17O) water (H217O) using 17O-MRI. Cerebral blood flow (CBF) was quantified using arterial spin-labeling (ASL) MRI. Capillary density was determined by immunohistochemical staining for glucose transporter-1 (GLUT1). Compared to wildtype control mice, AQP4-KO mice showed a significant reduction in peak and steady-state H217O uptake despite unaltered CBF. Interestingly, a 22% increase in cortical capillary density was observed in AQP4-KO mice. These results suggest that increased cerebral vascularization may be an adaptive response to chronic reduction in water exchange across BBB in AQP4-KO mice.

Aquaporin 4 Suppresses Neural Hyperactivity and Synaptic Fatigue and Fine-Tunes Neurotransmission to Regulate Visual Function in the Mouse Retina

The bidirectional water channel aquaporin 4 (AQP4) is abundantly expressed in the neural tissue. The advantages and disadvantages of AQP4 neural tissue deficiency under pathological conditions, such as inflammation, and relationship with neural diseases, such as Alzheimer’s disease, have been previously reported. However, the physiological functions of AQP4 are not fully understood. Here, we evaluated the role of AQP4 in the mouse retina using Aqp4 knockout (KO) mice. Aqp4 was expressed in Müller glial cells surrounding the synaptic area between photoreceptors and bipolar cells. Both scotopic and photopic electroretinograms showed hyperactive visual responses in KO mice, gradually progressing with age. Moreover, the amplitude reduction after frequent stimuli and synaptic fatigue was more severe in KO mice. Glutamine synthetase, glutamate aspartate transporter, synaptophysin, and the inward potassium channel Kir2.1, but not Kir4.1, were downregulated in KO retinas. KIR2.1 colocalized with AQP4 in Müller glial cells at the synaptic area, and its expression was affected by Aqp4 levels in primary Müller glial cell cultures. Intraocular injection of potassium in wild-type mice led to visual function hyperactivity, as observed in Aqp4 KO mice. Mitochondria molecules, such as Pgc1α and CoxIV, were downregulated, while apoptotic markers were upregulated in KO retinas. AQP4 may fine-tune synaptic activity, most likely by regulating potassium metabolism, at least in part, via collaborating with KIR2.1, and possibly indirectly regulating glutamate kinetics, to inhibit neural hyperactivity and synaptic fatigue which finally affect mitochondria and cause neurodegeneration.

Aquaporin 4 deficiency alleviates experimental colitis in mice.

Aquaporin (AQP) 4 is expressed in the basolateral membrane of colonic epithelial cells, and the purpose of this study was to explore the mechanistic role of AQP4 in experimental colitis. Experimental colitis was induced in AQP4 knockout (AQP4-/-) CD-1 mice and AQP4 wild-type (AQP4wt) mice by oral administration of dextran sulfate sodium (DSS). Experimental colitis was clinically established. Compared with AQP4wt mice, AQP4-/- mice showed increased tolerance to DSS-induced experimental colitis, including lesser degree of weight loss, diarrhea and bleeding, lower disease activity index scores, longer colon lengths, and lesser histologic scores. DSS-treated AQP4-/- mice had lower serum levels of IL-6 and TNF, higher IL-10 level, and lesser inflammatory cell infiltration. DSS-treated AQP4-/- mice also had lower immunostaining of NF-κB p65 as well as nuclear levels of p65 and phosphorylated p65. Sequencing of 16S rRNA indicated that DSS-treated AQP4-/- mice maintained intestinal microbial diversity and had higher Firmicutes/Bacteroidetes ratios and greater relative abundance of Erysipelotrichaceae species. These results suggested for the first time that AQP4 deficiency alleviates experimental colitis in mice. Our study helps to understand the pathogenesis of inflammatory bowel diseases, and blocking AQP4 may represent a novel therapeutic approach for ulcerative colitis.-Wang, L., Tang, H., Wang, C., Hu, Y., Wang, S., Shen, L. Aquaporin 4 deficiency alleviates experimental colitis in mice.

Aquaporin-4-dependent glymphatic solute transport in the rodent brain.

The glymphatic system is a brain-wide clearance pathway; its impairment contributes to the accumulation of amyloid-β. Influx of cerebrospinal fluid (CSF) depends upon the expression and perivascular localization of the astroglial water channel aquaporin-4 (AQP4). Prompted by a recent failure to find an effect of Aqp4 knock-out (KO) on CSF and interstitial fluid (ISF) tracer transport, five groups re-examined the importance of AQP4 in glymphatic transport. We concur that CSF influx is higher in wild-type mice than in four different Aqp4 KO lines and in one line that lacks perivascular AQP4 (Snta1 KO). Meta-analysis of all studies demonstrated a significant decrease in tracer transport in KO mice and rats compared to controls. Meta-regression indicated that anesthesia, age, and tracer delivery explain the opposing results. We also report that intrastriatal injections suppress glymphatic function. This validates the role of AQP4 and shows that glymphatic studies must avoid the use of invasive procedures.

Noninvasive, Targeted Creation of Neuromyelitis Optica Pathology in AQP4-IgG Seropositive Rats by Pulsed Focused Ultrasound.

Neuromyelitis optica spectrum disorders (herein called NMO) is an autoimmune disease of the CNS characterized by astrocyte injury, inflammation, and demyelination. In seropositive NMO, immunoglobulin G autoantibodies against aquaporin-4 (AQP4-IgG) cause primary astrocyte injury. A passive transfer model of NMO was developed in which spatially targeted access of AQP4-IgG into the CNS of seropositive rats was accomplished by pulsed focused ultrasound through intact skin. Following intravenous administration of microbubbles, pulsed ultrasound at 0.5 MPa peak acoustic pressure was applied using a 1 MHz transducer with 6-cm focal length. In brain, the transient opening of the blood-brain barrier (BBB) in an approximately prolate ellipsoidal volume of diameter ∼3.5 mm and length ∼44 mm allowed entry of IgG-size molecules for up to 3-6 hours. The ultrasound treatment did not cause erythrocyte extravasation or inflammation. Ultrasound treatment in AQP4-IgG seropositive rats produced localized NMO pathology in brain, with characteristic astrocyte injury, inflammation, and demyelination after 5 days. Pathology was not seen when complement was inhibited, when non-NMO human IgG was administered instead of AQP4-IgG, or in AQP4-IgG seropositive AQP4 knockout rats. NMO pathology was similarly created in cervical spinal cord in seropositive rats. These results establish a noninvasive, spatially targeted model of NMO in rats, and demonstrate that BBB permeabilization, without underlying injury or inflammation, is sufficient to create NMO pathology in AQP4-IgG seropositive rats.

Complement-dependent bystander injury to neurons in AQP4-IgG seropositive neuromyelitis optica

BackgroundAquaporin-4-immunoglobulin G (AQP4-IgG) seropositive neuromyelitis optica spectrum disorder (herein called NMO) is an autoimmune disease of the central nervous system in which AQP4-IgG binding to AQP4 on astrocytes results in complement-dependent astrocyte injury and secondary inflammation, demyelination, and neuron loss. We previously reported evidence for a complement bystander mechanism for early oligodendrocyte injury in NMO. Herein, we tested the hypothesis that complement bystander injury, which involves diffusion to nearby cells of activated soluble complement components from complement-injured astrocytes, is a general phenomenon that may contribute to neuronal injury in NMO.MethodsPrimary cocultures of rat astrocytes and cortical neurons were established to study complement-dependent cell death after exposure to AQP4-IgG and complement. In animal experiments, AQP4-IgG was delivered to adult rats by intracerebral injection. Cell cultures and rat brain were studied by immunofluorescence.ResultsIn primary astrocyte-neuron cocultures, addition of AQP4-IgG and complement resulted in death of neurons nearby astrocytes. Deposition of complement membrane attack complex C5b-9 was seen on neurons nearby astrocytes, whereas C1q, the initiating protein in the complement pathway, was seen only on astrocytes. Neuron death was not seen with a complement inhibitor, with C1q- or C6-depleted complement, in pure neuron cultures exposed to AQP4-IgG and complement or in cocultures exposed to an astrocyte toxin. Intracerebral injection in rats of AQP4-IgG and a fixable dead cell fluorescent marker produced death of neurons near astrocytes, with C5b-9 deposition. Neuron death was not seen in rats receiving a complement inhibitor or in AQP4-IgG-injected AQP4 knockout rats.ConclusionThese results support a novel mechanism for early neuron injury in NMO and provide evidence that complement bystander injury may be a general phenomenon for brain cell injury following AQP4-IgG-targeted astrocyte death.

The Importance of Aquaporin 1 in Pancreatitis and Its Relation to the CFTR Cl- Channel.

Aquaporins (AQPs) facilitate the transepithelial water flow involved in epithelial fluid secretion in numerous tissues; however, their function in the pancreas is less characterized. Acute pancreatitis (AP) is a serious disorder in which specific treatment is still not possible. Accumulating evidence indicate that decreased pancreatic ductal fluid secretion plays an essential role in AP; therefore, the aim of this study was to investigate the physiological and pathophysiological role of AQPs in the pancreas. Expression and localization of AQPs were investigated by real-time PCR and immunocytochemistry, whereas osmotic transmembrane water permeability was estimated by the dye dilution technique, in Capan-1 cells. The presence of AQP1 and CFTR in the mice and human pancreas were investigated by immunohistochemistry. Pancreatic ductal HCO3- and fluid secretion were studied on pancreatic ducts isolated from wild-type (WT) and AQP1 knock out (KO) mice using microfluorometry and videomicroscopy, respectively. In vivo pancreatic fluid secretion was estimated by magnetic resonance imaging. AP was induced by intraperitoneal injection of cerulein and disease severity was assessed by measuring biochemical and histological parameters. In the mice, the presence of AQP1 was detected throughout the whole plasma membrane of the ductal cells and its expression highly depends on the presence of CFTR Cl- channel. In contrast, the expression of AQP1 is mainly localized to the apical membrane of ductal cells in the human pancreas. Bile acid treatment dose- and time-dependently decreased mRNA and protein expression of AQP1 and reduced expression of this channel was also demonstrated in patients suffering from acute and chronic pancreatitis. HCO3- and fluid secretion significantly decreased in AQP1 KO versus WT mice and the absence of AQP1 also worsened the severity of pancreatitis. Our results suggest that AQP1 plays an essential role in pancreatic ductal fluid and HCO3- secretion and decreased expression of the channel alters fluid secretion which probably contribute to increased susceptibility of the pancreas to inflammation.

Aquaporin-4 knockout mice exhibit increased hypnotic susceptibility to ketamine.

PurposeThis study examines anesthetic/hypnotic effects of ketamine in AQP4 knockout (KO) and wild‐type (WT) mice with the particular focus on neurotransmission.Materials and MethodsKetamine (100 mg/kg) was intraperitoneally injected in 16 WT and 16 KO mice. The hypnotic potencies were evaluated by the loss of the righting reflex (LORR). The amino acids neurotransmitter levels in prefrontal cortex were measured by microdialysis.ResultsThis study demonstrated that AQP4 knockout significantly shortened the latency compared with WT mice (98.0 ± 4.2 vs. 138.1 ± 15.0 s, p < .05) and prolonged duration of LORR (884.7 ± 58.6 vs. 562.0 ± 51.7 s, p < .05) compared with WT mice in LORR induced by ketamine. Microdialysis showed that lack of AQP4 markedly decreased glutamate level within 20 min (p < .05) and increased γ‐aminobutyric acid (GABA) level within 30–40 min (p < .05) after use of ketamine. Moreover, the levels of taurine were remarkably higher in KO mice than in WT mice, but no obvious differences in aspartate were observed between two genotypes.Conclusion AQP4 deficiency led to more susceptibility of mice to ketamine, which is probably due to the modulation of specific neurotransmitters, hinting an essential maintenance of synaptic activity mediated by AQP4 in the action of ketamine.

Water transport and homeostasis as a major function of erythrocytes.

Erythrocytes have long been known to change volumes and shapes in response to different salt concentrations. Aquaporin-1 (AQP1) was discovered in their membranes more than 20 yr ago. The physiological roles of volume changes and AQP1 expression, however, have remained unclear. We propose that rapid water exchange through AQP1 coupled with large capacity for volume change may allow erythrocytes to play an important role in water regulation. In this study, we showed that erythrocytes in situ gradually reduced their volumes by 39% in response to the hyperosmotic corticomedullary gradient within mouse kidneys. AQP1 knockout (KO) erythrocytes, however, displayed only minimal reduction. Constructing a microfluidic device resembling capillary flow with an extracellular fluorescent reporter demonstrated that water exchanges between erythrocytes and their hypotonic or hypertonic surroundings in vitro reached steady state in ~60 ms. AQP1 KO erythrocytes, however, did not show significant change. To simulate the water transport in circulation, we built basic units consisting of three compartments (i.e., erythrocyte, plasma, and interstitial fluid) using Kedem-Katchalsky equations for membrane transport, and connected multiple units to account for the blood flow. These simulations agreed with experimental results. Importantly, volume-changing erythrocytes in capillaries always "increase" the osmotic gradient between plasma and interstitial fluid, making them function as "micropumps" to speed up the regulation of local osmolarity. Trillions of these micropumps, mobile throughout the body, may further contribute to water homeostasis. These insights suggest that the enhanced exchange of water, in addition to O2 and CO2, may well be the third major function of erythrocytes. NEW & NOTEWORTHY Physiological roles of erythrocyte volume change and aquaporin-1 were proposed and investigated here. We conclude that fast water transport by aquaporin-1 coupled with large volume-change capacity allows erythrocytes to enhance water exchange with local tissues. Furthermore, their huge number and mobility allow them to contribute to body water homeostasis.

Loss or Mislocalization of Aquaporin-4 Affects Diffusion Properties and Intermediary Metabolism in Gray Matter of Mice.

The first aim of this study was to determine how complete or perivascular loss of aquaporin-4 (AQP4) water channels affects membrane permeability for water in the mouse brain grey matter in the steady state. Time-dependent diffusion magnetic resonance imaging was performed on global Aqp4 knock out (KO) and α-syntrophin (α-syn) KO mice, in the latter perivascular AQP4 are mislocalized, but still functioning. Control animals were corresponding wild type (WT) mice. By combining in vivo diffusion measurements with the effective medium theory and previouslymeasured extra-cellular volume fractions, the effects of membrane permeability and extracellular volume fraction were uncoupled for Aqp4 and α-syn KO. The second aim was to assess the effect of α-syn KO on cortical intermediary metabolism combining in vivo [1-13C]glucose and [1,2-13C]acetate injection with ex vivo 13C MR spectroscopy. Aqp4 KO increased the effective diffusion coefficient at long diffusion times by 5%, and a 14% decrease in membrane water permeability was estimated for Aqp4 KO compared with WT mice. α-syn KO did not affect the measured diffusion parameters. In the metabolic analyses, significantly lower amounts of [4-13C]glutamate and [4-13C]glutamine, and percent enrichment in [4-13C]glutamate were detected in the α-syn KO mice. [1,2-13C]acetate metabolism was unaffected in α-syn KO, but the contribution of astrocyte derived metabolites to GABA synthesis was significantly increased. Taken together, α-syn KO mice appeared to have decreased neuronal glucose metabolism, partly compensated for by utilization of astrocyte derived metabolites.

Maximal Oxygen Consumption Is Reduced in Aquaporin-1 Knockout Mice.

We have measured maximal oxygen consumption (O2,max) of mice lacking one or two of the established mouse red-cell CO2 channels AQP1, AQP9, and Rhag. We intended to study whether these proteins, by acting as channels for O2, determine O2 exchange in the lung and in the periphery. We found that O2,max as determined by the Helox technique is reduced by ~16%, when AQP1 is knocked out, but not when AQP9 or Rhag are lacking. This figure holds for animals respiring normoxic as well as hypoxic gas mixtures. To see whether the reduction of O2,max is due to impaired O2 uptake in the lung, we measured carotid arterial O2 saturation (SO2) by pulse oximetry. Neither under normoxic (inspiratory O2 21%) nor under hypoxic conditions (11% O2) is there a difference in SO2 between AQP1null and WT mice, suggesting that AQP1 is not critical for O2 uptake in the lung. The fact that the % reduction of O2,max is identical in normoxia and hypoxia indicates moreover that the limitation of O2,max is not due to an O2 diffusion problem, neither in the lung nor in the periphery. Instead, it appears likely that AQP1null animals exhibit a reduced O2,max due to the reduced wall thickness and muscle mass of the left ventricles of their hearts, as reported previously. We conclude that very likely the properties of the hearts of AQP1 knockout mice cause a reduced maximal cardiac output and thus cause a reduced O2,max, which constitutes a new phenotype of these mice.