Deletion Of Aquaporin-4 Research Articles

The deletion of AQP4 and TRPV4 affects astrocyte swelling/volume recovery in response to ischemia-mimicking pathologies.

Astrocytic Transient receptor potential vanilloid 4 (TRPV4) channels, together with Aquaporin 4 (AQP4), are suspected to be the key players in cellular volume regulation, and therefore may affect the development and severity of cerebral edema during ischemia. In this study, we examined astrocytic swelling/volume recovery in mice with TRPV4 and/or AQP4 deletion in response to in vitro ischemic conditions, to determine how the deletion of these channels can affect the development of cerebral edema. We used three models of ischemia-related pathological conditions: hypoosmotic stress, hyperkalemia, and oxygenglucose deprivation (OGD), and observed their effect on astrocyte volume changes in acute brain slices of Aqp4-/-, Trpv4-/- and double knockouts. In addition, we employed single-cell RT-qPCR to assess the effect of TRPV4 and AQP4 deletion on the expression of other ion channels and transporters involved in the homeostatic functioning of astrocytes. Quantification of astrocyte volume changes during OGD revealed that the deletion of AQP4 reduces astrocyte swelling, while simultaneous deletion of both AQP4 and TRPV4 leads to a disruption of astrocyte volume recovery during the subsequent washout. Of note, astrocyte exposure to hypoosmotic stress or hyperkalemia revealed no differences in astrocyte swelling in the absence of AQP4, TRPV4, or both channels. Moreover, under ischemia-mimicking conditions, we identified two distinct subpopulations of astrocytes with low and high volumetric responses (LRA and HRA), and their analyses revealed that mainly HRA are affected by the deletion of AQP4, TRPV4, or both channels. Furthermore, gene expression analysis revealed reduced expression of the ion transporters KCC1 and ClC2 as well as the receptors GABAB and NMDA in Trpv4-/- mice. The deletion of AQP4 instead caused reduced expression of the serine/cysteine peptidase inhibitor Serpina3n. Thus, we showed that in AQP4 or TRPV4 knockouts, not only the specific function of these channels is affected, but also the expression of other proteins, which may modulate the ischemic cascade and thus influence the final impact of ischemia.

The effect of aquaporin‐4 mis‐localization on Aβ pathology in mice

AbstractBackgroundThe reduced clearance of amyloid‐beta (Aβ) is thought to contribute to the development of Alzheimer’s disease (AD) pathology, characterized by the deposition of Aβ plaques. Previous studies have shown that Aβ is cleared via the glymphatic system, a brain‐wide network of perivascular pathways that support exchange between cerebrospinal fluid and interstitial fluid within the brain. Such exchange is mediated by the water channel aquaporin‐4 (AQP4), localized at astrocytic endfeet. Research has found that the deletion or mislocalization of Aqp4 is associated with reduced Aβ clearance and, in turn, Aβ plaque formation.MethodIn this study, we evaluated how the deposition of Aβ plaques within a 5xFAD mouse model is impacted by either the mislocalization or deletion of perivascular AQP4. We utilized both the AQP4 knockout mouse and α‐syntrophin (Snta1) knockout mouse, which lacks perivascular AQP4 localization, and crossed them with a mouse model line of amyloidosis (5xFAD) to determine the effect on Aβ deposition. Mice were perfused at 18 weeks and amyloid‐beta, AQP4, and lectin were assessed by immunofluorescence. Amyloid β distribution was evaluated anterior to posterior through the brain utilizing regions of interests for entire slice, cortex, corpus callosum, hippocampus, subiculum, sub cortical structures, and amygdala when applicable.ResultWe observed that both the absence (Aqp4KO) and mislocalization (Snta1KO) of AQP4 significantly increases amyloid distribution across the brain, when compared with 5xFAD littermate controls. Further, the mislocalization of AQP4 resulted in a significant increase in the size of Aβ plaque deposition not present with AQP4 deletion.Conclusions highlights that the mislocalization of perivascular AQP4 has a greater impact on increased amyloid burden than the deletion of AQP4. In suggesting that the mechanism of AQP4 mislocalization may lie upstream of amyloid or tau pathology, targeting perivascular AQP4 localization may be provide a novel pathway toward secondary, or potentially primary prevention of Alzheimer’s disease.

Inhibition of NADPH oxidase 2 improves cognitive abilities by modulating aquaporin-4 after traumatic brain injury in mice

Traumatic brain injury (TBI) is caused by acquired damage that includes cerebral edema after a mechanical injury and may cause cognitive impairment. We explored the role of nicotinamide adenine dinucleotide phosphate oxidase 2 (NADPH oxidase 2; NOX2) and aquaporin-4 (AQP4) in the process of edema and cognitive abilities after TBI in NOX2−/− and AQP4−/− mice by using the Morris water maze test (MWM), step-down test (STD), novel object recognition test (NOR) and western blotting. Knockout of NOX2 in mice decreased the AQP4 and reduce edema in the hippocampus and cortex after TBI in mice. Moreover, inhibiting AQP4 by 2-(nicotinamide)-1,3,4-thiadiazole (TGN-020) or genetic deletion of AQP4 could attenuate neurological deficits without changing reactive oxygen species (ROS) levels after TBI in mice. Taken together, we suspected that inhibiting NOX2 could improve cognitive abilities by modulating ROS levels, then affecting AQP4 levels and brain edema after in TBI mice. Our study demonstrated that NOX2 play a key role in decreasing edema in brain and improving cognitive abilities by modulating AQP4 after TBI.

Aquaporin-4 Deficiency is Associated with Cognitive Impairment and Alterations in astrocyte-neuron Lactate Shuttle.

Cognitive impairment refers to notable declines in cognitive abilities including memory, language, and emotional stability leading to the inability to accomplish essential activities of daily living. Astrocytes play an important role in cognitive function, and homeostasis of the astrocyte-neuron lactate shuttle (ANLS) system is essential for maintaining cognitive functions. Aquaporin-4 (AQP-4) is a water channel expressed in astrocytes and has been shown to be associated with various brain disorders, but the direct relationship between learning, memory, and AQP-4 is unclear. We examined the relationship between AQP-4 and cognitive functions related to learning and memory. Mice with genetic deletion of AQP-4 showed significant behavioral and emotional changes including hyperactivity and instability, and impaired cognitive functions such as spatial learning and memory retention. 18F-FDG PET imaging showed significant metabolic changes in the brains of AQP-4 knockout mice such as reductions in glucose absorption. Such metabolic changes in the brain seemed to be the direct results of changes in the expression of metabolite transporters, as the mRNA levels of multiple glucose and lactate transporters in astrocytes and neurons were significantly decreased in the cortex and hippocampus of AQP-4 knockout mice. Indeed, AQP-4 knockout mice showed significantly higher accumulation of both glucose and lactate in their brains compared with wild-type mice. Our results show that the deficiency of AQP-4 can cause problems in the metabolic function of astrocytes and lead to cognitive impairment, and that the deficiency of AQP4 in astrocyte endfeet can cause abnormalities in the ANLS system.

Loss of aquaporin-4 results in glymphatic system dysfunction via brain-wide interstitial fluid stagnation.

The glymphatic system is a fluid transport network of cerebrospinal fluid (CSF) entering the brain along arterial perivascular spaces, exchanging with interstitial fluid (ISF), ultimately establishing directional clearance of interstitial solutes. CSF transport is facilitated by the expression of aquaporin-4 (AQP4) water channels on the perivascular endfeet of astrocytes. Mice with genetic deletion of AQP4 (AQP4 KO) exhibit abnormalities in the brain structure and molecular water transport. Yet, no studies have systematically examined how these abnormalities in structure and water transport correlate with glymphatic function. Here, we used high-resolution 3D magnetic resonance (MR) non-contrast cisternography, diffusion-weighted MR imaging (MR-DWI) along with intravoxel-incoherent motion (IVIM) DWI, while evaluating glymphatic function using a standard dynamic contrast-enhanced MR imaging to better understand how water transport and glymphatic function is disrupted after genetic deletion of AQP4. AQP4 KO mice had larger interstitial spaces and total brain volumes resulting in higher water content and reduced CSF space volumes, despite similar CSF production rates and vascular density compared to wildtype mice. The larger interstitial fluid volume likely resulted in increased slow but not fast MR diffusion measures and coincided with reduced glymphatic influx. This markedly altered brain fluid transport in AQP4 KO mice may result from a reduction in glymphatic clearance, leading to enlargement and stagnation of fluid in the interstitial space. Overall, diffusion MR is a useful tool to evaluate glymphatic function and may serve as valuable translational biomarker to study glymphatics in human disease.

A tyrosine-based YXXΦ motif regulates the degradation of aquaporin-4 via both lysosomal and proteasomal pathways and is functionally inhibited by a 10-amino-acid sequence within its C-terminus.

Aquaporin-4 (AQP4) is a dominant water channel in the brain and is expressed on astrocytic end-feet, mediating water homeostasis in the brain. AQP4 is a target of an inflammatory autoimmune disease, neuromyelitis optica spectrum disorders (NMOSD), that causes demyelination. An autoantibody recognizing the extracellular domains of AQP4, called NMO-IgG, is critically implicated in the pathogenesis of the disease. Complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) in astrocytes are the primary causes of the disease, preceding demyelination and neuronal damage. Additionally, some cytotoxic effects of binding of NMO-IgG to AQP4, independent of CDC/ADCC, have been proposed. Antibody-induced endocytosis of AQP4 is thought to be involved in CDC/ADCC-independent cytotoxicity induced by the binding of NMO-IgG to AQP4. To clarify the mechanism responsible for antibody-induced endocytosis of AQP4, we investigated the subcellular localization and trafficking of AQP4, focusing on its C-terminal domain, by making a variety of deletion and substitution mutants of mouse AQP4. We found that a tyrosine-based YXXΦ motif in the C-terminal domain of AQP4 plays a critical role in the steady-state subcellular localization/turnover and antibody-induced endocytosis/lysosomal degradation of AQP4. Our results indicate that the YXXΦ motif has to escape the inhibitory effect of the C-terminal 10-amino-acid sequence and be located at an appropriate distance from the plasma membrane to act as a signal for lysosomal degradation of AQP4. In addition to lysosomal degradation, we demonstrated that the YXXΦ motif also functions as a signal to degrade AQP4 using proteasomes under specific conditions.

Glymphatic system clears extracellular tau and protects from tau aggregation and neurodegeneration.

Accumulation of tau has been implicated in various neurodegenerative diseases termed tauopathies. Tau is a microtubule-associated protein but is also actively released into the extracellular fluids including brain interstitial fluid and cerebrospinal fluid (CSF). However, it remains elusive whether clearance of extracellular tau impacts tau-associated neurodegeneration. Here, we show that aquaporin-4 (AQP4), a major driver of the glymphatic clearance system, facilitates the elimination of extracellular tau from the brain to CSF and subsequently to deep cervical lymph nodes. Strikingly, deletion of AQP4 not only elevated tau in CSF but also markedly exacerbated phosphorylated tau deposition and the associated neurodegeneration in the brains of transgenic mice expressing P301S mutant tau. The current study identified the clearance pathway of extracellular tau in the central nervous system, suggesting that glymphatic clearance of extracellular tau is a novel regulatory mechanism whose impairment contributes to tau aggregation and neurodegeneration.

Inhibition of Aquaporin 4 Decreases Amyloid A\u03b240 Drainage Around Cerebral Vessels

Aquaporin-4 (AQP4) is located mainly in the astrocytic end-feet around cerebral blood vessels and regulates ion and water homeostasis in the brain. While deletion of AQP4 is shown to reduce amyloid-β (Aβ) clearance and exacerbate Aβ peptide accumulation in plaques and vessels of Alzheimer’s disease mouse models, the mechanism and clearing pathways involved are debated. Here, we investigated how inhibiting the function of AQP4 in healthy male C57BL/6 J mice impacts clearance of Aβ40, the more soluble Aβ isoform. Using two-photon in vivo imaging and visualizing vessels with Sulfurodamine 101 (SR101), we first showed that Aβ40 injected as a ≤ 0.5-μl volume in the cerebral cortex diffused rapidly in parenchyma and accumulated around blood vessels. In animals treated with the AQP4 inhibitor TGN-020, the perivascular Aβ40 accumulation was significantly (P < 0.001) intensified by involving four times more vessels, thus suggesting a generalized clearance defect associated with vessels. Increasing the injecting volume to ≥ 0.5 ≤ 1 μl decreased the difference of Aβ40-positive vessels observed in non-treated and AQP4 inhibitor-treated animals, although the difference was still significant (P = 0.001), suggesting that larger injection volumes could overwhelm intramural vascular clearance mechanisms. While both small and large vessels accumulated Aβ40, for the ≤ 0.5-μl volume group, the average diameter of the Aβ40-positive vessels tended to be larger in control animals compared with TGN-020-treated animals, although the difference was non-significant (P = 0.066). Using histopathology and ultrastructural microscopy, no vascular structural change was observed after a single massive dose of TGN-020. These data suggest that AQP4 deficiency is directly involved in impaired Aβ brain clearance via the peri-/para-vascular routes, and AQP4-mediated vascular clearance might counteract blood-brain barrier abnormalities and age-related vascular amyloidopathy.

Deletion of aquaporin-4 aggravates brain pathology after blocking of the meningeal lymphatic drainage

The glymphatic pathway and meningeal lymphatic vessels are involved in clearance of metabolic macromolecules from the brain. However, the functional interaction between the two systems in the maintenance of brain homeostasis remains unclear. Here we reported that deletion of aquaporin-4 (AQP4), a functional regulator of glymphatic clearance, aggravated brain pathology of 3 month-old mice after blocking of the meningeal lymphatic drainage for 2 weeks via ligation of the deep cervical lymphatic nodes (LdcLNs). LdcLNs increased total and phosphorylated Tau protein levels in the hippocampus of both genotype mice, but increased hippocampal amyloid beta 1–40 and 1–42 levels only in AQP4 null mice, with up-regulation of beta-site amyloid precursor protein-cleaving enzyme 1 and down-regulation of insulin degrading enzyme. Consistently, LdcLNs caused microglial reactivity and activation of nod-like receptor protein-3 inflammasomes in the AQP4 null hippocampus. These mice also showed hippocampal neuronal apoptosis and declines in exploring and cognitive abilities. Deletion of AQP4, but not LdcLNs, increased brain water content. Together, these findings have revealed respective and interactive roles of the glymphatic system and the dural lymphatic system in maintaining amyloid beta, Tau proteins and water homeostasis in the brain, helping to understand the pathogenesis of neurological diseases associated with mis-accumulation of brain macromolecules.

Hippocampal and Cortical Pyramidal Neurons Swell in Parallel with Astrocytes during Acute Hypoosmolar Stress.

Normal nervous system function is critically dependent on the balance of water and ions in the extracellular space (ECS). Pathological reduction in brain interstitial osmolarity results in osmotically-driven flux of water into cells, causing cellular edema which reduces the ECS and increases neuronal excitability and risk of seizures. Astrocytes are widely considered to be particularly susceptible to cellular edema due to selective expression of the water channel aquaporin-4 (AQP4). The apparent resistance of pyramidal neurons to osmotic swelling has been attributed to lack of functional water channels. In this study we report rapid volume changes in CA1 pyramidal cells in hypoosmolar ACSF (hACSF) that are equivalent to volume changes in astrocytes across a variety of conditions. Astrocyte and neuronal swelling was significant within 1 min of exposure to 17 or 40% hACSF, was rapidly reversible upon return to normosmolar ACSF, and repeatable upon re-exposure to hACSF. Neuronal swelling was not an artifact of patch clamp, occurred deep in tissue, was similar at physiological vs. room temperature, and occurred in both juvenile and adult hippocampal slices. Neuronal swelling was neither inhibited by TTX, nor by antagonists of NMDA or AMPA receptors, suggesting that it was not occurring as a result of excitotoxicity. Surprisingly, genetic deletion of AQP4 did not inhibit, but rather augmented, astrocyte swelling in severe hypoosmolar conditions. Taken together, our results indicate that neurons are not osmoresistant as previously reported, and that osmotic swelling is driven by an AQP4-independent mechanism.

Deletion of Aquaporin-4 Curtails Extracellular Glutamate Elevation in Cortical Spreading Depression in Awake Mice.

Cortical spreading depression (CSD) is a phenomenon that challenges the homeostatic mechanisms on which normal brain function so critically depends. Analyzing the sequence of events in CSD holds the potential of providing new insight in the physiological processes underlying normal brain function as well as the pathophysiology of neurological conditions characterized by ionic dyshomeostasis. Here, we have studied the sequential progression of CSD in awake wild-type mice and in mice lacking aquaporin-4 (AQP4) or inositol 1,4,5-triphosphate type 2 receptor (IP3R2). By the use of a novel combination of genetically encoded sensors that a novel combination - an unprecedented temporal and spatial resolution, we show that CSD leads to brisk Ca2+ signals in astrocytes and that the duration of these Ca2+ signals is shortened in the absence of AQP4 but not in the absence of IP3R2. The decrease of the astrocytic, AQP4-dependent Ca2+ signals, coincides in time and space with a decrease in the duration of extracellular glutamate overflow but not with the initial peak of the glutamate release suggesting that in CSD, extracellular glutamate accumulation is extended through AQP4-dependent glutamate release from astrocytes. The present data point to a salient glial contribution to CSD and identify AQP4 as a new target for therapy.

Removal of aquaporin-4 from glial and ependymal membranes causes brain water accumulation.

There is a constitutive production of water in brain. The efflux routes of this excess water remain to be identified. We used basal brain water content as a proxy for the capacity of water exit routes. Basal brain water content was increased in mice with a complete loss of aquaporin-4 (AQP4) water channels (global Aqp4-/- mice), but not in mice with a selective removal of perivascular AQP4 or in a novel mouse line with a selective deletion of ependymal AQP4 (Foxj1-Cre:Aqp4flox/flox mice). Unique for the global Aqp4-/- mice is the loss of the AQP4 pool subjacent to the pial membrane. Our data suggest that water accumulates in brain when subpial AQP4 is missing, pointing to a critical role of this pool of water channels in brain water exit.

Increased Cerebral Capillary Density in Aquaporin‐4 Knockout Mouse

In the brain, Aquaporin 4 (AQP4) is highly expressed in the blood–brain barrier and brain–CSF interfaces. AQP4 functions as an efficient water‐selective transporting protein and plays an important role in cerebral water balance. It has been shown that knocking out of AQP4 in mice reduces brain edema after acute water intoxication and ischemic stroke. The specific localization of AQP4 to these anatomical and cellular regions of the central nervous system may also suggest a role for AQP4 as a gas channel to facilitate oxygen transport. In this study we investigated the role of AQP4 on cerebral capillary density and cerebral blood flow using AQP4 knockout (AQP4‐KO) mice in comparison with the wild‐type (WT) mice. Cerebral blood flow was measured using arterial spin labeling (ASL) MRI in WT mice (n = 7) and AQP4‐KO mice (n = 4). Mice were then perfusion fixed and cerebral capillary density was identified by GLUT‐1 immunohistochemistry staining and was quantified from the number of the positive counts per unit area (number/mm2). Results showed that the cerebral blood flows were similar in the WT and the AQP4‐KO groups (Mean ± SD, 182 ± 30 vs. 178 ± 52, ml/min/100g tissue). The AQP4‐KO mice exhibited a 24% increase in capillary density as compared to the WT mice (Mean ± SD, 504 ± 49 vs. 406 ± 32, N/mm2). Our results showed that the deletion of AQP4 increased the capillary density without altered blood flow in mouse brain, suggesting a compensation for decreased gas transport in the KO mice.Support or Funding InformationNIH R01 NS 38632

Deletion of aquaporin-4 in APP/PS1 mice exacerbates brain Aβ accumulation and memory deficits.

BackgroundPreventing or reducing amyloid-beta (Aβ) accumulation in the brain is an important therapeutic strategy for Alzheimer’s disease (AD). Recent studies showed that the water channel aquaporin-4 (AQP4) mediates soluble Aβ clearance from the brain parenchyma along the paravascular pathway. However the direct evidence for roles of AQP4 in the pathophysiology of AD remains absent.ResultsHere, we reported that the deletion of AQP4 exacerbated cognitive deficits of 12-moth old APP/PS1 mice, with increases in Aβ accumulation, cerebral amyloid angiopathy and loss of synaptic protein and brain-derived neurotrophic factor in the hippocampus and cortex. Furthermore, AQP4 deficiency increased atrophy of astrocytes with significant decreases in interleukin-1 beta and nonsignficant decreases in interleukin-6 and tumor necrosis factor-alpha in hippocampal and cerebral samples.ConclusionsThese results suggest that AQP4 attenuates Aβ pathogenesis despite its potentially inflammatory side-effects, thus serving as a promising target for treating AD.Electronic supplementary materialThe online version of this article (doi:10.1186/s13024-015-0056-1) contains supplementary material, which is available to authorized users.

How astrocyte networks may contribute to cerebral metabolite clearance.

The brain possesses an intricate network of interconnected fluid pathways that are vital to the maintenance of its homeostasis. With diffusion being the main mode of solute transport in cerebral tissue, it is not clear how bulk flow through these pathways is involved in the removal of metabolites. In this computational study, we show that networks of astrocytes may contribute to the passage of solutes between tissue and paravascular spaces (PVS) by serving as low resistance pathways to bulk water flow. The astrocyte networks are connected through aquaporin-4 (AQP4) water channels with a parallel, extracellular route carrying metabolites. Inhibition of the intracellular route by deletion of AQP4 causes a reduction of bulk flow between tissue and PVS, leading to reduced metabolite clearance into the venous PVS or, as observed in animal studies, a reduction of tracer influx from arterial PVS into the brain tissue.

Deletion of aquaporin-4 is neuroprotective during the acute stage of micro traumatic brain injury in mice

Micro traumatic brain injury (TBI) is the most common type of brain injury, but the mechanisms underlying it are poorly understood. Aquaporin-4 (AQP4) is a water channel expressed in astrocyte end-feet, which plays an important role in brain edema. However, little is known about the role of AQP4 in micro TBI. Here, we examined the role of AQP4 in the pathogenesis of micro TBI in a closed-skull brain injury model, using two-photon microscopy. Our results indicate that AQP4 deletion reduced cell death, water content, astrocyte swelling and lesion volume during the acute stage of micro TBI. Our data revealed that astrocyte swelling is a decisive pathophysiological factor in the acute phase of this form of micro brain injury. Thus, treatments that inhibit AQP4 could be used as a neuroprotective strategy for micro TBI.

Deletion of aquaporin-4 increases extracellular K+ concentration during synaptic stimulation in mouse hippocampus

The coupling between the water channel aquaporin-4 (AQP4) and K+ transport has attracted much interest. In this study, we assessed the effect of Aqp4 deletion on activity-induced [K+]o changes in acute slices from hippocampus and corpus callosum of adult mice. We show that Aqp4 deletion has a layer-specific effect on [K+]o that precisely mirrors the known effect on extracellular volume dynamics. In CA1, the peak [K+]o in stratum radiatum during 20 Hz stimulation of Schaffer collateral/commissural fibers was significantly higher in Aqp4 −/− mice than in wild types, whereas no differences were observed throughout the [K+]o recovery phase. In stratum pyramidale and corpus callosum, neither peak [K+]o nor post-stimulus [K+]o recovery was affected by Aqp4 deletion. Our data suggest that AQP4 modulates [K+]o during synaptic stimulation through its effect on extracellular space volume.

Glial-conditional deletion of aquaporin-4 ( Aqp4 ) reduces blood–brain water uptake and confers barrier function on perivascular astrocyte endfeet

Tissue- and cell-specific deletion of the Aqp4 gene is required to differentiate between the numerous pools of aquaporin-4 (AQP4) water channels. A glial-conditional Aqp4 knockout mouse line was generated to resolve whether astroglial AQP4 controls water exchange across the blood-brain interface. The conditional knockout was driven by the glial fibrillary acidic protein promoter. Brains from conditional Aqp4 knockouts were devoid of AQP4 as assessed by Western blots, ruling out the presence of a significant endothelial pool of AQP4. In agreement, immunofluorescence analysis of cryostate sections and quantitative immunogold analysis of ultrathin sections revealed no AQP4 signals in capillary endothelia. Compared with litter controls, glial-conditional Aqp4 knockout mice showed a 31% reduction in brain water uptake after systemic hypoosmotic stress and a delayed postnatal resorption of brain water. Deletion of astroglial Aqp4 did not affect the barrier function to macromolecules. Our data suggest that the blood-brain barrier (BBB) is more complex than anticipated. Notably, under certain conditions, the astrocyte covering of brain microvessels is rate limiting to water movement.

Deletion of aquaporin‐4 renders retinal glial cells more susceptible to osmotic stress

The glial water channel aquaporin-4 (AQP4) is implicated in the control of ion and osmohomeostasis in the sensory retina. Using retinal slices from AQP4-deficient and wild-type mice, we investigated whether AQP4 is involved in the regulation of glial cell volume under altered osmotic conditions. Superfusion of retinal slices with a hypoosmolar solution induced a rapid swelling of glial somata in tissues from AQP4 null mice but not from wild-type mice. The swelling was mediated by oxidative stress, inflammatory lipid mediators, and sodium influx into the cells and was prevented by activation of glutamatergic and purinergic receptors. Distinct inflammatory proteins, including interleukin-1 beta, interleukin-6, and inducible nitric oxide synthase, were up-regulated in the retina of AQP4 null mice compared with control, whereas cyclooxygenase-2 was down-regulated. The data suggest that water flux through AQP4 is involved in the rapid volume regulation of retinal glial (Müller) cells in response to osmotic stress and that deletion of AQP4 results in an inflammatory response of the retinal tissue. Possible implications of the data for understanding the pathophysiology of neuromyelitis optica, a human disease that has been suggested to involve serum antibodies to AQP4, are discussed.

Aquaporin-4 is expressed in basolateral membranes of proximal tubule S3 segments in mouse kidney.

Because of the availability of knockout mouse models to examine renal transport mechanisms, it has become increasingly important to describe the cellular distribution of major renal transporters in mice. We have used immunocytochemistry and freeze-fracture electron microscopy to compare the renal distribution of aquaporin-4 (AQP4) with that previously described in rat. In rat kidney AQP4 is present exclusively in basolateral membranes of collecting duct principal cells. In mice, however, AQP4 was also detected by immunocytochemistry in basolateral membranes of proximal tubule S3 segments, and not detected in S1 and S2 segments of proximal tubule. Freeze-fracture electron microscopy revealed orthogonal arrays of intramembrane particles (OAPs) on the basolateral membranes of the S3 segment. In AQP4-knockout mice, immunostaining was absent and OAPs were found neither in collecting ducts nor in the S3 segment of the proximal tubule. The urinary concentrating capacity after deletion of both AQP1 and AQP4 was further reduced compared with that of AQP1 or AQP4 null mice, suggesting an additive effect of AQP1 and AQP4 in the concentrating mechanism. The functional significance of the apparent species-dependent expression of AQP4 in proximal tubules is unknown, but may relate to physiological differences between rats and mice.