Three distinct roles of aquaporin-4 in brain function revealed by knockout mice

Aquaporin-4 (AQP4) is expressed in astrocytes throughout the central nervous system, particularly at the blood-brain and brain-cerebrospinal fluid barriers. Phenotype analysis of transgenic mice lacking AQP4 has provided compelling evidence for involvement of AQP4 in cerebral water balance, astrocyte migration, and neural signal transduction. AQP4-null mice have reduced brain swelling and improved neurological outcome in models of (cellular) cytotoxic cerebral edema including water intoxication, focal cerebral ischemia, and bacterial meningitis. However, brain swelling and clinical outcome are worse in AQP4-null mice in models of vasogenic (fluid leak) edema including cortical freeze-injury, brain tumor, brain abscess and hydrocephalus, probably due to impaired AQP4-dependent brain water clearance. AQP4 deficiency or knock-down slows astrocyte migration in response to a chemotactic stimulus in vitro, and AQP4 deletion impairs glial scar progression following injury in vivo. AQP4-null mice also manifest reduced sound- and light-evoked potentials, and increased threshold and prolonged duration of induced seizures. Impaired K⁺ reuptake by astrocytes in AQP4 deficiency may account for the neural signal transduction phenotype. Based on these findings, we propose modulation of AQP4 expression or function as a novel therapeutic strategy for a variety of cerebral disorders including stroke, tumor, infection, hydrocephalus, epilepsy, and traumatic brain injury.     

Three distinct roles of aquaporin-4 in brain function revealed by knockout mice

Aquaporin-4 (AQP4) is expressed in astrocytes throughout the central nervous system, particularly at the blood-brain and brain-cerebrospinal fluid barriers. Phenotype analysis of transgenic mice lacking AQP4 has provided compelling evidence for involvement of AQP4 in cerebral water balance, astrocyte migration, and neural signal transduction. AQP4-null mice have reduced brain swelling and improved neurological outcome in models of (cellular) cytotoxic cerebral edema including water intoxication, focal cerebral ischemia, and bacterial meningitis. However, brain swelling and clinical outcome are worse in AQP4-null mice in models of vasogenic (fluid leak) edema including cortical freeze-injury, brain tumor, brain abscess and hydrocephalus, probably due to impaired AQP4-dependent brain water clearance. AQP4 deficiency or knock-down slows astrocyte migration in response to a chemotactic stimulus in vitro, and AQP4 deletion impairs glial scar progression following injury in vivo. AQP4-null mice also manifest reduced sound- and light-evoked potentials, and increased threshold and prolonged duration of induced seizures. Impaired K+ reuptake by astrocytes in AQP4 deficiency may account for the neural signal transduction phenotype. Based on these findings, we propose modulation of AQP4 expression or function as a novel therapeutic strategy for a variety of cerebral disorders including stroke, tumor, infection, hydrocephalus, epilepsy, and traumatic brain injury.     

Erythrocyte water permeability and renal function in double knockout mice lacking aquaporin-1 and aquaporin-3

Aquaporin (AQP) water channel AQP3 has been proposed to be the major glycerol and non-AQP1 water transporter in erythrocytes. AQP1 and AQP3 are also expressed in the kidney where their deletion in mice produces distinct forms of nephrogenic diabetes insipidus. Here AQP1/AQP3 double knockout mice were generated and analyzed to investigate the functional role of AQP3 in erythrocytes and kidneys. 53 double knockout mice were born out of 756 pups from breeding double heterozygous mice. The double knockout mice had reduced survival and impaired growth compared with the single knockout mice. Erythrocyte water permeability was 7-fold reduced by AQP1 deletion but not further reduced in AQP1/AQP3 null mice. AQP3 deletion did not affect erythrocyte glycerol permeability or its inhibition by phloretin. Daily urine output in AQP1/AQP3 double knockout mice (15 ml) was 9-fold greater than in wild-type mice, and urine osmolality (194 mosm) was 8.4-fold reduced. The mice remained polyuric after DDAVP administration or water deprivation. The renal medulla in most AQP1/AQP3 null mice by age 4 weeks was atrophic and fluid-filled due to the severe polyuria and hydronephrosis. Our data provide direct evidence that AQP3 is not functionally important in erythrocyte water or glycerol permeability. The renal function studies indicate independent roles of AQP1 and AQP3 in countercurrent exchange and collecting duct osmotic equilibration, respectively.     

Gastric blood flow and acid secretion

Prerequisites for proper measurement of gastric mucosal blood flow (MBF) are different according to whether the blood supply of the stomach is being recorded in experimental animals or in humans. In this review the pros and cons of the methods to measure MBF are analysed systematically. Although it is easier to find a procedure for experimental animals none of the method hitherto published proved to be quantitatively accurate for the determination of MBF changes and simultaneous variations in gastric acid production. It is even more difficult to elaborate a method for human studies which should not cause serious inconvencience for the patient. The most one can achieve is a sufficiently accurate estimate of the MBF and its changes in the course of the study. With the help of such procedures it is possible to clarify the interrelation between MBF and acid secretion using interventions or bioactive substances which influence either the mucosal circulation or parietal cell function or both.     

Defective secretion of saliva in transgenic mice lacking aquaporin-5 water channels.

Aquaporin-5 (AQP5) is a water-selective transporting protein expressed in epithelial cells of serous acini in salivary gland. We generated AQP5 null mice by targeted gene disruption. The genotype distribution from intercross of founder AQP5 heterozygous mice was 70:69:29 wild-type:heterozygote:knockout, indicating impaired prenatal survival of the null mice. The knockout mice had grossly normal appearance, but grew approximately 20% slower than litter-matched wild-type mice when placed on solid food after weaning. Pilocarpine-stimulated saliva production was reduced by more than 60% in AQP5 knockout mice. Compared with the saliva from wild-type mice, the saliva from knockout mice was hypertonic (420 mosM) and dramatically more viscous. Amylase and protein secretion, functions of salivary mucous cells, were not affected by AQP5 deletion. Water channels AQP1 and AQP4 have also been localized to salivary gland; however, pilocarpine stimulation studies showed no defect in the volume or composition of saliva in AQP1 and AQP4 knockout mice. These results implicate a key role for AQP5 in saliva fluid secretion and provide direct evidence that high epithelial cell membrane water permeability is required for active, near-isosmolar fluid transport.     

Topiramate reduced sweat secretion and aquaporin-5 expression in sweat glands of mice

Decreased sweat secretion is a primary side effect of topiramate in pediatric patients, but the mechanism underlying this effect remains unclear. This study aimed to better understand how topiramate decreases sweat secretion by examining its effect on the expression of carbonic anhydrase (CA) II and aquaporin-5 (AQP5), total CA activity, as well as on tissue morphology of sweat glands in mice. Both developing and mature mice were treated with a low (20 mg/kg/day) and high dose (80 mg/kg/day) of topiramate for 4 weeks. Sweat secretion was investigated by an established technique of examining mold impressions of hind paws. CA II and AQP5 expression levels were determined by immunofluorescence and immunoblotting and CA activity by a colorimetric assay. In mature mice, topiramate treatment decreased the number of pilocarpine reactive sweat glands from baseline in both the low and high dose groups by 83% and 75%, respectively. A similar decrease was seen in developing mice. Mature mice with reactive sweat glands that declined more than 25% compared to baseline were defined as anhidrotic mice. These mice did not differ from controls in average secretory coil diameter, CA II expression and CA activity. In contrast, anhidrotic mice did show a reduction in membrane AQP5 expression in sweat glands after topiramate delivery. Thus, sweat secretion and membrane AQP5 expression in mouse sweat glands decreased following topiramate administration. These results suggest dysregulation of AQP5 may be involved in topiramate-induced hypohidrosis and topiramate may serve as a novel therapy for hyperhidrosis.     

Impaired hearing in mice lacking aquaporin-4 water channels.

A role for aquaporins (AQPs) in hearing has been suggested from the specific expression of aquaporins in inner ear and the need for precise volume regulation in epithelial cells involved in acoustic signal transduction. Using mice deficient in selected aquaporins as controls, we localized AQP1 in fibrocytes in the spiral ligament and AQP4 in supporting epithelial cells (Hensen's, Claudius, and inner sulcus cells) in the organ of Corti. To determine whether aquaporins play a role in hearing, auditory brain stem response (ABR) thresholds were compared in wild-type mice and transgenic null mice lacking (individually) AQP1, AQP3, AQP4, and AQP5. In 4-5-week-old mice in a CD1 genetic background, ABR thresholds in response to a click stimulus were remarkably increased by >12 db in AQP4 null mice compared with wild-type mice (p < 0.001), whereas ABR thresholds were not affected by AQP1, AQP3, or AQP5 deletion. In a C57/bl6 background, nearly all AQP4 null mice were deaf, whereas ABRs could be elicited in wild-type controls. ABRs in AQP4 null CD1 mice measured in response to tone bursts (4-20 kHz) indicated a frequency-independent hearing deficit. Light microscopy showed no differences in cochlear morphology of wild-type versus AQP4 null mice. These results provide the first direct evidence that an aquaporin water channel plays a role in hearing. AQP4 may facilitate rapid osmotic equilibration in epithelial cells in the organ of Corti, which are subject to large K(+) fluxes during mechano-electric signal transduction.     

Ghrelin-induced food intake and growth hormone secretion are altered in melanocortin 3 and 4 receptor knockout mice

Ghrelin stimulates food intake in part by activating hypothalamic neuropeptide Y (NPY) neurons/agouti related peptide (AGRP) neurons. We investigated the role of AGRP/melanocortin signaling in ghrelin-induced food intake by studying melanocortin 3 and 4 receptor knockout (MC3R KO and MC4R KO) mice. We also determined whether reduced ghrelin levels and/or an altered sensitivity to the GH-stimulating effects of ghrelin accompany the obesity syndromes of MC3R KO and MC4R KO mice. Compared to wild-type (WT) mice, the effects of ghrelin on food intake were reduced in MC3R KO and MC4R KO mice and circulating ghrelin levels were reduced in female MC4R KO mice. Female MC3R KO and MC4R KO mice exhibited a diminished responsiveness to the GH-releasing effects of ghrelin. Thus, deletion of the MC3R or MC4R results in a decreased sensitivity to ghrelin and verifies the involvement in the melanocortin system in ghrelin-induced food intake.     

Skeletal muscle function and water permeability in aquaporin-4 deficient mice

It has beenproposed that aquaporin-4 (AQP4), a water channel expressed at theplasmalemma of skeletal muscle cells, is important in normal musclephysiology and in the pathophysiology of Duchenne's musculardystrophy. To test this hypothesis, muscle water permeability andfunction were compared in wild-type and AQP4 knockout mice. Immunofluorescence and freeze-fracture electron microscopy showed AQP4protein expression in plasmalemma of fast-twitch skeletal muscle fibersof wild-type mice. Osmotic water permeability was measured inmicrodissected muscle fibers from the extensor digitorum longus (EDL) and fractionated membrane vesicles from EDLhomogenates. With the use of spatial-filtering microscopy to measureosmotically induced volume changes in EDL fibers, half times(t_1/2) for osmotic equilibration (7.5-8.5 s)were not affected by AQP4 deletion. Stopped-flow light-scatteringmeasurements of osmotically induced volume changes in plasmalemmavesicles also showed no significant differences in water permeability.Similar water permeability, yet ~90% decreased AQP4 proteinexpression was found in EDL from mdx mice that lack dystrophin.Skeletal muscle function was measured by force generation in isolatedEDL, treadmill performance time, and in vivo muscle swelling inresponse to water intoxication. No differences were found in EDL forcegeneration after electrical stimulation [42 ± 2 (wild-type) vs. 41 ± 2 (knockout) g/s], treadmill performance time (22 vs. 26 min; 29 m/min, 13° incline), or muscle swelling (2.8 vs. 2.9% increasedwater content at 90 min after intraperitoneal water infusion). Togetherthese results provide evidence against a significant role of AQP4 inskeletal muscle physiology in mice.

     

Colon water transport in transgenic mice lacking aquaporin-4 water channels

Transgenic null mice were used to test the hypothesis that water channel aquaporin-4 (AQP4) is involved in colon water transport and fecal dehydration. AQP4 was immunolocalized to the basolateral membrane of colonic surface epithelium of wild-type (+/+) mice and was absent in AQP4 null (-/-) mice. The transepithelial osmotic water permeability coefficient (P(f)) of in vivo perfused colon of +/+ mice, measured using the volume marker (14)C-labeled polyethylene glycol, was 0.016 +/- 0.002 cm/s. P(f) of proximal colon was greater than that of distal colon (0.020 +/- 0.004 vs. 0. 009 +/- 0.003 cm/s, P < 0.01). P(f) was significantly lower in -/- mice when measured in full-length colon (0.009 +/- 0.002 cm/s, P < 0. 05) and proximal colon (0.013 +/- 0.002 cm/s, P < 0.05) but not in distal colon. There was no difference in water content of cecal stool from +/+ vs. -/- mice (0.80 +/- 0.01 vs. 0.81 +/- 0.01), but there was a slightly higher water content in defecated stool from -/- mice (0.68 +/- 0.01 vs. 0.65 +/- 0.01, P < 0.05). Despite the differences in water permeability with AQP4 deletion, theophylline-induced secretion was not impaired (50 +/- 9 vs. 51 +/- 8 microl. min(-1). g(-1)). These results provide evidence that transcellular water transport through AQP4 water channels in colonic epithelium facilitates transepithelial osmotic water permeability but has little or no effect on colonic fluid secretion or fecal dehydration.     

Glial cell aquaporin-4 overexpression in transgenic mice accelerates cytotoxic brain swelling

Aquaporin-4 (AQP4) is a water transport protein expressed in glial cell plasma membranes, including glial cell foot processes lining the blood-brain barrier. AQP4 deletion in mice reduces cytotoxic brain edema produced by different pathologies. To determine whether AQP4 is rate-limiting for brain water accumulation and whether altered AQP4 expression, as occurs in various pathologies, could have functional importance, we generated mice that overexpressed AQP4 in brain glial cells by a transgenic approach using the glial fibrillary acid protein promoter. Overexpression of AQP4 protein in brain by approximately 2.3-fold did not affect mouse survival, appearance, or behavior, nor did it affect brain anatomy or intracranial pressure (ICP). However, following acute water intoxication produced by intraperitoneal water injection, AQP4-overexpressing mice had an accelerated progression of cytotoxic brain swelling, with ICP elevation of 20 +/- 2 mmHg at 10 min, often producing brain herniation and death. In contrast, ICP elevation was 14 +/- 2 mmHg at 10 min in control mice and 9.8 +/- 2 mmHg in AQP4 knock-out mice. The deduced increase in brain water content correlated linearly with brain AQP4 protein expression. We conclude that AQP4 expression is rate-limiting for brain water accumulation, and thus, that altered AQP4 expression can be functionally significant.     

Tendon properties in interleukin-4 and interleukin-6 knockout mice

Cytokines are known to play an important role in normal tendon development, function, and maintenance through interactions with fibroblasts and extracellular matrix proteins. However, the role of interleukins on normal tendon activity remains poorly understood. Previous studies that have researched the role of specific cytokines by exogenously applying them have often reported conflicting results. Therefore, a knockout mouse model was used to investigate the role of interleukins 4 and 6 on normal tendon organizational and biomechanical properties. It was hypothesized that interleukin-6 knockout (IL6 -/-) mice will display more organized collagen orientation and greater cross-sectional area and mechanical properties when compared to that of control mice. In addition, interleukin-4 knockout (IL4 -/-) mice will display the most disorganized collagen orientation and lowest cross-sectional area and mechanical properties. As hypothesized, IL6 -/- mice show a trend towards lower angular deviation (more organized) (p<0.1) when compared to IL4 -/- mice. In addition, the IL6 -/- mice show a trend towards a higher percent relaxation (p<0.1) and a significantly higher modulus (p<0.01) when compared to CTL and IL4 -/- mice. Unexpectedly, the IL6 -/- mice exhibited no significant differences in collagen fiber distribution and maximum stress from the other groups and actually had a smaller cross-sectional area than CTL mice (p<0.1). This study supports transgenic mice as an animal model for investigating how cytokines affect normal tendon properties. In addition, this study demonstrates that interleukins may play an important role in tendon development, function, and maintenance.     

Bleomycin-induced lung fibrosis in IL-4-overexpressing and knockout mice

The role of IL-4 in the development of lung fibrosis is as yet unclear. Bleomycin (Bleo) or saline (Sal) was injected intratracheally into three groups of C57BL/6J mice: transgenic animals that overexpressed IL-4 (IL-4 TG, n = 14), mice with a targeted knockout mutation of the IL-4 gene (IL-4 KO, n = 11), and wild-type (WT, n = 13) mice. At 14 days, lung fibrosis was evaluated by hydroxyproline measurement and by quantitative image analysis of fibrosis fraction and alveolar wall area fraction. Bronchoalveolar lavage cell counts in all Bleo-treated groups demonstrated an increased percentage of lymphocytes with a corresponding decrease in the percentage of macrophages. Comparing Bleo- to Sal-treated controls within each group of mice showed increases in all lung fibrosis parameters in IL-4 KO and WT, but not in any of the parameters in IL-4 TG mice. The severity of Bleo-induced fibrotic response was decreased in overexpressed IL-4 TG compared with IL-4 KO mice. These data negate a critical profibrotic role for IL-4 in Bleo-induced lung fibrosis.     

Tendon healing in interleukin-4 and interleukin-6 knockout mice

Cytokines have been shown to play an important role in tendon and ligament healing by regulating cellular differentiation and activity. The majority of studies that have investigated the role of cytokines in tendon and ligament healing have added them to injured tissue and assessed their effect. Because the efficacy of exogenously applying cytokines is dependent upon many factors such as the correct dosage, timing, and frequency, conflicting results are often reported. To avoid these factors, this study used transgenic mice with knockouts of interleukin-4 (IL4 -/-) and interleukin-6 (IL6 -/-) to investigate their role in tendon healing. Because of the reported roles of both of these cytokines in inflammation and fibroplasia, it was hypothesized that the order of organizational, geometric, and mechanical properties would be (greatest to least) injured IL6 -/-, injured control, and injured IL4 -/- mice. In addition, it was hypothesized that specific cytokines would be upregulated in each knockout group, but not compensate for the lack of IL-4 or IL-6. Mechanical and organizational properties of injured tendons from IL6 -/- mice were inferior to that of control and IL4 -/- mice despite the upregulation of the pro-inflammatory cytokine TNF-alpha. Temporal levels of IL-10 and IL-13 in the IL4 -/- mice resulted in comparable and even superior properties when compared to CTL mice. This study shows that IL-6 could not be compensated for and plays an important role in tendon healing. This study also supports the use of this animal model to further investigate tendon healing.     

Disease mechanisms and neuroprotection by tauroursodeoxycholic acid in Rpgr knockout mice

Mutations in the retinitis pigmentosa GTPase regulator (RPGR) gene are the predominant cause of retinitis pigmentosa. RPGR plays a critical role as a scaffold protein in the regulation of protein trafficking from the basal body to the axoneme, where the cargoes are transported to the outer segments (OSs) of photoreceptors. This trafficking process is controlled directly by intraflagellar transport complexes and regulated by the RPGR protein complex, although the precise mechanisms have yet to be defined. We used an Rpgr conditional knockout (cko) mouse model to investigate the disease mechanisms during retinal degeneration and to evaluate the protective effects of tauroursodeoxycholic acid (TUDCA). Rhodopsin, cone opsins and transducin were mislocalized in Rpgr cko photoreceptors, while localization of NPHP4 to connecting cilia was absent, suggesting that RPGR is required for ciliary protein trafficking. Microglia were activated in advance of retinal degeneration in Rpgr cko mouse retinas. TUDCA treatment suppressed microglial activation and inflammation and prevented photoreceptor degeneration in Rpgr cko mice. Our data demonstrated that TUDCA has therapeutic potential for RPGR-associated RP patients.