Cerebral edema contributes significantly to morbidity and death associated with many common neurological disorders. However, current treatment options are limited to hyperosmolar agents and surgical decompression, therapies introduced more than 70 years ago. Here we show that mice deficient in aquaporin-4 (AQP4), a glial membrane water channel, have much better survival than wild-type mice in a model of brain edema caused by acute water intoxication. Brain tissue water content and swelling of pericapillary astrocytic foot processes in AQP4-deficient mice were significantly reduced. In another model of brain edema, focal ischemic stroke produced by middle cerebral artery occlusion, AQP4-deficient mice had improved neurological outcome. Cerebral edema, as measured by percentage of hemispheric enlargement at 24 h, was decreased by 35% in AQP4-deficient mice. These results implicate a key role for AQP4 in modulating brain water transport, and suggest that AQP4 inhibition may provide a new therapeutic option for reducing brain edema in a wide variety of cerebral disorders.

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 ϳ20% 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 wildtype 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.The family of molecular water channels (aquaporins) numbers 10 in mammals and many more in plants and lower organisms. There has been considerable recent interest in the role of aquaporins in mammalian physiology and disease mechanisms. In humans, mutation of the vasopressin-regulated water channel of kidney collecting, AQP2, 1 causes hereditary nephrogenic diabetes insipidus in which patients are unable to concentrate their urine (1). Recent phenotype characterization of transgenic knockout mice lacking AQP1 and AQP4 has been very informative in defining the roles of these water channels in the urinary concentrating mechanism, lung fluid transport, and gastrointestinal physiology (2-6). However the phenotype studies indicated that the tissue expression of an aquaporin does not ensure its functional significance.AQP5 is a water channel with a unique tissue expression pattern (7). Immunocytochemical studies from several laboratories showed AQP5 expression in the apical membranes of serous acinar cells in salivary and lacrimal glands, type I alveolar epithelial cells, and surface corneal epithelial cells (8 -11). AQP5 appears to function as an unregulated waterselective channel with comparable intrinsic water permeability to AQP1 (12). The human AQP5 gene contains 4 exons with exon-intron boundaries at identical locations to those several other aquaporins (13); the genes for AQP5, AQP2, and AQP6 are clustered in a small 27-kb region at chromosome locus 12q13 (14). It was proposed that AQP5 plays an important role in glandular secretions of saliva and tears and that abnormalities in AQP5 might occur in some forms of Sjogren's syndrome (15,16). Aquaporin gene delivery to salivary gland has been proposed to increase fluid secretion (15). However, these possibilities are based on the unproven assu...

Water channel aquaporin-1 (AQP1) is strongly expressed in kidney in proximal tubule and descending limb of Henle epithelia and in vasa recta endothelia. The grossly normal phenotype in human subjects deficient in AQP1 (Colton null blood group) and in AQP4 knockout mice has suggested that aquaporins (other than the vasopressin-regulated water channel AQP2) may not be important in mammalian physiology. We have generated transgenic mice lacking detectable AQP1 by targeted gene disruption. In kidney proximal tubule membrane vesicles from knockout mice, osmotic water permeability was reduced 8-fold compared with vesicles from wild-type mice. Although the knockout mice were grossly normal in terms of survival, physical appearance, and organ morphology, they became severely dehydrated and lethargic after water deprivation for 36 h. Body weight decreased by 35 ؎ 2%, serum osmolality increased to >500 mOsm, and urinary osmolality (657 ؎ 59 mOsm) did not change from that before water deprivation. In contrast, wild-type and heterozygous mice remained active after water deprivation, body weight decreased by 20 -22%, serum osmolality remained normal (310 -330 mOsm), and urine osmolality rose to >2500 mOsm. Urine [Na ؉ ] in water-deprived knockout mice was <10 mM, and urine osmolality was not increased by the V2 agonist DDAVP. The results suggest that AQP1 knockout mice are unable to create a hypertonic medullary interstitium by countercurrent multiplication. AQP1 is thus required for the formation of a concentrated urine by the kidney.There are a family of related water transporting proteins (water channels, aquaporins) whose members are expressed widely in animals, plants, and bacteria. Eight aquaporin-type water channels (AQP1-AQP8) 1 with homology to the major intrinsic protein of lens fiber have been cloned in mammals to date (1-3). AQP2 is the only mammalian water channel shown to be important physiologically. AQP2 functions as a vasopressin-stimulated water transporter in kidney collecting duct epithelium (4, 5). Mutations in AQP2 cause the urinary concentrating defect in hereditary nephrogenic diabetes insipidus (6). In contrast, humans lacking AQP1 were reported to be phenotypically normal (7), although formal clinical evaluation was not done. Recently, we reported that transgenic mice lacking AQP4 were phenotypically similar to wild-type mice except for a mild decrease in maximum urinary concentration (8).AQP1 is the erythrocyte water transporter (9) that is expressed strongly in kidney (10 -12), as well as in choroid plexus, ciliary body, alveolar microvessels, gallbladder, placenta, and various other epithelia and endothelia (13,14). In kidney, AQP1 is found at the apical and basolateral membranes of epithelial cells in proximal tubule and thin descending limb of Henle (10 -12) and in endothelial cells of descending vasa recta (15). The density of AQP1 is exceptionally high in thin descending limb (16), where Ͼ25% of membrane protein has been attributed to AQP1. AQP1 functions as a water-selective transporting pro...

Secretory diarrhea is the leading cause of infant death in developing countries and a major cause of morbidity in adults. The cystic fibrosis transmembrane conductance regulator (CFTR) protein is required for fluid secretion in the intestine and airways and, when defective, causes the lethal genetic disease cystic fibrosis. We screened 50,000 chemically diverse compounds for inhibition of cAMP/flavone-stimulated Cl(-) transport in epithelial cells expressing CFTR. Six CFTR inhibitors of the 2-thioxo-4-thiazolidinone chemical class were identified. The most potent compound discovered by screening of structural analogs, CFTR(inh)-172, reversibly inhibited CFTR short-circuit current in less than 2 minutes in a voltage-independent manner with K(I) approximately 300 nM. CFTR(inh)-172 was nontoxic at high concentrations in cell culture and mouse models. At concentrations fully inhibiting CFTR, CFTR(inh)-172 did not prevent elevation of cellular cAMP or inhibit non-CFTR Cl(-) channels, multidrug resistance protein-1 (MDR-1), ATP-sensitive K(+) channels, or a series of other transporters. A single intraperitoneal injection of CFTR(inh)-172 (250 micro g/kg) in mice reduced by more than 90% cholera toxin-induced fluid secretion in the small intestine over 6 hours. Thiazolidinone CFTR inhibitors may be useful in developing large-animal models of cystic fibrosis and in reducing intestinal fluid loss in cholera and other secretory diarrheas.

To investigate the role of aquaporin-1 (AQP1) water channels in proximal tubule function, in vitro proximal tubule microperfusion and in vivo micropuncture measurements were done on AQP1 knockout mice. The knockout mice were generated by targeted gene disruption and found previously to be unable to concentrate their urine in response to water deprivation. Unanesthetized knockout mice consumed 2.8-fold more f luid than wild-type mice and had lower urine osmolality (505 ؎ 40 vs. 1081 ؎ 68 milliosmolar). An important function of the kidney proximal tubule is the near-isosmolar reabsorption of a significant fraction of fluid that is filtered by the glomerulus. The proximal tubule also reabsorbs nearly all of the filtered glucose, amino acids, and bicarbonate. The apical and basolateral plasma membranes of proximal tubule cells contain water channel protein aquaporin-1 (AQP1), which is thought to provide an important water-selective pathway for transcellular fluid transport (1-3). However, there is conflicting evidence that significant paracellular water transport occurs (4), and it has been suggested that other water channels (AQP7, ref. 5) and transporters (glucose transporter GLUT1, refs. 6, 7; sodium-glucose cotransporter SGLT1, ref. 8) might contribute to transcellular water movement. It is generally believed, but without direct evidence, that high proximal tubule water permeability is important to permit the efficient coupling of solute and water transport to accomplish near-isosmolar fluid absorption.The AQP1 water channel is a water-selective transporter (9, 10) that is found in membranes as tetramers (11) in which each functionally independent monomer (12) contains six transmembrane, tilted helical domains surrounding a putative aqueous pore (13-15). In kidney, AQP1 is strongly expressed in apical and basolateral plasma membranes of epithelial cells in proximal tubule and thin descending limb of Henle and in endothelial cells of descending vasa recta (1-3, 16, 17). Recently, a transgenic AQP1 knockout mouse was generated by targeted gene disruption and shown to manifest a severe defect in urinary concentrating ability (18). When given access to water, the mice appeared grossly normal except for mild growth retardation compared with wild-type mice. When deprived of water, the mice were unable to concentrate their urine and conserve fluid, resulting in marked dehydration and serum hyperosmolality in 1-2 days.The purpose of this study was to define the role of AQP1 in proximal tubule water transport and fluid reabsorption. Isolated tubule microperfusion was used to measure transepithelial osmotic water permeability and fluid absorption under defined in vitro conditions. Free-flow micropuncture was used to determine the in vivo consequences of decreased proximal tubule water permeability. A remarkable decrease in proximal tubule water permeability and fluid reabsorption was found in the AQP1 knockout mice. The results have important implications regarding the mechanisms of proximal tubule fluid reabsorpt...

Aquaporin-4 (AQP4) is a mercurial-insensitive, water-selective channel that is expressed in astroglia and basolateral plasma membranes of epithelia in the kidney collecting duct, airways, stomach, and colon. A targeting vector for homologous recombination was constructed using a 7-kb SacI AQP4 genomic fragment in which part of the exon 1 coding sequence was deleted.

Aquaporin-3 (AQP3) is a water channel expressed at the basolateral plasma membrane of kidney collecting-duct epithelial cells. The mouse AQP3 cDNA was isolated and encodes a 292-amino acid water͞glycerol-transporting glycoprotein expressed in kidney, large airways, eye, urinary bladder, skin, and gastrointestinal tract. The mouse AQP3 gene was analyzed, and AQP3 null mice were generated by targeted gene disruption. The growth and phenotype of AQP3 null mice were grossly normal except for polyuria. AQP3 deletion had little effect on AQP1 or AQP4 protein expression but decreased AQP2 protein expression particularly in renal cortex. Fluid consumption in AQP3 null mice was more than 10-fold greater than that in wild-type litter mates, and urine osmolality (<275 milliosmol) was much lower than in wild-type mice (>1,200 milliosmol). After 1-desamino-8-D-arginine-vasopressin administration or water deprivation, the AQP3 null mice were able to concentrate their urine partially to Ϸ30% of that in wild-type mice. Osmotic water permeability of cortical collecting-duct basolateral membrane, measured by a spatial filtering optics method, was >3-fold reduced by AQP3 deletion. To test the hypothesis that the residual concentrating ability of AQP3 null mice was due to the inner medullary collecting-duct water channel AQP4, AQP3͞AQP4 double-knockout mice were generated. The double-knockout mice had greater impairment of urinary-concentrating ability than did the AQP3 single-knockout mice. Our findings establish a form of nephrogenic diabetes insipidus produced by impaired water permeability in collecting-duct basolateral membrane. Basolateral membrane aquaporins may thus provide blood-accessible targets for drug discovery of aquaretic inhibitors.water transport ͉ AQP3 ͉ kidney ͉ urinary-concentrating mechanism ͉ polyuria A quaporin-3 (AQP3, originally called glycerol intrinsic protein, GLIP, based on its glycerol-transport function) was cloned from rat kidney by our laboratory (1) as well as by Ishibashi et al. (2) and Echevarria et al. (3). AQP3 is a relatively weak transporter of water but functions as an efficient glycerol transporter (4). Reflection coefficient measurements (5) and mutagenesis studies (6) suggested that water and glycerol share a common pathway through the AQP3 protein, although inhibition experiments were interpreted as suggesting different pathways (7). Immunocytochemistry in rat showed AQP3 protein expression in basolateral membrane of kidney collecting duct and large airways, as well as in several tissues that are thought not to have an important water-transporting role including urinary bladder, conjunctiva, and epidermis (8-11). Recent studies report strong AQP3 expression in various regions of the gastrointestinal tract including small intestine (12). Another unique feature of AQP3 is its gene structure, which is different from the water-selective mammalian aquaporins (13). AQP3, AQP7, and AQP9 have been called ''aquaglyceroporins'' because of their relatively broad solute specificity and sequence ho...

Deletion of the epidermal water/glycerol transporter aquaporin-3 (AQP3) in mice reduced superficial skin conductance by ϳ2-fold (Ma, T., Hara, M., Sougrat, R., Verbavatz, J. M., and Verkman, A. S. (2002) J. Biol. Chem. 277, 17147-17153), suggesting defective stratum corneum (SC) hydration. Here, we demonstrate significant impairment of skin hydration, elasticity, barrier recovery, and wound healing in AQP3 null mice in a hairless (SKH1) genetic background and investigate the cause of the functional defects by analysis of SC morphology and composition. Utilizing a novel 3 H 2 O distribution method, SC water content was reduced by ϳ50% in AQP3 null mice. Skin elasticity measured by cutometry was significantly reduced in AQP3 null mice with ϳ50% reductions in elasticity parameters Uf, Ue, and Ur. Although basal skin barrier function was not impaired, AQP3 deletion produced an ϳ2-fold delay in recovery of barrier function as measured by transepidermal water loss after tape stripping. Another biosynthetic skin function, wound healing, was also ϳ2-fold delayed by AQP3 deletion. By electron microscopy AQP3 deletion did not affect the structure of the unperturbed SC. The SC content of ions (Na ؉ , K ؉ , Ca 2؉ , Mg 2؉ ) and small solutes (urea, lactic acid, glucose) was not affected by AQP3 deletion nor was the absolute amount or profile of lipids and free amino acids. However, AQP3 deletion produced significant reductions in glycerol content in SC and epidermis (in nmol/g protein: 5.5 ؎ 0.4 versus 2.3 ؎ 0.7 in SC; 0.037 ؎ 0.007 versus 0.022 ؎ 0.005 in epidermis) but not in dermis or blood. These results establish hydration, mechanical, and biosynthetic defects in skin of AQP3-deficient mice. The selective reduction in epidermal and SC glycerol content in AQP3 null mice may account for these defects, providing the first functional evidence for physiologically important glycerol transport by an aquaporin.Hydration of the stratum corneum (SC), 1 the non-viable outermost layer of skin, is an important determinant of skin appearance, metabolism, mechanical properties, and barrier function (1-3). Water is continuously exchanged among the SC, the underlying viable epidermis, and the external atmosphere. SC water content depends on external humidity, the capacity of the epidermis to replace evaporative water losses, and the intrinsic SC "water holding capacity" (4). The determinants of SC water holding capacity are thought to include SC structure and composition, particularly the content of small molecule osmolytes or "humectants" such as free amino acids (5, 6). Decreased SC water content is found is a number of common skin diseases such as atopic dermatitis (7), eczema (8), psoriasis (9), senile xerosis (10), and hereditary ichthyosis (11).The water/glycerol transporting protein aquaporin-3 (AQP3) is expressed in the basal (innermost) layer of keratinocytes in mammalian epidermis as originally shown in rat skin (12) and then in human (13) and mouse (14) skin. AQP3 facilitates the transmembrane transport of water in response t...