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...

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...

Urea transporter UT-B has been proposed to be the major urea transporter in erythrocytes and kidney-descending vasa recta. The mouse UT-B cDNA was isolated and encodes a 384-amino acid urea-transporting glycoprotein expressed in kidney, spleen, brain, ureter, and urinary bladder. The mouse UT-B gene was analyzed, and UT-B knockout mice were generated by targeted gene deletion of exons 3-6. The survival and growth of UT-B knockout mice were not different from wild-type littermates. Urea permeability was 45-fold lower in erythrocytes from knockout mice than from those in wild-type mice. Daily urine output was 1.5-fold greater in UT-B-deficient mice (p < 0.01), and urine osmolality (U osm ) was lower (1532 ؎ 71 versus 2056 ؎ 83 mosM/ kgH 2 O, mean ؎ S.E., p < 0.001). After 24 h of water deprivation, U osm (in mosM/kgH 2 O) was 2403 ؎ 38 in UT-B null mice and 3438 ؎ 98 in wild-type mice (p < 0.001). Plasma urea concentration (P urea ) was 30% higher, and urine urea concentration (U urea ) was 35% lower in knockout mice than in wild-type mice, resulting in a much lower U urea /P urea ratio (61 ؎ 5 versus 124 ؎ 9, p < 0.001). Thus, the capacity to concentrate urea in the urine is more severely impaired than the capacity to concentrate other solutes. Together with data showing a disproportionate reduction in the concentration of urea compared with salt in homogenized renal inner medullas of UT-B null mice, these data define a novel "ureaselective" urinary concentrating defect in UT-B null mice. The UT-B null mice generated for these studies should also be useful in establishing the role of facilitated urea transport in extrarenal organs expressing UT-B.

Hereditary non-X-linked nephrogenic diabetes insipidus (NDI) is caused by mutations in the aquaporin-2 (AQP2) water channel. In transfected cells, the human disease-causing mutant AQP2-T126M is retained at the endoplasmic reticulum (ER) where it is functional and targetable to the plasma membrane with chemical chaperones. A mouse knock-in model of NDI was generated by targeted gene replacement using a Cre-loxP strategy. Along with T126M, mutations H122S, N124S, and A125T were introduced to preserve the consensus sequence for N-linked glycosylation found in human AQP2. Breeding of heterozygous mice yielded the expected Mendelian distribution with 26 homozygous mutant offspring of 99 live births. The mutant mice appeared normal at 2-3 days after birth but failed to thrive and generally died by day 6 if not given supplemental fluid. Urine/serum analysis showed a urinary concentrating defect with serum hyperosmolality and low urine osmolality that was not increased by a V2 vasopressin agonist. Northern blot analysis showed up-regulated AQP2-T126M transcripts of identical size to wild-type AQP2. Immunoblots showed complex glycosylation of wild-type AQP2 but mainly endoglycosidase H-sensitive core glycosylation of AQP2-T126M indicating ER-retention. Biochemical analysis revealed that the AQP2-T126M protein was resistant to detergent solubilization. Kidneys from mutant mice showed collecting duct dilatation, papillary atrophy, and unexpectedly, some plasma membrane AQP2 staining. The severe phenotype of the AQP2 mutant mice compared with that of mice lacking kidney water channels AQP1, AQP3, and AQP4 indicates a critical role for AQP2 in neonatal renal function in mice. Our results establish a mouse model of human autosomal NDI and provide the first in vivo biochemical data on a diseasecausing AQP2 mutant.The formation of concentrated urine by the kidney requires high osmotic water permeability across the collecting duct epithelium. Collecting duct epithelial cells express aquaporin water channels AQP2, 1 AQP3, and AQP4 (1-4). AQP2 is the vasopressin (antidiuretic hormone)-regulated water channel (5, 6). Vasopressin induces the fusion of intracellular vesicles containing AQP2 with the apical plasma membrane resulting in increased water permeability (7-9). AQP2 is of considerable clinical importance in fluid and electrolyte balance. Mutations in human AQP2 cause hereditary non-X-linked nephrogenic diabetes insipidus (10 -13). Down-regulation of AQP2 expression occurs in several forms of acquired NDI (14), and AQP2 up-regulation is important in the pathophysiology of fluidretaining states such as congestive heart failure (15, 16). AQP3 and AQP4 are expressed constitutively at the basolateral membrane of collecting duct epithelial cells, with AQP3 mainly in cortical collecting duct and AQP4 in inner medullary collecting duct. Transgenic mice lacking AQP3 have low water permeability in cortical collecting duct and manifest NDI with marked polyuria and decreased urine osmolality (17). In contrast, mice lacking AQP4 show onl...

The regulation of MUC2, a major goblet cell mucin gene, was examined by constructing transgenic mice containing bases −2864 to +17 of the human MUC25′-flanking region fused into the 5′-untranslated region of a human growth hormone (hGH) reporter gene. Four of eight transgenic lines expressed reporter. hGH message expression was highest in the distal small intestine, with only one line expressing comparable levels in the colon. This contrasts with endogenous MUC2 expression, which is expressed at its highest levels in the colon. Immunohistochemical analysis indicated that goblet cell-specific expression of reporter begins deep in the crypts, as does endogenous MUC2 gene expression. These results indicate that the MUC2 5′-flanking sequence contains elements sufficient for the appropriate expression of MUC2 in small intestinal goblet cells. Conversely, elements located outside this region appear necessary for efficient colonic expression, implying that the two tissues utilize different regulatory elements. Thus many, but not all, of the elements necessary for MUC2 gene regulation reside between bases −2864 and +17 of the 5′-flanking region.

We have developed a transgenic mouse expressing enhanced green fluorescent protein (EGFP) in virtually all type II (TII) alveolar epithelial cells. The CBG mouse (SPC-BAC-EGFP) contains a bacterial artificial chromosome modified to express EGFP within the mouse surfactant protein (SP)-C gene 3' untranslated region. EGFP mRNA expression is limited to the lung. EGFP fluorescence is both limited to and exhibited by all cells expressing pro-SP-C; fluorescence is uniform throughout all lobes of the lung and does not change as mice age. EGFP(+) cells also express SP-B but do not express podoplanin, a type I (TI) cell marker. CBG mice show no evidence of lung disease with aging. In 3 hours, TII cells can be isolated in >99% purity from CBG mice by FACS; the yield of 3.7 ± 0.6 × 10(6) cells represents approximately 25 to 60% of the TII cells in the lung. By FACS analysis, approximately 0.9% of TII cells are in mitosis in uninjured lungs; after bleomycin injury, 4.1% are in mitosis. Because EGFP fluorescence can be detected for >14 days in culture, at a time that SP-C mRNA expression is essentially nil, this line may be useful for tracking TII cells in culture and in vivo. When CBG mice are crossed to transgenic mice expressing rat podoplanin, TI and TII cells can be easily simultaneously identified and isolated. When bred to other strains of mice, EGFP expression can be used to identify TII cells without the need for immunostaining for SP-C. These mice should be useful in models of mouse pulmonary disease and in studies of TII cell biology, biochemistry, and genetics.