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Severely Impaired Urinary Concentrating Ability in Transgenic Mice Lacking Aquaporin-1 Water Channels*

Tonghui Ma, Baoxue Yang, Annemarie Gillespie, Elaine J. Carlson, Charles J. Epstein, and A. S. VerkmanDagger

From the Departments of Medicine, Physiology, and Pediatrics, Cardiovascular Research Institute, University of California, San Francisco, California 94143-0521

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results & Discussion
References

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.

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results & Discussion
References

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 >25% of membrane protein has been attributed to AQP1. AQP1 functions as a water-selective transporting protein (17, 18) that excludes small solutes including urea, protons, and monovalent ions.

The high expression of AQP1 in kidney suggests an essential role in the renal concentrating mechanism. We have generated transgenic mice lacking AQP1 and demonstrate that these mice have low water permeability in proximal tubule membrane vesicles and are unable to concentrate their urine.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results & Discussion
References

Generation of AQP1 Null Mouse-- The mouse AQP1 gene was isolated from a C57BL6/J genomic library (CLONTECH ML1043j) and analyzed. A targeting vector was constructed using an 8-kb genomic DNA fragment containing exon 1 and intron 1. Part of the exon 1 coding sequence into ~1.3 kb of intron 1 was replaced by a 1.8-kb PolIIneobpA cassette for positive selection, and a PGK-tk cassette was inserted upstream for negative selection. The vector was linearized at a unique downstream NotI site and electroporated into CB1-4 embryonic stem (ES) cells (19). Transfected ES cells were selected with G418 and FIAU for 7 days, yielding seven targeted clones out of 232 doubly resistant colonies upon screening by polymerase chain reaction using a neo-specific sense primer and an AQP1 gene-specific antisense primer located 50 base pairs downstream of the targeting region. Homologous recombination was confirmed by Southern hybridization: 10 µg of genomic DNAs were digested with ApaI, electrophoresed, transferred to a nylon membrane, and hybridized with the 0.8-kb 32P-labeled genomic fragment indicated in Fig. 1A. ES cells were injected into PC 2.5-day 8-cell morula stage CD1 zygotes, cultured overnight to blastocysts, and transferred to pseudopregnant B6D2 females. Offspring were genotyped by polymerase chain reaction followed by Southern blot analysis as described above. Heterozygous mice were intercrossed to produce homozygous AQP1 knockout mice.

Water Permeability in Apical Vesicles from Proximal Tubule-- Sealed apical membrane vesicles from kidney proximal tubule were isolated by a magnesium aggregation procedure (20, 21). 1-mm slices of kidney cortex were homogenized and centrifuged to remove unbroken cells. Solid MgCl2 was added to the homogenate to a final concentration of 10 mM. Vesicles were isolated by differential centrifugation and suspended in 50 mM mannitol-Tris buffer, pH 7.4. Osmotic water permeability was measured by stopped-flow light scattering (17, 20). Apical membrane vesicles (~0.1 mg of protein/ml) were suspended in 50 mM mannitol-2 mM Tris (55 mOsm) and mixed in <1 ms with an equal volume of hyperosmolar buffer (155 mOsm) to give a 50 mOsm inwardly directed osmotic gradient. Ninety degree light scattering was measured at 520 nm wavelength. Pf was computed using a vesicle surface-to-volume ratio of 2.5 × 105 cm-1.

Urine Chemistries-- Urine osmolalities and serum chemistries of 32-35-day-old mice were measured by the University of California, San Francisco Clinical Chemistry Laboratory before and after water deprivation. To collect urine samples, mice were placed on a wire mesh platform in a clean glass beaker until spontaneous voiding was observed. Fresh urine sample was collected and kept on ice until assay. In some experiments, 0.4 µg/kg DDAVP was injected intraperitoneally after obtaining the 36-h urine sample, and urine was collected at 1-2 h after injection.

Northern and Immunoblot Analysis-- RNAs from kidney, lung, eye, spleen, and heart were isolated using TRIzol reagent (Life Technologies, Inc.). RNAs (10 µg/lane) were resolved on a formaldehyde-agarose denaturing gel, transferred to a Nylon membrane, and hybridized at high stringency with 32P-labeled probes corresponding to each of the cDNA coding regions of AQPs 1-4. For immunoblot analysis tissues were homogenized in 250 mM sucrose, 10 mM Tris-HCl, pH 7.4, containing 1 µg/ml leupeptin, 1 µg/ml pepstatin A, and 4 µg/ml antipain. Proteins were resolved on a 12% polyacrylamide gel, electrotransferred to a polyvinylidene difluoride membrane, and blotted with a rabbit polyclonal anti-AQP1 antibody raised against purified erythrocyte AQP1 protein.

    RESULTS AND DISCUSSION
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Abstract
Introduction
Materials & Methods
Results & Discussion
References

AQP1 knockout mice were generated by targeted gene disruption. A targeting vector for homologous recombination was designed to delete a critical part of the AQP1 coding sequence (Fig. 1A). Intercross of heterozygous founder mice showed wild-type, heterozygous, and knockout genotypes (Fig. 1B). The knockout mice did not express detectable full-length AQP1 transcript or protein in kidney (Figs. 1, C and D) or in tissues where it is known to be strongly expressed (lung, eye, spleen, and heart; not shown). As expected from previous studies on rat kidney (10-12), AQP1 was localized in wild-type mice to plasma membranes of proximal tubule (Fig. 1E) and descending limb of Henle epithelia and vasa recta endothelia (not shown). Specific AQP1 staining was not detected in kidneys from knockout mice (Fig. 1F).


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Fig. 1.   A, targeting strategy for AQP1 gene interruption. Homologous recombination results in replacement of the last 24 base pairs of exon 1 into 1.3 kb of intron 1 by the 1.8-kb PolIIneo selectable marker. B, Southern blot of mouse liver genomic DNA digested with ApaI and probed as indicated in A. C, Northern blot of kidney probed with the mouse AQP1 coding sequence. D, immunoblot of kidney homogenate probed with AQP1 antibody. E, immunofluorescence of AQP1 in kidney cortex of wild-type mouse. F, immunofluorescence of kidney in AQP1 knockout mouse.

Genotype analysis of 166 births showed 45 wild-type, 84 heterozygote, and 37 knockout mice, consistent with 1:2:1 Mendelian inheritance. The AQP1 knockout mice were not obviously different from wild-type mice in survival, gross physical appearance, and organ morphology, except for mild growth retardation.

Functional analysis of AQP1 in kidney was done using purified apical membrane vesicles from proximal tubule epithelium. Osmotic water permeability (Pf) was measured from the time course of 90° scattered light intensity in response to a rapidly imposed osmotic gradient. Pf was decreased strongly in membranes from knockout versus wild-type mice with intermediate Pf for heterozygotes (Fig. 2A, Pf = 0.018 ± 0.002 cm/s wild-type; 0.010 ± 0.001 cm/s heterozygous; 0.002 ± 0.0003 cm/s knockout). After inhibition by 0.2 mM HgCl2, Pf in membranes from wild-type and heterozygous mice was similar to Pf in knockout mice (0.001-0.002 cm/s, Fig. 2B). This low Pf is typical of lipid bilayer membranes not containing water channels, indicating that AQP1 is the principal functional water transporter of proximal tubule apical membrane. Stopped-flow measurements also revealed an ~8-fold decrease in Pf in erythrocytes from knockout versus wild-type mice (not shown).


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Fig. 2.   Stopped-flow light scattering measurements of osmotic water permeability in apical membrane vesicles from kidney proximal tubule. Vesicles were subjected to a 50 mM inwardly directed sucrose gradient at 10 °C. Data are plotted using three contiguous times scales to show the complete osmotic equilibration. A, water permeabilities in kidney vesicles from wild-type, heterozygous, and AQP1 knockout mice. Pf values are reported in the text. B, same as in A. except that vesicles were incubated with 0.2 mM HgCl2 for 5 min prior to measurements.

To determine whether AQP1 is required for formation of a concentrated urine, freshly voided urine specimens were collected before and after water deprivation. Whereas the wild-type and heterozygous mice were active and appeared grossly normal after being deprived of water for 36 h, the knockout mice became remarkably lethargic and in some cases unresponsive. Average body weight decreased by 35% in the knockout mice compared with 20-22% in wild-type and heterozygous mice (Fig. 3A), and serum osmolality increased to 517 mOsm compared with 311-325 mOsm in wild-type and heterozygous mice. For comparison, body weight decreased by 22% for age-matched AQP4 knockout mice. Interestingly, despite the marked hyperosmolality in water-deprived AQP1 knockout mice, nearly every mouse could be resuscitated by oral water administration without morbidity.


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Fig. 3.   Body weight (A) and urine osmolality (B) in mice before versus after water deprivation for 36 h. * indicates p < 0.001.

Fig. 3B shows a remarkably low urine osmolality in AQP1 knockout mice prior to water deprivation (580-610 mOsm) that did not increase after deprivation. Measurement of urine osmolalities of the knockout mice every 8 h showed values consistently less than 650 mOsm. In contrast, average urine osmolality in wild-type and heterozygous mice was 1400 mOsm before water deprivation and increased to ~3000 mOsm after deprivation. In four out of five water-deprived AQP1 knockout mice, urine sodium concentration was less than 10 mM. Intraperitoneal injection of the V2 receptor agonist DDAVP caused no further increase in urine osmolality in knockout mice, indicating that the urinary concentrating defect associated with AQP1 knockout involves the kidney and not central osmoreceptor sensing.

To investigate the possibility that AQP1 knockout is associated with a change in the expression of other renal aquaporins that might be involved in kidney fluid balance, expression of AQP2, AQP3, and AQP4 was quantified by Northern and immunoblot analysis. AQP1 deletion did not change the expression of AQP3 and AQP4, but was associated with a mild increase (20-50% over that in wild-type mice) in expression of AQP2 transcript and protein.

The inability of AQP1 knockout mice to form a concentrated urine can be the consequence of distinct defects acting in synergy: impairment of near-isosmolar fluid reabsorption in proximal tubule leading to fluid overload of the distal nephron and disruption of the medullary countercurrent multiplication mechanism because of low water permeability in descending limb of Henle and vasa recta (22-24). The absence of a detectable defect in urinary concentrating ability in heterozygous mice indicates that the kidney expresses a considerable excess of AQP1 over that needed to maintain fluid homeostasis. The low urine sodium concentration in water-deprived AQP1 knockout mice suggests that renal salt transporting function is intact, which is consistent with urine > serum osmolality prior to water deprivation. The absence of DDAVP effect, which should equalize urine and medullary interstitial osmolalities, indicates that the medullary interstitium of AQP1 knockout mice is not appropriately hypertonic. Taken together, these results suggest that the primary renal defect in AQP1 knockout mice is the inability to generate a hypertonic medullary interstitium by countercurrent multiplication.

The data above implicate an important role for AQP1 in the renal urinary concentrating mechanism. The report of apparently normal phenotype in three humans lacking AQP1 (7) did not include analysis of fluid consumption or physiological response to stresses such as dehydration. The strong expression of AQP1 in choroid plexus, ciliary body, pulmonary capillary endothelia, biliary tract epithelia, and reproductive organs indicates the need for rigorous evaluation of cerebrospinal and aqueous humor fluid balance, lung interstitial fluid clearance, and gastrointestinal and reproductive function. The dramatically abnormal phenotype associated with AQP1 deletion raises the possibility that other mammalian aquaporins have important physiological function. Deletion of each remaining aquaporin in mice is likely be informative, as might the screening for aquaporin mutations in genetic diseases associated with abnormalities of fluid balance.

    FOOTNOTES

* This work was supported by National Institutes of Health Grants DK35124, HL42368, and HL51854; Grant R613 from the National Cystic Fibrosis Foundation; and National Institutes of Health Gene Therapy Core Center Grant DK47766.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: 1246 Health Sciences East Tower, Cardiovascular Research Institute, University of California, San Francisco, CA 94143-0521. Tel.: 415-476-8530; Fax: 415-665-3847; E-mail: verkman{at}itsa.ucsf.edu.

1 The abbreviations used are: AQP, aquaporin; kb, kilobase(s); ES, embryonic stem; DDAVP, desmopressin.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results & Discussion
References

  1. Verkman, A. S., van Hoek, A. N., Ma, T., Frigeri, A., Skach, W. R., Mitra, A., Tamarappoo, B. K., Farinas, J. (1996) Am. J. Physiol. 270, C12-C30[Abstract/Free Full Text]
  2. Agre, P., Preston, B. M., Smith, B. L., Jung, J. S., Raina, S., Moon, C., Guggino, W. B., Nielsen, S. (1993) Am. J. Physiol. 265, F463-F476[Abstract/Free Full Text]
  3. Van Os, C. H., Deen, P. M. T., and Dempster, J. A. (1994) Biochim. Biophys. Acta 1197, 291-309[Medline] [Order article via Infotrieve]
  4. Fushimi, K., Uchida, S., Hara, Y., Hirata, Y., Marumo, F., and Sasaki, S. (1993) Nature 361, 549-552[CrossRef][Medline] [Order article via Infotrieve]
  5. Nielsen, S., Chou, C. L., Marples, D., Christensen, E. I., Kishore, B. K., Knepper, M. A. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 1013-1017[Abstract]
  6. Deen, P. M., Verkijk, M. A, Knoers, N. V., Wieringa, B., Monnens, L. A., van Os, C. H., van Oost, B. A. (1994) Science 264, 92-95[Medline] [Order article via Infotrieve]
  7. Preston, G. M., Smith, B. L., Zeidel, M. L., Moulds, J. J., Agre, P. (1994) Science 265, 1585-1587[Medline] [Order article via Infotrieve]
  8. Ma, T., Yang, B., Gillespie, A., Carlson, E. J., Epstein, C. J., Verkman, A. S. (1997) J. Clin. Invest. 100, 957-962[Abstract/Free Full Text]
  9. Preston, G. M., and Agre, P. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 11110-11114[Abstract]
  10. Sabolic, I., Valenti, G., Verbavatz, J. M., van Hoek, A. N., Verkman, A. S., Ausiello, D. A., Brown, D. (1992) Am. J. Physiol. 263, C1225-C1233[Abstract/Free Full Text]
  11. Nielsen, S., Digiovani, S. R., Christensen, E. I., Knepper, M. A., Harris, H. W. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 11663-11667[Abstract]
  12. Zhang, R., Skach, W. R., Hasegawa, H., van Hoek, A. S., Verkman, A. S. (1993) J. Cell Biol. 120, 359-369[Abstract]
  13. Hasegawa, H., Lian, S. C., Finkbeiner, W. E., Verkman, A. S. (1994) Am. J. Physiol. 266, C893-C903[Abstract/Free Full Text]
  14. Nielsen, S., Smith, B. L., Christensen, E. I., Agre, P. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 7275-7279[Abstract]
  15. Pallone, T. L., Kishore, B. K., Nielsen, S., Agre, P., and Knepper, M. A. (1997) Am. J. Physiol. 272, F587-F596[Abstract/Free Full Text]
  16. Verbavatz, J. M., Brown, D., Sabolic, I., Valenti, G., Ausiello, D. A., van Hoek, A. N., Ma, T., Verkman, A. S. (1993) J. Cell Biol. 123, 605-618[Abstract]
  17. Van Hoek, A. N., and Verkman, A. S. (1992) J. Biol. Chem. 267, 18267-18269[Abstract/Free Full Text]
  18. Zeidel, M. L., Ambudkar, S. V., Smith, B. L., Agre, P. (1992) Biochemistry 31, 7436-7440[Medline] [Order article via Infotrieve]
  19. Li, Y., Huang, T. T., Carlson, E. J., Melov, S., Ursell, P. C., Olson, J. L., Novel, L. J., Yoshimura, M. P., Berger, C., Chan, P. J., Wallace, D. C., Epstein, C. J. (1995) Nat. Genet. 11, 376-381[Medline] [Order article via Infotrieve]
  20. Verkman, A. S., Dix, J. A., and Seifter, J. L. (1985) Am. J. Physiol. 248, F650-F655[Medline] [Order article via Infotrieve]
  21. Booth, A. G., and Kenny, A. J. (1974) Biochem. J. 142, 575-583[Medline] [Order article via Infotrieve]
  22. Knepper, M. A. (1997) Am. J. Physiol. 272, F3-F12[Abstract/Free Full Text]
  23. Jen, J. R., and Stephenson, L. B. (1994) Bull. Math. Biol. 56, 491-514[Medline] [Order article via Infotrieve]
  24. Chou, C.-L., Digiovanni, S. R., Mejia, R., Nielsen, S., and Knepper, M. A. (1995) Am. J. Physiol. 268, F70-F77


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