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Erythrocyte Water Permeability and Renal Function in Double Knockout Mice Lacking Aquaporin-1 and Aquaporin-3

2001; Elsevier BV; Volume: 276; Issue: 1 Linguagem: Inglês

10.1074/jbc.m008664200

1083-351X

Baoxue Yang, Tonghui Ma, A.S. Verkman,

Magnesium in Health and Disease

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. 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. aquaporin phosphate-buffered saline The route for water movement across the erythrocyte plasma membrane has been a subject of longstanding interest. Erythrocyte osmotic water permeability is inhibited by ∼90% by mercurial sulfhydryl compounds and has biophysical properties of a pore pathway including a low Arrhenius activation energy and a high ratio of osmotic to diffusional water permeability (1Macey R.I. Am. J. Physiol. 1984; 246: C195-C203Crossref PubMed Google Scholar). The water-selective transporter AQP11 is the major erythrocyte water transporter as proven by the reduced water permeability in erythrocytes from Colton −/− humans lacking AQP1 (2Macey R.I. Mori S. Smith B.L. Preston G.M. Mohandas N. Collins M. van Zili P.C.M. Zeidel M.L. Agre P. J. Biol. Chem. 1996; 271: 1309-1313Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar) and transgenic AQP1 null mice (3Ma T. Yang B. Gillespie A. Carlson E.J. Epstein C.J. Verkman A.S. J. Biol. Chem. 1998; 273: 4296-4299Abstract Full Text Full Text PDF PubMed Scopus (505) Google Scholar). Recently, immunocytochemical evidence has suggested that the “aquaglyceroporin” AQP3 provides the major non-AQP1 pathway for water transport across the erythrocyte plasma membrane as well as the transport pathway for glycerol (4Roudier N. Verbavatz J.-M. Maurel C. Ripoche P. Tacnet F. J. Biol. Chem. 1998; 273: 8407-8412Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). Erythrocyte glycerol permeability is substantially greater than that across lipid bilayers and is inhibited by phloretin (5Macey R.I. Farmer R.E. Biochim. Biophys. Acta. 1970; 211: 104-106Crossref PubMed Scopus (304) Google Scholar); however, the molecular identity of the putative glycerol transporter has not been established. The principal goal of this study was to determine the functional role of AQP3 in erythrocyte water and glycerol permeability. Transport measurements were done on erythrocytes lacking AQP1 and AQP3 individually and AQP1/AQP3 together. We reasoned that the very low water permeability of AQP1-deficient erythrocytes would permit the detection of even small amounts of functional AQP3 as a further decrease in permeability in AQP1/AQP3-deficient erythrocytes. A secondary goal of this study was to investigate the roles of AQP1 and AQP3 in the urinary-concentrating mechanism. AQP1 and AQP3 are expressed in the kidney: AQP1 in proximal tubule, thin descending limb of Henle, and medullary vasa recta (6Sabolic I. Valenti G. Verbavatz J.M. Van Hoek A.N. Verkman A.S. Ausiello D.A. Brown D. Am. J. Physiol. 1992; 263: C1225-C1233Crossref PubMed Google Scholar, 7Nielsen S. Smith B.L. Christensen E.I. Knepper M.A. Agre P. J. Cell Biol. 1993; 120: 371-383Crossref PubMed Scopus (451) Google Scholar) and AQP3 at the basolateral membrane of collecting duct principal cells (8Frigeri A. Gropper M. Turck C.W. Verkman A.S. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4328-4331Crossref PubMed Scopus (375) Google Scholar, 9Ecelbarger C.A. Terris J. Frindt G. Echevarria M. Marples D. Nielsen S. Knepper M.A. Am. J. Physiol. 1995; 269: F663-F672PubMed Google Scholar). Wild-type mice have base-line urine osmolalities of 1000–1500 mosm that increase to >3000 mosm after water deprivation. Mice lacking AQP1 are polyuric and have urine osmolalities of 500–700 mosm that do not increase after water deprivation or DDAVP administration (3Ma T. Yang B. Gillespie A. Carlson E.J. Epstein C.J. Verkman A.S. J. Biol. Chem. 1998; 273: 4296-4299Abstract Full Text Full Text PDF PubMed Scopus (505) Google Scholar). In vivo micropuncture revealed defective proximal tubule fluid absorption in AQP1 null mice (10Schnermann J. Chou C.L. Ma T. Traynor T. Knepper M.A. Verkman A.S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9660-9664Crossref PubMed Scopus (395) Google Scholar), and isolated tubule microperfusion showed remarkably reduced osmotic water permeability in thin descending limb of Henle (11Chou C.L. Knepper M.A. van Hoek A.N. Brown D. Yang B. Ma T. Verkman A.S. J. Clin. Invest. 1999; 103: 491-496Crossref PubMed Scopus (195) Google Scholar) and outer medullary descending vasa recta (12Pallone T.L. Edwards A. Ma T. Silldorff E. Verkman A.S. J. Clin. Invest. 2000; 105: 2686-2692Crossref Scopus (121) Google Scholar). Mice lacking AQP3 also manifest nephrogenic diabetes insipidus but with a very different pattern (13Ma T. Song Y. Yang B. Gillespie A. Carlson E.J. Epstein C.J. Verkman A.S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4386-4391Crossref PubMed Scopus (337) Google Scholar). AQP3 null mice are remarkably polyuric with base-line urine osmolalities of 90% of living AQP1 and AQP3 null mice that were genotyped at 5 days remained alive at 8 weeks, only 50% of the double knockout mice were alive at 8 weeks. Osmotic water permeability was measured in erythrocytes from wild-type mice and mice lacking AQP1 and AQP3 individually and AQP1/AQP3 together. Fig. 1 A shows the time course of osmotic cell shrinking in response to a 100 mm inwardly directed osmotic gradient of sucrose. The data are plotted using three contiguous time scales to show the full time course of decreasing cell volume. Erythrocyte water permeability was remarkably reduced by AQP1 deletion but not further reduced by AQP3 deletion. Fig. 1 B shows the inhibition of water transport by the mercurial HgCl2. Water transport was strongly inhibited in erythrocytes from wild-type and AQP3 null mice and was inhibited to a lesser extent in erythrocytes from AQP1 null mice and AQP1/AQP3 double knockout mice. Fig. 2 summarizes osmotic water permeability coefficients (Pf) determined in four mice of each genotype. AQP3 deletion did not reduce Pf in wild-type or AQP1 null mice nor did it affect the inhibitory potency of HgCl2. Temperature dependence measurements were performed to determine the Arrhenius activation energy for erythrocyte water transport. Computed activation energies (10–37 °C) were 8 kcal/mol for AQP1 null mice and the double knockout mice. Erythrocyte glycerol permeability was measured from the time course of cell swelling in response to a 100 mm inwardly directed gradient of glycerol. As shown in Fig.3 A, the glycerol gradient produced an initial rapid decrease in cell volume due to osmotically induced water efflux followed by slower cell swelling that was due to glycerol and secondary water influx. At 20 °C, glycerol equilibrated across erythrocytes from wild-type mice with a half-time of ∼20 s, giving a permeability coefficient (PGly) of 2.63 × 10−6 cm/s (top curve). Glycerol permeability was inhibited by 64% by 0.5 mmphloretin (second curve) and was strongly temperature-sensitive (bottom curves), increasing 2.8-fold from 10 to 30 °C that was consistent with a facilitated transport pathway. Fig. 3 B shows glycerol permeability in erythrocytes from AQP3 null mice in the absence and presence of phloretin. Results were similar to those in erythrocytes from wild-type mice. Data from a series of mice showed that AQP3 deletion did not reduce erythrocyte glycerol permeability or its inhibition by phloretin (Fig.3 C). Together the permeability measurements indicate that AQP3 does not contribute measurably to erythrocyte water or glycerol permeability. Immunocytochemistry was done to look for AQP3 protein in erythrocytes. Fig. 4 A shows little AQP3 antibody labeling of permeabilized erythrocyte smears from humans (left) and wild-type mice (middle). Similar low levels of labeling were detected in erythrocytes from AQP3 null mice (right). In contrast, AQP3 was readily detected in the kidney-collecting duct in wild-type mice (Fig. 4 B,left) but not in AQP3 null mice (right). Urinary-concentrating function was compared in the wild-type, single knockout, and double knockout mice. Fig.5 A shows daily fluid consumption and urinary output in the mice. Polydipsia and polyuria were greater in AQP3 than in AQP1 null mice and further increased in the AQP1/AQP3 double knockout mice. The difference in fluid intake and urinary output, primarily representing insensible respiratory losses, was similar in all groups. Fig. 5 B summarizes urine osmolalities in mice given free access to food and water and in mice deprived of food and water for 24 h. Base-line urine osmolality was high (>1500 mosm) in wild-type mice and nearly doubled after water deprivation. Base-line urine osmolality was much lower in the AQP1 null mice and changed little after water deprivation. Urine osmolality was lower in AQP3 null mice but increased 2.4-fold after water deprivation. Interestingly, the deletion of AQP1 and AQP3 together resulted in an even lower base-line urine osmolality, which unlike that in AQP1 null mice increased 2.9-fold after water deprivation (see “Discussion”). Most adult AQP1/AQP3 double knockout mice showed marked tumor-like swelling of the flanks bilaterally (Fig.6 A, left), which was never seen in wild-type mice. The flank swelling was caused by kidney enlargement (Fig. 6 A, middle and right). Examination of the morphology in kidneys from wild-type mice showed well demarcated cortex and papilla (Fig.6 B, top panels). In contrast, kidneys from AQP3 null mice (Fig. 6 B, bottom panels) and AQP1/AQP3 double knockout mice (not shown) showed medullary atrophy and cortical thinning. At 4 weeks of age, >50% of kidneys from AQP3 null mice and >90% of kidneys from AQP1/AQP3 double knockout mice showed these changes. Many adult mice showed hydronephrosis. The kidneys with severe hydronephrosis were markedly enlarged and transparent enough to reveal dilated renal blood vessels (Fig. 6 A, right panel). Similar changes in renal morphology have been seen in polyuria causing increased intrarenal pressures (15Takahashi N. Chernavvsky D.R. Gomez R.A. Igarashi P. Gitelman H.J. Smithies O. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5434-5439Crossref PubMed Scopus (214) Google Scholar). The medullary atrophy appeared to be an age-dependent phenomenon that was infrequently seen in mice under the age of 2 weeks but found in ∼50% of mice at age 4 weeks (Fig. 6 B). The mice with flank swelling and renal enlargement had serum azotemia (blood urea nitrogen 78 ± 27 mg/dl, normal 500 mosmol. Together these results indicate distinct renal defects involving medullary countercurrent exchange (AQP1) and cortical collecting duct water permeability (AQP3). These data should be very useful in testing mathematical models of the urinary-concentrating mechanism. We thank Liman Qain for mouse breeding and genotype analysis.