点击显示 收起
【摘要】 Transgenic mouse models of defective urinary concentrating ability produced by deletion of various membrane transport or receptor proteins, including aquaporin-2 (AQP2), are associated with neonatal mortality from polyuria. Here, we report an inducible mouse model of AQP2 gene deletion with severe polyuria in adult mice. LoxP sequences were inserted into introns 1 and 2 in the mouse AQP2 gene by homologous recombination in embryonic stem cells. Mating of germ-line AQP2-loxP mice with tamoxifen-inducible Cre-expressing mice produced offspring with inducible homozygous Cre-AQP2-loxP, which had a normal phenotype. Tamoxifen injections over 10 days resulted in AQP2 gene excision, with undetectable 95% reduction in immunoreactive AQP2 protein. Urine osmolality decreased from 2,000 to <500 mosmol/kgH 2 O after 4-5 days, with urine output increasing from 2 to 25 ml/day. Urine osmolality did not increase after water deprivation. Interestingly, AQP3 protein expression in the collecting duct was increased by about fivefold after AQP2 gene excision. Mild renal damage was seen after 6 wk of polyuria, with collecting duct dilatation, yet normal creatinine clearance and serum chemistries. These results establish the first adult model of nephrogenic diabetes insipidus (NDI) caused by AQP2 deficiency, with daily urine output comparable to body weight, although remarkable preservation of renal function compared with non-inducible NDI models.
【关键词】 water transport water channel transgenic mouse NDI polyuria
AQUAPORIN -2 ( AQP 2) IS THE antidiuretic hormone (vasopressin)-regulated water channel expressed in the mammalian kidney collecting duct. Apical plasma membrane AQP2 targeting is regulated by a vesicular transport mechanism in which vasopressin acting through cAMP causes the exocytic insertion of AQP2 ( 1, 26 ). AQP2 is of clinical importance in acquired disorders of urinary concentrating function, both in nephrogenic diabetes insipidus (NDI), as produced by lithium therapy ( 20 ), and the syndrome of inappropriate antidiuretic hormone ( 7 ). Mutations in the AQP2 gene can cause the very rare disorder of hereditary NDI ( 5, 8, 12 ). Several AQP2 mutations causing autosomal recessive NDI, such as AQP2-T126M, are associated with defective AQP2 folding and retention at the endoplasmic reticulum ( 2, 23, 36 ). There is great interest in identifying small-molecule correctors of defective protein folding as potential therapy for human hereditary diseases, such as NDI caused by V2 receptor mutation ( 22 ) and cystic fibrosis caused by CFTR mutation ( 28 ). We found that small-molecule chemical chaperones correct the defective processing of AQP2 mutants in transfected cell models, including AQP2-T126M ( 35 ).
Transgenic mouse models of NDI caused by AQP2 mutation are needed to evaluate the in vivo efficacy of small-molecule therapies that are active in cell culture models. To this end, we generated transgenic AQP2-T126M knock-in mice in which the T126M mutation was introduced into the mouse AQP2 gene by targeted gene replacement ( 39 ). However, the mice manifested a urinary concentrating defect with polyuria early in life, resulting in neonatal mortality even if fluid replacement was given. Similar neonatal mortality was found in transgenic mouse models of defective urinary concentrating function caused by deletion/mutation of the V2 receptor ( 41 ), ROMK1 K + channel ( 15, 16 ), and Na + -K + -2Cl - cotransporter ( 6, 34 ).
As a first step in creating adult mouse models of NDI caused by defective AQP2, we have created and characterized an inducible mouse model of AQP2 deficiency. Essentially complete AQP2 protein deletion in the kidney collecting duct was accomplished by tamoxifen-inducible Cre-recombinase expression in homozygous mice in which loxP sites were introduced in introns of the mouse AQP2 gene. The mice developed severe polyuria with daily urine output approximately equal to their body weight.
METHODS
Generation of AQP2 conditional knockout mice. Based on mouse AQP2 gene structure ( 40 ), a gene replacement targeting vector was constructed as shown in Fig. 1 A. The vector contained a 5-kb mouse genomic DNA fragment containing exons 1-3 of the AQP2 gene. Exon 2 and a Pol2neobpA selection cassette were flanked by loxP sites for conditional gene deletion and positive selection with G418, and a PGKtk cassette was inserted at the 3'-end of the AQP2 targeting sequence for negative selection with FIAU. The vector was linearized at a unique Not I site and electroporated into CB1-4 embryonic stem (ES) cells. Transfected ES cells were selected with G418 and FIAU for 7 days, yielding 4 targeted clones of 96 doubly resistant colonies on PCR screening using a sense primer specific for the neo cassette (5'-CAGTTCACCGCTGAATATGCAT-3') and an antisense primer specific for the AQP2 gene (5'-GCAAAGCTCTGAGAAAACCGC-3') located beyond the 3'-end of the construct. Homologous recombination was confirmed by Southern blot analysis using a 1-kb genomic fragment as probe as indicated in Fig. 1 A. 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 PCR followed by Southern blot analysis. Heterozygous floxed AQP2 females were mated with male Cre/Esr1 transgenic mice (Jackson Laboratories, Bar Harbor, ME) ( 10 ) to yield mice heterozygous for both genes. The second crossing of heterozygous mice yielded homozygous floxed AQP2 mice with the Cre/Esr1 gene. Protocols were approved by University of California San Francisco Committee on Animal Research.
Fig. 1. Gene targeting strategy for conditional aquaporin-2 (AQP2) gene knockout. A, top : organization and restriction map of the mouse AQP2 gene. Rectangles indicate exon segments, with coding regions shaded. Middle (2 lines): targeting strategy for floxed AQP2 gene deletion. Homologous recombination results in replacement of the indicated segments (thick lines) of the AQP2 gene. Exon 2 and a 1.8-kb polII-neo-selectable marker were flanked by loxP sites (arrowheads). Bottom : AQP2 gene structure after conditional deletion by Cre-recombinase. Probe used for Southern blot analysis is indicated ("probe") along with expected sizes of hybridized fragments after Apa I digestion. B : Southern blot of genomic DNA from kidney of wild-type (WT) mouse and of liver, kidney, testes, and tail from AQP2 flox mice (flox), digested with Apa I and probed as indicated in A. Where indicated, mice were treated for 10 days with tamoxifen (4OH-TM).
Induction of AQP2 gene deletion. A stock solution of tamoxifen (4-hydroxytamoxifen, Sigma, St. Louis, MO) in ethanol (250 mg/ml) was diluted in corn oil to 5 mg/ml. Adult mice (age 5-8 wk) were given intraperitoneal injections of the tamoxifen suspension (0.1 ml of 5 mg/ml) over 10 days.
Southern and Northern blot analysis. AQP2 gene targeting and deletion were confirmed by Southern hybridization in which 10 µg of genomic DNA were digested with Apa I, electrophoresed, transferred to a Nylon+ membrane (Amersham Biosciences, Piscataway, NJ), and hybridized with a 1-kb genomic fragment (indicated in Fig. 1 A ). For Northern blot analysis, total RNA from kidney, inner ear, colon, testes, and vas deferens was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA). RNAs (10 µg/lane) were resolved on a 1.2% formaldehyde-agarose denaturing gel, transferred to a Nylon+ membrane, and hybridized at high stringency with a 32 P-labeled probe corresponding to the mouse AQP2 cDNA coding sequence.
Immunofluorescence and immunoblot analysis. Kidney, inner ear (decalcified), testis, vas deferens, and colon tissue samples were fixed with 4% paraformaldehyde in PBS for 4 h, infiltrated with 30% sucrose in PBS overnight, frozen in OCT with liquid nitrogen, and cut into 3-µm-thick sections using a cryostat. Tissues were incubated using a rabbit polyclonal antibody against the 30-amino acid COOH terminus of AQP2 as described ( 40 ). Immunoblot analysis of the tissue homogenate was carried out with the same polyclonal serum. Tissues were homogenized with a glass Dounce homogenizer in 250 mM sucrose containing 1 mM EDTA, 20 µg/ml PMSF, 1 µg/ml pepstatin A, and 1 µg/ml leupeptin (pH 7.4), and centrifuged at 4,000 g for 15 min to remove whole cells, nuclei, and mitochondria. Total protein was assayed in the supernatant fractions using a Bio-Rad DC protein assay kit (Bio-Rad, Richmond, CA) and loaded on a 12% SDS-PAGE gel (10 µg/lane). Proteins were transferred to polyvinylidene difluoride membranes (Gelman Scientific, Ann Arbor, MI) and immunoblotted by standard procedures ( 40 ).
Fluorescence-based real-time RT-PCR. Total RNA from whole mouse kidney was isolated by homogenization in TRIzol reagent (Invitrogen), and mRNA was extracted using an Oligotex mRNA mini-kit (Qiagen, Valencia, CA). cDNA was reverse transcribed from mRNA with oligo(dT) (SuperScript II preamplification kit, Invitrogen). Fluorescence-based real-time RT-PCR was carried out using the LightCycler instrument (Roche Diagnostics, Indianapolis, IN) with a LightCycler FastStart DNA Master PLUS SYBR Green I kit. Primers were designed, and real-time PCR was carried out as described previously ( 11 ). -Actin was used as the reference gene, and pooled wild-type cDNA as the calibrator. Data are reported as normalized, calibrated ratios in which all samples were normalized to the reference gene.
Kidney histology. Kidney tissue was fixed in Bouin's fixative overnight, embedded in paraffin, and cut into 3-µm-thick sections. Kidney sections, including cortex, medulla and papilla, were stained with hematoxylin and eosin.
Urinary concentrating studies. Urine samples were collected by placing mice on a wire mesh platform in a clean glass beaker until spontaneous voiding was observed. In some experiments, urine samples were obtained from the same mice under basal conditions (unrestricted access to food and water), and after 18-h deprivation of food and water. Twenty-four-hour urine output and water consumption were measured in metabolic cages adapted for mice (Harvard Apparatus, Holliston, MA). Blood samples were collected in heparinized glass tubes by puncture of the periorbital venous sinus. Plasma was separated from blood cells by centrifugation. Urine osmolality was measured by freezing point osmometry (microosmometer, Precision Systems, Natick, MA). Urine and plasma chemistries were measured by the University of California San Francisco Clinical Chemistry Laboratory.
Statistical analysis. Statistical analysis was performed using Student's t -test.
RESULTS
Figure 1 A shows the gene-targeting strategy for generation of an inducible mouse model of AQP2 gene deletion. LoxP sequences (arrowheads) were inserted in intronic segments in the AQP2 gene, as well as Pol2neobpA and PGK-tk selection cassettes. Cre-recombinase-induced excision of the gene sequence between the loxP sites ("floxed" gene), which includes an exon sequence, is predicted to produce a truncated AQP2 transcript and no functional protein. Gene targeting was done in ES cells. Homologous recombination was confirmed by Southern blot analysis of ES cell genomic DNA using a 1-kb genomic fragment as a probe, as indicated in Fig. 1 A. Apa I digestion produced a fragment at 3.2 kb corresponding to the replaced gene containing an Apa I site in the Pol2neobpA cassette, and a fragment at 6 kb corresponding to wild-type gene, indicating correct AQP2 gene targeting with the floxed targeting sequence (AQP2 flox ). Heterozygous AQP2 flox mice were bred with Cre/Esr1 mice (tamoxifen-inducible Cre-recombinase-expressing mice) to generate AQP2 flox mice containing multiple Cre/Esr1 expression cassettes.
Homozygous AQP2 flox mice carrying the Cre/Esr1 transgene were generated by breeding double heterozygous mice containing AQP2 flox and Cre/Esr1. To assess the efficiency of induced Cre-mediated deletion of the loxP-flanked AQP2 gene segment containing exon 2 in the AQP2 flox mice, Southern blot analysis was done with genomic DNA from different tissues before and after intraperitoneal injections of tamoxifen over 10 days. After Apa I digestion, fragments corresponding to wild-type AQP2, floxed AQP2, and deleted AQP2 genes were predicted at sizes of 6.0, 3.2, and 5.5 kb, respectively. Figure 1 B shows a single band at 6.0 kb (in kidney DNA) of a wild-type mouse, and a single band at 3.2 kb (in tail DNA) in the homozygous AQP2 flox mice, as expected. Tamoxifen treatment caused the appearance of a 5.5-kb band and the partial disappearance of the 3.2-kb band, with an efficiency 90% in kidney and 80% in liver and testis.
Northern blot analysis in Fig. 2 A shows the 1.7-kb AQP2 transcript in kidney, inner ear, testis, and vas deferens of wild-type mice, with no detectable transcript in tamoxifen-treated AQP2 flox mice. No AQP2 transcript was found in the colon of wild-type or AQP2 flox mice. RT-PCR with primers designed to amplify the full-length AQP2 coding sequence gave strong 0.8-kb amplified fragments in cDNAs of kidney, inner ear, testis, and vas deferens of wild-type mice and smaller, weak (0.6 kb) fragments in the tamoxifen-treated AQP2 flox mice ( Fig. 1 B ). Very weak fragments at 0.8 kb were seen in the tamoxifen-treated AQP2 flox mice when the PCR amplification was carried out for 40 instead of 30 cycles (not shown). Immunoblot analysis showed the expected 29-kDa band in kidney, vas deferens, and testis of wild-type mice, which was very faint in the tamoxifen-treated AQP2 flox mice ( Fig. 1 C ). A diffuse band migrating at higher molecular size, corresponding to glycosylated AQP2, was seen in kidneys of wild-type mice. No specific band was found in the colon.
Fig. 2. AQP2 gene expression after Cre-induced deletion of the floxed AQP2 gene. A : Northern blot of indicated tissues from tamoxifen-treated WT and AQP2 flox mice (flox) probed with the mouse AQP2 coding sequence. B : RT-PCR amplification of cDNA from indicated tissues with primers amplifying the full-length AQP2 coding region. C : immunoblot analysis using purified polyclonal AQP2 antibody. def., Deferens.
AQP2 protein was localized in mouse tissues by immunofluorescence. Figure 3 shows specific AQP2 immunostaining in kidney and vas deferens of wild-type mice ( left ), with little AQP2 immunostaining in tamoxifen-treated treated AQP2 flox mice ( right ). Residual AQP2 immunostaining in the cortex was less than in the medulla of kidneys of tamoxifen-treated AQP2 flox mice. No specific AQP2 staining was detected in testis or in inner ear and colon (not shown).
Fig. 3. AQP2 immunofluorescence. AQP2 antibody staining of kidney cortex and inner medulla, testis, and vas deferens of tamoxifen-treated WT ( left ) and AQP2 flox (flox; right ) mice. Scale bar = 100 µm. Arrowheads indicate retained AQP2 expression in kidney. Arrows indicate AQP2 expression in vas deferens.
An analysis of mouse growth by body weight (age 1-5 wk) before tamoxifen administration showed no differences among the genotypes. As expected, the AQP2 flox mice had grossly normal appearance, activity, and behavior, and normal renal function.
The urinary concentrating function was compared in wild-type and AQP2 flox mice by measuring urine osmolality before and after tamoxifen treatment. There was no significant difference in urine osmolality between wild-type mice (1,984 ± 91 mosmol/kgH 2 O) and AQP2 flox mice (1,967 ± 237 mosmol/kgH 2 O) before tamoxifen administration. Urine osmolality in AQP2 flox mice decreased remarkably from 2,000 to <500 mosmol/kgH 2 O by 4-5 days after the first tamoxifen injection, and remained low thereafter at 200 mosmol/kgH 2 O ( Fig. 4 A ). Daily urinary output in wild-type and AQP2 flox mice was measured while they were housed in metabolic cages before and after tamoxifen treatment. The AQP2 flox mice were severely polyuric, excreting 10-fold greater fluid than litter-matched wild-type mice ( Fig. 4 B ). Urinary concentrating ability was measured in response to an 18-h water deprivation. Urine osmolality in wild-type mice increased from 1,860 to 2,720 mosmol/kgH 2 O, whereas no significant increase was seen in tamoxifen-treated AQP2 flox mice ( Fig. 4 C, top ). Marked total body weight loss was seen in these AQP2 flox mice after water deprivation ( Fig. 4 C, bottom ), as expected from their inability to concentrate their urine.
Fig. 4. Urinary concentrating function in tamoxifen-treated AQP2 flox mice. A : urine osmolality in tamoxifen-treated WT mice ( ) and AQP2 flox mice ( ) given free access to food and water (means ± SE, 3 mice/group). Arrows indicate tamoxifen injections. B : 24-h urine output in untreated ( left ) and tamoxifen-treated ( right ) WT and AQP2 flox mice (arrows indicate the urine level). C, top : urine osmolality before (basal) and after (water-deprived) 18-h water deprivation. Bottom : body weight loss for the same mice after water deprivation.
Table 1 summarizes urine and plasma chemistries of wild-type and tamoxifen-treated AQP2 flox mice at 6 wk after tamoxifen treatment. Plasma concentrations of electrolyte, urea, and creatinine were not significantly different in wild-type and AQP2 flox mice, nor was creatinine clearance. As expected, daily urine volume was much greater and osmolality lower in the AQP2 flox mice, and the concentrations of various urinary solutes were reduced.
Table 1. Urine and plasma chemistries in wild-type and AQP2 flox mice
Despite normal renal function at 6 wk after induction of polyuria, the kidneys of AQP2 flox mice showed evidence of structural damage from the sustained polyuria. Many kidneys showed evidence of medullary atrophy ( Fig. 5 A ). Histological examination showed dilatation of collecting ducts in the renal cortex and medulla ( Fig. 5 B ). Massively enlarged hydroureters and bladders were seen in the 4OH-TM-treated AQP2 flox mice (not shown).
Fig. 5. Dilatation of collecting ducts after AQP2 gene deletion. A : gross morphology of kidney slices from WT and AQP2 flox mice at 6 wk after AQP2 gene excision. Scale bar = 2 mm. B : kidney histology in hematoxylin- and eosin-stained sections of renal cortex and medulla. Scale bar = 100 µm.
Quantitative real-time RT-PCR analysis of kidney homogenates was carried out to examine the effect of induced AQP2 gene deletion on the expression of other renal aquaporins. Figure 6 A shows a marked increase in AQP3 transcript but no significant effect on transcripts for AQPs1, -4, -6, and -7 in the tamoxifen-treated AQP2 flox mice. Immunoblot analysis confirmed increased (6 ± 3-fold by quantitative densitometry) AQP3 protein expression in these mice ( Fig. 6 B ). The increased AQP3 protein expression was seen in collecting duct basolateral membranes throughout the cortex and medulla ( Fig. 6 C ).
Fig. 6. Increased renal AQP3 expression at 6 wk after AQP2 gene deletion. A : fluorescence-based real-time RT-PCR. Shown is aquaporin transcript expression expressed relative to that in WT kidneys. Values are means ± SE; n = 3 (see METHODS ). * P < 0.001. B : immunoblot detection of AQP1, AQP2, AQP3, and AQP4 protein. Each lane was loaded with protein (10 µg) from whole kidney homogenates. C : AQP3 immunofluorescence of kidney cortex ( top ) and medulla ( bottom ). Scale bar = 100 µm.
DISCUSSION
We report here the first inducible mouse model of NDI. The motivation for this work was the neonatal mortality seen in several noninducible models of NDI, as reviewed in the introduction. A surviving adult mouse model of NDI has applications for the study of the pathophysiology of NDI in mice and for the evaluation of the in vivo efficacy of NDI therapies such as molecular and chemical chaperones. Our strategy was the introduction of LoxP sites into the mouse AQP2 gene by targeted gene replacement in ES cells and inducible excision of LoxP-flanked gene sequences in adult mice. The LoxP insertions in introns 1 and 2 did not affect AQP2 gene expression or function, as non-tamoxifen-treated homozygous AQP2 flox mice carrying the Cre/Esr1 transgene had a normal phenotype and AQP2 protein expression. After evaluating several Cre-recombinase-expressing mice, including TgN (Mx1-Cre) (Jackson Laboratories), we found adequate collecting duct Cre expression in Cre/Esr1 mice to efficiently excise the LoxP-flanked AQP2 gene sequence and reduce renal AQP2 protein expression to near zero.
Because the Cre/Esr1 fusion protein is expressed in all cells of homozygous AQP2 flox mice, the efficiency of AQP2 gene excision depends in part on tissue/cell-specific tamoxifen delivery. Strong Cre-recombinase activity has been shown in Cre/Esr1 mice in highly vascular tissues following intraperitoneal injection of tamoxifen, including in brain, heart, kidney, lung, pancreas, and intestine ( 10 ). However, Cre-recombinase activity was lower 90% of AQP2 gene excision was found in kidneys of tamoxifen-treated homozygous AQP2 flox mice, with near complete absence of full-length AQP2 transcript by Northern blot and RT-PCR analysis, and absent AQP2 protein by immunoblot analysis. The efficient renal AQP2 gene excision may be due, in part, to the excellent tamoxifen penetration in collecting duct epithelial cells from both their blood- and urine-facing surfaces.
Tamoxifen-treated AQP2 flox adult mice developed a severe urinary concentrating defect with marked polyuria, urine hyposmolality, and unresponsiveness to water deprivation. This pattern of NDI is substantially more severe and differs from that in other aquaporin knockout mouse models. Mice lacking AQP1 manifest relatively mild polyuria with daily urine output of 5-6 ml, urine osmolality of 500-600 mosmol/kgH 2 O, and vasopressin insensitivity ( 19 ). The NDI in AQP1 null mice is due to a combination of defective near-isosmolar fluid absorption in the proximal tubule ( 31 ) and defective countercurrent multiplication produced by reduced water permeability in thin descending limb of Henle ( 3 ) and outer medullary descending vasa recta ( 27 ). Mice lacking AQP7, which is expressed only in the S3 segment of the proximal tubule, have little if any defect in urinary concentrating ability ( 32 ). AQP3 null mice, which have reduced water permeability in collecting duct basolateral membrane, also manifest NDI with low urine osmolality (260 mosmol/kgH 2 O) and moderate polyuria (8 ml/day), but with partial vasopressin sensitivity ( 17 ). Thus, as is generally believed, the luminal membrane in collecting duct epithelial cells normally constitutes the rate-limiting barrier for transcellular water transport. AQP4 null mice, which have reduced water permeability in the inner medullary collecting duct, manifest only a mild urinary concentrating defect ( 18 ), because in antidiuresis the majority of fluid leaving the ascending limb is absorbed in the cortical collecting duct.
Our previous analysis of AQP2-T126M mutant mice indicated a critical role of AQP2 in neonatal renal function ( 39 ). Mice were polyuric and unresponsive to vasopressin and manifested rapidly progressive renal insufficiency with papillary atrophy and death within 6 days after birth. As expected from cell culture experiments ( 35 ), AQP1-T126M protein was retained at the endoplasmic reticulum in collecting duct principal cells of the homozygous mutant mice. It was not possible to examine effects of "correctors" of AQP2-T126M cellular processing because of the early neonatal pathology. The inducible AQP2 null mice generated here, when appropriately mated with AQP2-T126M knock-in mice, are predicted to generate conditional knock-in mice suitable for such studies. Adult mice with AQP2-T126M expression would be produced by deletion of the wild-type AQP2 allele in hemozygous mice having one inducible wild-type AQP2 allele and one AQP2-T126M mutant allele. Recently, AQP2-F204V mutant mice were identified by forward genetic screening of ethylnitrosourea-mutagenized mice ( 14 ). AQP2-F204V is a relatively mild mutation compared with AQP2-T126M (or the null mutant), permitting these mice to survive beyond the neonatal period with a milder form of NDI. When additional mouse models of AQP2 mutation become available, it will be interesting to compare collecting duct water permeability with urinary concentrating function and vasopressin sensitivity.
Studies of renal aquaporin expression indicated significant upregulation of AQP3 transcript and protein in tamoxifen-treated homozygous AQP2 flox mice but no differences in the expression of AQP1, AQP4, AQP6, and AQP7. Prior studies have shown increased AQP3 expression by vasopressin ( 4, 37 ), although not as high as found here. Interestingly, polyuria significantly reduced AQP3 expression in some rat models, including water-loading ( 24 ), bilateral ureteral obstruction ( 13 ), hypercalcemia ( 38 ), and lithium administration ( 29 ). In the present study, tamoxifen-treated AQP2 flox mice have marked polyuria, likely associated with chronically elevated serum vasopressin concentration and hence AQP3 upregulation.
A secondary purpose of this study was to evaluate extrarenal AQP2 expression. Previous studies have reported AQP2 expression in the rat inner ear ( 21 ), colon ( 9 ), testis ( 25 ), and vas deferens ( 33 ). In the rat inner ear, AQP2 expression in the endolymphatic sac was found to be increased by vasopressin ( 30 ) and proposed to play a important role vestibular function and possibly in the pathogenesis of Meniere's disease. Immunocytochemistry in rat testis showed constitutive AQP2 expression in apical membrane of distal vas deferens principal cells ( 33 ). The significance of these observations is unclear as NDI subjects with AQP2 mutations have no impairment of vestibular or reproductive functions. AQP2 expression has also been reported in the apical membrane of rat distal colon epithelium ( 9 ). Utilizing tamoxifen-treated AQP2 flox mice as a control, AQP2 transcript expression was found in mouse kidney, inner ear, testes, and vas deferens, but not in colon.
In summary, we have generated and characterized the first inducible mouse model of severe NDI. Unlike prior models of noninducible NDI, adult mice with severe polyuria are able to thrive for more than 6 wk without plasma/urine evidence of renal insufficiency. The strategy of inducible gene deletion should thus be useful in generating mice suitable for testing of therapeutic strategies aimed at correcting defective proteins causing human NDI.
GRANTS
This work was supported by National Institutes of Health Grants DK-35124, HL-59198, EY-13574, EB-00415, DK-72517, and HL-73856 (to A. S. Verkman) and DK-66194 (to B. Yang), a Research Development Program Grant (R613) from the Cystic Fibrosis Foundation (to A. S. Verkman), and Grant 0365027Y from the American Heart Association (to B. Yang).
【参考文献】
Brown D. The ins and outs of aquaporin-2 trafficking. Am J Physiol Renal Physiol 284: F893-F901, 2003.
Buck TM, Eledge J, and Skach WR. Evidence for stabilization of aquaporin-2 folding mutants by N-linked glycosylation in endoplasmic reticulum. Am J Physiol Cell Physiol 287: C1292-C1299, 2004.
Chou CL, Knepper MA, Hoek AN, Brown D, Yang B, Ma T, and Verkman AS. Reduced water permeability and altered ultrastructure in thin descending limb of Henle in aquaporin-1 null mice. J Clin Invest 103: 491-496, 1999.
Combet S, Geffroy N, Berthonaud V, Dick B, Teillet L, Verbavatz JM, Corman B, and Trinh-Trang-Tan MM. Correction of age-related polyuria by dDAVP: molecular analysis of aquaporins and urea transporters. Am J Physiol Renal Physiol 284: F199-F208, 2003.
Deen PM, Verdijk MA, Knoers NV, Wieringa B, Monnens LA, van Os CH, and van Oost BA. Requirement of human renal water channel aquaporin-2 for vasopressin-dependent concentration of urine. Science 264: 92-95, 1994.
Flagella M, Clarke LL, Miller ML, Erway LC, Giannella RA, Andringa A, Gawenis LR, Kramer J, Duffy JJ, Doetschman T, Lorenz JN, Yamoah EN, Cardell EL, and Shull GE. Mice lacking the basolateral Na-K-2Cl cotransporter have impaired epithelial chloride secretion and are profoundly deaf. J Biol Chem 274: 26946-26955, 1999.
Fujita N, Ishikawa SE, Sasaki S, Fujisawa G, Fushimi K, Marumo F, and Saito T. Role of water channel AQP-CD in water retention in SIADH and cirrhotic rats. Am J Physiol Renal Fluid Electrolyte Physiol 269: F926-F931, 1995.
Fujiwara TM and Bichet DG. Molecular biology of hereditary diabetes insipidus. J Am Soc Nephrol 16: 2836-2846, 2005.
Gallardo P, Cid LP, Vio CP, and Sepulveda FV. Aquaporin-2, a regulated water channel, is expressed in apical membranes of rat distal colon epithelium. Am J Physiol Gastrointest Liver Physiol 281: G856-G863, 2001.
Hayashi S and McMahon AP. Efficient recombination in diverse tissues by a tamoxifen-inducible form of Cre: a tool for temporally regulated gene activation/inactivation in the mouse. Dev Biol 244: 305-318, 2002.
Klein JD, Sands JM, Qian L, Wang X, and Yang B. Upregulation of urea transporter UT-A2 and water channels AQP2 and AQP3 in mice lacking urea transporter UT-B. J Am Soc Nephrol 15: 1161-1167, 2004.
Knoers NV and Deen PM. Molecular and cellular defects in nephrogenic diabetes insipidus. Pediatr Nephrol 16: 1146-1152, 2001.
Li C, Wang W, Kwon TH, Isikay L, Wen JG, Marples D, Djurhuus JC, Stockwell A, Knepper MA, Nielsen S, and Frøkiær J. Downregulation of AQP1, -2, and -3 after ureteral obstruction is associated with a long-term urine-concentrating defect. Am J Physiol Renal Physiol 281: F163-F171, 2001.
Lloyd DJ, Hall FW, Tarantino LM, and Gekakis N. Diabetes insipidus in mice with a mutation in aquaporin-2. PLoS Genet 1: 171-178, 2005.
Lorenz JN, Baird NR, Judd LM, Noonan WT, Andringa A, Doetschman T, Manning PA, Liu LH, Miller ML, and Shull GE. Impaired renal NaCl absorption in mice lacking the ROMK potassium channel, a model for type II Bartter's syndrome. J Biol Chem 277: 37871-37880, 2002.
Lu M, Wang T, Yan Q, Yang X, Dong K, Knepper MA, Wang W, Giebisch G, Shull GE, and Hebert SC. Absence of small conductance K + channel (SK) activity in apical membranes of thick ascending limb and cortical collecting duct in ROMK (Bartter's) knockout mice. J Biol Chem 277: 37881-37887, 2002.
Ma T, Song Y, Yang B, Gillespie A, Carlson EJ, Epstein CJ, and Verkman AS. Nephrogenic diabetes insipidus in mice lacking aquaporin-3 water channels. Proc Natl Acad Sci USA 97: 4386-4391, 2000.
Ma T, Yang B, Gillespie A, Carlson EJ, Epstein CJ, and Verkman AS. Generation and phenotype of a transgenic knockout mouse lacking the mercurial-insensitive water channel aquaporin-4. J Clin Invest 100: 957-962, 1997.
Ma T, Yang B, Gillespie A, Carlson EJ, Epstein CJ, and Verkman AS. Severely impaired urinary concentrating ability in transgenic mice lacking aquaporin-1 water channels. J Biol Chem 273: 4296-4299, 1998.
Marples D, Christensen S, Christensen EI, Ottosen PD, and Nielsen S. Lithium-induced downregulation of aquaporin-2 water channel expression in rat kidney medulla. J Clin Invest 95: 1838-1845, 1995.
Mhatre AN, Jero J, Chiappini I, Bolasco G, Barbara M, and Lalwani AK. Aquaporin-2 expression in the mammalian cochlea and investigation of its role in Meniere's disease. Hear Res 170: 59-69, 2002.
Morello JP, Salahpour A, Laperriere A, Bernier V, Arthus MF, Lonergan M, Petaja-Repo U, Angers S, Morin D, Bichet DG, and Bouvier M. Pharmacological chaperones rescue cell-surface expression and function of misfolded V2 vasopressin receptor mutants. J Clin Invest 105: 887-895, 2000.
Mulders SM, Knoers NV, Van Lieburg AF, Monnens LA, Leumann E, Wuhl E, Schober E, Rijss JP, Van Os CH, and Deen PM. New mutations in the AQP2 gene in nephrogenic diabetes insipidus resulting in functional but misrouted water channels. J Am Soc Nephrol 8: 242-248, 1997.
Murillo-Carretero MI, Ilundain AA, and Echevarria M. Regulation of aquaporin mRNA expression in rat kidney by water intake. J Am Soc Nephrol 10: 696-703, 1999.
Nelson RD, Stricklett P, Gustafson C, Stevens A, Ausiello D, Brown D, and Kohan DE. Expression of an AQP2 Cre recombinase transgene in kidney and male reproductive system of transgenic mice. Am J Physiol Cell Physiol 275: C216-C226, 1998.
Noda Y and Sasaki S. Trafficking mechanism of water channel aquaporin-2. Biol Cell 97: 885-892, 2005.
Pallone TL, Edwards A, Ma T, Silldorff EP, and Verkman AS. Requirement of aquaporin-1 for NaCl-driven water transport across descending vasa recta. J Clin Invest 105: 215-222, 2000.
Pedemonte N, Lukacs GL, Du K, Caci E, Zegarra-Moran O, Galietta LJ, and Verkman AS. Small-molecule correctors of defective F508-CFTR cellular processing identified by high-throughput screening. J Clin Invest 115: 2564-2571, 2005.
Rojek A, Nielsen J, Brooks HL, Gong H, Kim YH, Kwon TH, Frøkiær J, and Nielsen S. Altered expression of selected genes in kidney of rats with lithium-induced NDI. Am J Physiol Renal Physiol 288: F1276-F1289, 2005.
Sawada S, Takeda T, Kitano H, Takeuchi S, Kakigi A, and Azuma H. Aquaporin-2 regulation by vasopressin in the rat inner ear. Neuroreport 13: 1127-1129, 2002.
Schnermann J, Chou CL, Ma T, Traynor T, Knepper MA, and Verkman AS. Defective proximal tubular fluid reabsorption in transgenic aquaporin-1 null mice. Proc Natl Acad Sci USA 95: 9660-9664, 1998.
Sohara E, Rai T, Miyazaki J, Verkman AS, Sasaki S, and Uchida S. Defective water and glycerol transport in the proximal tubules of AQP7 knockout mice. Am J Physiol Renal Physiol 289: F1195-F1200, 2005.
Stevens AL, Breton S, Gustafson CE, Bouley R, Nelson RD, Kohan DE, and Brown D. Aquaporin 2 is a vasopressin-independent, constitutive apical membrane protein in rat vas deferens. Am J Physiol Cell Physiol 278: C791-C802, 2000.
Takahashi N, Chernavvsky DR, Gomez RA, Igarashi P, Gitelman HJ, and Smithies O. Uncompensated polyuria in a mouse model of Bartter's syndrome. Proc Natl Acad Sci USA 97: 5434-5439, 2000.
Tamarappoo BK and Verkman AS. Defective aquaporin-2 trafficking in nephrogenic diabetes insipidus and correction by chemical chaperones. J Clin Invest 101: 2257-2267, 1998.
Tamarappoo BK, Yang B, and Verkman AS. Misfolding of mutant aquaporin-2 water channels in nephrogenic diabetes insipidus. J Biol Chem 274: 34825-34831, 1999.
Terris J, Ecelbarger CA, Nielsen S, and Knepper MA. Long-term regulation of four renal aquaporins in rats. Am J Physiol Renal Fluid Electrolyte Physiol 271: F414-F422, 1996.
Wang W, Li C, Kwon TH, Knepper MA, Frøkiær J, and Nielsen S. AQP3, p-AQP2, and AQP2 expression is reduced in polyuric rats with hypercalcemia: prevention by cAMP-PDE inhibitors. Am J Physiol Renal Physiol 283: F1313-F1325, 2002.
Yang B, Gillespie A, Carlson EJ, Epstein CJ, and Verkman AS. Neonatal mortality in an aquaporin-2 knock-in mouse model of recessive nephrogenic diabetes insipidus. J Biol Chem 276: 2775-2779, 2001.
Yang B, Ma T, Xu Z, and Verkman AS. cDNA and genomic cloning of mouse aquaporin-2: functional analysis of an orthologous mutant causing nephrogenic diabetes insipidus. Genomics 57: 79-83, 1999.
Yun J, Schoneberg T, Liu J, Schulz A, Ecelbarger CA, Promeneur D, Nielsen S, Sheng H, Grinberg A, Deng C, and Wess J. Generation and phenotype of mice harboring a nonsense mutation in the V2 vasopressin receptor gene. J Clin Invest 106: 1361-1371, 2000.
作者单位：Departments of Medicine and Physiology, Cardiovascular Research Institute, University of California, San Francisco, California