Background Urine-concentrating ability is regulated by vasopressin.Recently, the specific water-channel protein of the renal collectingduct, known as aquaporin-2, was cloned. However, it is not certainwhether this molecule is responsive to vasopressin.
Methods We measured the urinary excretion of aquaporin-2 andits response to vasopressin in 11 normal subjects and 9 patientswith central or nephrogenic diabetes insipidus. The urine sampleswere collected during periods of dehydration and hydration andafter the administration of vasopressin. Urine samples wereanalyzed for aquaporin-2 by the Western blot assay and immunogoldlabeling, and the amount of aquaporin-2 was determined by radioimmunoassay.
Results Aquaporin-2 was detectable in the urine in both solubleand membrane-bound forms. In the five normal subjects tested,the mean (±SE) urinary excretion of aquaporin-2 was 11.2±2.2pmol per milligram of creatinine after a period of dehydration,and it decreased to 3.9±1.9 pmol per milligram of creatinine(P = 0.03) during the second hour after a period of hydration.In the six other normal subjects, an infusion of desmopressin(1-desamino-8-d-arginine vasopressin) increased the urinaryexcretion of aquaporin-2 from 0.8±0.3 to 11.2±1.6pmol per milligram of creatinine (P<0.001). The five patientswith central diabetes insipidus also had increases in urinaryexcretion of aquaporin-2 in response to the administration ofvasopressin, but the four patients with X-linked or non–X-linkednephrogenic diabetes insipidus did not.
Conclusions Aquaporin-2 is detectable in the urine, and changesin the urinary excretion of this protein can be used as an indexof the action of vasopressin on the kidney.
The permeability of the collecting ducts of the kidney to waterincreases rapidly in response to vasopressin. In what is calledthe shuttle hypothesis, this increase is believed to be mediatedby the movement of a vasopressin-regulated water-channel proteinto the apical membranes of the cells of the collecting ducts.1,2,3,4The aquaporins are a family of membrane proteins that functionas water-selective channels in many water-transporting tissues.5The first aquaporin to be identified, aquaporin-1 (initiallytermed an aquaporin of channel-forming integral protein), ispresent in red cells, proximal and descending tubules of thekidney, and other tissues.6,7
We recently cloned a water-channel protein specific to the collectingduct of the kidney, aquaporin-2, first termed aquaporin-CD,from rats and humans. It is a 271-amino-acid protein with amolecular weight of 29,000. This aquaporin is localized to theapical region of collecting-duct cells, which suggests thatit is the vasopressin-regulated water channel.8,9 To determinethe responsiveness of this protein to vasopressin, we measuredthe urinary excretion of aquaporin-2 and its response to vasopressinin normal subjects and patients with central and nephrogenicdiabetes insipidus.
Methods
Normal Subjects and Patients
We studied four groups of subjects. The protocol was approvedby the appropriate institutional review boards, and all subjectsgave informed consent.
The first group consisted of five normal men ranging in agefrom 31 to 46 years. At 9 a.m., after an overnight period ofdehydration during which they were not allowed to drink water,the subjects were asked to urinate and then to drink 1 literof water in a 20-minute period. Starting at 10 a.m., urine sampleswere collected every 60 minutes for 3 hours. The samples obtainedin this and the three subsequent studies were analyzed immediatelyor stored at -80°C until their use.
The second group consisted of five patients (three women andtwo men, ranging in age from 40 to 72 years) who had idiopathiccentral diabetes insipidus and regularly took desmopressin (1-desamino-8-d-argininevasopressin, or DDAVP) intranasally twice daily. The patientsdiscontinued desmopressin therapy 24 hours before the study.After an overnight fast (during which free intake of water wasallowed), the patients were asked to urinate, after which theywere given 10 U of arginine vasopressin (Sankyo Pharmaceutical,Tokyo, Japan) subcutaneously. Urine samples were collected atone-hour intervals for three hours after the injection.
The third study group consisted of four male patients with nephrogenicdiabetes insipidus — three with the X-linked form andone with the non–X-linked form. All three patients withX-linked nephrogenic diabetes insipidus (who ranged in age from37 to 50 years) had mutations in the gene for the vasopressinV2 receptor.10 The patient with non–X-linked nephrogenicdiabetes insipidus was hospitalized at the age of five monthswith fever and dehydration, at which time his plasma sodiumconcentration was 161 mmol per liter. He had normal fibrinolyticand vasodilatory responses to desmopressin, but his urinaryosmolality did not increase, indicating a functional abnormalitydistal to the vasopressin V2 receptor.11 We have sequenced thecoding regions of both the aquaporin-2 gene and the vasopressinV2 receptor gene in this patient and have found no mutations(unpublished data). The three patients with X-linked nephrogenicdiabetes insipidus were not taking any medications. The patientwith non–X-linked nephrogenic diabetes insipidus discontinuedhis regular medication (hydrochlorothiazide) 36 hours beforethe desmopressin-infusion test.
The fourth group consisted of six normal subjects (two womenand four men, ranging in age from 23 to 35 years). All the patientsin the third and fourth groups received intravenous infusionsof desmopressin (0.3 µg per kilogram of body weight, upto a maximal dose of 24 µg; Ferring Pharmaceuticals, Malmö,Sweden) in 100 ml of saline over a 20-minute period.12 Urinesamples were collected at 30-minute intervals before the startof infusion and for 3 hours afterward.
Western Blot Analysis
A polyclonal antibody against a synthetic portion of the C-terminalof human aquaporin-2 raised in rabbits9 was used in the Westernblot analysis. This portion of the aquaporin-2 molecule hasno sequence homology with other aquaporins. Samples of urineor suspensions of normal human renal medullary membranes9 werediluted in a half volume of a loading buffer containing 9 percentsodium dodecyl sulfate (SDS), 195 mM Tris–hydrochloricacid, 30 percent glycerol, and 15 percent 2-mercaptoethanoland heated at 70°C for 10 minutes. The samples were subjectedto SDS–polyacrylamide-gel electrophoresis (SDS-PAGE) with10 to 20 percent gradient gels and were then transferred topolyvinyl membranes (Immobilon, Millipore, Bedford, Mass.).The membranes were incubated with antibody (dilution, 1:500),and the reaction products were visualized with protein A labeledwith iodine-125 (ICN Biochemicals, Irvine, Calif.). Some urinesamples were concentrated by ultrafiltration (cutoff, a molecularweight of 3000; Microcon-3, Amicon, Beverly, Mass.) before SDS-PAGE.The renal medullary tissue used was tumor-free tissue from kidneysremoved during surgery to treat renal-cell carcinoma.
Radioimmunoassay
Urinary aquaporin-2–like immunoreactivity was measuredby a specific radioimmunoassay that used the same antibody thatwas employed in the Western blot analysis. A synthetic peptide(tyr0.aquaporin-2[V257-A271]) corresponding to the 15-amino-acidsequence of the C-terminal of aquaporin-2 was radioiodinatedwith iodine-125 (New England Nuclear, Boston) by the lactoperoxidasemethod, as described elsewhere.13 For the assay, 0.1 ml of theurine sample or of a standard, 0.1 ml of assay buffer (0.05M sodium phosphate [pH 7.4], 0.08 M sodium chloride, 0.01 MEDTA, 0.1 percent bovine serum albumin, 0.1 percent Triton X-100,and 0.01 percent sodium azide), and 0.1 ml of the antibody (finaldilution, 1:12,000) were incubated at 4°C for 24 hours,followed by the addition of 0.1 ml of the radiolabeled syntheticpeptide (approximately 10,000 cpm) and further incubation at4°C for 48 hours. Bound and free quantities of radiolabeledligand were separated by the double-antibody method. The serial-dilutioncurve of the urine samples was parallel to that of the standard(data not shown). Each sample was analyzed in duplicate, andthe intra- and interassay coefficients of variation were lessthan 10 percent. The minimal detectable quantity of aquaporin-2was 25 pg per tube, and an amount equivalent to 600 pg per tubecaused 50 percent inhibition of binding of the radiolabeledligand. Urinary creatinine concentrations were measured withan AutoAnalyzer.
Immunolabeling of Urinary Aquaporin-2
Fresh samples of urine were first centrifuged at low speed (2000rpm) for 15 minutes to remove cellular elements, then the supernatantswere centrifuged at high speed (120,000 x g) for 30 minutes.The resulting sediments were suspended in 7.5 mM phosphate buffer(pH 7.4) containing 20 µg of phenylmethylsulfonyl fluorideper milliliter of solution, 1 µg of pepstatin A per milliliter,and 1 µg of leupeptin per milliliter. An equal volumeof 0.5 M sucrose was added to each suspension, and the resultingmixture was then mixed with an equal volume of a twofold concentrationof periodate–lysine–paraformaldehyde fixative at4°C for 40 minutes. After three washings, the sediment wassuspended in an equal volume of a solution containing 20 percentpolyvinylpyrrolidone and 1.84 M sucrose in phosphate-bufferedsaline. The sediment was frozen in an immersion cryofixationsystem (KF-80, Reichert–Jung, Vienna, Austria), and ultrathincryosections were cut at -100°C and collected on coppergrids. The grids were placed on droplets of the antibody solution(dilution, 1:200) at room temperature for one hour. After beingrinsed, the grids were incubated for 30 minutes with a 1:100dilution of a F(ab')2 fragment of a goat antirabbit IgG antiserumto which 10-nm particles of gold had been conjugated. The gridswere rinsed with phosphate-buffered saline, fixed with 2 percentglutaraldehyde, and stained with 2 percent uranyl acetate afteradsorption staining with a mixture of polyvinyl alcohol anduranyl acetate. The sections were examined at 100 kV in an electronmicroscope (H 7100, Hitachi, Ibaraki, Japan).
Statistical Analysis
The results are expressed as means ±SE. Statistical datawere calculated by analysis of variance. All P values are two-sided.
Results
Western Blot Analysis of Urine
Immunoblots of urine samples obtained after a 12-hour overnightdehydration showed aquaporin-2 with molecular sizes of 29 kdand 40 to 50 kd (Figure 1, lane 2), as did the samples of membranesfrom renal medullary tissue (Figure 1, lane 1). The broad bandat 40 to 50 kd represents a glycosylated form of the 29-kd protein.9The amount of aquaporin-2 detected increased when the urinewas concentrated threefold by ultrafiltration (Figure 1, lane3). These results suggest that intact aquaporin-2 is excretedin the urine. Aquaporin-2 was not detected in the cellular urinarysediment obtained by low-speed centrifugation (Figure 1, lane4), ruling out the possibility that collecting-duct cells inthe urine are the source of urinary aquaporin-2. The sedimentsobtained by high-speed ultracentrifugation of urine (at 120,000x g) contained abundant amounts of aquaporin-2 protein (Figure 1,lanes 5 and 6), indicating that some of the aquaporin-2 inurine is bound to membrane structures. Quantification by radioimmunoassayindicated that 42.3±3.8 percent of the aquaporin-2 inthe urine of the nine normal subjects studied was depositedas sediment by this ultracentrifugation procedure. Recently,Preston et al. reported finding aquaporin-1 in cellular urinarysediment obtained by low-speed centrifugation.14 The lack ofstaining in the sediment obtained at low speed in our Westernblot analysis (Figure 1, lane 4) indicated that our antibodydid not cross-react with aquaporin-1.
Figure 1. Western Blot Analysis of Urine Samples from Normal Subjects and Normal Renal Medullary Tissue.
Lane 1 shows a suspension of renal medullary tissue (10 µg); lane 2, a urine sample obtained after the subject underwent a 12-hour period of dehydration (10 µl); lane 3, a urine sample concentrated threefold (10 µl); lane 4, cellular sediment obtained after low-speed centrifugation of a urine sample (10 µg); and lanes 5 and 6, membrane fractions of urine after ultracentrifugation (lane 5, 0.2 µg; lane 6, 0.5 µg). The membranes were incubated with anti–aquaporin-2 antibody and visualized with 125I-labeled protein A. Markers of molecular weight were subjected to electrophoresis simultaneously and detected by staining with Coomassie blue (not shown).
Immunoelectron-Microscopical Analysis of the Sediment of Urine Centrifuged at High Speed
Immunoelectron-microscopical analysis of the urinary sedimentobtained by ultracentrifugation revealed membrane structuresforming vesicle-like shapes. Immunogold labeling with specificantibody against aquaporin-2 indicated the localization of aquaporin-2along these membranes (Figure 2A); staining with nonimmune serumwas negative (Figure 2B).
Figure 2. Immunogold Labeling of Urinary Sediment Obtained by Ultracentrifugation.
The urine sample was obtained from a normal subject after a 12-hour period of dehydration. The sediment was immunostained with antibody against aquaporin-2 (Panel A) and nonimmune serum (Panel B). After being rinsed, the sediment samples were incubated with a 1:100 dilution of a F(ab')2 fragment of goat antirabbit IgG antiserum to which 10-nm particles of gold had been conjugated. After staining with 2 percent uranyl acetate, the sections were examined at 100 kV in an electron microscope. The particles of gold appeared as black dots. The bars represent 200 nm.
Urinary Excretion of Aquaporin-2 in Normal Subjects and Patients with Diabetes Insipidus
Effect of Dehydration and Water Loading in Normal Subjects
After overnight dehydration, the mean (±SE) urinary excretionof aquaporin-2 by five normal subjects was 11.2±2.2 pmolper milligram of creatinine. The mean values decreased to 3.9±1.9and 3.5±1.5 pmol per milligram of creatinine two andthree hours, respectively, after water loading (P = 0.03 andP = 0.02) (Figure 3). The mean urinary osmolality decreasedfrom 933±55 mOsm per kilogram of water to 436±272and 627±246 mOsm per kilogram of water after two andthree hours, respectively.
Figure 3. Effect of Dehydration and Water Loading on Urinary Excretion of Aquaporin-2 in Normal Subjects.
Urine samples were collected after the subjects had undergone dehydration overnight (base line) and were then collected hourly for 3 hours after the subjects had drunk 1 liter of water in a 20-minute period. The open circles represent the values for each subject, and the solid squares and bars the means (±SE). P = 0.03 and P = 0.02 for the comparisons of the mean base-line value with the mean values after two and three hours, respectively.
Effect of Arginine Vasopressin in Normal Subjects and Patients with Diabetes Insipidus
The mean basal excretion of aquaporin-2 by the five patientswith central diabetes insipidus was 0.4±0.05 pmol permilligram of creatinine (Figure 4). A subcutaneous injectionof arginine vasopressin increased urinary excretion of aquaporin-2by four to six times (P<0.001) during the first hour afterthe injection. The values decreased thereafter in most patients.The mean urinary osmolality increased from 142±16 to334±65 mOsm per kilogram of water after one hour, to520±83 mOsm after two hours, and to 548±57 mOsmafter three hours.
Figure 4. Effect of an Injection of Arginine Vasopressin on Urinary Excretion of Aquaporin-2 in Patients with Central Diabetes Insipidus.
Urine samples were collected after the patients had undergone fasting overnight (base line) and were then collected hourly for three hours after a subcutaneous injection of 10 U of arginine vasopressin. The open circles represent the values for each patient, and the solid squares and bars the means (±SE). P<0.001, P = 0.002, and P = 0.003 for the comparisons of the mean base-line value with the mean values after one, two, and three hours, respectively.
The six normal subjects who received 20-minute infusions ofdesmopressin had a rapid 14-fold increase in mean urinary excretionof aquaporin-2, from 0.8±0.3 to 11.2±1.6 pmolper milligram of creatinine (P<0.001). Although the valuesdecreased subsequently, they remained elevated for three hoursafter the start of the infusion (Figure 5A). The mean urinaryosmolality increased from 592±113 mOsm per kilogram ofwater to 762±87, 803±94, and 875±121 mOsmper kilogram of water in these subjects after one, two, andthree hours, respectively. In contrast, neither urinary excretionof aquaporin-2 nor osmolality (approximately 130 mOsm per kilogramof water) increased during or after the infusion of desmopressinin any of the four patients with nephrogenic diabetes insipidus(Figure 5B).
Figure 5. Effect of an Infusion of Desmopressin on Urinary Excretion of Aquaporin-2 in Normal Subjects (Panel A) and Patients with Nephrogenic Diabetes Insipidus (Panel B).
All the subjects received intravenous infusions of desmopressin (0.3 µg per kilogram) for 20 minutes. The urine samples were collected at the start of the infusion (base line) and at 30-minute intervals for 3 hours. In Panel A, the open circles represent the values for each subject, and the solid squares and bars the means (±SE). (P<0.001, P = 0.003, P = 0.03, and P = 0.01 for the comparisons of the mean base-line value with the mean values after 1/2 hour, 1 hour, 21/2 hours, and 3 hours, respectively.) In Panel B, the open circles represent the values in the three patients with X-linked nephrogenic diabetes insipidus, and the solid circles represent the values in a patient with non–X-linked nephrogenic diabetes insipidus.
Discussion
According to the shuttle hypothesis,1,2,3,4 the vasopressin-regulatedwater channel is localized in the apical plasma membrane andthe membranes of the cytoplasmic vesicles of collecting-ductcells in the kidney. Cytoplasmic vesicles carrying the water-channelproteins fuse to the apical membrane in response to vasopressin,thereby increasing the permeability of the membrane to water.Immunohistochemical studies using specific antibodies againstaquaporin-2 showed that the localization of aquaporin-2 in tissueis consistent with that predicted by the shuttle hypothesis.5,6,15,16Endocytosis of apical membranes containing water-channel proteinsis known to be stimulated by vasopressin in collecting-ducttissue and toad bladders. This retrieval of membranes is acceleratedby the influx of water into the cell, and it may be involvedin readjustment of the permeability of vasopressin-responsivetissues to water.17,18,19 Thus, vasopressin appears to stimulatethe turnover of membranes in two directions; one is the fusionof vesicles carrying water-channel proteins to the apical membrane,and the other is the endocytotic retrieval of apical membranecontaining the water-channel proteins. In this study we demonstratedthat aquaporin-2 is present in the urine of humans and thatit is present on the membranes of vesicle-like structures inurinary sediments obtained by high-speed centrifugation. Thelatter findings indicate that some particulate aquaporin-2 isshed into the luminal fluid. The possibility of the disposalof water-channel proteins has not been considered in the frameworkof the shuttle hypothesis.
The urinary excretion of aquaporin-2 increased during antidiuresisin normal subjects, whether it was induced by a period of dehydrationor by the infusion of vasopressin. The best explanations forthis increase are that vasopressin increases the movement ofaquaporin-2–carrying vesicles to the apical membrane andthat a fraction of the apical membrane reaches the urine byexocytosis. Alternatively, the endocytotic vesicles that carrythe water-channel proteins may be released directly into theurine. Whatever the mechanism or mechanisms, the increased urinaryexcretion of aquaporin-2 induced by vasopressin probably resultsfrom the increased movement of aquaporin-2 to the apical membrane.
The patients with central diabetes insipidus, like the normalsubjects, had increased urinary excretion of aquaporin-2 inresponse to vasopressin, suggesting that the urine-concentratingmechanisms in collecting-duct cells in the kidney were intact,but this increase was less than that in normal subjects. Thisfinding may have occurred because the patients lacked endogenousvasopressin, which is important for the expression of aquaporin-2messenger RNA and protein. The 5' flanking region of the aquaporin-2gene contains an element that is responsive to cyclic AMP20(vasopressin increases the intracellular concentration of cyclicAMP in collecting-duct cells), and long-term administrationof vasopressin increases the content of aquaporin-2 proteinin the kidney medulla.21,22
Congenital nephrogenic diabetes insipidus is a rare, inheriteddisorder characterized by renal unresponsiveness to vasopressin.In most families it is transmitted as an X-linked recessivedisorder caused by mutations in the vasopressin V2–receptorgene, which is located on the X chromosome.10,23,24,25 In patientswith non–X-linked nephrogenic diabetes insipidus, whohave normal V2 receptors,11,24,26 the disorder may be causedby abnormalities in the aquaporin-2 protein.27,28 The patientswe studied with either form of the disorder had no increasesin urinary excretion of aquaporin-2 in response to vasopressin.The absence of such an increase in patients with nephrogenicdiabetes insipidus may help differentiate this disorder fromcentral diabetes insipidus.
Supported by research grants from the Japanese Ministry of Education,Science, and Culture and by the Uehara Memorial Foundation.Dr. Bichet is a Career Investigator of the Fonds de la Rechercheen Santé du Québec.
We are indebted to Dr. A. Fujita, Mr. H. Kishida, and Mrs. K.Sato for technical assistance.
Source Information
From the Second Department of Internal Medicine, School of Medicine, Tokyo Medical and Dental University, Tokyo (K.K., S.S., Y.H., K.F., F.M.); the Department of Endocrinology and Metabolism, Jichi Medical School, Tochigi (S.I.); and Pharmaceutical Basic Research Laboratories, Central Pharmaceutical Research Institute, JT Inc., Kanagawa (S.N.) — all in Japan; and the Centre de Recherche, Hôpital du Sacré-Coeur, University of Montreal, Montreal (D.G.B).
Address reprint requests to Dr. Sasaki at the Second Department of Internal Medicine, School of Medicine, Tokyo Medical and Dental University, 1-5-45, Yushima, Bunkyo-Ku, Tokyo 113, Japan.
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