1 Service de Biologie Cellulaire, Commissariat à l'Énergie Atomique/Saclay, Gif-sur-Yvette; 2 Assistance-Publique-Hôpital de Paris, Hôpital Ste. Périne, Paris 16; and 3 Institut National de la Santé et de la Recherche Médicale U-430, Hôpital Broussais, Paris 14, France
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ABSTRACT |
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The mechanisms underlying age-related polyuria were investigated in 10- and 30-mo-old female WAG/Rij rats. Urinary volume and osmolality were 3.9 ± 0.3 ml/24 h and 2,511 ± 54 mosmol/kgH2O in adult rats and 12.8 ± 0.8 ml/24 h and 1,042 ± 44 mosmol/kgH2O in senescent animals. Vasopressin V2 receptor mRNA did not significantly differ between 10 and 30 mo, and [3H]vasopressin binding sites in membrane papilla were reduced by 30%. The cAMP content of the papilla was unchanged with age, whereas papillary osmolality was significantly lowered in senescent animals. The expression of aquaporin-1 (AQP1) and -4 was mostly unaltered from 10 to 30 mo. In contrast, aquaporin-2 (AQP2) and -3 (AQP3) expression was downregulated by 80 and 50%, respectively, and AQP2 was markedly redistributed into the intracellular compartment, in inner medulla of senescent animals, but not in renal cortex. These results indicate that age-related polyuria is associated with a downregulation of AQP2 and AQP3 expression in the medullary collecting duct, which is independent of vasopressin-mediated cAMP accumulation.
aquaporins
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INTRODUCTION |
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IMPAIRED ABILITY OF THE AGING kidney to retain water has been documented in humans as well as in experimental models. It is characterized by a reduced capacity to concentrate urine during dehydration or after vasopressin administration. In rodents given free access to water, this is also accompanied by mild polyuria and a corresponding decrease in urine osmolality (3, 8, 25, 28, 31, 34). Such age-related changes in water homeostasis may be due to inappropriate secretion of vasopressin along the hypothalamo-hypophyseal axis or to kidney vasopressin escape, or both.
An age-associated defect in vasopressin release has been documented in some strains of rats (32, 38). In other strains, however, polyuria is independent of variation in plasma vasopressin concentration, suggesting that age-related diuresis is mainly caused by changes in kidney function or structure (16, 34). Chronic progressive nephrosis, loss of nephrons, and hyperfiltration of the remaining glomeruli have frequently been invoked to explain this altered water homeostasis. Although this hypothesis would apply to animals with severe renal diseases, it has been challenged by several investigations, which evidenced water diuresis in aging rats free of glomerulosclerosis, indicating resistance to vasopressin of the kidney (2).
Renal concentrating ability is mainly determined by proximal water reabsorption, corticopapillary osmotic gradient, and collecting duct water permeability, all of which involve molecular water channels of the aquaporin family. Aquaporin-1 (AQP1) is primarily responsible for the high constitutive water permeability of proximal tubules and thin descending limbs of Henle (29). Aquaporin-2 (AQP2) is implicated in the water permeability of the collecting duct through vasopressin V2 receptor activation, intracellular cAMP accumulation, and insertion of AQP2-carrying vesicles into apical plasma membrane of principal cells (17, 21, 30). In addition, the constitutive localization of the aquaporin-3 (AQP3) and aquaporin-4 (AQP4) water channels in basolateral plasma membranes of principal cells confers the epithelium a high water permeability, in concert with AQP2 insertion in the luminal membrane. Changes in expression of aquaporins, in constitution of the corticopapillary gradient or in vasopressin-dependent AQP2 trafficking may all contribute to the altered water homeostasis reported with age.
The possible involvement of these parameters in age-related polyuria was presently investigated in WAG/Rij rats in which vasopressin secretion, number of nephrons, and single filtration rates are maintained in the course of aging. Vasopressin V2 receptor mRNA content was measured in dissected collecting tubules and was compared with vasopressin binding sites, cAMP content, and osmolality of the papilla. AQP1, AQP2, AQP3, and AQP4 were quantified by Western blot analysis and their distributions within the kidney were examined by immunofluorescence staining. Although V2 receptor expression and cAMP content of the medulla were barely modified in aging rats, papillary osmolality was significantly reduced and AQP2 and AQP3 were markedly downregulated in the inner medullary collecting duct. This may account for a vasopressin-independent age-related acquired nephrogenic diabetes insipidus.
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MATERIALS AND METHODS |
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Animals. Female WAG/Rij rats were born and maintained in the animal care facility of Commissariat à l'Énergie Atomique/Saclay on a 12:12 light-dark cycle, 50% humidity, and a temperature of 20°C, as previously described (19). The animals were housed four per cage and had free access to water. From weaning to 3 mo, they were fed ad libitum a commercial growth diet (DO3, UAR, Villemoisson, France) composed of 9% fish protein, 16% vegetable protein, and a total of 3,500 kcal/kg. At the age of 3 mo, animals were switched for life to a diet containing 2% fish and 15% vegetable protein and a total of 2,900 kcal/kg (DO4, UAR).
Survival, growth rate, and tumor incidence of the inbred WAG/Rij strain have previously been published by Bureck (4). Mean survival of female rats fed ad libitum is close to 30 mo. In this strain of rat, body weight of females is ~200 g during most of their life, and the incidence of pituitary tumors or renal disease is very low (4). The present experiments were performed in virgin adult (10-mo-old) and senescent (30-mo-old) female rats.Food and water intake. Daily food and water intakes as urinary volume were measured in individual metabolic cages, where the animals were acclimated for 2 days before the 24-h period collection. Urine was collected under oil to avoid evaporation, and its osmolality was determined with a Roebling Automatik Osmometer.
Papillary cAMP content. Animals were killed by decapitation, and the kidneys were rapidly excised and chilled in an ice-cold saline solution. The papilla of each kidney was dissected, weighed, and homogenized with a manual potter in 500 µl of a 5% formic acid-ethanol solution. The samples were evaporated overnight and resuspended in an appropriate volume of enzyme immunoassay buffer. The cAMP content of these samples was determined by enzyme immunoassay according to Pradelles et al. (26). The results were expressed as picomoles per milligram of wet papilla.
Papillary osmolality. Renal papilla of 10- and 30-mo-old animals were isolated as described above, weighed, and homogenized in 200 µl of distilled water, and the osmolality of the solution was measured with a Roebling Automatik Osmometer. The osmolality of papilla was calculated from their initial weight. As suggested by Apostol et al. (1), the final values were corrected assuming that 80% of the wet weight is water.
Vasopressin V2 receptor mRNA quantification. V2 vasopressin receptor mRNA was quantified in microdissected outer medullary collecting tubules. Rats were anesthetized by 10 mg/100 g body wt intraperitoneal injection of Inactin (Byk-Gulden, Constance, Germany), the left kidney was perfused in situ, and pieces of medulla were incubated with a dissection solution containing 0.32 unit/mg collagenase A (Boehringer). Outer medullary collecting tubules were dissected under a binocular magnifier, and their lengths were measured under an inverted microscope before addition to the RNA extraction buffer. In each animal, tubular segments corresponding to 20-40 mm were pooled. The RNA was subsequently prepared by the method of Chomczynski and Sacchi (5). V2 vasopressin receptor mRNA content was quantified in these samples by RT-PCR as previously described, with the difference that 32P radioactivity was determined with a phosphorimager (Storm 840, Molecular Dynamics) on an electrophoresis gel (14, 20, 23). RT-PCR quantifications of the vasopressin V2 receptor mRNA were performed in triplicate in all adult and senescent animals, and the data were expressed as molecules mRNA per millimeter of tubule.
Papillary membrane isolation. Adult and senescent rats were killed by decapitation. Their kidneys were rapidly excised and placed in ice-cold buffer containing 10 mM Tris, 10 mM MgCl2, 1 mM EGTA, 250 mM sucrose, and 0.01% bacitracine (pH 7.4). Papilla from kidneys of three adult or three senescent animals were dissected, weighed, and pooled in 2 ml of 10 mM Tris, 10 mM MgCl2, 1 mM EGTA, and 0.01% bacitracine (pH 7.4). They were subsequently homogenized with 15 strokes of a motor-driven Teflon pestle. The homogenate was centrifuged at 500 g for 5 min at 4°C, and the pellet was discarded. The supernatant was centrifuged at 20,000 g for 20 min at 4°C. The resulting pellet was resuspended in 500 µl of the same solution and once again centrifuged at 20,000 g for 20 min at 4°C. The final pellet was suspended in 1 ml of binding buffer: 50 mM Tris, 3 mM MgSO4, 120 mM NaCl, 5 mM KCl, and 1 mM EGTA, 0.01% bacitracine, pH 7.4, and the protein content was determined according to Bradford with BSA (fraction V, Sigma Chemical) as a standard. Thereafter, 0.1% BSA was added to the membrane suspension, which was used for binding experiments on the day of preparation.
[3H]vasopressin binding experiments.
The [3H]vasopressin binding sites of the papillary
membrane suspensions were determined by filtration. Whatman GF/C glass
microfiber filters (ref. 1822 025) were previously equilibrated at room
temperature for 1 h in binding buffer containing 1% BSA. Aliquots
of membrane suspensions corresponding to 100 µg of protein were
incubated for 1 h at room temperature with gentle rocking in 600 µl of binding buffer containing 108 M
[3H]vasopressin (59 Ci/mM, NEN, Boston, MA). In
preliminary experiments, this concentration of tritiated vasopressin
was found saturating for vasopressin receptor binding sites.
Nonspecific binding was determined by addition of 4 × 10
6 M unlabeled vasopressin to the radioactive solution.
At the end of the incubation period, 600 µl of membrane suspension
were mixed with 4 ml of ice-cold binding buffer without tritiated
vasopressin and spotted onto GF/C filters on a Millipore vacuum
manifold. The filters were washed three times with 3 ml of binding
buffer free of [3H]vasopressin and dried, and
radioactivity was measured over 60 min in a Packard scintillation
counter. The results, calculated from the difference between total and
nonspecific binding, were expressed in ficomoles of vasopressin per
milligram membrane protein.
Western blotting. Animals were anesthetized with Inactin, and the right kidney was ligatured for isolation. The kidney was dissected on ice into cortex, outer medulla, and inner medulla, which were homogenized in PBS. The protein concentration of each sample was determined by the method of Bradford. Samples were diluted to a final concentration of 1 mg/ml and solubilized in Laemmli buffer. SDS-PAGE (12%) was performed with equal amounts (10 µg/lane) of kidney samples from 10- and 30-mo-old animals, and proteins were transferred to polyvinylidene fluoride membranes. Western blots were preincubated in PBS containing 5% nonfat dry milk (PBS/milk), incubated for 1 h in primary polyclonal antibodies against AQP1, AQP2, AQP3, or AQP4 and washed 3 × 15 min in PBS/milk. Membranes were then incubated in a 1:5,000 dilution of peroxidase-conjugated anti-rabbit polyclonal antibodies in PBS/milk, washed 2 × 15-min and 1 × 1 h in PBS/milk, followed by 2 × 5-min washes in PBS containing 0.02% Tween-20. Antibody staining was revealed on Hyperfilm by enhanced chemiluminesence. For semiquantitative analysis of antibody staining, films were scanned and band densities were integrated. Densities obtained with samples from 30-mo-old rats were expressed as a percentage ± SE of the average density obtained from 10-mo-old animals.
Immunofluorescence. For indirect immunofluorescence, the left kidney was fixed by perfusion at a pressure of 120 mmHg through the abdominal artery with a PBS solution containing 4% paraformaldehyde warmed to 37°C. The kidney was excised, sliced, and postfixed overnight in PBS-4% paraformaldehyde and washed extensively. Fixed kidney slices were infiltrated overnight in 30% sucrose, frozen in liquid nitrogen, and 4-µm cryostat sections were collected on SuperFrost+ glass slides. Sections were preincubated in PBS containing 1% BSA (PBS/BSA) for 5 min, incubated in PBS/BSA for 1 h with rabbit polyclonal antibodies raised against AQP1, AQP2, AQP3, or AQP4, and then washed 3 × 10 min in PBS. Slides were then incubated for 45 min in FITC-conjugated goat anti-rabbit antibodies (Promega) 10 µg/ml, washed 3 × 10 min and mounted in 50% glycerol solution containing 2% n-propyl-gallate before observation under fluorescence microscope (Olympus Van OX).
Electron microscopy. Animals were anesthetized as above, kidneys were fixed by perfusion of PBS containing 4% paraformaldehyde and 0.1% glutaraldehyde, sliced, postfixed overnight, and washed several times. Small tissue pieces from either the outer medulla or the inner medulla were dehydrated in ethanol, embedded in unicryl, and 90-nm sections were cut at the ultramicrotome and collected on electron microscopy grids. Sections were preincubated for 30 min in 20 mM Tris buffer (pH 7.5) containing 0.1% BSA, 0.1% fish gelatin, 0.05% Tween-20, incubated in Tris buffer for 2 h in primary, affinity-purified, polyclonal antibodies against a C-ter peptide of AQP2, and washed 6 × 5 min. Grids were then incubated in 10-nm colloidal gold-conjugated goat-anti-rabbit antibodies in Tris buffer for 1 h and washed 6 × 5 min. Sections were stained and observed under an electron microscope (Philips EM 400).
Statistics. The results were analyzed by ANOVA, and the significance was set at P < 0.05.
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RESULTS |
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Data from metabolic cage experiments are reported in Table
1. Body weight and daily food intake were
slightly greater in 30- than in 10-mo-old animals. Urinary volume was
much larger in senescent than in adult animals, and the age-related
difference in water loss was compensated for by a proportional increase
in water intake. Urine osmolality was inversely correlated to urinary volume: the larger the volume, the lower the osmolality.
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Vasopressin V2 receptor expression and cAMP accumulation. The V2 receptor mRNA contents in outer medullary collecting tubules were comparable in adult and senescent animals: 21,602 ± 1,491 molecules mRNA/mm tubule (n = 6) in 10-mo-old rats and 22,606 ± 1,377 mRNA/mm tubule (n = 5) in 30-mo-old rats, with n corresponding to the number of animals.
Tritiated vasopressin binding experiments were performed on papillary membranes prepared from the kidneys of three rats in each assay. The total amount of membranes obtained from papilla of adult and senescent rats was comparable as determined from protein concentration (931 ± 52 µg, n = 6, in 10-mo-old and 827 ± 50 µg, n = 6, in 30-mo-old animals). The individual results of binding experiments are shown in Fig. 1. The mean value for [3H]vasopressin binding was significantly (P = 0.017) greater in the 10-mo-old (81.9 ± 9.4 fmol/mg protein, n = 6) than in the 30-mo-old animals (51.7 ± 4.9 fmol/mg protein, n = 6).
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AQP1, AQP2, AQP3, and AQP4 expression.
In renal cortex, where AQP1 is expressed in proximal tubules and where
AQP2 and AQP3 are expressed in connecting segments and collecting
ducts, no significant difference in staining between adult and
senescent rats was observed for any of the three aquaporins (AQP2 in
Fig. 2A). In the outer
medulla, although staining for AQP1, AQP3, and AQP4 was similar in 10- and 30-mo-old animals (Fig. 2C for AQP3), staining for AQP2
was clearly weaker in the 30-mo-old rats (Fig. 2B).
Semiquantification of this staining by densitometry did not reveal any
significant reduction in the expression of AQP1, AQP3 ,and AQP4 in the
outer medulla between adult and senescent rats but demonstrated a 70%
decrease in the signal for AQP2 in senescent rats (Fig. 4).
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Cellular localization of AQP1, AQP2, AQP3, and AQP4.
AQP1 was abundant and localized to plasma membranes of the proximal
tubules and thin descending limbs of Henle's loop in both groups (Fig.
5, a and b). AQP4
was localized in principal cell basolateral membranes of the medullary
collecting ducts (Fig. 5, c and d). As expected
from Western blotting studies, there was no difference in staining for
AQP1 and AQP4 between 10- and 30-mo-old rats. AQP3 was localized in
principal cell basolateral membranes of the collecting duct. The
staining intensity, distribution, or localization of AQP3 were
comparable between adult (Fig. 6, a and c) and senescent (Fig. 6, b and
d) rat in the outer and inner medulla despite a lower level
of expression found with Western blotting in the inner medulla.
Immunostaining for AQP2 (Fig. 7) was
abundant in the apical region of all collecting duct principal cells in
the outer medulla from both 10- and 30-mo-old rat kidneys (Fig. 7,
a and b). In contrast, staining for AQP2 in inner
medullary collecting duct was much weaker in senescent than in younger
animals (Fig. 7, c and d). A similar decrease was
observed in all cells of every collecting duct and was not restricted
to a tubular or cellular subpopulation.
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DISCUSSION |
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Aging rats with free access to water exhibited a larger urinary volume and a lower urine osmolality than adults, in the present study as in previous ones. Different mechanisms have been proposed to explain theses changes in renal function. One emphasizes a possible loss of nephrons with age and hyperfiltration of the remaining glomeruli, which would decrease kidney concentrating ability. Although this would apply to rats that develop severe chronic progressive nephrosis, it does not explain the diuresis of WAG/Rij rats with a constant number of nephrons and single nephron filtration rates (8, 9). A decrease in vasopressin secretion, resulting in reduced collecting duct osmotic permeability and increased water excretion, was also proposed as a mechanism. Such reduced pituitary and plasma concentration of vasopressin have been reported in some, but not all, aging rats (25, 28, 38). However, the unchanged plasma vasopressin concentration measured in 30-mo-old WAG/Rij rats suggests that age-related polyuria and decreased urine osmolality are mostly linked to an intrarenal defect in urinary concentration (16).
A possible age-linked renal resistance to vasopressin has previously been investigated in different in vivo and in vitro experiments. Micropunctures performed in superficial nephrons of adult and old rats have shown that the diluting capacity of Henle's loop and the tubular flow rate in distal tubules did not change from 10 to 30 mo (10). In mice, in vitro microperfusion experiments indicated that baseline transport capacity of medullary thick ascending limb is unaltered in the course of aging (12). However, the vasopressin-induced increase in salt transport was reduced by one-half in the tubules isolated from senescent mice compared with those from adults. This is consistent with the impaired vasopressin-mediated cAMP accumulation reported in the medullary thick ascending limb of aging rats and could induce an age-related decrease in interstitial osmolality along the corticopapillary axis (20). Such a hypothesis was tested in the present study by measuring the average osmolality of the inner medulla dissected from adult and senescent animals. A significantly lower papillary osmolality was found in aging rats, indeed suggesting impaired medullary solute reabsorption and recycling. This could play a role in the polyuria reported in senescent animals by decreasing the driving force for osmotic water reabsorption along the collecting tubule.
A reduced vasopressin-induced water permeability of the collecting duct may also contribute to the age-related polyuria and decreased urine osmolality, in addition to a lower interstitial osmolality. In WAG/Rij rats, we presently found that V2 receptor mRNA expression was not significantly different in microdissected collecting ducts between 10- and 30-mo-old rats. Although these data suggest that gene transcription of V2 receptors is maintained with age, as was that of intrarenal renin in the same animals, they do not exclude changes in protein expression (7). Determination of vasopressin binding sites in membrane fractions from the papilla showed a significant 35% decrease in membrane vasopressin receptor density in senescent rats, with some overlap in the results between 10- and 30-mo-old animals (Fig. 1). This decrease in the number of vasopressin V2 receptors in female WAG/Rij rats is much smaller than that reported for male Brown Norway or Fisher 334 rats from binding or immunocytochemistry experiments (18, 25, 28) but rather compares with the close number of receptors found in adult and old rabbits (37). The consequences of V2 receptor decreased density on intracellular cAMP were assessed by measuring baseline cAMP contents of kidney papilla. Results from right and left kidneys indicated similar cAMP concentrations in the papilla of adult and senescent rats. This contrasts with the data of Beck and Yu (3) in Fisher 344 rats but agrees with the report of Davidson et al. (11), who did not find any age-related change in basal cAMP contents of renal cell suspensions. It also fits with the constant basal cAMP levels we previously found in the medullary thick ascending limb of aging rats (20). This suggests that the significant, but relatively small, decrease in vasopressin binding sites reported in the papilla of 30-mo-old WAG/Rij rats has a minor physiological impact on baseline intracellular signaling under these experimental conditions. The present results, together with previous findings, therefore suggest that the polyuria observed in senescent rats is not directly related to a change in intracellular cAMP levels of the collecting tubules. It does not exclude, however, that other intracellular signaling pathways would be involved in expression of aquaporins and water permeability of the collecting duct.
The parameters responsible for collecting duct water permeability downstream of cAMP production were further investigated by comparing aquaporins expression, distribution, and intracellular localization in adult and senescent rats. Expression of AQP1, which is located in proximal tubules and thin descending limbs, is unaffected by age, in agreement with the constant water permeability of brush-border membranes and the unchanged proximal water reabsorption previously reported in senescent rats (9, 27). In contrast, Western blotting and immunocytochemistry results demonstrated a marked downregulation of AQP2 expression in medullary collecting tubules but not in cortical collecting ducts. Immunolocalization of AQP2 showed that principal cells from all collecting tubules were similarly affected by this decrease in AQP2 expression, indicating that it did not result from focal chronic nephrosis. Western blotting also showed a 50% downregulation of AQP3 expression in the inner medullary collecting tubules but not in the cortical or outer medullary collecting ducts, whereas little or no change was observed in the expression of AQP4 in this segment of the nephron. Despite its significance, the physiological relevance to water reabsorption of AQP3 downregulation is unclear in view of the predominant role of AQP4 in the transepithelial water permeability of collecting duct in the inner medulla (6, 36).
In contrast, the role of AQP2 in the regulation of urine concentration is well established both in rats and humans. Brattleboro rats, which do not produce vasopressin, and humans with mutations of either the V2 receptor or AQP2, all exhibit nephrogenic diabetes insipidus as a result of defective AQP2 trafficking to principal cell apical plasma membranes (21, 33, 35). Downregulation of AQP2 expression has also been described under various experimental conditions, so-called "acquired nephrogenic diabetes insipidus," and was correlated with increased urinary volume and decreased water permeability of the collecting tubule (21, 24, 30, 33). Conversely, dehydration, water deprivation, and other experimental conditions with reduced urinary output and increased osmolalities were associated with upregulation of AQP2 expression. Under most experimental conditions, these changes were attributable to vasopressin-mediated cAMP accumulation and the regulation of the cAMP-responsive element present in the AQP2 promoter (23, 30).
In the present work, however, downregulation of AQP2 expression occurred without a change in baseline plasma vasopressin or cAMP. Such vasopressin-independent regulation of AQP2 expression has already been proposed (13, 22). A frequently quoted mechanism involves interstitial tonicity as a key parameter in aquaporin expression and is supported by upregulation of AQP2 mRNA in hypertonic medium (15). Regulation by interstitial osmolality within the kidney would fit with the reported changes in AQP2 and AQP3 expression in aging rats. Papillary osmolality was lower in senescent animals than in the adult rats, and the extent of the age-related downregulation of AQP2 was progressive from the cortex to the inner medulla, where changes in interstitial tonicity are more likely to occur. This is consistent with the low AQP2 detection by electron microscopy gold labeling in the deepest regions of the medulla and could also explain the impaired concentrating ability of aging kidney in response to vasopressin administration or acute dehydration.
In summary, aging is associated with a progressive downregulation of collecting duct AQP2 and AQP3 expression from cortex to medulla of rat kidney. This is independent of circulating vasopressin or basal intracellular cAMP level but may be related to changes in medullary tonicity. The large decrease in AQP2 expression in the inner medulla suggests a reduced collecting duct water permeability, which could account for the polyuria and the reduced concentrating ability of the aging kidney.
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ACKNOWLEDGEMENTS |
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This work was funded by the Institut de Recherches International Servier, France, and supported by a Training and Mobility of Research ERB 4061PL97-0406 from the European Union. Laurent Teillet was supported by a grant from the Assistance-Publique-Hôpitaux de Paris, Direction de la Recherche Clinique, Paris, France.
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FOOTNOTES |
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Address for reprint requests and other correspondence: B. Corman, Service de Biologie Cellulaire, CEA/Saclay, Gif-sur-Yvette, 91191, France (E-mail: corman{at}dsvidf.cea.fr).
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. §1734 solely to indicate this fact.
Received 5 August 1999; accepted in final form 3 March 2000.
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