1 Institute of Anatomy, University of Zurich, CH-8057 Zurich, Switzerland; 2 Division of Nephrology, Vanderbilt University, Nashville, Tennessee 37232; 3 Laboratory of Kidney and Electrolyte Metabolism, National Heart, Lung, and Blood Institute, Bethesda, Maryland 20892; and 4 Institut de Pharmacologie et de Toxicologie, Université de Lausanne, CH-1005 Lausanne, Switzerland
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ABSTRACT |
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The amiloride-sensitive epithelial sodium channel (ENaC) and the vasopressin-dependent water channel aquaporin-2 (AQP2) mediate mineralocorticoid-regulated sodium- and vasopressin-regulated water reabsorption, respectively. Distributions of ENaC and AQP2 have been shown by immunohistochemistry in rats. Functional data from rabbits suggest a different distribution pattern of these channels than in rats. We studied, by immunohistochemistry in the rabbit kidney cortex, the distributions of ENaC and AQP2, in conjunction with marker proteins for distal segments. In rabbit cortex ENaC is restricted to the connecting tubule (CNT) cells and cortical collecting duct (CCD) cells. The intracellular distribution of ENaC shifts from the apical membrane in the most upstream CNT cells to a cytoplasmic location further downstream in the CNT and in the CCD cells. AQP2 is detected in the CCD cells exclusively. The anatomic subdivisions in the rabbit distal nephron coincide exactly with distributions of apical transport systems. The differences between rabbits and rats in the distribution patterns of ENaC and AQP2 may explain functional differences in renal salt and water handling between these species.
immunohistochemistry; morphology; intercalated cells; bumetamide-sensitive sodium-potassium chloride cotransporter; thiazide-sensitive sodium chloride cotransporter; diuretics
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INTRODUCTION |
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MINERALOCORTICOIDS AND VASOPRESSIN are the major
hormones involved in renal control of salt and water excretion. In the
renal cortex one target, among others, for mineralocorticoids to
regulate sodium reabsorption is the amiloride-sensitive epithelial
sodium channel (ENaC; for review, see Ref. 19). The target for
vasopressin-regulated water reabsorption is the water channel,
aquaporin-2 (AQP2; for review, see Ref. 38). In the cortical distal
nephron of rats ENaC was located in the connecting tubule (CNT) and
cortical collecting duct (CCD) cells, and, additionally, in the second
half of the distal convoluted tubule (DCT 2; 11, 44). In that latter
segment, ENaC and the thiazide-sensitive sodium chloride cotransporter (NCC) are coexpressed in the DCT cells (44). The distribution of ENaC
in the rat coincides with the distributions of mineralocorticoid receptors and of 11- hydroxysteroid dehydrogenase (4). Former biochemical studies using isolated tubule preparations of rats and mice
assessed vasopressin sensitivity in both segments of the cortical
collecting system, the CNT and CCD (25). In rats the
immunohistochemical demonstration of AQP2 in the CNT (31) and in the
CCD confirmed these findings (15, 37).
Because a large body of physiological and pharmacological data are available concerning the transport of water and electrolytes in segments of the distal rabbit nephron (for review, see Ref. 36), it would be particularly important to know the precise distribution of ENaC and AQP2 in the rabbit nephron. Because of marked interspecies differences in the segmentation of the cortical distal nephron, data from rat may not be applicable to rabbit. Therefore, the aim of the present study was to assess by immunohistochemistry (IHC) the locations of the amiloride-sensitive sodium- and of the vasopressin-dependent water channel in the rabbit cortical distal nephron.
Our results demonstrate in rabbits that ENaC is easily detectable in the CNT and CCD but undetectable all along the DCT. AQP2 is detectable in the CCD exclusively. Moreover, the data show that the distributions of the apical transport systems (NCC, ENaC, AQP2) coincide exactly with the structural segmentation of the cortical distal nephron. The differential distributions along the cortical distal nephron of the transporters in rabbits and rats suggest a different handling of renal cortical salt and water reabsorption in these species.
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MATERIALS AND METHODS |
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Rabbits
Kidneys from three adult untreated white New Zealand rabbits (2 males, 1 female; between 3 and 5 kg body wt; BRL, Füllinsdorf, Switzerland) were investigated.Fixation and tissue processing. Rabbits were anesthetized with urethan (1.5 g/kg body wt, ip). Kidneys were fixed by vascular perfusion via the abdominal aorta, as previously described (28). The fixative consisted of 3% paraformaldehyde (PFA) and 0.05% picric acid. The fixative was dissolved in a 3:2 mixture of 0.1 M cacodylate buffer (pH 7.4, adjusted to 300 mosM with sucrose) and 10% hydroxyethyl starch in saline (HAES sterile; Fresenius, Germany). After 5 min of fixation in situ the kidneys were removed and cut into coronal slices. For IHC and in situ hybridization (ISH) tissue slices were stored for 2 h in cacodylate buffer. The fixed tissue slices were mounted onto thin cork plates and frozen in liquid propane (cooled with liquid nitrogen). Five-micrometer-thick serial sections were cut in a cyrostat and mounted alternately for IHC on chromalum/gelatin-coated glass slides, and for ISH on silanized glass slides. For light microscopy perfusion-fixed tissue was postfixed for at least 24 h by immersion in the 3% PFA fixative solution, to which 0.5% glutaraldehyde was added. Thereafter, the tissue was embedded in epoxy resin and cut into 1-µm sections following routine procedures.
Primary Antibodies Used for IHC, and cDNA, for ISH
Rabbits.
For detection of ENaC we used rabbit antisera, directed against the rat
-,
- and
-subunits of the channel (11). AQP2 was detected with
a rabbit-anti-human AQP2 antiserum (12) that also recognized the rabbit
and rat isoforms. The polyclonal rabbit antisera were diluted 1:2,000.
All antibodies have been extensively characterized in previous
publications (11, 12).
Rats. For comparison of the locations of AQP2 in rabbits and rats, some slices of perfusion-fixed rat kidneys were immunostained with the rabbit-anti-human AQP2 antiserum as well as with a rabbit anti-rat AQP2 antiserum (dilutions 1:2,000). Consecutive rat kidney sections were immunostained with a rabbit anti-rat NCC (1:8,000) antiserum. The rabbit anti-rat AQP2 antiserum and the rabbit anti-rat NCC antiserum have been characterized previously (37, 42).
Incubations For IHC
The cryostat sections were stored in PBS until use and then preincubated for 10 min with 10% normal goat serum in PBS. Incubations with the primary antibodies, diluted in PBS-BSA to the given concentration, took place overnight in a humidified chamber at 4°C. After repeated rinsing in PBS, binding sites of the primary antibodies were detected with a 1:40 dilution of FITC-conjugated swine-anti-rabbit IgG (Dakopatts, Glostrup, Denmark) or with a 1:1,000 dilution of Cy3-conjugated goat-anti-rabbit IgG (Jackson Immuno Research Laboratories, West Grove, PA), and with FITC-conjugated goat-anti-mouse IgG (Jackson Immuno Research Laboratories) diluted 1:40.Finally, the sections were rinsed with PBS and coverslips were mounted, using DAKO-Glycergel (Dakopatts), to which 2.5% 1,4-diazabicyclo[2.2.2]octane (Sigma Chemical) was added, as a fading retardant. The preparations were studied by epifluorescence microscopy (Polyvar, Reichert-Jung, Vienna, Austria).
To discriminate the binding of the secondary FITC- or Cy3-labeled
anti-rabbit antibodies to IgGs of the specific rabbit antisera from
binding to native IgGs, present in the rabbit tissue, on some slides
the specific antiserum was replaced by PBS-BSA or nonimmune rabbit
serum. On these slides any epithelial cells were unlabeled; however,
all basement membranes and mesangial cells were more or less strongly
stained. This staining could be significantly reduced (Fig.
1) by immersing the slides in 0.01 M
sodium-citrate buffer (pH 6.0) and placing them into a microwave oven
for 10 min before incubation with the primary antiserum. This
treatment, often used as an antigen-retrieval technique (for review,
see Ref. 50), also improved the histochemical detection of -ENaC, but not of
- and
-ENaC.
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ISH
Digoxigenin-11-UTP-labeled riboprobes were synthesized by in vitro transcription [DIG RNA labeling kit (SP6/T7); Boehringer, Mannheim, Germany] from the full-length rNCC cDNA (18). After linearization by BamH I and Sal I, sense and antisense riboprobes were generated by T7 and SP6 RNA polymerases, respectively. RNA probes were degraded by alkaline hydrolysis to fragments of ~200 bases in length.The cryostat sections were postfixed by 4% PFA in PBS for 20 min. After two brief rinsing steps the sections were pretreated for 10 min at room temperature with a solution containing 50 µg/ml proteinase K (Sigma Chemical), 0.1% collagenase (Sigma Chemical) and 0.1 unit/µl RNase inhibitor (Boehringer) in H20. After two brief rinses in PBS the slides were acetylated for 20 min with 0.1 M triethanolamine and 0.25% acetic anhydrate. Subsequently, slides were rinsed twice in PBS, dehydrated in a graded series of ethanol (70, 80, and 95%) and air dried for 20 min at room temperature. Prehybridization was carried out at 42°C for 2 h in 50% formamide (Sigma Chemical), 50 mM Tris · HCl (pH 7.6), 25 mM EDTA (pH 8.0), 20 mM NaCl, 0.2% SDS, 0.25 mg/ml tRNA, and 2.5× Denhardt's. Hybridization was performed at 42°C for 18 h in 50% formamide, 25 mM Tris · HCl (pH 7.6), 1.25 mM EDTA (pH 8.0), 40.5 mM NaCl, 10 mM 1,4-dithiothreitol, 0.75 mg/ml tRNA, 0.1 mg/ml salmon sperm DNA, 1.25× Denhardt's, 12.5% dextran-sulfate, and 10 ng/µl hydrolyzed sense and antisense riboprobes, respectively. Subsequently, the sections were rinsed for 10 min at room temperature in 2× sodium chloride-sodium citrate solution (SSC), followed by three rinses at 49°C in 50% formamide with decreasing SSC concentrations (1×, 0.5×, and 0.25×) in each rinsing step. Digoxigenin-labeled hybridized probes were detected by using the DIG DNA labeling and detection kit (Boehringer) according to the manufacturer's instructions. The alkaline phosphatase-linked sheep-anti-digoxigenin antibody was diluted 1:500. Nitroblue tetrazolium salt and 5-bromo-4-chloro-3-indolyl phosphate served as chromogenic substrates for the alkaline phophatase-catalyzed color reaction.
Documentation
Micrographs were acquired with a charge-coupled device camera (Visicam 1280, Visitron Systems, Puching, Germany) and processed by Image-Pro and Photoshop software. ![]() |
RESULTS |
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Rabbits
Light microscopy.
The cortical distal nephron of the rabbit kidney reveals a clear-cut
structural segmentation (41) into the DCT, CNT, and CCD (27). The
latter two represent the cortical collecting system. In 1-µm sections
of epon-embedded tissue, the transition from the TAL of Henle's loop
to the DCT is evident by the steep, severalfold increase in epithelial
height (Fig. 2). The DCT is composed
exclusively of "DCT cells." The DCT cells are abruptly replaced
by structurally different "CNT cells," which are intermingled
with IC cells (Fig. 3A). The DCT
and the CNT segments are both located in the cortical labyrinth. The
CNTs of deep and intermediate nephrons form arcades that ascend in
close proximity to the cortical radial vessels and that drain tubular
fluid from several nephrons to the CCDs. Shortly before the arcades
open into CCDs, situated within the medullary rays, the structure of
the segment-specific cells abruptly changes to CCD cells, formerly
called "principal" cells (Fig. 3D). IC cells are
interspersed also among the CCD cells.
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Distribution of marker proteins for distal cell types. To correlate immunostaining for ENaC and AQP2 in rabbits to the distal segments and cell types, we performed double labelings, or labeling of parallel sections, for proteins with established distribution patterns.
Calbindin D28k and proton ATPase. In rabbit renal cortex the calcium-binding protein calbindin D28k is present in the cytoplasm and in the nuclei of all three segment-specific cell types, the DCT, CNT, and CCD cells (46), although in differential abundance. Immunostaining for calbindin is weak in DCT cells (Figs. 2C and 3B), particularly abundant in CNT cells, and intermediate in CCD cells (Fig. 3F). The distinct changes in calbindin D28k abundance coincide exactly with the morphological segmentation (Fig. 3, A,C, D, and F).
IC cells in the cortical rabbit nephron do not bind calbindin D28k antibodies. They appear as dark, sharply, demarkated patches among the brightly stained CNT cells and among the CCD cells. In addition, IC cells are discriminated by their strong binding to antibodies against proton ATPase (Fig. 3E).Bumetamide-sensitive Na-K-2Cl cotransporter (NKCC2) and thiazide-sensitive Na-Cl cotransporter (NCC). The transport proteins, functionally characterizing the TAL cells and the DCT cells, are NKCC2 (29, 40) and NCC (2, 39, 42), respectively. Immunostaining for NKCC2 (Fig. 2B) was present on the apical cell membrane of TAL cells and ended at the morphologically well-marked transition from the TAL to the DCT (Fig. 2A ). ISH for the NCC, combined with immunostaining for calbindin D28k, revealed that the extension of the ISH signal for the NCC coincides exactly with the extension of the very low calbindin D28k abundance (Fig.3, B and C); i.e., it started at the morphologically well-marked transition from the TAL to the DCT and stopped suddenly at the morphological transition to the CNT. The very weak binding of the DCT to calbindin D28k was also seen in simply immunostained sections.
Distribution of ENaC.
ENaC is composed by -,
- and
-subunits (5). From the available
antisera, raised in rabbits (11), only the one against the
-subunit
reacted well with the rabbit tissue. That against the
-subunit
provided the same distribution pattern as that for the
-subunit but
yielded high background staining. Binding of the anti-
-subunit
antiserum was undetectable.
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Distribution of AQP2. In overviews, on the renal cortex AQP2-related fluorescence was seen exclusively in the medullary rays (Fig. 4C), where it was confined to the CCD cells. IC cells were negative. The AQP2 immunostaining started sharply in the short branches of the CCDs that join with the CNT at the site of the sudden decrease in calbindin D28k labeling and the structural change from the CNT to the CCD cell type (Fig. 5, D and E). The AQP2 immunostaining was prominent in the apical membrane and in the subapical cytoplasm of CCD cells. A weaker, although consistent, AQP2 staining was present at the base of CCD cells, which comprises the infolded basolateral cell membranes (Fig. 5).
Rats
In agreement with results of other authors (42, 44), binding of anti-NCC was detected in the apical membrane of DCT cells. In the terminal portion of the DCT, the NCC immunostaining was markedly lower than in earlier portions and the epithelial apical membrane staining became discontinuous (Fig. 6A). The binding patterns of anti-rat and anti-human AQP 2 antiserum were identical. With both sera strong apical and weaker basolateral membrane staining were observed all along the rat cortical collecting system, i.e., in cells of the CNT and CCD (Fig. 6B), in full agreement with results of Kishore et al. (31). At the transition from the DCT to the CNT, immunostaining for NCC and AQP2 bordered on each other; occasionally, AQP2-positive and NCC-positive cells were intermingled over a short distance. At the beginning of the AQP2- positive segment, AQP2-related immunostaining was weaker than in more downstream portions of the CNT and than in the CCD (Fig. 6B). Cells, staining neither for NCC nor ENaC, are IC cells.
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In summary, in rabbit renal cortex, ENaC immunostaining is restricted to CNT and CCD cells, NCC-expressing segments lacking any ENaC immunoreactivity. ENaC immunostaining shifted along the CNT from the apical membrane to the cytoplasm of CNT and CCD cells. AQP2 is restricted in rabbits to CCD cells, in marked contrast to rats, where both segments of the cortical collecting system, CNT and the CCD, show AQP2 staining.
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DISCUSSION |
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The cortical distal nephron downstream of the TAL is involved in the fine tuning of renal electrolyte and water excretion and is the target for several diuretic drugs. Anatomically, this nephron portion displays three segments: the DCT, the CNT, and the CCD. The segmentation is based on differences in epithelial cell structure, which are the most prominent in the rabbit kidney. The morphological demarcations served as "landmarks" for dissecting isolated rabbit nephrons into segments, used for investigations in vitro (reviewed in Refs. 36 and 43). The in vitro perfusion techniques were particularly helpful for analyzing the transport characteristics of segments as the TAL, the CNT, or the CCD, which are not directly accessible to functional studies in vivo by micropuncture. However, the precise functional roles of some segments, in particular the CNT, remained unclear or discrepant data were obtained in rabbits (1, 18, 45, 49, 53) and rats (7, 8, 13, 48).
The present study reveals some major differences between rabbits and rats in the distribution of ENaC and AQP2. Moreover, our data demonstrate the exact congruency between the distribution of the apical transport systems and the anatomic subdivision of the distal nephron in rabbits.
ENaC-Expressing Epithelia and IC
In the rabbit we and others (10) detected ENaC in the CNT and CCD, but not in the DCT. The absence of immunoreactive ENaC in the DCT correlates with recent observations from rabbits that, in the DCT, ENaC activity is lacking (49). Yet, in the second half of the DCT in rat kidney (DCT-2), ENaC is well detectable by IHC (44). The DCT cells in DCT-2 express ENaC, additionally to the DCT-characteristic NCC (33, 44). The appearance of ENaC-expressing cells in the nephron epithelia of rabbits, rats (44), and mice (33) is associated with the appearance of IC cells that are intermingled with the segment-specific cell types. In contrast to sodium chloride reabsorption by the NCC, ENaC-mediated reabsorption is tighty coupled to apical potassium secretion (17). The latter proceeds most likely across the recently identified ROMK channel (22), present in the apical membrane of several cell types, including ENaC-expressing cells (35, 52, 54). Thus increased sodium reabsorption by the ENaC-positive tubular epithelia obligatorily entails increased potassium secretion into the tubular fluid. IC cells possess a H-K-ATPase in their apical membrane (reviewed in Refs. 16 and 51), in addition to the electrogenic proton ATPase (3), and can reabsorb potassium from the tubular fluid. Thus the regular presence of IC cells with the potential for potassium reabsorption among the ENaC-expressing cells may constitute an important means for maintaining potassium balance under conditions of altered ENaC-mediated sodium reabsorption.Intracellular Distribution of ENaC in the CNT and CCD
Interestingly, the intracellular distribution of ENaC shifts from the apical membrane at the beginning of the CNT to the cytoplasm further downstream in the CNT and in the CCD. The apparently decreasing abundance of ENaC channels in the apical plasma membrane of CNT cells, together with the marked axial reduction of the "transport machinery" [i.e., mitochondrial volume, basolateral membrane area, and Na-K-ATPase activity per unit tubular length (28, 30, 32)], suggests a corresponding reduction of sodium transport activity in direction of tubular flow. Net sodium reabsorption in isolated rabbit CNTs seems to be three to four times higher than in the CCD (1). The exclusive intracellular localization of ENaC in the CCD, as determined by IHC, is in accordance with the fact that functional ENaC channels are virtually undetectable by electrophysiological techniques in the CCD (14), and that in isolated rat CCD, sodium transport across the epithelium is nearly nonexistent unless the animal is pretreated with mineralocorticoids (47).Distribution of AQP2 in Distal Segments
Analogous to the structural and functional changes along the water-impermeable TAL (20), the axial reduction of the transport machinery along the rabbit CNT had been taken as an indication for possible water impermeability of the CNT epithelium (27, 28). This assumption was directly supported by some transport studies in isolated rabbit tubules (1, 24), and indirectly by the lack of vasopressin-dependent rises in cAMP concentration in isolated rabbit CNTs (6, 25). The absence of the vasopressin-dependent water channel AQP2 in the rabbit CNT now definitey confirms that, in rabbits, this segment plays no role in vasopressin-regulated water reabsorption. Moreover, in rabbits epithelial sodium channels are dissociated from vasopressin-dependent water channels in the greatest part of the cortical distal nephron. This points to a major difference in handling of cortical water reabsorption and regulation of extracellular fluid volume between rabbits and rats or mice. In the latter two species, a much greater proportion of the cortical tubular system is available for vasopressin-regulated water reabsorption. In addition, in these species the potentials for water reabsorption and mineralocorticoid-regulated Na reabsorption are associated all along the cortical collecting system, as suggested by the colocalization of ENaC and AQP2 in the rat and mice CNT and CCD (11, 31, 33, 37, 44). Coordinated regulation of osmotic water flow via AQP2 and of sodium entry via ENaC might be an important mechanism in transepithelial solute transport as well as in cell volume regulation in vasopressin responsive-epithelia (26, 34).Organization of the Distal Nephron
Among all investigated mammalian species the structural and functional organization of the distal nephron is the most evident in rabbits. In each of the four distal segments one major salt transport system prevails (Fig. 7): 1) in the TAL, NKCC2 (40), 2) in the DCT, NCC (2), 3) in the CNT, ENaC, which 4) in the CCD is colocalized with the AQP2 . The arrangement in flow direction of differential salt-reabsorbing mechanisms with probably increasing affinities for salt reabsorption might be interpreted to ensure the most efficient recovery of vital solutes from the tubular fluid.
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In rabbits the distribution of the given apical sodium- and water-transport proteins or channels, but not that of other apical [e.g., epithelial calcium channel (23)] or basolateral proteins (e.g., Na-K-ATPase), coincides exactly with the obvious morphological segmentation of the distal nephron, on the basis of differences mainly in the basolateral organization of the cells. The more gradual structural transformations from the DCT to the CNT and then to the CCD in the rat distal nephron, as described already by Crayen and Thoenes (9), are also paralleled by more gradual replacements of the apical transport systems (Figs. 5 and 6). Yet, regardless of the species, cells that differ with respect to apical sodium transport proteins differ also in their basolateral organization. So far, the interdependence of apical transport systems and basolateral organization of the cells is unknown.
In conclusion, in rabbits ENaC is found from the CNT, AQP2 from the CCD downstream. This differs from rats, in which ENaC and AQP2 are associated all along the CNT and CCD. The sharp changes from one apical transport system to the other in the rabbit distal nephron coincide exactly with the morphological segmentation. As pointed out by Karl Peter in 1909 (41) for the overall organization of the rabbit kidney, we assume that the organization of the rabbit distal nephron might represent the basic pattern of the mammalian distal nephron. It might be helpful for understanding this nephron portion in more complicated kidneys, in particular the human kidney, of which the precise functional and structural distal nephron organization are still largely unknown.
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ACKNOWLEDGEMENTS |
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We acknowledge the technical assistance of M. Mueller and L. Klaeusli; we thank S. Gluck for supplying the monoclonal antibody against proton ATPase.
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FOOTNOTES |
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This study was supported by Swiss National Science Foundation Grant 31-47742.96 (to B. Kaissling)
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.
Address for reprint requests and other correspondence: B. Kaissling, Institute of Anatomy, Univ. of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland (E-mail: bkaissl{at}anatom.unizh.ch).
Received 3 September 1999; accepted in final form 12 November 1999.
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