1 Second Department of Internal Medicine, Tokyo Medical and Dental University, Tokyo 113-8519; 2 Shigei Medical Research Institute, Okayama 701-0202, Japan; and 3 Department of Cell Biology, Institute of Anatomy, University of Aarhus, DK-8000 Aarhus, Denmark
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ABSTRACT |
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To determine the immunolocalization of ClC-5 in the mouse kidney, we developed a ClC-5-specific rat monoclonal antibody. Immunoblotting demonstrated an 85-kDa band of ClC-5 in the kidney and ClC-5 transfected cells. Immunocytochemistry revealed significant labeling of ClC-5 in brush-border membrane and subapical intracellular vesicles of the proximal tubule. In addition, apical and cytoplasmic staining was observed in the type A intercalated cells in the cortical collecting duct. In contrast, the staining was minimal in the outer and inner medullary collecting ducts and the thick ascending limb. Western blotting of vesicles immunoisolated by the ClC-5 antibody showed the presence of H+-ATPase, strongly indicating that these two proteins were present in the same membranes. Double labeling with antibodies against ClC-5 and H+-ATPase and analysis by confocal images showed that ClC-5 and H+-ATPase colocalized in these ClC-5-positive cells. These findings suggest that ClC-5 might be involved in the endocytosis and/or the H+ secretion in the proximal tubule cells and the cortical collecting duct type A intercalated cells in mouse kidney.
proximal tubule; endocytosis; proton pump; Dent's disease; chloride channel
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INTRODUCTION |
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THE CLC-5 CHLORIDE CHANNEL (human gene symbol: CLCN5) is a molecule responsible for four hereditary kidney stone diseases: Dent's disease (33), X-linked recessive hypophosphatemic rickets (XLRH) (1), X-linked recessive nephrolithiasis (XRN) (7), and idiopathic low-molecular-weight proteinuria (15). All four of these diseases are associated with mutations in the ClC-5 gene and have common phenotypic features characterized by excessive low-molecular-weight proteinuria, hypercalciuria, and either nephrocalcinosis or nephrolithiasis (19, 20, 23, 25, 28). Accordingly, these four related X-linked syndromes have been considered phenotypic variants of a single disease. However, other renal tubular dysfunctions such as phosphaturia, glycosuria, uricosuria, and impairment of urinary acidification may also occur, and the mutation in the ClC-5 gene does not always correlate with the severity of the phenotype. To date, we have no pathophysiological explanation of how and why a molecular dysfunction of the single channel ClC-5 causes such different renal tubular dysfunctions in these related syndromes. Localization of ClC-5 expression along the nephron is of particular importance in understanding the physiological roles of ClC-5 in the kidney.
In a RT-PCR study performed on microdissected tubules, Steinmeyer et al. (29) first demonstrated that the ClC-5 message was ubiquitously expressed along the nephron, and this expression was predominant in the cortical collecting tubules, the S3 segment of the proximal tubules, and the thick ascending limb. However, contamination of the samples in the dissection procedure might disturb the accurate localization. A subsequent in situ hybridization study of rat kidney showed expression of ClC-5 in the collecting duct type A intercalated cells from cortex through the upper portion of the inner medulla, but not in the proximal tubule cells (24). The absence of the message in proximal tubule cells is not consistent with the presence of low-molecular-weight proteinuria in the ClC-5-defected syndromes. Recently, Gunther et al. (13) described the cellular and subcellular localization of ClC-5 in rat kidney using ClC-5-specific polyclonal antibodies. They further showed partly overlapping labeling patterns of ClC-5 and the vacuolar-type H+-ATPase. In the present study, we examined the localization of ClC-5 in the mouse kidney using a monoclonal antibody. In addition, we examined whether ClC-5 colocalizes with H+-ATPase in the same membrane vesicles and membrane domains. For this purpose, two series of experiments were carried out. First double-labeling confocal microscopy of ClC-5 and H+-ATPase was undertaken. Second, ClC-5-bearing vesicles were immunoisolated and subjected to immunoblotting.
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METHODS |
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Preparation of monoclonal antibodies. The anti-peptide antibodies were generated against a synthetic peptide (CC54: CKHIAQMANQDPDSILFN) corresponding to the COOH-terminal 17 amino acids of ClC-5, and cysteine was added for coupling to NH2 terminus. Eight-week-old female WKY/NCrj rats were injected via hind footpads with 1 mg/ml of authentic peptide (CC54) conjugated with keyhole limpet hemocyanin and Freud's complete adjuvant. After 2 wk, the rats were killed, and peritoneal lymph nodes were removed. Isolated lymphocytes (1 × 108) were fused with mouse myeloma cell line SP2/O-Ag14 as previously described (26). The cells were plated in four 96-well tissue culture plates (Becton-Dickinson Labware), and supernatants from individual wells were collected and screened by enzyme-linked immunosorbent assay (ELISA). The epitope of each isolated monoclonal antibody was mapped using twenty kinds of overlapping 10-residue peptides corresponding to the immunizing peptide (CC54) (11).
Preparation of membrane fractionations. Crude
membrane fractions were prepared from the mouse kidneys. The kidneys
were minced finely and homogenized in an isolation buffer (0.3 M
sucrose, 5 mM Tris · HCl, and 2 mM EDTA, pH 7.2, containing the following protease inhibitors: 1 mM phenylmethylsulfonyl
fluoride, 2 µg/ml leupeptin, and 2 µg/ml pepstatin) with five
strokes of a motor-driven Teflon-glass homogenizer at 1,000 rpm. The
homogenate was centrifuged at 4,000 g for 15 min at 4°C to
remove nuclei, mitochondria, and any remaining large cellular
fragments. The supernatant was rehomogenized and centrifuged at 100,000 g (Beckman Optima L-70K: SW41Ti rotor) for 60 min at 4°C.
The resultant pellet was resuspended in ~100 µl of resuspension
buffer, frozen in liquid N2, and stored at 80°C
until use. The cultured cells were harvested after three rinses with
ice-cold PBS. The cell suspension was centrifuged at 1,000 rpm for 5 min at 4°C and then treated in the same manner described above.
Immunoisolation of ClC-5-bearing vesicles. Membrane fractions enriched either for intracellular vesicles (high speed) or plasma membranes (low speed) from mouse kidney were prepared as described previously (22). Magnetic beads (Dynal M-450: Dynal, Oslo, Norway) precoated with anti-rat immunoglobulin G (IgG) antibodies were coated with the SS53 monoclonal antibody against ClC-5 and then incubated with the membrane fractions overnight at 4°C with continuous agitation in an incubation buffer containing PBS, 2 mM EDTA, and 0.1% BSA. After careful washing three times, the beads were mixed with 100 µl of Laemmli sample buffer and heated to 60°C for 15 min to solubilize the proteins. The beads were then removed magnetically, and the remaining sample buffer was used for immunoblotting to detect ClC-5 and H+-ATPase. The only difference in the treatment of the controls was the substitution of the anti-ClC-5 antibody with nonimmune rat IgG.
Electrophoresis and immunoblotting. The membrane samples were solubilized in the loading buffer (2% SDS, 30% glycerol, 10% 2-mercaptoethanol, and 250 mM Tris · HCl, pH 6.8). Samples were loaded at 10-20 µg/lane onto a 7.5% or 10-20% SDS-polyacrylamide gel and run on a minigel system, and the proteins were electrophoretically transferred to polyvinylidene difluoride (PVDF) membrane. After blocking with 5% nonfat milk and 0.1% Tween 20 in PBS (PBS-T) for 1 h, the blots were incubated overnight at 4°C with antibodies (1:1,000). Blots were visualized by the enhanced chemiluminescence procedure (ECL Plus; Amersham, Arlington Heights, IL). Controls in which the primary antibody was substituted with nonimmune rat IgG or the primary or secondary antibody was omitted revealed no labeling. To confirm the specificity of the primary antibody, it was preincubated with the immunizing peptide (1 mg/ml) overnight at 4°C prior to exposure to the PVDF membranes.
Immunohistochemistry. The mouse kidneys were perfusion-fixed with PLP fixative containing 2% paraformaldehyde, 75 mM lysine, and 10 mM sodium periodate in phosphate buffer (pH 7.4). The blocks were postfixed in the same fixative for an additional 4 h at 4°C and cryoprotected in 20% sucrose in a phosphate buffer. After freezing the blocks in liquid nitrogen, they were cut into 5-µm sections and mounted on Silane-coated slides and the slides were stained by antibodies. In addition, the transfected cultured cells expressing ClC-5 (J2702) or ClC-3 (C21) using dexamethasone (DEX)-inducible mammalian expression vector (pMAM-neo) were used to determine the specificity of anti-ClC-5 antibodies (18, 27). After overnight treatment with 5 µM DEX, the cells were fixed for 10 min in 2% paraformaldehyde in PBS, and permeabilized for 2-5 min in 0.1% Triton X-100 at room temperature. The primary antibody was visualized with secondary antibodies [FITC-conjugated F(ab')2 fragment rabbit anti-rat IgG (H+L) diluted 1:200, Cy5-conjugated goat anti-rabbit IgG (H+L) diluted 1:500, or Cy3-conjugated goat anti-mouse IgG (H+L) diluted 1:500]. The fluorescence signal of labeled specimens was observed first with a Zeiss Axivert microscope and then analyzed by a laser confocal microscope (Zeiss LSM 510). Digitized images were produced with a Mirus Film Printer GALLERIA using Raster Plus 95 software (version 1.01). In some studies, we used the affinity-purified polyclonal antibody against the aquaporin-2 (AQP2) water channel, an antibody which has been previously described and characterized (8). We also examined the staining of the monoclonal antibody against the 31-kDa subunit of H+-ATPase (E11, kindly provided by S. L. Gluck, Gainesville, FL) (14). E11 was diluted 1:1 and visualized with Cy3-conjugated goat anti-mouse IgG (H+L) diluted 1:500 (CyDye; Amersham, Buckinghamshire, UK).
Immunoelectron microscopy. For electron microscopy, 5- to 7-µm thick sections of kidneys fixed by the PLP fixative containing 2% paraformaldehyde were initially incubated with the SS53 antibody for 12 h and then with peroxidase-conjugated F(ab')2 fragment goat anti-rat IgG (Cappel, Aurora, OH) for 3 h. The sections were then fixed in 2.5% glutaraldehyde for 10 min at 4°C, and immersed in 3,3'-diaminobenzidine tetrahydrochloride (DAB) solution for 30 min and DAB containing 0.005% H2O2 for 10 min, respectively. Tissue slices were postfixed by 2% OsO4 and embedded in Quetol 812. Ultrathin sections were examined with a Hitachi transmission electron microscope.
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RESULTS |
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ClC-5-specific monoclonal antibodies. Rats were immunized with
the 18-amino-acid synthetic peptide CKHIAQMANQDPDSILFN. The last 17 amino acids of this sequence, KHIAQMANQDPDSILFN, are conserved among human (6), rat (27, 29), and mouse (unpublished data). The
criteria for selection of monoclonal antibodies were specificity for
ClC-5, epitope specificity and length, a good ability to stain the
mouse kidney sections, and classification as an IgG subclass. Two
monoclonal antibodies (SS53 and SS54) were selected and characterized (Table 1). Since ClC-5 is highly homologous
(~80% amino acid sequence identity) to ClC-3 and ClC-4
chloride channels (2, 16, 17, 31), the antibodies were evaluated for
the cross-reactivities to corresponding COOH-terminal peptides of
ClC-3, -4, and -5 (Table 1). The result showed that SS53 was selective
to ClC-5, whereas SS54 had a considerable cross-reactivity to ClC-3 and
ClC-4. The determined epitopes were DSILF and HIAQMA in SS53 and SS54,
respectively. In ClC chloride channel family, the alignment of DSILF is
not conserved except for the amino acid sequences in
ClC-5. Thus, we used SS53 for further studies.
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In the Western blots (Fig. 1), SS53
recognized a band of about 85 kDa in the crude membranes prepared from
both the mouse kidney and the stably ClC-5-transfected cultured cells
with DEX treatment. This size of the band conformed well with that
demonstrated by Gunther et al. (13). Indirect immunofluorescence
staining of the stably transfected CHO-K1 cells of rat ClC-3
(C21) or ClC-5 (J2702) also confirmed the isoform specificity of
our monoclonal antibodies. The SS53 antibody recognized J2702
cells (Fig. 2A) but not C21 cells
(Fig. 2B), indicating the isoform specificity of SS53 for
ClC-5.
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Cellular and subcellular localization of ClC-5 along the
nephron. Immunostaining of the mouse kidney by SS53 showed
that ClC-5 was strictly localized in the proximal tubule cells and
subpopulation of cortical collecting duct (CCD) cells. The
specificity of antibody (SS53) was evaluated using the preadsorbed
antibody with synthesized peptide (CC54) or nonimmune rat IgG (data are
not shown). No staining was seen, even though a high concentration of
the antibody was applied in the glomerulus and the other renal tubule
cells. ClC-5 appeared to be broadly expressed from the S1 to S3
segments in all proximal tubules examined. Subcellular localization
determined by a high magnification showed a marked staining beneath the
brush-border membrane, indicating the presence of ClC-5 in the apical
cytoplasmic vesicles. Staining was also found in the brush-border
membrane itself (Fig. 3A).
Transmission electron microscopy showed the presence of immunoreactive
material in the brush-border membrane and the subapical regions
containing endocytotic vesicles (Fig. 3, B and C).
These subcellular localizations were confirmed by immunogold labeling
(data are not shown).
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ClC-5 was localized broadly in the apical cytoplasmic regions of the
subpopulation of collecting duct cells in the cortex. Double staining
with FITC-conjugated SS53 and Cy5-conjugated anti-AQP2 antibody showed
the staining of ClC-5 (green) in the apical site of the cells which
lacked the AQP2 staining (red) (Fig. 4). No overlapping of the staining was observed (Fig. 4C). These
findings indicate that ClC-5 is strictly localized in the intercalated cells and not in the principal cells in the CCD. Most of
the ClC-5-positive intercalated cells were stained as apical
predominant pattern, indicating that these cells were type A
intercalated cells. This was confirmed using an antibody to the
H+-ATPase in parallel sections demonstrating strong apical
labeling in type A intercalated cells, as described previously (3). Occasionally, a reverse pattern, i.e., staining of ClC-5 and
H+-ATPase at the basolateral region, was observed (see Fig.
7). In contrast, the antibody did not stain intercalated cells in the
outer medullary region, and positive staining of the proximal S3
segments in the same section confirmed that this was not due to a
technical error (Fig. 5A). In the
other nephron segments, we could not detect any significant staining
with anti-ClC-5 antibody compared with those with preadsorbed antibody
with synthesized peptide (CC54) or with nonimmune rat IgG (Fig. 5,
B and C).
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Colocalization of ClC-5 with
H+-ATPase. Since the
localization of ClC-5 was similar to that of H+-ATPase,
especially in the rat (3) but also to some extent in the mouse (30), we
examined whether these two proteins colocalize. For this purpose double
immunostaining was performed, and two-color confocal images of ClC-5
(green) and H+-ATPase (red) demonstrated overlapping
labeling patterns (resulting in a yellow color; Figs.
6 and 7) in proximal tubule and type A
intercalated cells. In the proximal tubule cells, the region beneath
the brush border was extensively stained (Fig. 6). In addition, green
color at the brush-border membrane was evident after merging two color
images, indicating presence of ClC-5 in this region in addition to the
presence in subapical cytoplasmic vesicles.
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In the collecting duct, the staining pattern for H+-ATPase
and ClC-5 was essentially the same. The intercalated cells
characterized by their protuberance into the lumen predominantly
expressed both H+-ATPase and ClC-5 (Fig.
7).
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To test the colocalization of ClC-5 and H+-ATPases in the
same membranes, membrane vesicles were immunoisolated with the SS53 antibody and then analyzed by immunoblotting. As shown in Fig. 8, vesicles immunoisolated by the
anti-ClC-5 antibody showed abundant ClC-5 labeling in low-speed and
high-speed sedimentated fractions, whereas no ClC-5 or
H+-ATPase was recovered when nonimmune rat IgG was used for
vesicle isolation (Fig. 8, A and B). Immunoblotting of
ClC-5-immunoisolated vesicles revealed a clear presence of the 31-kDa
H+-ATPase in both fractions (Fig. 8B). Although the
separation of the membrane fraction is not perfect, these results
indicate that H+-ATPase and ClC-5 colocalize in the same
membranes in the plasma membrane and intracellular vesicle membrane.
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DISCUSSION |
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Localization of ClC-5 along the nephron in the mouse kidney. In this study, we successfully made a ClC-5-specific rat monoclonal antibody and examined its cellular and subcellular localization in the mouse kidney by use of immunoblotting and light- and electron-microscopic immunohistochemistry.
Immunoblotting demonstrated selective and specific labeling of ClC-5 using membrane fraction of mouse kidney. In mouse ClC-5 localized throughout all proximal tubule segments (S1, S2, and S3 segments). In the proximal tubule cells, the intense staining of ClC-5 was evident in both the brush-border membrane and subapical region (Figs. 3 and 6). The intercalated cells in the CCD were also clearly stained. In contrast, the staining of thick ascending limb of Henle's loop was minimal. According to a recent report by Teng-umnuay et al. (30), intercalated cells in the cortical and outer medullary collecting ducts of the mouse are composed of three morphologically and immunologically distinct cell types in a pattern similar to that of the rat. Most of the ClC-5-positive intercalated cells were type A intercalated cells characterized by an apical staining of H+-ATPase.
The immunohistological properties of ClC-5 localization along the nephron in the mouse kidney were basically similar to those of rat recently reported by Gunther et al. (13). However, the cellular and subcellular localization was somewhat different in two points: 1) in the proximal tubule cells, the labeling of the brush-border membrane was stronger in our study, suggesting that the physiological roles of ClC-5 are important in the brush-border membrane as well as in the cytoplasmic vesicles; 2) the ClC-5 expression was evident in the intercalated cells in the cortex, but the intercalated cells in outer medullary collecting duct (OMCD) almost completely lacked immunoreactive ClC-5 (Fig. 5). However, Gunther et al. showed the staining of ClC-5 in the type A intercalated cells in OMCD as well as in CCD. Although the reason for this discrepancy between rat and mouse is not clear, we should keep in mind the following possibilities: 1) the partitioning of membrane transport protein between brush-border membrane and subapical vesicles is quite dynamic, 2) the differences in relative distribution among these two membranes between different studies may be differences in the conditions under which the animals are kept (diet, hydration, etc.). In addition, our data may also suggest that there is heterogeneity between the type A intercalated cells in CCD and OMCD in the mouse kidney.
The similarity between the localizations of ClC-5 and H+-ATPase reported in the literature led us to examine whether they are colocalized (3, 30). According to the analysis by confocal microscope, H+-ATPase was always present in the tubular cells in which ClC-5 was expressed, but ClC-5 was not always stained in the tubular cells in which H+-ATPase was expressed. The intercalated cells in OMCD also showed this character. It is tempting to speculate that there are other ClC chloride channels together with H+-ATPase in these cells. Other outwardly rectifying chloride channels are known to be expressed in the kidney, e.g., we have recently demonstrated that ClC-3 is expressed in the kidney and that it evokes outwardly rectified currents in the same manner as ClC-5 (17).
Physiological roles of ClC-5 in relation to Dent's disease. When we consider that mutations in the CLCN5 gene cause Dent's disease and that functional expression of these mutations causes the loss of function, we gain insights into the physiological roles of ClC-5. Low-molecular-weight proteinuria is a distinct characteristic of patients associated with mutations of the CLCN5 gene. This phenotypic characteristic implies that ClC-5 might play an important role in the reabsorption of low-molecular-weight protein in the proximal tubule. Previous studies have demonstrated that the low-molecular-weight proteins filtered by the glomerulus were reabsorbed by receptor-mediated endocytic uptake in the proximal tubule cells (4, 21). In this process, the endosomal acidification mediated by H+-ATPase has been considered to play important roles in maintaining the endocytic activity (5, 9, 12, 32). Chloride channel(s) has been regarded as an indispensable element of this process in the dissipation of the electrical gradient generated by H+ pump in the endosomes vesicles, but the molecular identity of such chloride channels remains elusive (10). The striking colocalization of ClC-5 and H+-ATPase in the apical cytoplasmic vesicles in the proximal tubule strongly suggests that ClC-5 plays such a role.
However, another unsolved question remains about the channel
characteristic of ClC-5. In the heterologous expression system using
Xenopus oocytes (19, 29) or CHO cells (27), ClC-5 channel elicits strong outwardly rectifying Cl currents
passing large currents only above +20 mV. Its inward currents were
almost completely inactivated at negative potentials in the
Xenopus oocytes, whereas small inward currents could be detected in the stably transfected mammalian cultured CHO cells. If
ClC-5 was the channel to shunt the voltage created by the
H+ pump, then the expected orientation of
Cl
currents should be inward (Cl
efflux). Thus, the strict outward rectification of the
Cl
currents evoked in the expression studies were
oriented in the direction opposite to that needed for endosomes. The
reason for this discrepancy is not clear at present, and we speculate
that some undefined factor(s) that regulates the
rectification of the ClC-5 channel is missing in the heterologous
expression system.
In summary, ClC-5 was abundantly present not only in proximal tubule cells, but also in type A intercalated cells of CCD in parallel to H+-ATPase. ClC-5 seems likely to work cooperatively with H+ pump as an essential element for acidification of endosomes and proton secretion into the lumen. These findings suggest that the molecular abnormalities of the ClC-5 channel cause the dysfunction of proximal tubule and type A intercalated cells.1
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ACKNOWLEDGEMENTS |
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We thank Dr. Fumihiro Shigei for encouragement of this study and Ms. M. Goto for technical assistance.
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FOOTNOTES |
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This work was supported in part by Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan.
1 During the submission and revision of this article, two independent studies were published reporting the immunolocalization of ClC-5 in rat kidney [Luyckx, V. A., F. O. Goda, D. B. Mount, T. Nishio, A. Hall, S. C. Hebert, T. G. Hammond, and A. S. L. Yu. Am. J. Physiol. 275 (Renal Physiol. 44): F761-F769, 1998] and human kidney (Deduyst, O., P. T. Christie, P. J. Courtoy, R. Beauwens, and R. V. Thakker. Hum. Mol. Genet. 8: 247-257, 1999). In these studies, polyclonal antibodies were raised against the ClC-5 peptides, in which ClC-5 isoform-specific antibody fractions were prepared by immunoadsorption against the corresponding fusion proteins or synthetic peptides of ClC-3 and ClC-4. Our finding is quite different in immunolocalization in the thick ascending limb of Henle's loop and/or intercalated cells of the collecting ducts. It is not clear whether the difference is due to the species and/or methodology, especially the modification of antibody properties following repeat immunoadsorption.
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: H. Sakamoto, Second Department of Internal Medicine, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8519, Japan (E-mail: hsakamoto.med2{at}med.tmd.ac.jp).
Received 20 November 1998; accepted in final form 1 July 1999.
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