1 Department of Nephrology, Göteborg University, SE-405 30 Göteborg, Sweden; and 2 Division of Nephrology, Albert Einstein College of Medicine, Bronx, New York 10461
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ABSTRACT |
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It has been suggested that proteinuria is
caused by alterations of the charge selectivity of the basement
membrane and/or the epithelial cell layer (podocytes). However, recent
findings suggest that the endothelial luminal surface coat, consisting of proteoglycans with their connected glycosaminoglycan (GAG) branches
and glycoproteins, may contribute to the permselectivity. Therefore, we
wanted to investigate the effects on endothelial GAG synthesis during
normal and pathological conditions. We treated glomerular endothelial
cell cultures with puromycin aminonucleoside (PAN, a nephrosis-inducing
agent) or interleukin-1 (IL-1
) for a total of 72 h and
compared the metabolic turnover and incorporation of
[35S]sulfate during the last 2 days. In control cultures,
the GAG content in the media supernatants increased 66 ± 6%
(mean ± SE) between 12 and 42 h of incubation with
radioactivity (P < 0.01, n = 8). The
content of 35S-labeled GAGs in the media was reduced by
31 ± 1% by PAN (P < 0.001, n = 8) and increased by 141 ± 15% by 10 U/ml IL-1
(P < 0.01, n = 8). Treatment with
enzymes revealed a dominance of heparan, chondroitin, and dermatan
sulfate GAGs. Thus the glomerular endothelial cell production of GAGs
was increased by IL-1
and reduced by PAN. Therefore, it is
conceivable that certain nephrotic conditions may be due to endothelial
dysfunction, rather than other renal causes.
endothelium; kidney glomerulus; interleukin-1; proteoglycan; puromycin aminonucleoside
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INTRODUCTION |
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GLOMERULAR PERMSELECTIVITY is mainly thought to be regulated at the level of the glomerular basement membrane and/or the podocyte slit diaphragm. The endothelial cells of the glomerulus are highly fenestrated and, thus, have not been considered to have prominent restrictive properties. However, recent findings suggest that the glomerular endothelial fenestrae may indeed be covered by a diaphragm similar to the diaphragm that covers fenestrae of other capillary beds (33). The protein PV-1, a component of the diaphragms of fenestrae and caveolae, is not present in the glomerular endothelium (40-42). Therefore, the glomerular endothelium may have properties different from those of other fenestrated capillary beds.
Earlier studies show a thick cell coat (glycocalyx) covering the endothelial cells (25, 29, 32, 46). The cell coat is a matrix-like gel composed of proteoglycans, with negatively charged as well as neutral glycosaminoglycans (GAGs), glycoproteins, and plasma proteins. In particular, the plasma protein orosomucoid seems to be vital for maintaining normal capillary permeability (8, 11, 12). Previously, our laboratory also demonstrated that orosomucoid is produced by endothelial cells (37). The negatively charged components are abundant in this gel-like cell coat, which is suggested to constitute at least part of the charge barrier in the kidney and other organs (1, 38, 39, 46).
The production of GAGs by the glomerular cells was first reported in
the early 1980s (18). Most studies have dealt with the
mesangial and epithelial cells; only a few studies have focused on the
glomerular endothelial cells (GECs). Kasinath (21) studied sulfated GAGs produced by GECs under control conditions and in the
presence of transforming growth factor-1 (TGF-
1). TGF-
1 caused
an almost twofold increase in the synthesis of GAGs. Moreover, it was
shown that the endothelial cells synthesize not only heparan sulfate
but also chondroitin and dermatan sulfate (21, 35). Other
studies measured the GAG synthesis and turnover in vitro and in vivo
and in response to puromycin aminonucleoside (PAN). Akuffo et al.
(2) found that glomerular GAGs mainly in the glomerular
basement membrane and mesangial matrix have a more rapid turnover rate
than GAGs in other tissues. They also observed a decrease in GAG
turnover rate in rats in response to PAN at day 5 of
treatment, when proteinuria was significant. A similar finding was
reported earlier for the glomerular basement membrane by Garin and
Shirey (10). In another study, it was shown that the
glycocalyx covering the endothelial cells contains the GAG hyaluronan,
which is needed to maintain a selective barrier to macromolecules
(14).
Our concern was that because so few reports deal with the endothelial
cell coat as a part of the permselective barrier in the glomeruli,
its involvement in certain pathological conditions of the kidney could
have been overlooked. To address this concern, we treated cultures of
bovine GECs with PAN or interleukin (IL)-1 to assess whether the
synthesis of sulfated GAGs was altered in any way. PAN was chosen
because it has previously been extensively used to induce a
nephrosis-like condition in vivo in the rat (30, 34).
IL-1
is a cytokine with a wide variety of proinflammatory actions,
such as endothelial cell activation and induction of leukocyte adhesion
molecules and other cytokines, as well as inducible nitric oxide
synthase (9). Several studies have shown that IL-1
increases the amount of GAG synthesis by glomerular cells (20, 26, 44). In addition, it has been shown that
increased biosynthesis of GAGs enhances glomerular injury
(6, 45).
Our primary goal was to analyze the hydrodynamic size and chemical
composition of the molecules produced by the endothelial cells. The
rate of production over time was also studied and compared with that in
the presence of PAN or IL-1.
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METHODS |
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Medium and reagents. GECs were cultured in culture flasks (Costar, Cambridge, MA) in a humidified, 5% CO2 atmosphere at 37°C. The culture flasks were coated with attachment factor (gelatin; Cascade Biologics, Portland, OR). RPMI 1640 medium was supplemented as follows: penicillin, streptomycin, and amphotericin B (Cascade Biologics), 20% fetal calf serum (FCS), bovine fibroblast growth factor (acidic, 8 ng/ml for cloning medium and 4 ng/ml for growth medium; R & D Systems), bovine brain extract (700 µl from stock solution; BioWhittaker, Västra Frölunda, Sweden), and heparin (2 µg/ml; Lövens, Malmö, Sweden). Trypsin (0.125%; Life Technologies, Täby, Sweden) was used for passaging of the cells, and collagenase (type IV, Sigma, St. Louis, MO) was used to digest the whole glomeruli. Stainless steel sieves (180, 106, and 75 µm; Endecotts, London, UK) were used to extract the glomeruli.
Isolation of glomeruli. Kidneys from calves younger than 1 yr were obtained from a slaughterhouse; within minutes after harvest, they were placed in medium containing antibiotics and stored on ice. Within 1 h after harvesting of the kidney, glomeruli were isolated and treated with collagenase to obtain single cells and cell clusters according to the protocol described by Ballermann (5). The pellet containing single cells and cell clusters was resuspended in cloning medium and plated onto gelatin-coated petri dishes. When the endothelial cell colonies had reached ~300 cells, they were transferred to 24-well plates by the use of cloning cylinders. The cells were subcloned in the event that pure cultures were not obtained the first time. When the cells were confluent, they were passaged and moved to 25-cm2 flasks, and the medium was changed to growth medium.
Immunohistochemistry.
The cells were grown in chamber slides (Becton Dickinson) precoated
with gelatin (Cascade Biologics) until they reached 70% confluence.
The cells were rinsed briefly with 0.01 M PBS-0.01% Triton X-100, pH
7.4, and fixed with 4% paraformaldehyde-PBS, pH 7.4, for 15 min at
room temperature. After fixation, the cells were rinsed with PBS-Triton
X-100 for 20 min. The cells were incubated in 50 mM NH4Cl
in 0.01 M PBS-0.15 M NaCl, pH 7.4, to block aldehyde groups. After they
were rinsed, the cells were treated with 0.1% Triton X-100-PBS for 30 min and incubated with 5% low-fat milk in PBS for 1 h at room
temperature. Primary human von Willebrand factor antibody (rabbit
monoclonal; catalog no. F-3520, Sigma), diluted 1:500 in the blocking
solution, was applied, and the cells were incubated for 1 h at
room temperature. As a control for contaminating cells in the culture,
smooth muscle -actin antibody (mouse monoclonal; catalog no. A-2547,
Sigma), diluted 1:400, was used under the conditions described above.
As a negative control, we used cells in which incubation with the
primary antibody was replaced by incubation with rabbit or mouse
nonspecific IgG (Sigma), and a different cell type (rat cardiomyocytes)
was incubated as described above for the GECs. The cells were incubated
with secondary antibodies conjugated with horseradish peroxidase (HRP;
Amersham Pharmacia Biotech, Uppsala, Sweden) for 1 h and then
developed with diaminobenzidine-H2O2.
Further characterization of the cells using acetylated low-density lipoprotein. The cells were treated with trypsin and seeded onto chamber slides (Falcon, Becton Dickinson, Meylan Cedex, France) precoated with gelatin (Cascade Biologics). The cells were allowed to grow under normal conditions until they reached 70% confluence. The medium was removed, and 0.3 ml of acetylated low-density lipoprotein (Dil-Ac-LDL; Biomedical Technologies, Stroughton, MA), diluted to 10 µg/ml in RPMI medium, was added to each well. After incubation for 3 h at 37°C, the cells were washed briefly three times with 0.01 M PBS, pH 7.4. The cells were fixed with 3% formaldehyde for 20 min on a rotary shaker at room temperature and then washed once with PBS. The PBS and the chamber wells were removed, and the cells were mounted. The cells were examined by fluorescence microscopy at 570 nm.
Biosynthesis of GAGs.
GAG biosynthesis by GECs was measured by a modification of the method
of Arisaka et al. (3). The cells (P6) were
allowed to grow to 70% confluence in 75-cm2 culture flasks
(Costar, Cambridge, MA) before they were stimulated with 1 nM PAN
(Sigma) or 10 or 20 U/ml IL-1 (Sigma). Eight 75-cm2
culture flasks were used for each concentration. As control, we used
eight flasks of nonstimulated cells grown under normal conditions.
Stimulation was performed for 24 h at 37°C in a humidified, 5%
CO2 atmosphere. The cells were "starved" in standard
medium with 2% FCS and half of the amount of bovine brain
extract during the study. For metabolic labeling of GAGs,
35S-labeled sodium sulfate (10 µCi/ml; NEX041, NEN,
Boston, MA) was added to each flask after initial treatment with the
test substances for 24 h. Conditioned media from the cell flasks
were collected 12, 24, 36, and 48 h after the
35S-labeled sodium sulfate was added. At 72 h, after
the cells were washed extensively with Hanks' balanced salt solution,
cell layers were scraped off and the resultant cell suspension was
centrifuged. The protein content of the cell fraction was measured by
the method of Lowry et al. (24).
HPLC analysis of the cell culture media. The samples from the cell culture media at different hours of stimulation were fractionated (BioSep SEC-S3000 column, Phenomenex, Torrance, CA) according to molecular size as follows: 41-50, 50-59, 59-69, 69-79, 79-90, 90-102, and >102 Å. Then each sample was placed in a vial with fresh quenching fluid and analyzed for 10 min in a beta counter (model L6 6500, Beckman). Calibrated standards were analyzed after every 16th sample to allow accurate estimations of Stokes-Einstein (SE) radii.
Enzyme digestion of proteoglycans. Three different enzymes known to degrade proteoglycans were used to treat the final media samples (taken at 72 h). After digestion, the samples were fractionated in an HPLC (see HPLC analysis of the cell culture media). After fractionation, the samples were analyzed using a scintillation counter for assessment of the amount of degraded GAGs compared with the same sample without enzyme treatment.
Chondroitin ABC lyase (EC 4.2.2.4, Sigma; dissolved in 1 M Tris · HCl, pH 8.0), which degrades chondroitin and dermatan sulfate, in a final concentration of 0.1 U/ml was incubated with the samples for 2 h at 37°C. Heparinase III (EC 4.2.2.8, Sigma; dissolved in 0.1 M sodium acetate, pH 7.0) in a final concentration of 50 mU/ml was incubated with the samples for 4 h at 37°C. Hyaluronidase (EC 3.2.1.35, Sigma; dissolved in sodium phosphate buffer + 0.15 M NaCl, pH 6.0) in a final concentration of 1 mg/ml was incubated with the samples for 1 h at 37°C. This enzyme degrades hyaluronan and chondroitin sulfate (A and C), but because hyaluronan does not contain sulfate, the treatment will reveal the chondroitin sulfate component of the GAGs. All three enzyme reactions were ended by lowering the temperature toWestern blot. The proteins were denatured and then separated on a 10% Bis-Tris polyacrylamide gel (Nupage, Novex, San Diego, CA), which was run at 200 V for 35 min. The proteins were then blotted onto a polyvinylidene difluoride membrane (Novex) in a standard manner (3 h at 25 V and 0.25 A). After transfer, the membrane was blocked for 1 h at room temperature in 5% nonfat dry milk-0.25% gelatin dissolved in Tris-buffered saline (10 mM Tris · HCl, pH 7.5, and 150 mM NaCl) with 0.1% Tween. The membrane was incubated for 1 h at room temperature with a primary bovine heparan sulfate proteoglycan antibody (mouse monoclonal IgG1, Biogenesis, Poole, UK) diluted 1:100 in the blocking solution. After the membrane was rinsed in TBS-0.1% Tween, it was incubated with a secondary antibody conjugated with HRP (anti-mouse HRP, Amersham). The protein-antibody complexes were visualized by enhanced chemiluminescence (ECL+ and Hyperfilm ECL, Amersham) according to the manufacturer's instructions.
RT-PCR. Synthesis of cDNA was carried out with 1 µg of RNA from GEC; as positive control, we used RNA from bovine kidney. The RT reaction was carried out for 50 min at 42°C and for 5 min at 70°C in RT buffer (Promega, Madison, WI) in the presence of 1 µg of random primer (Promega), 15 U of avian myeloblastosis virus RT (Promega), 20 U of RNasin (Promega), and dNTP mix (1.5 mM of each base) in a total volume of 20 µl. PCR amplification was performed in a 50-µl reaction buffer with 5 µl of the cDNA used as a template, PCR buffer [50 mmol/l KCl, 10 mmol/l Tris · HCl (pH 8.3), 1.5 mmol/l MgCl2, and 0.001% (wt/vol) gelatin (Perkin-Elmer Cetus, Norwalk, CT)], 0.2 µmol/l of each forward and reverse heparan sulfate primer (oci5), dNTP (10 nM each), and 2.5 U of Taq DNA polymerase (Promega). The following PCR program was used: 94°C for 4 min (94°C for 15 s, 54°C for 15 s, 72°C for 30 s) for 30 cycles and 72°C for 10 min (GeneAmp PCR System 2400, Perkin-Elmer). The sequences of the heparan sulfate primers were as follows: 5' AAC TAC CCA AGC CTG ACT CCA C 3' (forward) and 5' ATC TCC ACC ACA CCT GCC ATA C 3' (reverse) with a product size of 479 bp. The primers were located between 541-562 (forward) and 998-1,019 (reverse) bp in the human heparan sulfate cDNA sequence.
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RESULTS |
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GEC culture.
After ~7 days, the glomerular cells started to appear as small
colonies of different morphology, of which about one of five was of
endothelial origin. After 12 days, we observed clones large enough for
transfer to a 24-well plate (Fig.
1A). The cells were of
endothelial origin as judged by light microscopy and by endocytosis of
the endothelial cell-specific marker Dil-Ac-LDL as observed by
fluorescence microscopy (Fig. 1B) and expression of factor VIII-related antigen. The cells stained negatively for smooth muscle
-actin, and no staining was observed for Dil-Ac-LDL or factor
VIII-related antigen in the negative controls.
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GAG synthesis.
The fractionated cell media showed that most incorporated radioactivity
in the control cultures was in the hydrodynamic radius range of
70-90 Å. During the control period, there was a gradual increase
in the concentration of incorporated radioactivity from 12 to 42 h
(mean of 36- and 48-h samples) of 66 ± 6% (n = 8, P < 0.01). At 12 h, three groups contained
similar amounts of sulfated GAGs, namely, controls 3,180 ± 270 cpm/ml, puromycin 3,900 ± 270 cpm/ml, and IL-1 (10 U/ml)
3,820 ± 590 cpm/ml, while IL-1B (20 U/ml) was significantly
increased already at 10 min (5,800 ± 660 cpm/ml). Treatment with
PAN reduced the concentration by 31 ± 1%, P < 0.001, n = 8, during the same time period. IL-1
, on
the other hand, increased the synthesis of sulfated macromolecules significantly compared with control during the entire time period (Fig.
2). At 48 h, the concentration of
35S-labeled GAGs was twice as high as at 12 h for
both IL-1 treatment groups (10 and 20 U/ml, P < 0.01, n = 8). Moreover, treatment with IL-1
(10 U/ml) gave a value of 9,200 ± 570 cpm/ml at 42 h.
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Enzyme treatments.
After enzyme treatment, we found most of the cell medium activity in
the fractions containing small molecules, confirming that the large
proteoglycans had been degraded (Fig. 4).
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Protein synthesis.
The protein concentrations were similar for the different groups
(n = 8 in each group), indicating similar numbers of
cells and no apparent general toxicity of the drugs (Fig.
6).
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PCR. The expression of heparan sulfate proteoglycan mRNA from the different groups of GEC and cortex from whole kidney was examined by RT-PCR. Transcripts of the expected size (479 bp) were found in all samples (data not shown).
Western blot.
Western blot analysis of heparan sulfate proteoglycan was performed,
and 55-kDa bands in the protein extracts from the GECs indicate that
heparan sulfate core protein was present in all treatment groups (Fig.
7).
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DISCUSSION |
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The production of GAGs by GECs has been far less studied than GAG
synthesis by epithelial and mesangial cells. In this study, we
demonstrate that PAN and IL-1 affect GAG synthesis in the GECs. Thus
the amount of metabolic labeling was decreased in the PAN-treated cell
cultures. When the cells were stimulated with IL-1
, an increase in
metabolic labeling was observed. Because the protein concentrations
were similar in the different treatment groups (Fig. 6), it was mainly
the degree of sulfation that was affected. The 35S-labeled
GAG content of the culture medium was fractionated according to size by
HPLC. When examining the size distribution of the samples, we found a
peak at 50-90 Å. In all molecular size fractions, we found a
decreased synthesis of GAGs after treatment with PAN and an increased
synthesis after IL-1
treatment. The pattern was most pronounced in
the fractions of the largest molecules (>70 Å). This fits well with
the notion that proteoglycans are large molecules, consisting of a core
protein with GAG chains, such as heparan sulfate, attached (13,
23). The sulfation is carried out by several isoenzymes, and the
reactions are often incomplete, leading to a large variation in
sulfation of the heparan sulfate GAGs (22, 31).
The time-course study showed that the PAN effect had a slower onset
than the IL-1 effect. The significant reduction induced by PAN after
36 h remained at this low level at 48 h. The high dose of
IL-1
induced a significant increase at 12 h (P < 0.05), and the concentration of sulfated GAGs continued to rise
during the time course of the study (P < 0.01). The
lower dose of IL-1
(10 U/ml) resulted in no significant increase at
12 h, but thereafter the pattern was similar to the higher dose
(20 U/ml). For the control cells, the concentration of sulfated GAGs
increased slightly during the experiment (Fig. 2). In addition, we
examined the presence of mRNA for heparan sulfate in the RNA obtained
from the cells. All groups showed expression of heparan sulfate mRNA.
We also obtained evidence for heparan sulfate core protein synthesis by the cells.
It is known that human umbilical vein endothelial cells produce mainly heparan sulfate (29). However, in a study investigating GECs, it was shown that mainly chondroitin sulfate chains were attached to the core proteins of the proteoglycans (35). This difference in expression of GAGs could be interpreted as macro- and microvascular endothelium reflecting different characteristics, as previously noted by Zetter (48). We found that three main GAGs were present in bovine GECs: heparan, chondroitin, and dermatan sulfate (Fig. 5).
PAN causes nephrotic syndrome in rats when administered intraperitoneally (19, 27, 30, 34). It is therefore interesting to note that the drug also downregulates the incorporation and synthesis of GAGs by the endothelial cells. The endothelial cell coat (27, 30, 34) has been suggested to contribute to the permselective properties of the capillaries (1, 17, 38, 39, 47). A decreased synthesis of GAGs could increase permeability of the layer covering the fenestrae and, thus, increase the sieving of macromolecules (14). It has previously been shown that PAN has effects on the GEC fenestrations, which were decreased in size and amount during PAN-induced nephrosis (4). Thus, during certain pathological conditions, the charge barrier may be reduced as a result of a decreased amount of negatively charged components of the endothelial cell coat. This is in line with previous observations from our group where we showed that orosomucoid, a glycoprotein known to be important for the glomerular permselectivity [probably because of its high negative charge (1, 11, 12)], was synthesized by the endothelial cells (37). Thus it seems that the GECs can modify their expression and turnover of cell coat components in response to different stimuli. The importance of the GAGs is illustrated in a study where the gene for the enzyme responsible for sulfation (heparan sulfate 2-O-sulfotransferase) of heparan sulfate was knocked out. The mice did not develop kidneys as a result of failure of ureteric bud branching and mesencymal condensation, and they died shortly after birth (7).
In this study, we found marked changes in the degree of sulfation with
PAN and IL-1 treatment but no significant changes in protein
concentrations. This is in accordance with the observations of
Holthöfer et al. (16), who found similar effects of
PAN on the sulfation of podocalyxin.
IL-1 is a proinflammatory cytokine that is involved in the
acute-phase reaction during inflammation and is known to activate endothelial cells. In this study, we used one of the two available molecular forms of IL-1, IL-1
. The two forms (IL-1
and IL-1
) have similar effects and bind to the same receptors (9).
Our choice of IL-1
was based on a previous study on endothelial cell activation (44). In that study, it was also shown that
GECs synthesize IL-1
constitutively, but the major effects from
IL-1
are usually thought to come from infiltrating macrophages.
IL-1
has been shown to strongly and rapidly upregulate inflammatory mediators such as intercellular adhesion molecule-1 and monocyte chemoattractant protein-1 (15, 43). It has also been shown that IL-1
upregulates the expression of the neutral GAG hyaluronan in microvascular endothelium (28). In addition to
upregulating inflammatory mediators, we found that IL-1
also induced
an increased synthesis of sulfated GAGs by the GECs, an effect that can
be a part of the cell defense to maintain the permselective properties of the glomerular barrier (20). There may, however, be
differences between micro- and macrovascular endothelium, because the
synthesis of GAGs was downregulated when porcine aortic endothelial
cells were stimulated with IL-1
(36).
In summary, the GECs produced sulfated GAGs with a hydrodynamic size of
50-90 Å. Heparan, chondroitin, and dermatan sulfate were the
three main GAG components synthesized by bovine GECs. The rate of
production of GAGs decreased during PAN treatment and increased during
IL-1 treatment. We propose that the GAGs produced by the GECs may be
a vital part of the glomerular charge barrier. Indeed, certain
nephrotic conditions may actually be due to endothelial dysfunction,
rather than pure renal disorders.
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ACKNOWLEDGEMENTS |
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We appreciate the gift of calf kidneys from M. Larsson (Dalsjöfors Slaughterhouse).
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FOOTNOTES |
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This study was supported by Swedish Medical Research Council Grants 9898 and 2855, the Knut and Alice Wallenberg Research Foundation, the Ingabritt and Arne Lundberg Research Foundation, the National Association for Kidney Diseases, the Swedish Society for Medicine, and Sahlgrenska University Hospital Grant LUA-S71133.
Address for reprint requests and other correspondence: J. Sörensson, Dept. of Nephrology, Göteborg Univ., Box 432, SE-405 30 Göteborg, Sweden (E-mail: jenny.sorensson{at}kidney.med.gu.se).
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. Section 1734 solely to indicate this fact.
First published October 8, 2002;10.1152/ajprenal.00257.2002
Received 17 July 2002; accepted in final form 3 October 2002.
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