Departments of 1 Pathology and 2 Cell Physiology, Academic Medical Center, University of Amsterdam, 1105 AZ Amsterdam, The Netherlands; and 3 Institute of Physiological Chemistry, Martin Luther University, Halle-Wittenberg, Halle, Germany D-06097
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ABSTRACT |
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Minimal change nephrosis (MCN) is characterized by massive proteinuria and ultrastructural alterations of glomerular visceral epithelial cells (GVEC). MCN has been associated with elevated production of interleukin (IL)-13 by circulating T lymphocytes and with T helper 2 lymphocyte-dependent conditions. We recently showed that GVEC express IL-4 and IL-13 receptors and that IL-4 and IL-13 increase transcellular ion transport over GVEC monolayers. We therefore hypothesized that IL-13 may directly injure GVEC. Here we demonstrate that IL-4 and IL-13 induce bafilomycin A1-sensitive basolateral proton secretion by cultured GVEC, indicating involvement of vacuolar H+-ATPase. The effects of IL-4 and IL-13 were accompanied by redistribution of the small GTPases Rab5b and Rab7, as shown by confocal immunofluorescence studies. Furthermore, Western blot analysis and assays for cysteine proteinase activity revealed basolateral secretion of the lysosomal proteinase procathepsin L by cultured GVEC, stimulated by IL-4 and IL-13. We speculate that IL-4 and IL-13 influence intracellular trafficking of proteins and promote proteolysis at the basolateral surface of GVEC, which may play a pathogenic role in altered glomerular permeability.
podocytes; minimal change nephrosis; vacuolar hydrogen-adenosinetriphosphatase; Rab guanosinetriphosphatases; cathepsins
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INTRODUCTION |
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GLOMERULAR VISCERAL
EPITHELIAL cells (GVEC), or podocytes, are highly specialized
cells that contribute to the specific characteristics of the glomerular
filtration barrier. They cover the negatively charged glomerular
basement membrane (GBM) with multiple interdigitating foot processes
that are interconnected by slit diaphragms. In the nephrotic syndrome,
GVEC are flattened and have retracted their foot processes. This
so-called foot process effacement is thought to be a common reaction
pattern of GVEC to extrinsic damage, such as direct intoxication by
puromycin aminonucleoside or adriamycin or damage secondary to immune
complex deposition (19). Alternatively, foot process
effacement can be the result of an intrinsically altered expression of
proteins that are indispensable for the formation or maintenance of
foot processes. In this respect, somatic mutations or deletions of
genes encoding nephrin (20, 28), p130Cas
ligand with multiple SH3 domains/CD2-associated protein (CMS/CD2AP) (30), podocin (7), and
4-actinin (18) cause hereditary nephrotic
syndromes with renal lesions restricted to GVEC. Also, modification of the GBM composition by genetic knockout of the S-laminin gene, can lead to foot process effacement and proteinuria (26).
In nonfamilial nephrotic syndromes with renal lesions restricted to GVEC, such as minimal change nephrosis (MCN) and idiopathic focal segmental glomerulosclerosis (FSGS), it is unknown whether the primary cause is extrinsic damage to GVEC or an intrinsically altered expression or distribution of certain proteins involved in GVEC function. MCN and FSGS may be related to each other (14), and for both diseases, factors that are potentially noxious to GVEC have been characterized in the peripheral blood of these patients. T lymphocytes from patients with MCN have been shown to express more interleukin (IL)-13 than T lymphocytes from healthy controls or from patients in remission (21, 35). IL-13 is a pleiotropic cytokine that, like IL-4, is produced by activated CD4+ T helper 2(Th2) lymphocytes. Activation of Th2 lymphocytes plays a key role in development of atopy and allergy. A possible role for Th2 cytokines in the induction of proteinuria in MCN is supported by clinical observations on the association of MCN with atopy and the apparent induction of MCN by allergic events in some patients (1).
We showed previously (33) that GVEC in vivo and in vitro express specific receptors for IL-4 and IL-13 and that GVEC in vitro are susceptible to these cytokines. In the present work, we show that both IL-4 and IL-13 induce a bafilomycin-sensitive transport of protons toward the basolateral surface of GVEC in vitro and may affect intracellular trafficking of proteins and degradation of basal membrane proteins by cysteine proteinases.
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METHODS |
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Reagents. DMEM (catalog no. 1233254) was purchased from ICN (Costa Mesa, CA). Ham's F-10 (catalog no. 31550), Hanks' balanced salt solution (HBSS), and antibiotics were obtained from Life Technologies (Paisley, UK), Nu serum was from Becton Dickinson (Bedford, MA), and collagen (Vitrogen 100) was from Collagen Corp. (Palo Alto, CA). All other culture supplements and collagenase were from Sigma (St. Louis, MO). Recombinant rat IL-4 was obtained from Serotec (Oxford, UK). Hybridoma cells producing a blocking monoclonal antibody directed against rat IL-4 (OX81, mouse IgG1) were kindly provided by Dr. D. Mason (Sir William Dunn School of Pathology, Oxford, UK). Hybridoma cells producing a control monoclonal antibody TS2/9.1.4.3 (TS2, anti-human CD58, mouse IgG1) were obtained from the American Type Culture Collection (Manassas, VA). OX81 and TS2 were purified from hybridoma culture supernatants by protein A chromatography. Recombinant rat IL-13 and blocking anti-rat IL-13 antibodies (polyclonal rabbit IgG) were a kind gift of Dr. F. G. Lakkis (Emory University and Veterans Affairs Medical Center, Atlanta, GA). Bafilomycin A1 and DIDS were purchased from Sigma, and ethylisopropylamiloride (EIPA) was from Molecular Probes (Eugene, OR). Affinity-purified rabbit polyclonal anti-Rab7 antibodies were kindly provided by Dr. M. Zerial (European Molecular Biology Laboratories, Heidelberg, Germany). Rabbit polyclonal anti-Rab5b antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). FITC-conjugated goat anti-rabbit antibodies were from Jackson Immuno Research Laboratories (West Grove, PA), and TRITC-conjugated phalloidin was from Molecular Probes. Synthetic cathepsin substrates were purchased from Bachem (Bubenhorf, Switzerland), E-64 was from Sigma, and CA-074 Me was from Calbiochem (La Jolla, CA). Heparin was purchased from Leo Pharmaceutical Products (Weesp, The Netherlands). Cathepsin L was purified from rat liver lysosomes as described previously (23). Rabbit polyclonal antibodies against rat procathepsin L and monoclonal antibodies against human procathepsin L (CPLH 33/2, mouse IgG1) were generated as described previously (23, 34).
Cells.
Established lines of rat GVEC (29), which express the GVEC
differentiation marker synaptopodin (33), were cultured at 37°C in a humidified 5% CO2-95% air incubator on a type
I collagen gel. Collagenase type IA in HBSS (660 U/ml) was used to
detach cells before each passage. Culture medium consisted of a 1:1
ratio of DMEM and Ham's F-10, supplemented with 5% Nu serum, 25 ng/ml prostaglandin E1, 0.5 nM triiodothyronine, 10 nM sodium
selenite, 5 µg/ml transferrin, 50 nM hydrocortisone, 5 µg/ml
insulin, 100 U/ml penicillin, and 100 µg/ml streptomycin. For
monolayer experiments, cells were seeded on Transwell porous filters
(Costar, Cambridge, MA) with a pore diameter of 0.4 µm and a growth
area of 0.33 cm2 at a density of 150,000 cells/cm2. The inner and outer chambers contained 0.25 and
1.2 ml of medium, respectively. Transepithelial electrical resistance
(TER) was measured with a Millipore apparatus (Millipore, Bedford, MA). After reaching TER values >2.0 k · cm2, the
confluent monolayers were exposed to rat IL-4 (10 ng/ml), rat-IL-13 (1 U/ml), and/or blocking reagents in the basolateral compartment. In all
experiments, TER was monitored every 24 h. In some experiments,
basolateral medium was collected at various time points. These samples
were used to analyze pH of the medium in a Radiometer ABL505
(Radiometer, Copenhagen, Denmark) or to analyze presence of cysteine
proteinases as described in Fluorometric microassays for
cathepsin B, K, L, and S.
Immunofluorescence. Rat GVEC cultured on Transwell filters were fixed in situ with 4% paraformaldehyde in PBS, pH 7.4, for 20 min and permeabilized with 0.1% Triton X-100 in PBS for 1 min. The fixed cell monolayers were preincubated with 10% normal goat serum for 20 min, followed by incubation with the primary antibodies for 16 h at 4°C. Subsequently, antibody binding was detected with FITC-conjugated goat anti-rabbit antibodies. Cells were double-stained for F-actin with TRITC-conjugated phalloidin. Monolayers were viewed with a Leitz laser scanning confocal microscope (Leica, Heidelberg, Germany). FITC- and TRITC-derived images were corrected for cross-talk and merged using the Multicolor Analysis Software (Leica) and a look-up table to convert FITC signals to green, TRITC to red, and overlapping areas to white. Monolayers that had been basolaterally exposed to IL-4 or to IL-13 for up to 3 days were compared with untreated monolayers of the same age.
Fluorometric microassays for cathepsin B, K, L, and S.
Cathepsin activity in GVEC and in basolateral GVEC culture media was
determined by fluorometric microassays using
Z-Arg-Arg-4-methoxy--naphthylamide (MNA) as the substrate for
cathepsin B, Z-Leu-Arg-MNA as the substrate for cathepsin K, and
Z-Phe-Arg-MNA as the substrate for cathepsin B, L, and S. Medium was collected and concentrated 10-fold using Centricon
centrifugal filters (Millipore). Cells were incubated overnight with
extraction buffer containing 10 mM sodium cacodylate, 1 M NaCl, 0.1 mg/ml Triton X-100, 1 µM ZnCl2, and 0.1 mg/ml
NaN3. Aliquots (10 µl) of the cell extracts or of the
medium were incubated in 90 µl of 100 mM sodium phosphate buffer (pH
6.0 for cathepsin B and K, pH 5.5 for cathepsin L, and pH 7.5 for
cathepsin S) containing 1.3 mM EDTA, 1 mM freshly prepared
dithiothreitol, 2.7 mM freshly prepared L-cysteine, and 1 mg/ml of the substrate. In some experiments, heparin (final
concentration 150 U/ml) or cathepsin inhibitors E-64 (50 µM) or
CA-074 Me (40 µM) were added to the incubation medium. Fluorescence
of free MNA was measured in a Wallac 1420 VICTOR2
multilabel counter (Perkin Elmer, Wellesley, MA), applying excitation with a 355-nm wavelength and detecting fluorescence emission at a
430-nm wavelength, at 37°C, every 5 min. Enzyme activities in GVEC
culture medium were compared with the activity of a dilution curve of
purified rat cathepsin L.
Western blot analysis of procathepsin L secretion. Presence of procathepsin L protein in the basolateral culture medium was determined by Western blotting. Culture media were 10-fold concentrated as described in Fluorometric microassays for cathepsin B, K, L, and S. Subsequently, proteins in the concentrated samples were separated by 10% SDS-polyacrylamide gel electrophoresis and were blotted onto polyvinylidene difluoride membranes (Millipore). Staining with Ponceau S confirmed effective and equal protein transfer. Blocking was carried out with 5% blocking agent (Amersham, Little Chalfont, UK) in Tris-buffered saline containing 0.1% Tween 20 (Sigma) (TBSTM) for 1 h at room temperature. The membranes were subsequently incubated for 1 h with rabbit anti-rat procathepsin L antibodies, for 1 h with fluorescein-conjugated donkey anti-rabbit antibodies, and for 1 h with alkaline-phosphatase-conjugated anti-fluorescein antibodies, with extensive washing steps in TBSTM between all incubations. Binding was visualized using a chemifluorescence detection system (ECF; Amersham) and analyzed with an ImaGo imaging system (B&L Systems, Maarssen, The Netherlands).
Immunohistochemistry. The protein expression of (pro)cathepsin L in kidney was analyzed by immunohistochemistry on normal human kidney tissue (donor kidney unsuitable for renal transplantation) and on archival renal biopsies of six cases of MCN. Deparaffinized 5-µm-thick sections were preincubated with 10% normal goat serum (Sera Lab) and then incubated with monoclonal anti-human (pro)cathepsin L antibody at room temperature for 3 h. Subsequently, antibody binding was detected with horseradish peroxidase-conjugated goat anti-mouse IgG1 antibodies (Southern Biotechnology Associates, Birmingham, AL); 10% normal human ABO serum (CLB, Amsterdam, The Netherlands) was added to the secondary antibody to inhibit possible cross-reactivity to human immunoglobulins. Peroxidase activity was detected with 3-amino-9-ethylcarbazole (Sigma) and 0.03% H2O2. Negative controls were performed by replacing the first-step antibody with incubation buffer only.
Statistical analysis. Values are expressed as means ± SD when appropriate. To determine overall differences between the groups with respect to the variable considered, the Kruskall-Wallis rank-sum test was applied with correction for ties. For subsequent comparison of specific groups with the corresponding control group, differences in rank sum were analyzed using the Dunn procedure, applying correction for ties, for variable group size, and for multiple comparison of groups (11). Differences were considered statistically significant when P values were <0.05.
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RESULTS |
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Induction of bafilomycin-sensitive basolateral
H+ transport.
Within 3 days after seeding onto filters, rat GVEC formed confluent
monolayers with TER values of ~2.5 k · cm2,
rising to a maximum of 6.0 k
· cm2. As reported
previously (33), IL-4 and IL-13, when added to the
basolateral compartment of the cell monolayer, dose dependently caused
a strong decrease in TER to ~40% of the TER value of untreated controls. In that previous report, we showed that the effect of IL-4 and IL-13 on TER is the result of increased ion flux across GVEC
monolayers, whereas IL-4 and IL-13 do not alter GVEC monolayer permeability to macromolecules. In the present study, we observed that
the pH of the medium in the basolateral compartment was lowered during
exposure to IL-4 or IL-13, whereas the pH of the apical medium did not
change measurably. In 4 days of exposure to IL-4 (10 ng/ml) or IL-13 (1 U/ml), the pH of the basolateral medium gradually decreased to
7.21 ± 0.03 and 7.22 ± 0.02, respectively, compared with pH
7.52 ± 0.01 without exposure to IL-4 or IL-13 (Fig.
1; P < 0.001). This pH
shift in the buffered basolateral medium corresponds to a net secretion
of ~54 µmol H+/cm2 cells after exposure to
IL-4 or IL-13. Both the decrease in TER and the basolateral
acidification could be specifically blocked by monoclonal antibodies to
IL-4 (0.1 ng/ml) and polyclonal antibodies to IL-13 (1%), respectively
(Fig. 1), but not by species-matched and isotype-matched nonspecific
antibodies in comparable concentrations (not shown).
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Redistribution of Rab5b and Rab7.
V-ATPases mediate the acidification of multiple intracellular
organelles in eukaryotic cells. In proton-transporting renal epithelial
cells, the distribution of V-ATPases is dynamic. In response to
physiological stimuli, V-ATPases can be recruited toward the plasma
membrane by vesicular traffic (15). In this process,
endocytic vesicles containing the V-ATPase finally fuse with the plasma
membrane, which leads to insertion of the V-ATPase. To examine the role
of endosomal vesicular traffic in the activation of V-ATPase by IL-4
and IL-13 in our system, we studied the distribution of Rab5b and Rab7,
which are associated with the early and late endosomal compartments,
respectively. Figure 2 depicts the
reproducible differences in the staining pattern for both Rab5b and
Rab7 after treatment with IL-4 or IL-13. Untreated controls showed a
regular, dotted staining pattern in the apical pole of the cells. After treatment with IL-4 or IL-13, groups of cells displayed diffuse distribution of Rab5b and Rab7, whereas other cells retained the dotted
staining pattern or were not stained at all (Fig. 2). After 48-72
h of incubation with IL-4 or IL-13, the changes observed were less
prominent. All these findings indicate that the recruitment of V-ATPase
in GVEC on exposure to IL-4 or IL-13 was associated with the
redistribution of markers of the endosomal compartment.
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Secretion and expression of cysteine proteinases.
We speculated that the observed proton flux induced by IL-4 and IL-13
would affect the integrity and composition of basal membrane matrix by
activating proteolytic enzymes at the basolateral membrane. We
therefore studied the secretion of lysosomal cysteine proteinases,
which are optimally active under acidic conditions, by GVEC. By
fluorometric microassays with specific substrates, we found cathepsin
B-, K- and L-like activities in cell lysates (not shown). In
basolateral culture media, only cathepsin L-like activity was detected.
This activity was absent in culture medium that had not been incubated
with GVEC. The cathepsin inhibitor E-64, but not the cathepsin B
inhibitor CA-074 Me, could block the detected cathepsin L-like
activity. Furthermore, cathepsin L-like activity was enhanced by the
addition of heparin to the incubation medium (Fig.
3A), suggesting basal activity
of procathepsin L and autocatalytic cleavage into mature cathepsin L on
incubation with heparin. Cathepsin L-like activity was elevated in
media of GVEC that had been exposed to IL-4 and IL-13 (Fig. 3).
Cathepsin L-like activity in GVEC medium was maximal in incubation
buffer with pH 4.5-5.5 and absent at pH >7.0 (Fig.
4), suggesting the presence of cathepsin
L rather than cathepsin S. Cathepsin S cleaves the same substrate as
cathepsin L but is stable and operational at pH 7.5, in contrast to
cathepsin L (24). Western blot analysis confirmed
secretion of procathepsin L protein by GVEC and revealed stimulation of
procathepsin L secretion by IL-4 and IL-13 (Fig. 5).
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DISCUSSION |
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Our study describes polarized H+ transport by GVEC via a bafilomycin A1-sensitive mechanism, induced by exposure to Th2 cytokines IL-4 and IL-13. Polarized H+ transport in renal tubular cells has been well described and involves mainly the action of the EIPA-sensitive Na+/H+ exchanger in the proximal tubule and of the bafilomycin A1-sensitive V-ATPase in the collecting duct. The role of tubular H+ transport in the maintenance of acid-base and mineral balances in vivo has been well established, but the role of H+ transport in glomerular epithelial cells is less straightforward and may be of pathological significance.
First, the recruitment of V-ATPases may alter GVEC physiology. V-ATPases present in endo- and/or lysosomal vacuoles can be redistributed to the cell membrane by vesicular traffic (15). To study the role of endosomal vesicular traffic in the activation of V-ATPase in our system, we studied the expression of Rab5b and Rab7. Rab proteins are small GTP-binding proteins that regulate membrane traffic in the endocytic and biosynthetic pathways. Among them, Rab5 is localized to the early endosomal compartment and is involved in membrane transport to early endosomes (8). Of the three different Rab5 isoforms that have been reported, Rab5b is most abundantly expressed (10). Rab7 is localized to the late endosomal compartment (9) and is involved in membrane transport between early endosomes and lysosomes (13). We found an altered distribution of both Rab5b and Rab7 during exposure to IL-4 or IL-13, which was most marked after 24 h. Basolateral acidification by V-ATPase was not seen until 48 h of exposure to the cytokines, but recruitment of the pump may be an earlier event that remains as yet unobserved because of the buffering capacity of the medium. This is supported by the finding that only early addition of bafilomycin A1 to the medium, i.e., within 24 h after start of incubation, results in inhibition of the effects of IL-4 or IL-13 on TER and acidification (data not shown). Involvement of Rab7 in the recruitment of V-ATPase in bone-resorbing osteoclasts has been shown previously (27). Our data suggest that IL-4- or IL-13-induced recruitment of V-ATPase in GVEC is associated with an alteration in the endosomal compartment. Such alterations may interfere with normal vesicular transport that is essential for the formation and maintenance of the normal GVEC structure (31).
Second, the observed H+ flux may affect the tightly regulated interaction of GVEC and extracellular matrix by influencing pH-dependent processes at the basolateral membrane. To investigate this in more detail, we studied whether GVEC secrete the lysosomal cysteine proteinases cathepsins B, K, and L. These proteinases are synthesized as low-activity proenzymes that autoprocess to the mature enzymes under acidic conditions. Among them, cathepsin B is most abundant and widely expressed (6). Cathepsin K is secreted by proton-secreting activated osteoclasts (12). Cathepsin L can be secreted from a variety of cells, and this secretion can be regulated by growth factors such as platelet-derived growth factor and epidermal growth factor, by phorbol 12-myristate 13-acetate, and by activated oncogenes [reviewed by Ishidoh and Kominami (16)]. We observed that GVEC in vitro spontaneously secrete procathepsin L and that IL-4 and IL-13 stimulate this secretion. The cathepsin L-like activity secreted by GVEC was enhanced by incubation with heparin, which is in line with autocatalytic cleavage of procathepsin L into its mature form in the presence of negatively charged cell surface proteins (17). Also in vivo, GVEC focally expressed (pro)cathepsin L. So far, we have not obtained evidence for upregulation of cathepsin L expression in a limited series of biopsies of patients with MCN. However, immunohistochemistry does not allow us to examine the amount or the activity of secreted (pro)cathepsin L. Cathepsin L potently degrades both soluble and basement membrane type IV and V collagen, laminin, and proteoglycans (32) and is able to degrade GBM in vitro (3, 5). As such, cathepsin L may be involved in remodeling of GBM in concert with secreted matrix metalloproteinase (MMP)-2 and MMP-9 and tissue inhibitors of metalloproteinase (TIMP)-I and TIMP-II, which are also secreted by GVEC (25). Proteolytic activity may be enhanced in a pathogenic way on exposure to IL-4 or IL-13, which create an acidic environment favorable for proteolysis by cathepsin L. Baricos et al. (2, 4) demonstrated that administration of a cathepsin L inhibitor significantly reduced proteinuria in rats with experimentally induced, neutrophil-independent anti-GBM antibody disease, thereby supporting the idea that cathepsin L can contribute to glomerular injury leading to proteinuria.
Altogether, we show here that IL-4 and IL-13 in vitro influence intracellular trafficking of proteins and induce a basolateral H+ flux in GVEC, which favors proteolysis by cathepsin L at the basolateral membrane. In MCN, circulating IL-13 produced by triggered T lymphocytes (21, 35) could directly act on GVEC as observed in this study and thus may play a pathogenic role in altered glomerular permeability.
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ACKNOWLEDGEMENTS |
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We thank A. M. Schoenmaker and D. C. Jansen (Department of Cell Biology, Academic Medical Center, Amsterdam) for technical assistance with the fluorometric cathepsin assays and Dr. J. van Marle (Center for Microscopical Research, Academic Medical Center, Amsterdam) for technical assistance with laser scanning confocal microscopy. We thank Dr. D. J. Salant (Boston University Medical Center, Boston, MA) and Dr. D. Mason for providing cell lines, Dr. F. G. Lakkis for providing recombinant rat IL-13 and antibodies against rat IL-13, and Dr. M. Zerial for providing antibodies against Rab7.
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FOOTNOTES |
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This work was supported by grants from the Dutch Kidney Foundation (C98.1720) and from The Netherlands Organization for Scientific Research (920-03-062).
Address for reprint requests and other correspondence: J. G. van den Berg, Dept. of Pathology, Academic Medical Center, Meibergdreef 9, L2-256, 1105 AZ Amsterdam, The Netherlands (E-mail j.g.vandenberg{at}amc.uva.nl).
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 August 8, 2001; 10.1152/ajprenal.00102.2002
Received 23 March 2001; accepted in final form 15 August 2001.
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