1 Membrane Biology Group and 2 Division of Respiratory Medicine, Department of Medicine, University of Toronto, Toronto, Ontario, Canada M5S 1A8
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ABSTRACT |
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Reactive oxygen species (ROS) initiate multiple pathological and physiological cellular responses, including tyrosine phosphorylation of proteins. In this study, we investigated the effects of ROS on cell-extracellular matrix interactions utilizing the floating three-dimensional collagen gel assay. Exposure of mesangial cells grown in three-dimensional culture to H2O2, 3-amino-1,2,4-triazole (a catalase inhibitor), or puromycin is associated with gel reorganization accompanied by tyrosine phosphorylation of multiple proteins, including focal adhesion kinase (FAK). Neutrophils cocultured with mesangial cells in three-dimensional culture also induce mesangial cell-collagen gel reorganization and initiate tyrosine phosphorylation of a similar set of proteins. Collectively, these results show that ROS of either endogenous or exogenous origin can modulate mesangial cell-extracellular matrix interactions through initiation of a phosphotyrosine kinase signaling cascade. Consequently, ROS may play a role as signaling molecules that regulate mesangial cell-extracellular matrix interactions in both physiological and pathological conditions.
reactive oxygen species; protein-tyrosine kinase; neutrophils; glomerular mesangium; puromycin
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INTRODUCTION |
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CONSTITUTIVE PRODUCTION of reactive oxygen species (ROS) by mesangial cells originates from an intrinsic NADPH oxidase (21) that normally functions at a low level but increases in response to inflammatory stimuli such as cytokines (20). Neutrophils, monocytes, and macrophages also possess an NADPH oxidase, but their production of ROS is considerably greater than that of mesangial cells. ROS are implicated as causative agents in glomerular disease, and the source of ROS may be either the mesangial cell itself or "passenger" leukocytes such as neutrophils, monocytes, or macrophages (22). ROS also play a role in the pathogenesis of puromycin aminonucleoside nephrosis (7, 22, 23). However, in this case, the generation of ROS is as a result of an anthracycline compound undergoing a single-electron reduction to a free radical semiquinone species, which is subsequently reoxidized in the presence of molecular oxygen to form reactive oxygen metabolites (22).
Although ROS are highly reactive molecules with the potential to cause cell injury, they also initiate a number of physiological responses, including tyrosine phosphorylation of growth factor receptors (15) and p42/p44MAPK in vascular smooth muscle cells and neutrophils (3, 5). Furthermore, both proliferative and migratory effects of platelet-derived growth factor (PDGF) on vascular smooth muscle cells are, at least in part, dependent on the production of ROS (26). In cultured mesangial cells, ROS induce tyrosine phosphorylation of the PDGF receptor pp60c-src and a 125-kDa protein thought to be focal adhesion kinase (FAK) (9). In addition, high concentrations of ROS act as phosphatase inhibitors, and in the presence of sodium vanadate, ROS induce focal adhesion formation (6). The chemical basis of these various effects of ROS is unknown but may be due to direct oxidation of critical protein sulfhydryl groups or through formation of transition metal complexes (14).
Normal function of the mesangium, which includes controlling glomerular filtration rate, is dependent on complex mesangial cell-extracellular matrix interactions. These cell-extracellular matrix interactions can be studied in a realistic in vitro system by growing mesangial cells in three-dimensional collagen gels in which the cells exhibit a phenotype resembling the in vivo state (16, 17, 29). When cells are placed in collagen, they attach to it via integrins within 30 min of being plated (10). By 4 h, they begin to spread and develop lamellipodia (11). As cells spread, the actin cytoskeleton generates force that is transmitted to the extracellular matrix via integrins, resulting in extracellular matrix remodeling (8, 18, 25). Once the extracellular matrix is remodeled, cells migrate and form new cell-substratum attachments. If the collagen gel in which the cells are embedded is not fixed to the culture dish, then the extracellular matrix remodeling results in the diameter of the gel becoming smaller. This decrease in size of the gel is referred to as gel contraction.
Remodeling of collagen gels is induced by many agonists, including fetal bovine serum (FBS) and PDGF (10, 11, 29). The cell attachment, spreading, and migration essential for gel reorganization is dependent on tyrosine phosphorylation of a number of proteins, including FAK (29). Although remodeling of the gels takes at least 6 h, the tyrosine phosphorylation of the necessary signaling molecules is maximal 45 min after addition of the agonist (29).
Because ROS increase tyrosine phosphorylation of a number of proteins,
including a 125-kDa protein thought to be FAK (9), and tyrosine kinase
phosphorylation is implicated in mesangial cell-collagen gel
reorganization (29), we investigated the effects of ROS on mesangial
cells embedded in floating three-dimensional collagen I gels. For
logistical reasons, we used collagen I rather than collagen IV, which
is the normal extracellular matrix found in the mesangium. This remains
a physiologically relevant model (17, 28) because mesangial cells
express 1
1,
2
1, and
3
1 integrins that bind both
collagen I and collagen IV (19). We show that low concentrations of
H2O2
induce mesangial cells to reorganize collagen I gels. This process is
dependent on tyrosine phosphorylation of several proteins, among which
FAK is prominent. Similarly, when ROS are produced by the mesangial
cells themselves in response to 3-amino-1,2,4-triazole (an endogenous
catalase inhibitor) or puromycin or, alternatively, when ROS are
generated exogenously by neutrophils, mesangial cells reorganize
collagen gels. Because collagen gel reorganization is dependent on cell attachment, spreading, and migration, these results imply that ROS are
signaling molecules that may influence cell-extracellular matrix
interactions in the mesangium under both physiological and pathological conditions.
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METHODS |
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Materials. MEM, Hanks' balanced salt solution (HBSS), and FBS were supplied by GIBCO (Grand Island, NY). Collagen type I, collagenase type Ia, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), protein G-sepharose 4B, puromycin aminonucleoside, 3-amino-1,2,4-triazole, N-formyl-Met-Leu-Phe (fMLP), and N-t-BOC-Met-Leu-Phe (t-BOC) were obtained from Sigma (St. Louis, MO). Diisopropyl fluorophosphate was purchased from Calbiochem (La Jolla, CA). Monoclonal anti-phosphotyrosine antibodies and monoclonal anti-FAK antibodies were purchased from Upstate Biotechnologies (Lake Placid, NY). Polyclonal anti-FAK antibodies were purchased from Santa Cruz (La Jolla, CA). Catalase was obtained from Worthington Laboratories. Diphenyleneiodonium (DPI) was a gift from Dr. Sergio Grinstein (Hospital for Sick Children, Toronto, Ontario, Canada).
Tissue culture. Rat glomeruli were isolated from kidneys of 3-wk-old male Sprague-Dawley rats by the graded-sieve technique (12). Mesangial cells isolated from these glomeruli were grown in 100-mm plates in MEM containing 15% FBS as previously described (2). Experiments were performed on cells between passages 5 and 15.
Neutrophil preparation. Human neutrophils (>98% pure) were isolated from citrated whole blood obtained by venipuncture using dextran sedimentation and discontinuous plasma-Percoll gradients as previously described (13). The cells were used for the gel contraction assays immediately after isolation. To minimize proteolysis of mesangial cell proteins after extraction in detergent, we pretreated the neutrophils for 30 min with 5 mM diisopropyl fluorophosphate and washed them three times in PBS before use.
Gel contraction assay.
The gel contraction assay is based on previously described methods (2).
Mesangial cells were placed in a solution of MEM and type I collagen
and allowed to gelate for 3 h. The substances tested were dissolved in
MEM and added to the gels. In a variety of cell types for which the gel
contraction assay has been utilized, FBS empirically causes the
greatest amount of gel contraction (10, 11). We elected to quantitate
the effects of ROS on gel contraction relative to those induced by FBS.
The diameter of the gel was measured at the specified time points using
a dissecting microscope (Wild Leitz) at ×16 magnification, and
the percentage of maximal contraction was calculated using the
following formula: %gel contraction = 100 × [(AMEM Atest)/(AMEM
AFBS)],
where AMEM is the
area of the MEM-treated gel,
Atest is the area
of the test substance-treated gel, and
AFBS is the area
of the FBS-treated gel.
Cell-viability studies. Cell viability was assessed by the tetrazolium salt (MTT) method, as well as by trypan dye exclusion, as described previously (28).
Immunoblotting. To ensure that protein loads were comparable in the various immunoblotting protocols, we added an equal number of cells to each collagen gel. Cells embedded in collagen gels were isolated by dissolving the collagen with collagenase type I (20 µg/ml) mixed with the phosphatase inhibitors (1 mM Na3VO4, 30 mM sodium pyrophosphate, and 50 mM NaF). Cells were then lysed for 20 min at 4°C in a lysis buffer [20 mM Tris, pH 8.0, 137 mM NaCl, 10% glycerol, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride (PMSF), 0.15 U/ml aprotonin, and 1 mM Na3VO4]. The lysates were cleared by centrifugation at 10,000 g for 10 min at 4°C, after which the supernatants were collected and equal protein loading was verified by the Bradford protein assay. The proteins were dissolved in a 5× SDS-PAGE sample buffer and electrophoresed on a 10% polyacrylamide slab gel. Proteins were transferred electrophoretically to nitrocellulose and blocked overnight (0.25% gelatin, 10% ethanolamine, and 0.1 M Tris). The nitrocellulose was probed with a monoclonal anti-phosphotyrosine (4G10) antibody at a concentration of 1:5,000 for 2 h, followed by a 1-h incubation with a 1:5,000 dilution of a horseradish peroxidase-conjugated sheep anti-mouse antibody. Immunoreactive bands were visualized by enhanced chemiluminescence. On each blot, there were lysates of mesangial cells extracted from collagen gel cells incubated in either MEM (negative control) or FBS (positive control) or treated with test ROS agonists. Molecular-weight standards were run in parallel. We prepared Figs. 1, 2, 5, 6, and 9 by making composites comparing selected individual lanes from the same slab gels.
Immunoprecipitation of FAK. Cells were prepared in a similar fashion to that used for immunoblotting, except that the lysis and solubilization was carried out in a modified RIPA buffer (Tris · HCl, pH 7.4, 1% NP-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EGTA, 1 mM PMSF, 1 µg/ml aprotonin, 1 µg/ml leupeptin, 1 µg/ml pepstatin, 1 mM Na3VO4, and 1 mM NaF) for 20 min at 4°C. The lysates were cleared by centrifugation at 10,000 g for 10 min at 4°C, and the supernatants were collected and equalized for protein loading by the Bradford protein assay. Equal volumes of lysate were immunoprecipitated with an anti-FAK polyclonal antibody for at least 1 h at 4°C, after which they were incubated with protein G-sepharose beads (precleared by 0.25% ovalbumin) for an additional 2 h. Immunoprecipitates were washed three times in the lysis buffer and divided into fractions, each of which was electrophoresed separately on a 10% polyacrylamide slab gel and subjected to immunoblotting using either a monoclonal anti-phosphotyrosine or a monoclonal anti-FAK antibody, as described in Immunoblotting. In this way, it was possible to monitor changes in phosphorylation and verify the equivalency of the amount of immunoprecipitated FAK.
Statistics. All gel contraction assays were performed in duplicate on at least three different occasions. The results of the gel contraction assays were normalized as shown in METHODS. Means ± SE of the pooled data of the contraction assays were calculated and are shown graphically. When single points were measured, the means ± SE of the assays were calculated, and the significance of the differences of their means was compared using the Student's t-test. The immunoblot and immunoprecipitation experiments were done at least three times each, and representative examples are shown.
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RESULTS |
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Short-term exposure of mesangial cells in
three-dimensional culture to high concentrations of
H2O2
induces anchorage-dependent tyrosine phosphorylation.
To determine if exposure of mesangial cells to
H2O2
in three-dimensional culture resulted in tyrosine phosphorylation of
cellular proteins, we prepared collagen gels as described in
METHODS and left them in MEM
overnight. The following day, the gels were exposed to increasing
concentrations of
H2O2
(2.5-10 mM) for 45 min, after which the cells were lysed,
solubilized, and subjected to immunoblotting with an
anti-phosphotyrosine antibody. As shown in Fig.
1A,
exposure of mesangial cells in three-dimensional culture to
H2O2
induced tyrosine phosphorylation of several proteins in a
dose-dependent manner (lanes
2-4). In a parallel set of experiments (Fig.
1B), cells were grown on tissue
culture plates, after which they were serum starved for 48 h,
trypsinized, suspended in MEM for 1 h, and then exposed to
H2O2.
These cells showed little evidence of increased protein tyrosine
phosphorylation relative to cells in three-dimensional culture (Fig.
1B). Lysates from the unattached cells in suspension, as well as from the three-dimensional culture systems, were immunoprecipitated with a polyclonal anti-FAK antibody. The immunoprecipitate was divided into equal fractions; one-half was
immunoblotted with a monoclonal anti-phosphotyrosine antibody, and the
other half was immunoblotted with a monoclonal anti-FAK antibody. As
shown in Fig. 1C, an equivalent amount
of FAK was immunoprecipitated from the lysates, but the degree of
phosphorylation after exposure to 10 mM
H2O2
increased for cells grown in three-dimensional culture (compare
lanes 1 and
2), but not for unattached mesangial cells in suspension (compare lanes 3 and 4).
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ROS induce gel contraction and tyrosine kinase
phosphorylation.
Mesangial cells grown in three-dimensional culture and exposed to
H2O2
demonstrated a pattern of protein tyrosine phosphorylation similar to
that which occurs during FBS- and PDGF-induced mesangial cell-collagen
gel contraction (29). It was therefore of interest to
examine whether exposure of mesangial cells grown in three-dimensional culture to ROS might also induce gel contraction. Because mesangial cells require several hours to significantly remodel collagen gels, an
experimental protocol that allowed cells to be exposed to ROS for a
sufficient length of time was designed. Concentrations of
H2O2
(2.5-10 mM) that resulted in protein tyrosine phosphorylation of
mesangial cells within 45 min (see Fig. 1) were cytotoxic to cells
after longer exposure, as determined by the MTT assay (data not shown).
Therefore, three-dimensional mesangial cell-collagen gels were exposed
for 6 h to much lower concentrations of
H2O2 (25-800 µM) augmented by a low concentration of the catalase
inhibitor 3-amino-1,2,4-triazole (0.625 mM). This concentration of
3-amino-1,2,4-triazole did not change cell viability as monitored by
the MTT assay. Under these conditions,
H2O2
stimulated gel contraction that exhibited a dose-response curve with
maximal gel contraction that occurred at 200 µM
H2O2
and returned to basal levels at 800 µM
H2O2
(Fig. 2A). The
percentage of gel contraction was measured at 6 h and is expressed as a
percentage of maximal gel contraction induced by 3% FBS (see
METHODS).
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3-Amino-1,2,4-triazole and puromycin induce protein
tyrosine phosphorylation along with stimulation of gel
contraction.
In earlier work (28), we demonstrated that mesangial cells produce ROS
when exposed to either puromycin or 3-amino-1,2,4-triazole. Given that
mesangial cells remodel collagen gels when exposed to low
concentrations of
H2O2,
we decided to evaluate the effects of low concentrations of both these
substances on mesangial cell-collagen gel contraction. Gels were
prepared and incubated overnight in MEM, after which they were exposed
to either 3-amino-1,2,4-triazole (0.625-10 mM) or puromycin
(2.5-20 µg/ml). The percentage of gel contraction was measured
at 6 h and is expressed as a percentage of maximal gel contraction
induced by 3% FBS. 3-Amino-1,2,4-triazole (Fig.
3) and puromycin (Fig.
4) both induced gel contraction in a
dose-dependent fashion, yielding similar biphasic curves (i.e., as the
concentration was increased, gel contraction also increased, peaked,
and then decreased back to control levels). Moreover, inspection of
Figs. 3 and 4 reveals that, in each case, gel contraction was inhibited
by addition of either catalase (12.5 µg/ml; Figs. 3A and
4A), which degrades
H2O2,
or the flavoprotein inhibitor DPI (0.625 µM; Figs.
3B and
4B), which inhibits NADPH oxidase (24).
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FMLP induces gel contraction and protein tyrosine
phosphorylation.
FMLP stimulates NADPH oxidase via a specific formylpeptide receptor of
the seven transmembrane type, resulting in ROS production and
activation of downstream effector pathways including kinases and
phosphatases (4). To test if FMLP had any effect on
three-dimensional mesangial cell cultures, we prepared gels as
described in METHODS, incubated them
overnight in MEM, and then exposed them to increasing concentrations of
FMLP. Figure
6A shows
that the maximal effect of FMLP (0.1 µM) induced 40% of the maximal
gel contraction caused by FBS over a 6-h period. No gel contraction
relative to FMLP was induced in the presence of the inactive receptor
competitor t-BOC FMLP (0.1 µM)
(P < 0.01), confirming the
specificity of the response. Catalase and DPI, respectively, decreased
FMLP-induced contraction to 17% and 16% of maximal contraction
induced by FBS, which was significantly less than the 40% of maximal
gel contraction induced by FMLP alone
(P < 0.01). Although gel contraction
by FMLP in the presence of catalase and DPI was significantly less than
by FMLP alone, the inhibition was only ~60% of maximal FMLP-induced contraction. This suggests that FMLP-induced gel contraction is only
partially due to stimulation of mesangial cell ROS by FMLP.
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Neutrophils induce mesangial cell-collagen gel contraction that is accompanied by protein tyrosine phosphorylation. Neutrophils produce a basal concentration of ROS, as demonstrated by the fact that 106 cells produce 0.5 nM H2O2/min when incubated in PBS (27). To assess whether this neutrophil-derived ROS could induce gel contraction through cell-cell interactions, we created a coculture system in which 2.5 × 105 mesangial cells embedded in collagen gels were prepared and left in MEM overnight. The following day, neutrophils (2 × 106/ml) were added to the gels and the amount of gel contraction was measured after 6 h and compared with maximal gel contraction induced by FBS.
As shown in Fig. 7, addition of neutrophils stimulated gel contraction to ~70% of the maximal gel contraction caused by FBS after 6 h. Inspection of Fig. 7 shows that "coculture" of neutrophils in the presence of catalase and DPI resulted in only 8% and 5% of maximal gel contraction, respectively, which is significantly less than the untreated gels (P < 0.01). In contrast to FMLP-induced contraction, this means that stimulation of mesangial cell spreading and migration by exposure to neutrophils is predominantly due to production of ROS. In control experiments, gel remodeling did not occur when neutrophils were added to gels that did not contain mesangial cells (data not shown).
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DISCUSSION |
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Until recently, the effects of ROS on biological tissue have been evaluated principally in terms of potential to cause cell damage because of their high reactivity. It is becoming increasingly evident, however, that ROS may also play a physiological role as signaling molecules influencing a variety of cellular functions. The present study supports a regulatory role for ROS in mesangial cell function. We found that, at low concentrations, ROS induced mesangial cell attachment, spreading, and migration, which, in a three-dimensional collagen culture system, leads to matrix remodeling and gel contraction. Moreover, the ROS-induced gel contraction is dependent on tyrosine phosphorylation of a number of proteins, including FAK. The data demonstrate that ROS, produced either endogenously by mesangial cells or exogenously from other cellular sources such as neutrophils, modulate the behavior of mesangial cells in a concentration-dependent manner. Regardless of whether the source of ROS was H2O2, 3-amino-1,2,4-triazole, puromycin, or neutrophils, the same biphasic dose dependency was observed. At low concentrations, ROS increasingly stimulated gel contraction until a threshold (peak) was reached. Above that threshold, the ROS effect waned and gel contraction reduced to background levels.
It was recently shown that exposure of mesangial cells grown in two-dimensional culture to high concentrations of H2O2 increases tyrosine phosphorylation of the PDGF receptor, pp60c-src, and a 125-kDa protein presumed to be FAK (9). We confirmed that short-term exposure of mesangial cells grown in two-dimensional culture results in increased tyrosine phosphorylation of a number of proteins (data not shown), and we also demonstrated that similar exposure of mesangial cells to high concentrations of H2O2 in three-dimensional culture leads to stimulation of tyrosine phosphorylation of multiple proteins, including FAK.
The present study demonstrates that low concentrations of H2O2, as well as generation of ROS by mesangial cells after exposure to 3-amino-1,2,4-triazole, puromycin, or FMLP, or by neutrophils, each separately induce collagen gel remodeling, presumably by increasing attachment, spreading, and migration of mesangial cells. Although FBS is the most potent stimulator of gel contraction (10, 11), the mechanism whereby it induces gel contraction is unknown. In the present investigation, we found that ROS produce ~50-70% of maximal gel contraction caused by FBS. This effect is as powerful as, or more powerful than, any mediator other than FBS that has been used to induce collagen gel contraction (29). Although this is an entirely in vitro empirical finding, it suggests that ROS can be quite potent modulators of mesangial cell remodeling of collagen gels.
The present study was carried out using a three-dimensional collagen gel culture system because mesangial cells behave differently in this environment than in the usual two-dimensional culture. In three-dimensional culture systems, mesangial cells adopt a nonproliferative phenotype similar to that found in vivo rather than the proliferative phenotype found in two-dimensional culture systems (16, 17, 29). Cells in three-dimensional culture also respond differently to growth factors like PDGF compared with cells in the two-dimensional state. Mesenchymal cells, including mesangial cells, express fewer PDGF receptors with decreased autophosphorylation when grown under these conditions (17, 29). This resembles the situation in the adult kidney where little PDGF and few PDGF receptors are found (1).
There are several possible mechanisms that would explain how low concentrations of ROS might induce gel contraction. H2O2 is a broad-spectrum phosphatase inhibitor (14); this phosphatase inhibition may result in tyrosine phosphorylation of FAK, paxilline, pp60c-src, tensin, or other tyrosine kinases that may, in turn, lead to focal adhesion formation, subsequent cell attachment, and migration. This would be consistent with the finding that exposure of cells in a two-dimensional culture system to high concentrations of H2O2, albeit in the presence of vanadate (another phosphatase inhibitor), results in formation of focal adhesions (6). On the other hand, when mesangial cells were trypsinized from plastic and then exposed to H2O2, tyrosine phosphorylation was not markedly increased. This latter observation implies that cellular signaling responses to H2O2 are anchorage dependent. Therefore, it is possible that H2O2 has a direct effect on integrins, causing a conformational change in either the extracellular or intracellular domain, leading to integrin-mediated cell attachment to ligand and subsequent focal adhesion formation with resultant tyrosine phosphorylation. Finally, there is the possibility that ROS activate some other intracellular signaling molecule(s) functionally linked to phosphorylation of cytoskeletal proteins, with secondary recruitment of FAK to focal adhesion complexes.
Our findings related to neutrophils cocultured with mesangial cells may be relevant to those situations in which there is a significant inflammatory presence in the glomerulus. The release of ROS from "passenger" neutrophils could affect the behavior of constituent mesangial cells, depending on the extent of the neutrophil infiltrate. Small numbers of inflammatory cells might lead to functional activation (i.e., through stimulation of intracellular signaling), whereas, in the presence of a large cellular infiltrate, the large amount of ROS might simply cause cytotoxicity and loss of function.
Further experiments are necessary to determine if it is possible to extrapolate a possible physiological role in the intact glomerulus from the modulatory role of ROS in mesangial cell spreading and migratory behavior that we have observed in vitro.
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ACKNOWLEDGEMENTS |
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We thank Gary Song for help in preparing the manuscript and Martin Schwartz for helpful comments.
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FOOTNOTES |
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R. Zent is the recipient of a Fellowship from The Kidney Foundation of Canada. The Kidney Foundation of Canada and the Medical Research Council of Canada funded this work.
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: M. Silverman, Room 7207, Medical Sciences Bldg., Univ. of Toronto, Toronto, Ontario, Canada M5S 1A8.
Received 27 April 1998; accepted in final form 22 October 1998.
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