From the Program in Matrix Biology, Divisions of Gastroenterology
and Nephrology, Department of Medicine and the Cancer Center, Beth
Israel Deaconess Medical Center and Harvard Medical School, Boston,
Massachusetts 02215, the ¶ Gene Expression Laboratory, Boystown
National Research Hospital, Omaha, Nebraska 68131, and the
Department of Pediatric Oncology, Dana-Farber Cancer Institute
and Harvard Medical School, Boston, Massachusetts 02215
Received for publication, January 13, 2003
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ABSTRACT |
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There are about 2.5 million glomeruli in the
kidneys each consisting of a barrel of glomerular basement membrane
surrounded by glomerular endothelial cells on the inside and glomerular
epithelial cells with established foot processes (podocytes) on the
outside. Defects in this filtration apparatus lead to glomerular
vascular leak or proteinuria. The role of vascular endothelial growth
factor (VEGF) in the regulation of glomerular vascular permeability is still unclear. Recent studies indicate that patients receiving anti-VEGF antibody therapy may have an increased incidence of proteinuria. In a different setting, pregnancies complicated by preeclampsia are associated with elevated soluble VEGF receptor 1 protein (sFlt-1), endothelial cell dysfunction and proteinuria. These
studies suggest that neutralization of physiologic levels of VEGF, a
key endothelial survival factor, may lead to proteinuria. In the
present study, we evaluated the potential of anti-VEGF neutralizing
antibodies and sFlt-1 in the induction of proteinuria. Our studies
demonstrate that anti-VEGF antibodies and sFlt-1 cause rapid glomerular
endothelial cell detachment and hypertrophy, in association with
down-regulation of nephrin, a key epithelial protein in the glomerular
filtration apparatus. These studies suggest that down-regulation or
neutralization of circulating VEGF may play an important role in the
induction of proteinuria in various kidney diseases, some forms of
cancer therapy and also in women with preeclampsia.
Vascular endothelial growth factor is a 43-46-kDa glycoprotein
that serves as a key survival factor for vascular endothelium (1-4).
Several splice variants of human
VEGF1 have been reported to
exist, and VEGF165 along with VEGF121 are key
circulating forms (5-7). Physiological levels of VEGF in the serum are
considered pivotal for maintaining vascular endothelial cell
homeostasis (8-10). The importance of VEGF is further illustrated by
the fact the deletion of even a single allele of VEGF in mice leads to
embryonic lethality (11). While the importance of VEGF as a survival
factor for vascular endothelium is well appreciated, its role in the
regulation of kidney glomerular vascular endothelium behavior is poorly
understood. Some studies have suggested that VEGF expression is reduced
during renal injury (12-14) and supplementing the injured kidney with
exogenous VEGF121 was shown to provide some improvement in
renal function and histology (15). Preeclampsia is also associated with
proteinuria, hypertension, and endothelial cell dysfunction (16-18).
In this regard, Vuorela et al. (19) demonstrate that VEGF is
bound by a circulating serum protein in the amniotic fluid and maternal
serum. Later, the same group demonstrates that soluble VEGF receptor 1 protein (sFlt-1) is significantly elevated in the amniotic fluid of
peeclamptic women, again suggesting a pathogenic significance for the
decrease of VEGF levels in the serum and amniotic fluid in this
condition (20). In a related study, Zhou et al. (21)
demonstrate that cytotrophoblasts from placentas of women whose
pregnancies were complicated by preeclampsia produce higher levels of
sFlt-1 in vitro as compared with control cells. Such
separate studies collectively suggest a causal connection between
proteinuria/hypertension and endothelial cell dysfunction, mediated by
sFlt-1.
The significance of physiological circulating levels of VEGF in the
regulation of glomerular vascular leak or proteinuria is important to
evaluate as anti-VEGF antibody therapy, and VEGF receptor antagonists
are being used in several clinical cancer trials (www.nci.nih.gov). In
this regard, recent reports from Phase I and Phase II human clinical
cancer trials using anti-VEGF antibodies (Bevacizumab) suggest that
proteinuria was associated with this treatment protocol (22). These
results suggest that anti-VEGF antibodies may induce proteinuria.
In the present study we evaluated the capacity of anti-mouse VEGF
neutralizing antibodies and sFlt-1 to induce proteinuria. Our results
indicate that a single intravenous infusion of anti-VEGF antibodies
into normal healthy mice results in excessive albumin excretion in the
urine (proteinuria) via massive glomerular endothelial cell
detachment/damage and suppression of the glomerular epithelial slit
diaphragm apparatus-associated protein, nephrin. Thus we propose that
circulating physiological levels of VEGF are important for the proper
function and survival of glomerular endothelial cells and appropriate
filtration of blood in the kidney glomeruli. Additionally, these
results suggest that long term proteinuria may be a significant side
effect of chronic anti-VEGF therapy.
Anti-VEGF Ab and sFlt-1/Fc Bolus Injection
Studies--
Anti-VEGF antibody and sFlt-1 bolus injection studies
were performed in wild-type CD1 mice. Each mice were injected with a single intravenous injection of anti-VEGF antibody or sFlt-1/Fc at a
concentration of 3.25 and 32.5 pM (picomole per liter).
This concentration corresponds to equivalent molar concentration to that of 65 pg/ml (3.25 pM) of normal plasma VEGF (1:1
ratio) and 10 times molar excess to that of 65 pg/ml (3.25 pM) of normal plasma VEGF concentration (1:10 ratio). Mouse
anti-VEGF antibody (IgG1) and sFlt-1/Fc chimera were
purchased from NeoMarkers (Fremont, CA) and R&D Systems (Minneapolis,
MN), respectively. For the in vivo inhibition experiments,
human recombinant VEGF165 was injected at a concentration
of 32.5 pM to counteract the anti-VEGF antibodies injected
at the same concentration. Since timed urine collection was essential
to assess protienuria, we used 10 µg of furosemide in 200 µl of PBS
for injection after the infusion of anti-VEGF antibodies or sFlt-1.
Mice injected with control IgG1 served a control.
One-hundred microliters of urine was collected 0, 1, 3, 5, and 24 h after the initial injection. Injections of furosemide were repeated
every 1 h before the collection of the urine. Albumin and
creatinine concentrations in the urine were estimated using a
colorimetric assay according to the manufacturer's recommendations (Sigma). Urine albumin excretion was estimated as the quotient of urine albumin and urine creatinine (23). For these experiments, five
mice per each group were used. Some mice were sacrificed 5 h after
to collect kidneys for immunohistochemistry. The entire experiment was
repeated three times.
Immunofluorescence Staining--
Immunofluorescence staining was
performed as described previously (24). Briefly, 4-µm cryosections
were fixed in acetone ( Western Blotting--
Equal weights of cortical portions of
kidneys from each groups were homogenized in liquid nitrogen and they
were solubilized in the lysis buffer (0.05 M Hepes, pH 7.5, 0.01 M CaCl2, 4 mM N-ethylmaleimide, 5 mM benzamidine HCl, 1 mM phenylmethanesulfonyl fluoride, and 25 mM
Transmission Electron Microscopy--
Transmission electron
microscopy was performed as described previously (25).
Statistical Analysis--
Analysis of variance was used to
determine statistical differences. As needed, further analysis was
carried out using t test with Bonferroni correction to
identify significant differences. A p value <0.05 was
considered statistically significant.
Circulating physiological levels of free VEGF in the normal
mouse plasma (VEGF164 and VEGF120) is about 65 pg/ml (3.25 pM) as determined by ELISA (data not shown). We
used neutralizing mouse anti-VEGF antibodies at two different
concentrations, equivalent molar amount to the free serum VEGF in
normal mice according to our measurement (3.25 pM) and also
10-fold molar excess (32.5 pM), to evaluate their capacity
to induce proteinuria (as measured by albumin content in the urine).
The amount of protein in the urine was estimated as the ratio of urine
protein to urine creatinine (Fig. 1). Our
results indicate that starting 3 h after the intravenous injection
of anti-VEGF antibody, the mice develop significant proteinuria and
maintain the same level of proteinuria for next 7 h and gradually
after 24 h the albumin content in the urine returns to normal
levels (Fig. 1A; data not shown). The amount of proteinuria
in mice with 1:1, VEGF:anti-VEGF antibody ratio (1:1 ratio), is less
compared with 1:10 ratio (Fig. 1A). The likely explanation
for this is the potential lack of sensitivity on part of the ELISA
assay to detect all of the circulating VEGF in the plasma.
Nevertheless, these experiments suggest that very low amounts of
anti-VEGF antibody (3.25 pM) can induce proteinuria. To
further establish the specificity of this antibody, we performed the
antibody infusion (1:10 ratio) experiments in conjunction with infusion
of equivalent molar amounts of exogenous human VEGF165 (32.5 pM). Such experiments show that human
VEGF165 can inhibit proteinuria inducing capacity of
anti-VEGF antibody, providing further proof that anti-VEGF antibody
specifically induces proteinuria (Fig. 1A).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
20 °C) for 3 min and dried at room
temperature. After incubation with primary antibodies to
nephrin, CD2AP, podocin, and
-actinin-4 for 2 h at room
temperature, the sections were washed three times with PBS and
incubated with fluorescein isothiocyanate-labeled secondary
antibodies. After washing with PBS the sections were covered with glass
slips using Vectashield mounting media (Vector Laboratories,
Burlingame, CA). The staining was analyzed using a fluorescence
microscope Eclipse TE300 (Nikon, Tokyo, Japan). Antibodies to nephrin,
CD2AP, podocin, and
-actinin-4 were reported by our laboratory in a
previous publication (24).
-aminohexanoic acid). The lysates were dialyzed with PBS and
electrophoresed on a 10% SDS-polyacrylamide gel. Immunoblots were
blocked with 5% skim milk-containing TBST buffer (0.1% Tween 20, 20 mM Tris, pH 7.6, 140 mM NaCl). Western blotting was performed by incubating with indicated antibodies in TBST buffer
followed by secondary antibodies conjugated with horseradish peroxidase
and then developed by ECL kit as an enhanced chemiluminescence system
(Amersham Biosciences).
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
View larger version (121K):
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Fig. 1.
Blocking of circulating VEGF leads to
proteinuria via the disruption of glomerular endothelial cells.
A and B, anti-VEGF antibody and sFlt-1/Fc bolus
injection studies. A, a bar graph shows urine
albumin excretion 1, 3, and 5 h after control mouse
IgG1 infusion (black columns), 3.25 pM anti-VEGF Ab (white columns), 32.5 pM anti-VEGF Ab (gray columns), and 32.5 pM anti-VEGF Ab + 32.5 pM VEGF (slashed
columns) intravenous infusion. B, a bar
graph shows urine albumin excretion 1, 3, and 5 h after
control mouse IgG1 (black columns), 3.25 pM
sFlt-1/Fc (white columns), 32.5 pM sFlt-1/Fc
(gray columns), and 32.5 pM sFlt-1/Fc + 32.5 pM VEGF (slashed columns) intravenous infusion.
The results are shown as the mean ± S.E. * and ** indicate,
p < 0.05 and p < 0.01, respectively, compared with control IgG1 group. indicates p < 0.05, compared with 32.5 pM
anti-VEGF Ab group. # indicates p < 0.05, compared
with 32.5 pM sFlt-1/Fc group. II indicates
p < 0.05, compared with 32.5 pM anti-VEGF
Ab group. C and D, transmission electron
microscopy analysis of kidney sections from control IgG1
injected mice (5 h post-injection). Arrowheads show normal
fenestrations of endothelial cells, and black and
white arrows indicate normal podocyte foot processes and
slit diaphragms. E-H, transmission electron microscopy
analysis of kidney sections from 32.5 pM anti-VEGF
Ab-injected mice. These sections exhibit glomerular endothelial cell
hypertrophy (E, dotted line), damage
(F, dotted line), detachment from GBM
(G, arrow), and occasional disruption/loss of
slit diaphragms (E-H, arrowhead). Vacuolation is
observed in these endothelial cells and shown with * in the figure
(E and G). The magnifications are as shown in the
figure.
Recent studies propose that sFlt-1 can also neutralize circulating VEGF and suggest the use of this protein as an anti-cancer agent (26-30). Therefore, in the present study we used mouse sFlt-1/Fc fusion protein to perform similar experiments as done using anti-VEGF antibodies. Again, consistent with anti-VEGF antibody experiments, we show that sFlt-1 can induce proteinuria in mice within 3 h of intravenous injection and this effect disappears by 24 h (Fig. 1B; data not shown). Additionally, as shown earlier, exogenous VEGF supplementation at equivalent molar concentration of sFlt-1 (32.5 pM) inhibits the proteinuria-inducing effect of sFlt-1 (Fig. 1B). These results collectively show that intravenous injection of a single dose of anti-VEGF antibodies and sFlt-1 to neutralize circulating VEGF leads to proteinuria.
We next performed ultrastructural transmission electron microscopy analysis of the kidney glomerular tissue sections from mice which developed proteinuria upon treatment with anti-VEGF antibodies and sFlt-1. The control kidney sections, 5 h after injected with 32.5 pM of IgG1, reveal normal ultrastructual histology with well defined endothelial layer (arrowhead) adjacent to the glomerular basement membrane (GBM), proper alignment of podocyte foot processes (black arrow), and slit diaphragms (white arrow) (Fig. 1, C and D). Starting 3 h and even at 24 h, anti-VEGF antibodies treatment reveal glomerular endothelial hypertrophy (Fig. 1E, dotted line), damage (Fig. 1F, dotted line), endothelial cell detachment from GBM (Fig. 1G, arrow) and occasional disruption/loss of slit diaphragms (Fig. 1, E-H, arrowhead). Similar results are observed when kidney from sFlt-1 injected mice are analyzed (data not shown). These results suggest that anti-VEGF antibody and sFlt-1 infusion leads to glomerular endothelial cell damage and also patchy yet significant glomerular epithelial cell damage (podocytes). Such defects could result in proteinuria as shown by recent studies (22, 24, 31).
Fenestrations associated with glomerular endothelial cells are a
characteristic feature of the kidney (32, 33). Such fenestrations are
considered to allow for the filtration property of the glomerulus (34-37). It is well established now that fenestrated endothelium does
not prevent albumin penetration and in general many large proteins can
pass through the fenestrations (34, 37, 38). Thus, presence of albumin
in the urine has to be due to a defect in the glomerular basement
membrane or podocyte slit diaphragm structure associated with
glomerular epithelial cells (34, 39-44). In the anti-VEGF antibody and
sFlt-1 experiments, the glomerular basement membrane is quite intact
but occasional defects in the glomerular podocyte architecture can be
detected (Fig. 1, E-H). Therefore, we examined four
recently identified glomerular podocyte-associated proteins considered
to be important for glomerular filtration. Human mutations in nephrin,
podocin, and -actinin-4 result in kidney diseases associated with
proteinuria (45-47). Recently, CD2AP has been implicated as critical
for glomerular podocyte function (48). Mice deficient in nephrin die 2 days after birth associated with massive proteinuria and kidney defects
(24, 49, 50). Other studies have shown that nephrin is key regulator of
glomerular filtration apparatus (51, 52). Thus, in this study we
examined the effect of anti-VEGF antibodies and sFlt-1 on the
expression of glomerular slit diaphragm/podocyte associated proteins.
The expression of nephrin, CD2AP, podocin, and -actinin-4 were
examined in the kidneys from mice infused with anti-VEGF antibodies (1:10 ratio). By immunofluoresence and Western blot using either kidney
sections or kidney extracts, we demonstrate that anti-VEGF antibodies
significantly reduce the expression of glomerular slit diaphragm
associated protein, nephrin (Fig. 2). The
expression of CD2AP, podocin, and
-actinin-4 was unchanged in these
kidneys (Fig. 2). Similar results are also obtained when kidneys from sFlt-1 injected mice were analyzed (data not shown). These results suggest that significant decrease in the expression of nephrin, a
component of glomerular slit diaphragm which constitutes an important
barrier to large serum proteins such as albumin, is a key mediator of
anti-VEGF antibody and sFlt-1 induced proteinuria in mice. These
results are consistent with other reports which suggest that nephrin
expression must be altered for induction of proteinuria (53, 54).
|
Collectively, these experiments provide evidence for the importance for
circulating physiological levels VEGF in the homeostasis of kidney
glomerulus. The glomerular endothelial cells are known to express VEGF
receptors 1 and 2, while glomerular epithelial cells do not express
these receptors (55, 56). Thus the effect on glomerular epithelial
cells is conceivably indirect and derived from the loss of glomerular
endothelial cells due to the lack of survival signals from circulating
VEGF (Fig. 2). This study also provides the necessary biochemical and
molecular proof that anti-VEGF antibodies and sFlt-1 can cause
proteinuria, offering a possible explanation for proteinuria observed
in some cancer patients on anti-VEGF antibody therapy and also
pregnancies complicated by preeclampsia (16, 17, 19-21). In this
regard, a possible method to neutralize the effects of anti-VEGF
antibodies and sFlt-1 needs to be explored.
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ACKNOWLEDGEMENTS |
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We thank Dr. Judah Folkman for his helpful discussion in the spring of 2002. We also thank Lori Siniski in the preparation of this manuscript.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health (NIH) Grants DK-51711, DK 55001, by NIH renal Training Grant T32DK07199-25, and by research funds from the Program in Matrix Biology at the Beth Israel Deaconess Medical Center.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.
These authors contributed equally to this work.
§ Supported by Japan Research Foundation for Clinical Pharmacology between 2001 and 2002. Currently supported by Stop & Shop Family Pediatric Brain Tumor Program.
** To whom correspondence should be addressed: Harvard Medical School, Director, Program in Matrix Biology, DANA 514, Beth Israel Deaconess Medical Center, 330 Brookline Ave., Boston MA 02215. Tel.: 617-667-0445; Fax: 617-975-5663; E-mail: rkalluri@bidmc. harvard.edu.
Published, JBC Papers in Press, January 21, 2003, DOI 10.1074/jbc.C300012200
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ABBREVIATIONS |
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The abbreviations used are: VEGF, vascular endothelial growth factor; Ab, antibody; ELISA, enzyme-linked immunosorbent assay; GBM, glomerular basement membrane; PBS, phosphate-buffered saline; sFlt-1, soluble vascular endothelial growth factor receptor 1.
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