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J. Biol. Chem., Vol. 278, Issue 15, 12605-12608, April 11, 2003

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ACCELERATED PUBLICATION
Neutralization of Circulating Vascular Endothelial Growth Factor (VEGF) by Anti-VEGF Antibodies and Soluble VEGF Receptor 1 (sFlt-1) Induces Proteinuria^*

Hikaru Sugimoto, Yuki Hamano^§, David Charytan, Dominic Cosgrove^¶, Mark Kieran, Akulapalli Sudhakar, and Raghu Kalluri^**

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

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

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.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

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.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

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 (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).

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 -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).

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.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

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).

View larger version (121K): [in this window] [in a new window]   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).

View larger version (40K): [in this window] [in a new window]   Fig. 2.   Blocking of circulating VEGF reduces the expression of nephrin. A, immunofluorescence staining of kidney sections from anti-VEGF Ab-injected mice. The renal tissues from the mice injected control IgG1 (Control), 32.5 pM anti-VEGF antibody (VEGF Ab), and 32.5 pM anti-VEGF Ab + 32.5 pM VEGF (VEGF Ab + VEGF) were stained with indicated antibodies. The magnification is as shown in the figure. B, Western blotting analysis in anti-VEGF Ab-injected mice. The renal extracts from the mice injected with control IgG1 (Control), 32.5 pM anti-VEGF Ab (VEGF Ab), and 32.5 pM anti-VEGF Ab + 32.5 pM VEGF (VEGF Ab + VEGF) were used for the immunoblotting with indicated antibodies. Relative density of nephrin is shown by a bar graph.

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.

	ACKNOWLEDGEMENTS

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.

	FOOTNOTES

^* 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

	ABBREVIATIONS

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.

	REFERENCES

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