Guillermo Valdez Yañez

Accelerated Publication

THE JOURNAL OF BIOLOGICAL CHEMISTRY

Vol. 278, No. 15, Issue of April 11, pp. 12605–12608, 2003

© 2003 by The American Society for Biochemistry and Molecular Biology, Inc.

Printed in U.S.A.

This paper is available on line at http://www.jbc.org 12605

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ated 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

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 _g of furosemide in 200 _l 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-_m 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 isothiocyanatelabeled 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

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 capac

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 (graycolumns), 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. 12606 Anti-VEGF Antibody and Proteinuria

Downloaded from www.jbc.org by guest, on July 26, 2010 ity of anti-VEGF antibody, providing further proof that anti-VEGF antibody specifically induces proteinuria (Fig. 1A).

Recent studies propose that sFlt-1 can also neutralize circulating VEGF and suggest the use of this protein as an anticancer 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 com-

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.

Anti-VEGF Antibody and Proteinuria 12607

Downloaded from www.jbc.org by guest, on July 26, 2010 ponent 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.

Acknowledgments—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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