Antibodies against Vascular Endothelial Growth ... - Semantic Scholar

J Am Soc Nephrol 12: 993–1000, 2001

Antibodies against Vascular Endothelial Growth Factor Improve Early Renal Dysfunction in Experimental Diabetes AN S. DE VRIESE,* RONALD G. TILTON,† MARLIES ELGER,‡ CLIFFORD C. STEPHAN,† WILHELM KRIZ,‡ and NORBERT H. LAMEIRE* *Renal Unit, Ghent University, Belgium; †Departments of Cell Biology and Pharmacology, Texas Biotechnology Corporation, Houston, Texas; and the ‡Institute of Anatomy and Cell Biology I, University of Heidelberg, Heidelberg, Germany.

Abstract. Vascular endothelial growth factor (VEGF) is a cytokine that potently stimulates angiogenesis, microvascular hyperpermeability, and endothelium-dependent vasodilation, effects that are largely mediated by endothelial nitric oxide synthase (eNOS). The expression of VEGF is pronounced in glomerular visceral epithelial cells, but its function in renal physiology and pathophysiology is unknown. VEGF expression is upregulated by high ambient glucose concentrations in several cell types in vitro and in glomeruli of diabetic rats. To assess the role of VEGF in the pathophysiology of early renal dysfunction in diabetes, monoclonal anti-VEGF antibodies (Ab) were administered to control and streptozotocin-induced diabetic rats for 6 wk after induction of diabetes. Based on in

vitro binding studies, an adequate serum VEGF inhibitory activity was achieved during the entire course of anti-VEGF Ab administration. Anti-VEGF Ab treatment but not administration of isotype-matched control Ab decreased hyperfiltration, albuminuria, and glomerular hypertrophy in diabetic rats. VEGF blockade also prevented the upregulation of eNOS expression in glomerular capillary endothelial cells of diabetic rats. Antagonism of VEGF had no effect on GFR and glomerular volume in control rats. These results identify VEGF as a pathogenetic link between hyperglycemia and early renal dysfunction in diabetes. Targeting VEGF may prove useful as a therapeutic strategy for the treatment of early diabetic nephropathy.

It is well established that hyperglycemia is a major risk factor for the development and progression of diabetic nephropathy. Hyperglycemia induces multiple cellular and molecular alterations that presage the development of renal vascular dysfunction. However, the exact sequence of events from exposure to high glucose concentrations to development of renal damage remains equivocal. Early alterations in the diabetic kidney include the development of glomerular hyperfiltration and glomerular hypertrophy, followed by thickening of the glomerular basement membrane, mesangial matrix accumulation, increased urinary albumin excretion rate (UAER), and ultimately progression to glomerular sclerosis. There is compelling evidence for the involvement of various growth factors, including insulin-like growth factor-I (IGF-I) and transforming growth factor-␤ (TGF-␤), in the early renal changes in diabetes (1,2). Vascular endothelial growth factor (VEGF) is a member of a family of heparin-binding growth factors that very potently stimulate endothelial cell proliferation and play a pivotal role in physiologic and pathologic angiogenesis (3). In addition, VEGF is one of the most potent vascular permeabilizing agents known and has the capacity to induce endothelium-dependent

vasodilation. Several lines of evidence indicate that endothelium-derived nitric oxide (NO) acts as a downstream mediator for VEGF (4,5). In cultured endothelial cells, VEGF stimulates endothelial NO synthase (eNOS) expression and activity, resulting in enhanced generation of bioactive NO (6). In the normal kidney, VEGF is strongly expressed in podocytes and its binding sites are localized mainly on glomerular endothelial cells (7–9). Because of this strategic anatomic location, it has been speculated that VEGF may play a role in the regulation of glomerular permeability and glomerular endothelial cell growth (10). Recent studies have demonstrated an upregulation of VEGF and its receptors in kidneys of diabetic rats (11,12). However, whether VEGF plays a causative role in the pathophysiology of diabetic nephropathy remains unknown. Nevertheless, in view of its known biologic properties, its high glomerular expression in physiologic circumstances, and its upregulation in diabetes, VEGF is an ideal candidate to provide a mechanistic link between hyperglycemia and glomerular hypertrophy, glomerular hyperfiltration, and increased glomerular permeability for macromolecules. We therefore investigated the possibility that inhibition of VEGF activity prevents the onset of early renal dysfunction in experimental diabetes. In addition, the effect of VEGF blockade on glomerular eNOS expression was studied.

Received May 5, 2000. Accepted October 10, 2000. Correspondence to Dr. An De Vriese, Renal Unit, University Hospital, OK12, De Pintelaan 185, B-9000 Ghent, Belgium. Phone: ⫹32-9-2404524; Fax: ⫹32-9-2404599; E-mail: [email protected] 1046-6673/1205-0993 Journal of the American Society of Nephrology Copyright © 2001 by the American Society of Nephrology

Materials and Methods Laboratory Animals The studies were performed with female Wistar rats (Iffa Credo, Brussels, Belgium) that received care in accordance with the national

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guidelines for animal protection. Diabetes was induced by intravenous injection of streptozotocin (65 mg/kg; Pfanstiel Europe LTD, Davenham, UK). Slow-release insulin implants (Linshin, Scarborough, Canada), with a release rate of 1 U/24 h, were used to maintain moderate hyperglycemia. Age-matched control rats received an intravenous injection of buffer solution and placebo implants composed of palmitic acid. Final experiments were carried out 6 wk after the induction of diabetes. At the beginning of each experiment, plasma samples were drawn for analysis of glucose, fructosamine, and total protein levels.

Anti-VEGF Antibody Treatment Monoclonal anti-VEGF antibodies (Ab) and isotype-matched control Ab were prepared as described previously (13). Briefly, female 8-wk-old BALB/c mice (Harlan Sprague Dawley, Inc., Indianapolis, IN) were immunized, then received a booster three times, 21 d apart, by intraperitoneal and subcutaneous injections of 50 ␮g of rhVEGF165, emulsified with an equal volume of Complete Freund’s Adjuvant for the primary immunization and Incomplete Freund’s Adjuvant for secondary immunizations. The mouse with the highest serum titer to rhVEGF165 as measured by enzyme-linked immunosorbent assay received an intravenous injection of an additional 30 ␮g of immunogen in phosphate-buffered saline (PBS), 21 d after the last immunization. Three d later, spleen cells were harvested for production of hybridomas to rhVEGF165. Two hybridoma cell lines with highest Ab titer and neutralizing Ab activity were cloned three to four times by limiting dilution in 96-well microtiter plates. Ascites fluid was collected from pristane (Sigma Chemical Co., St. Louis, MO) primed BALB/c mice that received intraperitoneal injections of each of the cloned hybridomas (107 cells), and purified IgG was prepared by Protein A chromatography (Sigma). The isotype and light chain composition of the Ab and the characterization of neutralizing activity were performed as described previously (13). Diabetic rats were either untreated (n ⫽ 11) or treated with antiVEGF Ab (n ⫽ 12) or with isotype-matched control Ab (n ⫽ 9). One mg of the appropriate Ab was injected intraperitoneally three times per week, starting 2 d after the streptozotocin injection until the final experiments. Age-matched control rats were either untreated (n ⫽ 12) or treated with anti-VEGF Ab (n ⫽ 7) during 6 wk.

VEGF Binding Studies To determine the efficiency of the anti-VEGF treatment, we obtained serum samples at baseline and after 2, 4, and 6 wk of treatment with anti-VEGF or control Ab. The samples were drawn from the tail vein just before the next Ab administration. Binding studies were performed using a fusion protein composed of the seven loop ectodomain of Flt-1 fused to the heavy chains of a mouse IgG2a Ab. The fusion protein was captured onto Immulon 4 strip wells with IgG2aspecific goat anti-mouse Ab (Sigma). Increasing concentrations of monoclonal anti-VEGF Ab (used as standards) and increasing dilutions of serum (1:50, 1:100, and 1:250) were incubated separately with 4 ng/ml 125I-VEGF (Biomedical Technologies Inc., Stoughton, MA) for 30 min at 37°C before adding to the receptor. Nonspecific binding was defined as the binding measured in the presence of a 100-fold molar excess of unlabeled VEGF. Incubations were terminated after 60 min at 22°C by washing the wells once with 400 ␮l of ice-cold PBS, then twice more with 200 ␮l of ice-cold PBS. Bound VEGF was measured in a gamma spectrometer (Life Technologies, Inc., Schaumburg, IL).

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Measurement of UAER and GFR For determination of UAER, rats were housed in metabolic cages two times for 24 h and the urine was collected. Urine samples were stored at ⫺20°C until analysis. Albumin was determined with an enzyme-linked immunosorbent assay kit specific for rat albumin (Nephrat, Exocell, Philadelphia, PA). To obtain an estimate of GFR, we measured inulin clearance. The rats were anesthetized with thiobutabarbital (Inactin, RBI, Natick, MA; 100 mg/kg intraperitoneally). The trachea was intubated, a jugular vein was cannulated for continuous infusion of isotonic saline at a rate matching diuresis, and a carotid artery was cannulated for drawing of blood samples. Inulin clearance was measured using the slope technique, as described previously (14). A single dose of FITC-Inulin (Sigma, 80 mg/kg) was administered intravenously as a bolus, and plasma samples were obtained at t ⫽ 3, 30, 120, 140, 160, and 180 min. FITC-inulin plasma levels were measured with a scanning fluorescence detector (Waters 474, Milford, MA). Inulin clearance was calculated as the ratio of administered dose and area under the curve of the inulin plasma levels.

Measurement of Kidney Weight and Estimation of Glomerular Volume Kidneys of six animals from each experimental group were rinsed with PBS by retrograde aortic perfusion for 1 min. The right kidney was perfusion-fixed with 2% glutaraldehyde in PBS, stored in the same fixative for 24 h, and rinsed in PBS. Kidneys were embedded in paraplast and stained with Masson-Goldner’s trichrome technique. Morphometric analysis was carried out with a semiautomatic image analysis system (VIDS IV, AiTectron, Du¨sseldorf, Germany) connected to a Zeiss photomicroscope. Cross-sectional glomerular tuft area (AT, minimal convex polygon) was determined from the mean of 80 random glomerular profiles per animal. Mean glomerular tuft volume (VT) was calculated as VT ⫽ ␤/k ⫻ (AT)(3/2), with ␤ ⫽ 1.38, the shape coefficient for spheres and k ⫽ 1.1, a size distribution coefficient (15).

Immunocytochemistry for eNOS The expression of eNOS in the glomeruli was investigated using indirect immunocytochemistry. After retrograde aortic perfusion with PBS for 1 min, the left kidney of six animals from each experimental group was removed and snap-frozen in melting isopentane cooled by liquid nitrogen. Five-␮m frozen sections were prepared with a 2800 Frigocut E cryostat microtome (Reichert-Jung GmbH, Nussloch, Germany) and fixed in acetone for 10 min. Nonspecific Ab staining was blocked by incubation with PBS containing 0.2% cold-water fish gelatin (Sigma), 2% bovine serum albumin (Sigma), and 2% fetal calf serum (Sigma) for 30 min at room temperature. Incubation with a mouse monoclonal Ab against human eNOS isotype IgG1 (Transduction Laboratories, Lexington, KY), diluted 1:1000 in PBS containing fish gelatin, was carried out overnight at 4°C. Ab binding was visualized with Cy3-conjugated goat anti-mouse IgG Ab (Jackson Laboratories, West Grove, PA). Sections were mounted and viewed with a Polyvar fluorescence microscope (Reichert-Jung). The specificity of the immunolabeling was confirmed by incubation without primary Ab and by incubation with nonspecific Ab. The intensity of the eNOS Ab staining in the kidneys was evaluated by two independent investigators who were unaware of the status of the animals. Because the receptors for VEGF are localized mainly on glomerular capillary endothelial cells (7–9), eNOS staining was assessed semiquantitatively in these cells. For each kidney, 20 glomerular profiles, cut in equatorial section planes and successively appear-

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VEGF and Renal Dysfunction in Diabetes

ing in the visual field of the microscope when moving the section through the entire depth of the cortex, were evaluated. The number of capillary profiles stained by eNOS Ab was determined and normalized for glomerular surface area.

Immunocytochemical Localization of the Anti-VEGF and Control Ab To exclude the possibility of precipitation of the Ab or Ab-antigen complexes in the glomeruli, we analyzed a separate group of kidneys (3 diabetes ⫹ anti-VEGF Ab, 1 control ⫹ anti-VEGF Ab, 2 diabetes ⫹ control Ab, 2 diabetes, 2 control). Kidneys were rinsed with PBS by retrograde aortic perfusion for 1 min, then perfusion-fixed with 2% paraformaldehyde and stored in the same fixative for 24 h before being washed with PBS and embedded in paraffin. To detect the murine anti-VEGF Ab, we deparaffinized 5-␮m kidney sections with xylene followed by 100% ethanol, rehydrated them with graded ethanol (100%, 95%, and 70%) followed by PBS, blocked them for 1 h at room temperature using 2% normal goat serum in PBS (Sigma), then incubated them for 1 h at room temperature with anti-mouse IgG, Cy3 conjugate (Sigma), diluted 1:500 in PBS containing 2% normal goat serum.

Statistical Analyses The data are presented as mean ⫾ SEM. ANOVA and unpaired t tests were used as appropriate to test statistical significance. The significance level was set at P ⬍ 0.05.

Results Characteristics of Laboratory Animals Diabetic animals had significantly higher plasma glucose and fructosamine levels as compared with the age-matched control rats (Table 1). There were no differences in metabolic control between the Ab-treated and the untreated diabetic rats. Body weights were significantly lower in diabetic rats as compared with control rats. There were no significant differences in body weight among the diabetic groups. Kidney weight was significantly higher in all diabetic animals as compared with the control groups. The kidney weight in the diabetic animals that were treated with anti-VEGF Ab tended to be lower than in the other diabetic groups, but the difference was not significant (Table 1). Food consumption was approximately 70% higher in diabetic animals as compared with controls, without significant differences between the diabetic groups (Table 1).

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VEGF Binding Studies VEGF inhibitory activities were measured after dilution of the serum at 1:50, 1:100, and 1:250. The results of the 1:50 dilution are displayed in Figure 1. At baseline, VEGF inhibitory activity was low and not different between experimental groups. A significantly increased serum inhibitory activity was found at 2, 4, and 6 wk of treatment with anti-VEGF Ab in both diabetic and control rats. At 4 wk, inhibitory activity was somewhat lower in anti-VEGF–treated control rats as compared with anti-VEGF–treated diabetic rats. Because the inhibitory activity of undiluted serum of anti-VEGF–treated rats is almost 100%, the clinical relevance of this difference may be minimal. No changes in VEGF inhibitory activity were seen in diabetic rats during treatment with control Ab, except at 6 wk, when a lower inhibitory activity was noted. The inhibitory activities at 1:100 and 1:250 dilution of the serum were lower than those at 1:50 dilution but followed a similar pattern (data not shown).

Measurement of UAER and GFR Untreated diabetic rats showed a marked elevation of the UAER after 6 wk of diabetes, which was partially reduced by anti-VEGF treatment but not by administration of control Ab (Figure 2). Inulin clearance was elevated in diabetic rats, as compared with control rats (Figure 3). The development of hyperfiltration in the diabetic rats was largely prevented by treatment with anti-VEGF Ab but not by injection of control Ab. The anti-VEGF Ab treatment did not significantly decrease GFR in control rats.

Glomerular Volume Glomerular volume was significantly higher in diabetic rats as compared with age-matched control rats. Anti-VEGF Ab treatment decreased glomerular volume in diabetic rats but not in control animals (Figure 4). Administration of control Ab did not affect glomerular volume in diabetic rats.

Immunocytochemical Study of eNOS Expression Positive eNOS staining was detectable in the endothelial cells of preglomerular vessels (arcuate arteries, interlobular arteries, afferent arterioles), of glomerular capillaries, and of postglomerular vessels (efferent arterioles, peritubular capillar-

Table 1. Gravimetric and biochemical characteristics of the experimental groupsa

Glycemia (mg/dl) Fructosamine (␮mol/g total protein) Body weight (g) Kidney weight (g) Food consumption (g) a b

Control (n ⫽ 12)

Control ⫹ AntiVEGF Ab (n ⫽ 7)

Diabetes (n ⫽ 11)

Diabetes ⫹ AntiVEGF Ab (n ⫽ 12)

Diabetes ⫹ Control Ab (n ⫽ 9)

144 ⫾ 12 1.78 ⫾ 0.04 285 ⫾ 4 0.91 ⫾ 0.03 14.0 ⫾ 1.4

117 ⫾ 8 1.79 ⫾ 0.06 273 ⫾ 5 0.88 ⫾ 0.02 14.1 ⫾ 1.3

430 ⫾ 36b 4.00 ⫾ 0.23b 253 ⫾ 6b 1.20 ⫾ 0.05b 24.5 ⫾ 1.5b

438 ⫾ 63b 4.17 ⫾ 0.31b 259 ⫾ 8b 1.12 ⫾ 0.03b 22.5 ⫾ 1.9b

459 ⫾ 31b 3.92 ⫾ 0.28b 254 ⫾ 7b 1.20 ⫾ 0.05b 23.5 ⫾ 1.4b

VEGF, vascular endothelial growth factor; Ab, antibody. P ⬍ 0.001 versus controls.

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Figure 3. FITC-inulin clearance in untreated (n ⫽ 12) and anti-VEGF Ab-treated (n ⫽ 7) control rats and in untreated (n ⫽ 11), anti-VEGF Ab-treated (n ⫽ 12), and control Ab-treated (n ⫽ 9) diabetic rats. *, P ⬍ 0.01 versus control; §, P ⬍ 0.05 versus diabetes; #, P ⬍ 0.01 versus diabetes ⫹ control Ab; $, P ⬍ 0.05 versus control ⫹ antiVEGF Ab. Figure 1. Serum inhibitory activities in diabetic rats ⫹ anti-vascular endothelial growth factor (VEGF) antibody (Ab) (n ⫽ 12, □), control rats ⫹ anti-VEGF Ab (n ⫽ 7, p) and diabetic rats ⫹ control Ab (n ⫽ 9, 䡵) before and 2, 4, and 6 wk after the start of treatment. Inhibitory activities are expressed as percentage of inhibition by 1:50 diluted serum of the binding between 125I-VEGF and Flt-1 in vitro. Comparison among time points: *, P ⬍ 0.001 versus 0 wk; **, P ⬍ 0.05 versus 0, 2, and 4 wk. Comparison among groups: #, P ⬍ 0.001 versus diabetic ⫹ anti-VEGF Ab and control ⫹ anti-VEGF Ab; §, P ⬍ 0.05 versus diabetic ⫹ anti-VEGF Ab.

ies, vascular bundles in the outer medulla). In the glomeruli, the number of stained capillary profiles per glomerulus, normalized for glomerular surface area, was significantly higher in diabetic rats compared with control rats (12.6 ⫾ 0.8 versus 9.1 ⫾ 0.1; P ⬍ 0.01). The increased staining for eNOS in the glomerular capillary endothelial cells was also present in diabetic rats that were treated with control Ab (13.1 ⫾ 1.33; P ⬍ 0.05 versus control) but not in anti-VEGF Ab-treated diabetic rats (8.1 ⫾ 0.4; P ⬍ 0.01 versus diabetes and diabetes ⫹ control Ab; Figure 5).

Immunocytochemical Localization of the Anti-VEGF and Control Ab No staining was detected in either glomeruli, tubules, or peritubular spaces, indicating that the anti-VEGF or control Ab did not accumulate in the kidney (data not shown).

Figure 2. Albuminuria in untreated (n ⫽ 12) and anti-VEGF Abtreated (n ⫽ 7) control rats and in untreated (n ⫽ 11), anti-VEGF Ab-treated (n ⫽ 12), and control Ab-treated (n ⫽ 9) diabetic rats. *, P ⬍ 0.0001 versus control; §, P ⬍ 0.001 versus diabetes; #, P ⬍ 0.02 versus diabetes ⫹ control Ab; $, P ⬍ 0.01 versus control ⫹ anti-VEGF Ab.

Discussion A potential role for VEGF in the pathophysiology of early diabetic nephropathy can be reasoned based on several lines of evidence. First, VEGF is constitutively expressed with a distinct glomerular localization, which suggests an important role in the regulation of glomerular permeability, blood flow, and endothelial cell growth (10). In the adult kidney, VEGF mRNA and protein expression is very pronounced in visceral epithelial cells (7–9). VEGF receptor mRNA and protein were detected in endothelial cells of glomerular capillaries (8,9,11) and preand postglomerular vessels (8). The finding that glomerular endothelial cells are a major binding site for exogenous 125IVEGF (9,11) is consistent with these observations. Although it remains unclear how VEGF crosses the glomerular basement membrane, it is reasonable to postulate the existence of a local regulatory mechanism, with generation of the peptide in podocytes, paracrine secretion, and binding on glomerular capillary endothelium. Second, VEGF expression is increased in several cell types and tissues by high ambient glucose concentrations. High glucose levels in the culture medium upregulate VEGF expression in vascular smooth muscle cells (16), in retinal epithelial cells (17), and in glomerular endothelial cells (18). Upregulation of VEGF has been demonstrated in the retina of diabetic patients (19) and experimental animals (20). An increased renal expression of VEGF mRNA and protein was reported in

Figure 4. Glomerular volume in untreated (n ⫽ 6) and anti-VEGF Abtreated (n ⫽ 6) control rats and in untreated (n ⫽ 6), anti-VEGF Ab-treated (n ⫽ 6), and control Ab-treated (n ⫽ 6) diabetic rats. *, P ⬍ 0.01 versus control; §, P ⬍ 0.01 versus diabetes; #, P ⬍ 0.001 versus diabetes ⫹ control Ab.

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experimental rat models of diabetes type I (11) and type II (12). In addition, the expression of VEGF receptors was found to be upregulated in kidneys of diabetic rats (11). Finally, interference with the VEGF-NO axis was shown to prevent microvascular dysfunction induced by high glucose levels in the dorsal skinfold chamber of the rat (13). Although these studies suggest a causal role for VEGF in the pathophysiology of diabetic nephropathy, the evidence remains circumstantial. The present data are the first to support an causative role for VEGF in the early renal changes in diabetes. Streptozotocin-induced diabetic rats and age-matched control rats were treated chronically with murine monoclonal antiVEGF Ab or with isotype-matched control Ab, the latter to exclude an effect of immunization. Based on in vitro binding studies, an adequate serum VEGF inhibitory activity was achieved during the entire course of anti-VEGF Ab administration. In diabetic rats, anti-VEGF treatment partially prevented early renal dysfunction. The decrease in GFR in the anti-VEGF Ab-treated diabetic rats could not merely be attributed to precipitation of antigen-Ab complexes in the glomerular basement membrane, because no extravascular murine Ab was detected at the end of the experiment by immunohistochemical analysis. The effects were specific for diabetes, because the anti-VEGF Ab did not affect filtration rate and glomerular volume in control rats. These findings are consistent with a previous report that a VEGF165 aptamer did not affect glomerular morphology in normal rats (21). The present results indicate that the upregulation of VEGF and its receptors in the diabetic kidney, as reported by others (11,12), contributes to the pathophysiology of early renal dysfunction in diabetes. The beneficial effect of VEGF-blockade does not exclude the involvement of other growth factors in the pathogenesis of early diabetic renal dysfunction. Inhibition of TGF-␤ (22,23) and interference with the growth hormone/ IGF-I system (24,25) were shown recently to prevent manifestations of early experimental diabetic nephropathy. Several diabetes-induced mediators interact in their adverse effects on the kidney; therefore, it is not surprising that correction of any of them results in amelioration of diabetic nephropathy. In addition, it should be noted that the deleterious effects of VEGF may be limited to early diabetes. When diabetic vascular disease is full-blown and results in tissue ischemia, VEGF may actually be essential for collateral vessel formation (26). Several recent studies provided evidence that VEGF exerts its angiogenic, vascular permeability, and hemodynamic ef-

Figure 5. Immunocytochemical staining for endothelial nitric oxide synthase (eNOS) in control (A), control Ab-treated diabetic (B), and anti-VEGF Ab-treated (C) kidneys. Staining is detectable in glomerular capillaries and in afferent and efferent arterioles, visible at the vascular pole of the glomeruli. As compared with control rats, the glomeruli of control Ab-treated diabetic rats are characterized by increased number of eNOS-positive capillary profiles. Treatment of diabetic rats with neutralizing anti-VEGF Ab results in a lower number of stained capillaries, which is not different from controls. Magnification, ⫻330.

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fects via upregulation of eNOS in endothelial cells (4 – 6). The action of NO as a downstream mediator of VEGF is commensurate with the upregulation of the NO system in early diabetes and its implication in the pathogenesis of early renal dysfunction. NOS blockade substantially reduces or even completely eliminates the hyperfiltration in experimental diabetes (27–29). Chronic administration of NG-nitro-L-arginine methyl ester to diabetic rats partially prevented the increase in glomerular volume in diabetic rats (30). In the present study, an increased expression of eNOS was documented in glomerular capillary endothelial cells of the diabetic rats, confirming earlier findings (27,30). VEGF blockade prevented the upregulation of eNOS in the diabetic glomeruli, thus supporting the contention that VEGF binds to its receptors on glomerular capillary endothelial cells and increases eNOS expression in these cells. The mechanism(s) by which VEGF may affect glomerular permeability and filtration rate remains speculative. VEGF is known to induce fenestrations in endothelial cells in vitro (31), and it has been hypothesized that VEGF is involved in the induction and maintenance of the fenestrae in the glomerular capillary endothelial cells (9). It is, however, generally acknowledged that the capillary fenestrations do not represent the ultimate barrier for filtration but rather that the glomerular basement membrane and the podocyte foot processes with their interconnecting slit diaphragms restrict the passage of proteins. Alternatively, VEGF could affect the filtration barrier by increasing the production of NO in glomerular endothelial cells, which in turn diffuses to the podocytes and acts on the slit pores. In support of this hypothesis, local NOS blockade is known to reduce glomerular ultrafiltration coefficient (32). In addition, VEGF may increase glomerular filtration surface area by stimulating glomerular capillary endothelial cell growth. Nyengaard and Rasch (33) demonstrated that the increased glomerular filtration surface in diabetes results from a formation of new glomerular capillaries, in addition to a slight elongation of existing capillaries. This phenomenon is analogous to the capillary proliferation observed in diabetic retinas and in other vascular beds and thus may be understood as one expression of a generalized diabetic microangiopathy. Our finding that blockade of VEGF prevented the increase in glomerular tuft volume in diabetic rats supports the contention that VEGF is an essential growth factor for glomerular capillaries in pathophysiologic circumstances. In accordance, administration of a VEGF165 aptamer was found to decrease glomerular endothelial cell regeneration in experimental glomerulonephritis (21). The cause of the upregulation of VEGF in diabetes remains speculative, but multiple factors may be implicated. Several factors relevant to the pathogenesis of diabetic complications have been shown to promote VEGF expression in various cell types and tissues, including advanced glycation end products (34), angiotensin II (35), reductive stress (13), reactive oxygen species (36), TGF-␤ (37), and IGF-I (38). Importantly, protein kinase C (PKC), which is increasingly recognized as a central mediator of the damaging effects of hyperglycemia, has been shown to upregulate VEGF (16,39). Direct (40) or indirect (41) inhibition of PKC activation in diabetic rats produced similar

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improvement of glomerular hyperfiltration and albuminuria as in our study, further supporting a link between PKC and VEGF. Taken together, many factors could act independently or in combination to increase VEGF expression in the diabetic kidney. However, the in vivo relevance of these pathways remains to be determined. In addition to increased renal expression of VEGF and its receptors (11,12), a disturbed feedback regulation of VEGF could contribute to the pathophysiologic effects of VEGF in the diabetic kidney. Because VEGF is a molecule with very high potency, the existence of powerful defense mechanisms may be assumed. One such mechanism may be the binding of VEGF to the heparin sulfate glycosaminoglycan side chains in the glomerular basement membrane (42). It is tempting to speculate that the reduced heparin content of the glomerular basement membrane in diabetes (43) may contribute to an increased access of VEGF to its receptors. In conclusion, blockade of VEGF has renoprotective effects in early streptozotocin-induced diabetes in rats. In comparison with untreated diabetic rats, animals that were treated with monoclonal Ab against VEGF exhibited a smaller increase in GFR, glomerular volume, and UAER. The present study demonstrates a new mechanism by which hyperglycemia causes renal dysfunction. Targeting VEGF may prove useful as a therapeutic strategy for the treatment of early diabetic nephropathy.

Acknowledgments The authors thank Rita De Smet, Julien Dupont, Bruni Haehnel, Inge Hartmann, Pascale Vogeleere, and Marie-Anne Waterloos for their expert technical assistance. A.S.D.V. is supported by a grant from the Fund for Scientific Research-Flanders (N20/0).

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