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*
Renal Unit, Ghent University, Belgium
Departments of Cell Biology and Pharmacology, Texas Biotechnology
Corporation, Houston, Texas
Institute of Anatomy and Cell Biology I, University of Heidelberg,
Heidelberg, Germany.
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: an.devriese{at}rug.ac.be
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 (7,8,9). 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.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 µg 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
µg 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 IgG2a-specific 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 µl of ice-cold PBS, then twice more with
200 µl of ice-cold PBS. Bound VEGF was measured in a gamma spectrometer
(Life Technologies, Inc., Schaumburg, IL).
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,
Düsseldorf, 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 = ßk x (AT)(3/2), with ß = 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-µm 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 (7,8,9), eNOS staining was assessed semiquantitatively in these cells. For each kidney, 20 glomerular profiles, cut in equatorial section planes and successively appearing 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-µm 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.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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).
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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.
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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.
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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 capillaries, 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).
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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).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 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 anti-VEGF 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-ß (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 effects via upregulation of eNOS in endothelial cells (4,5,6). 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 (27,28,29). 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-ß (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 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.
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-tocopherol.
J Am Soc Nephrol 8:426
-435, 1997[Abstract]
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), control rats +
anti-VEGF Ab (n = 7, [UNK]) and diabetic rats + control Ab
(n = 9, 
) 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.
