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*
Department of Endocrinology, Diabetes and Internal Medicine, Division of
Medicine, King's College London Guy's Hospital, London, United
Kingdom
Renal Pathology Unit GKT School of Medicine, King's College London Guy's
Hospital, London, United Kingdom.
Correspondence to Dr. Stephen Thomas, Department of Diabetes, Endocrinology and Internal Medicine, GKT School of Medicine KCL, 5th Floor, Thomas Guy House, Guy's Hospital, London SE1 9RT, United Kingdom. Phone : +44 0171 955 4826 ; Fax : +44 0171 955 2985 ; E-mail : stephen.thomas{at}kcl.ac.uk
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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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VEGF has been implicated in the induction of proteinuria in renal disease (19), and in vitro glomerular mesangial cells produce VEGF, which can be stimulated by fetal calf serum (FCS) (20), transforming growth factor-ß1 (5), angiotensin II (21), and the application of mechanical stretch (22). In addition, the upregulation of VEGF in the mesangium of mesangial proliferative disease was recently described (23). To date, VEGF receptors and VEGF action on HMC have not been studied. We therefore investigated VEGF receptors and the effect of VEGF165 on HMC in vitro and examined the presence of VEGF receptors in the mesangium from human kidney biopsy material to determine whether VEGF165 plays a role in mesangial cell pathophysiology.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cell Culture
HMC were isolated as described previously
(24). Briefly, normal renal
cortex for the in vitro studies was obtained from three kidneys. Two
were obtained from the opposite tumor-free pole of nephrectomy specimens,
removed for localized hypernephromas, and one was obtained from a donor
nephrectomy found to be unsuitable for transplantation on the basis of an
abnormal vascular supply. The cortical tissue was analyzed histologically to
confirm the absence of tumor cells or other renal pathology. Intact glomeruli
were collected from cortical homogenates by serial sieving. The isolated
glomeruli were digested with collagenase (type IV, 750 U/ml) and then seeded
in culture flasks. After the outgrowth of HMC, the glomeruli were removed by
washing and the cells were cultured in RPMI 1640, supplemented with
insulin-transferrin-selenium, 20% FCS, 7 mM glucose, 100 U/ml penicillin, and
100 µg/ml streptomycin in a humidified 5% CO2 incubator at
37°C. HMC were passaged using 0.25% trypsin and 0.5%
ethylenediaminetetra-acetic acid. The cells were stellate or fusiform in
appearance, grew in multilayers, formed hillocks in long-term culture, and
stained for 
-smooth muscle actin by direct immunofluorescence. Cells
did not stain for cytokeratin, factor VIII, common leukocyte antigen, and
Thy-1, excluding contamination of epithelial cells, endothelial cells,
lympho-monocytes, and fibroblasts
(25). Studies were performed
between passages 4 and 7, while the cells retained all of the morphologic and
immunofluorescent features described above. Human umbilical vein endothelial
cells (HUVEC) were obtained as a kind gift from Corrine Nevard (Department of
Pediatric Nephrology Guy's Hospital London United Kingdom).
VEGF Receptor Analysis
Immunofluorescence.
HMC were grown on glass slides and fixed in 100% methanol for 4 min at
-20°C. They were then incubated with 10% normal swine serum for 20 min,
followed by either a rabbit polyclonal anti-human flt-1 antibody (10 µg/ml
in phosphate-buffered saline-bovine serum albumin), a rabbit polyclonal
anti-KDR antibody (10 µg/ml), or a goat polyclonal anti-neuropilin-1
antibody (10 µg/ml) for 60 min at 37°C. After rinsing, the cells were
incubated with a TRITC-swine anti-rabbit antibody conjugate (TRITC-rabbit
anti-goat antibody conjugate for neuropilin-1) for 60 min. The cells were
examined using a fluorescence microscope (Olympus, Lake Success, NY).
A series of negative controls was performed in parallel : (1) the primary antibody was omitted, (2) the cells were preincubated for 30 min at 37°C with 100 ng/ml VEGF165 before addition of the primary antibody, (3) a blocking peptide—soluble flt-1 (100 µg/ml)—was added, and (4) a nonimmune IgG (rabbit for flt-1 and KDR, and goat for neuropilin-1) was used at the same concentration as the primary antibody.
Western Blotting.
Cells were washed twice with ice-cold phosphate-buffered saline and
harvested using a cell scraper. The cells were lysed and sonicated (MSE
Instruments) in 50 mM Tris-HCl with 1% sodium dodecyl sulfate and 1%
ß-mercaptoethanol. Lysates were then boiled for 1 min for protein
denaturation. Glycerol was added to a final concentration of 10%, and
bromphenol blue was added before loading onto a 7.5% sodium dodecyl
sulfate-polyacrylamide gel. After electrophoresis, the separated proteins were
electrotransferred onto a nitrocellulose membrane. The membrane was blocked in
2% nonfat milk in Tris-buffered saline (TBS), pH 7.6, with 0.1% Tween 20
(TBS-T) for 1 h at room temperature with shaking. The membrane was then
incubated for 1 h with either a polyclonal anti-human flt-1 antibody, a
polyclonal anti-human KDR antibody, or a polyclonal anti-human neuropilin
antibody (1 µg/ml in TBS-T and 0.5% nonfat milk), extensively washed in
TBS-T then incubated with horseradish peroxidase-linked antibodies (1 : 1500
dilution). Signal was performed by enhanced chemiluminescence. Negative
controls consisted of : (1) omission of the primary antibody,
(2) an flt-1 blocking peptide (100 µg/ml), and (3) goat
nonimmune IgG at the same concentration as the primary antibody.
Cell Proliferation
HMC were grown to confluence, passaged, and seeded at 35,000 cells per
well. After 48 h of serum deprivation, the cells were treated with recombinant
human VEGF165 for 24 h. Neutralization experiments were performed
in the presence or absence of a rabbit antihuman VEGF165 polyclonal
neutralizing antibody (100 µg/ml).
3H-Thymidine Incorporation.
3H-Thymidine incorporation was measured in TCA-precipitable
material (26).
3H-Thymidine (1 µCi/ml) was added to experimental media for 24
h. The cells were washed and incubated three times in ice-cold 0.6 M TCA at
4°C for 20 min each time. The cells were then solubilized by 0.2N
perchloric acid. Incorporated radioactivity was measured using a liquid
scintillation counter (Packard Tri-Carb Liquid Scintillation Analyzer model
1900 CA).
Cell Number.
The cell number was determined after cell separation using
trypsin-ethylenediaminetetra-acetic acid by Coulter counter analysis, using an
aperture size of 13 to 49 µM (model ZI ; Coulter Electronics, Hialeah,
FL).
Cell proliferation was also assessed using a tetrazolium dye assay based on the conversion of 3-(4,5-dimethyltriazol-2-yl)-2,5-diphenyltetrazolium bromide to formazan by living cells (27). The insoluble formazan was solubilized with 100% ethanol and measured spectrophotometrically at 560 nm.
Ex Vivo Studies : Immunohistochemistry
Subjects.
Normal cortical renal tissue was obtained from five localized tumor
nephrectomy samples for the ex vivo studies (patients age range, 50
to 75 yr) and processed in an identical manner to biopsy specimens from renal
diseases. After retrieval, the samples were immersed in liquid nitrogen for 1
h and then stored at -70°C until analysis. The cortical samples were taken
from the tumor-free pole, and normal pathology was confirmed by light
microscopy. Renal cortical samples were also analyzed from biopsy samples of
five patients (age range, 30 to 75) with proliferative renal diseases. Three
patients had IgA nephropathy and presented with microscopic hematuria and mild
renal impairment, with a serum creatinine level <150 µmol. One patient
had diffuse mesangial proliferative glomerulonephritis with mesangial IgM and
presented with nephrotic syndrome and a creatinine clearance of 29 ml/min, and
another had acute diffuse mesangial proliferative glomerulonephritis with
immune-complex deposits and presented with microscopic hematuria and
proteinuria (1.1 g/24 h).
Immunoperoxidase staining was carried out on 5-µm frozen tissue sections fixed in acetone at -4°C for 10 min. Tissue sections were washed in TBS-T, pH 7.4, then blocked for 30 min with 5% goat serum in TBS-T for flt-1 and 10% goat serum in TBS-T for KDR. Primary antibodies against anti-flt1 (2 µg/ml) and KDR (5 µg/ml) diluted in TBS-T with 5 to 10% goat serum were incubated for 1 h at room temperature. The samples were then incubated with a peroxidase-labeled polymer conjugated to goat anti-rabbit IgG (Envision System ; Dako) for 30 min. Intermediate washes were done with TTBS. For visualization, the sections were developed with 3,3'-diaminobenzidine to produce a brown color, counterstained with hematoxylin, and finally mounted in DePeX mounting medium. Control experiments were performed in parallel. The negative control was by omission of the first antibody and the positive control was factor VIII at a dilution of 1 : 300.
Statistical Analyses
Comparisons among groups were analyzed by ANOVA and between experiments by
the Student-Newman-Keuls test using Statistical Package for the Social
Sciences software. Differences were considered significant at P <
0.05. All data points were determined in duplicate. Data are reported as means
± SD.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Preincubation with VEGF165 (100 ng/ml) for 30 min resulted in virtually no specific staining, as did omission of the primary antibody (Figure 1D). Similarly, the addition of a specific anti-flt-1 antibody blocking peptide (100 mg/ml) also prevented staining of the flt-1 receptor, confirming the specificity of the anti-flt-1 antibody. There was no specific staining seen using nonimmune IgG as the primary antibody.
Western Blotting.
Total protein lysates, from HMC serum and insulin deprived for 48 h, were
analyzed. Using specific antibodies, all three VEGF receptors—KDR with a
molecular weight of 180 kD, flt-1 with a molecular weight of 180 kD, and
neuropilin-1, molecular weight 130 kD
(9,
29,
30)—were detected in HMC
(Figure 2). These findings were
consistent with the known molecular weights of these receptors and were
identical to those seen using HUVEC as a positive control. The specificity of
the band was confirmed by the absence of specific signal in the negative
controls.
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VEGF Induces HMC Proliferation
3H-Thymidine Incorporation.
In time-response experiments, serum- and insulin-deprived cells were
exposed to VEGF165 (100 ng/ml) for 6, 12, and 24 h.
There was a 1.6-fold increase in 3H-thymidine incorporation by 6 h, rising to 3.3-fold at 12 h and 4.8-fold at 24 h. The 24-h time point was thus chosen for further experiments.
In dose-response experiments, a dose-dependent increase in HMC 3H-thymidine incorporation was seen, with a peak 4.8-fold increase at 100 ng/ml (mean ± SD fold increases of 2.3 ± 0.99, 3.8 ± 1.69, 4.8 ± 2.3, and 3.4 ± 2.5 at concentrations of 25, 50, 100, and 200 ng/ml, respectively) (P = 0.016 ; n = 5) (Figure 3A).
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Cell Number.
To establish whether an increase in 3H-thymidine incorporation
was paralleled by an increase in cell number, serum- and insulin-deprived HMC
were seeded in equal number and VEGF165 was added at doses of 50,
100, and 200 ng/ml for 24 h. Cell number was determined by Coulter analysis
after 24 h.
The final cell number was VEGF165 concentration-dependent, with a peak 1.6-fold increase at a concentration of 100 ng/ml (mean ± SD fold increases of 1.2 ± 0.27, 1.6 ± 0.69, and 1.4 ± 0.33 at 50, 100, and 200 ng/ml, respectively) (P = 0.005 ; n = 6) (Figure 3B).
Similar results were also obtained in a subset of experiments, using a tetrazolium dye-based assay of cell proliferation that estimates numbers of viable cells. VEGF165 (100 ng/ml) induced a consistent 1.4-fold increase in HMC proliferation after 24 h (n = 3).
Neutralization Experiments.
The addition of a polyclonal VEGF-neutralizing antibody prevented
VEGF165-induced proliferation as assessed by both
3H-thymidine incorporation
(Figure 3C) and cell number
(Figure 3D).
Ex Vivo Studies : Immunohistochemistry
To exclude that the VEGF receptor expression was not solely related to
in vitro culture conditions and given the effect of VEGF on mesangial
cell proliferation, we examined expression of VEGF flt-1 and KDR, the
principal VEGF receptors, in normal cortical tissue and in proliferative
glomerulonephritis. In normal cortical tissue from nephrectomy specimens,
there was strong positive staining for both flt-1 and KDR in the glomerular
capillary walls. In the mesangium, there was no specific flt-1 staining, but
there was weak KDR immunoreactivity. In contrast, in proliferative
glomerulonephritis samples, in addition to the glomerular capillary staining,
there was strong specific flt-1 and KDR in the mesangium
(Figure 4). In both normal and
disease tissue, some staining of proximal tubular cells was also seen, as has
been described previously
(31). No staining was seen in
negative controls.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the present study, immunofluorescence revealed in HMC in vitro flt-1, KDR, and neuropilin-1 with a staining pattern similar to that seen in human umbilical vein endothelial cells used as a positive control. VEGF receptors colocalize with caveolin-1 in plasma membrane caveolae (28), which explains why the staining pattern was that of a membrane-bound receptor coupled with cytoplasmic staining. There was no nuclear staining as has been described previously with VEGF receptors (28). Preincubation with recombinant human VEGF prevented detection of the receptors, confirming the specificity of the staining for VEGF receptors. This was further confirmed by the addition of soluble flt-1 as a blocking peptide, which prevented staining detected using the anti flt-1 antibody. The receptors were further identified in HMC lysates by Western blotting.
The addition of VEGF to cultured HMC induced mesangial cell proliferation. VEGF at 25 ng/ml stimulated a 2.3-fold increase in HMC 3H-thymidine incorporation, with a peak 4.8-fold increase at a dose of 100 ng/ml. At this dose, there was also a significant 1.6-fold increase in cell number. Both 3H-thymidine incorporation and the increase in cell number were prevented by the addition of specific VEGF-neutralizing antibodies, indicating that these are specific effects of VEGF. Differences in magnitude of the effect as measured by the different methodologies probably reflect the fact that 3H-thymidine incorporation is not a direct estimate of cell number. Indeed, the two methods more directly estimating cell number, namely the Coulter counter method and the tetrazolium dye assay, gave very similar results. The maximum effect was seen at VEGF165 concentrations of approximately 100 ng/ml, higher than those that induce endothelial cell proliferation and collagenase production (34, 35), but comparable to those that induce monocyte migration (18) and pericyte proliferation (36). Notably, HMC in culture produce VEGF in concentrations of this order (20), suggesting that these concentrations may be pathophysiologically relevant. In our study, there was no further increase in either 3H-thymidine incorporation or cell number at doses of 200 ng/ml, a feature also seen in other cell types (34, 35), which may indicate saturation at the receptor or postreceptor level.
The present study did not determine which receptor is important in mediating VEGF-induced mesangial cell proliferation. The mitogenic and gross morphologic effects of VEGF on endothelial cells (37) appear to be mediated via KDR, whereas flt-1 appears important in angiogenesis (38) and in VEGF-induced monocyte migration (39). In vivo, both flt-1 and KDR appear to be upregulated by hypoxia, whereas in vitro hypoxia leads to strong transcriptional activity of the flt-1 promoter with no change, or even a downregulation, in KDR transcriptional activity (38). Hypoxia is an unlikely explanation for the finding of VEGF receptors in the present study, because the HMC were cultured at normal oxygen concentrations, well above those used in the studies of hypoxia.
Two of the kidney specimens used for mesangial cell culture were obtained from tumor nephrectomies. Although we used only tissue distant from a localized tumor, it was possible that this could influence our findings because VEGF and the VEGF receptors are overexpressed in renal cell carcinomas (40). To exclude this possibility, we obtained a normal cadaveric kidney unsuitable for transplantation. The results in cells derived from this kidney were superimposable on those of cells from tumor nephrectomies.
We confirmed our in vitro findings in human biopsy material. We studied "normal" kidneys and, as an example of a disease state, we chose mesangioproliferative glomerulonephritis because we had shown in vitro that VEGF stimulated mesangial cell proliferation. In the normal kidney, staining for VEGF receptors was detected in the capillary wall, but no flt-1 staining was seen in the mesangium and there was only weak KDR staining. This is consistent with the previously reported physiologic location of these receptors (32). In contrast, both flt-1 and KDR are both clearly present in the mesangium in biopsies of patients with mesangial proliferative glomerulonephritis. This is in accord with the observed upregulation of the KDR receptor seen in animal models of mesangioproliferative disease (41) and nephrosis (42), and human studies of early mesangioproliferative disease. Importantly, in both animal models of renal disease and in early human mesangioproliferative glomerulonephritis, upregulation of mesangial VEGF has also been described (23, 41).
Mesangial cell proliferation is a common response of the glomerulus to several diverse injuries in both human and experimental glomerular disease, and several growth factors are implicated in this process. There are several potential sources of glomerular VEGF. VEGF can be released by both resident glomerular cells and by infiltrating T lymphocytes (43) and monocytes (5). Of glomerular cells, epithelial (32), endothelial (44), and mesangial (5) cells produce VEGF in vitro. VEGF is upregulated by several growth factors, inflammatory cytokines (45), and vasoactive agents in the kidney (46). Similarly, known inducers of the VEGF receptor include serum, platelet-derived growth factor, and hypoxia (47). Thus, a series of stimuli involved in glomerular injury can induce both VEGF and its receptor. Our results raise the possibility that VEGF/VEGF receptor systems are upregulated in glomerular disease and may play an important role in mesangial cell pathophysiology.
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Acknowledgments |
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