Functional evidence that vascular endothelial growth
factor may act as an autocrine factor on human podocytes
Rebecca R.
Foster1,
Rachel
Hole2,
Karen
Anderson3,
Simon C.
Satchell3,
Richard J.
Coward4,
Peter W.
Mathieson3,
David A.
Gillatt5,
Moin A.
Saleem4,
David O.
Bates1, and
Steven J.
Harper1,3
1 Microvascular Research Laboratories, Department of
Physiology, University of Bristol, Preclinical Veterinary School,
Bristol BS2 8EJ; and 2 Department of Pathology,
3 Academic and 4 Children's Renal Unit, University
of Bristol, and 5 Bristol Urological Institute, Southmead
Hospital, Westbury on Trym, Bristol BS10 5NB, United Kingdom
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ABSTRACT |
Vascular endothelial growth
factor (VEGF) is expressed by renal glomerular epithelial cells
(podocytes) and is thought to be protective against nephrotoxic agents.
VEGF has been shown to be an autocrine survival factor in
neuropilin-1-positive, VEGF receptor-negative breast carcinoma cells.
Normal human podocytes are also known to express neuropilin-1, VEGF,
and are VEGF-R2 negative. Here, we investigated whether a similar
VEGF autocrine loop may exist in podocytes. Podocyte
cytosolic calcium concentration ([Ca2+]i) was analyzed in primary cultured
and conditionally immortalized podocytes using ratiometric fluorescence
measurement. Cytotoxicity was determined by lactate dehydrogenase
assay, proliferation by [3H]-thymidine incorporation, and
cell counts by hemocytometric assay. VEGF decreased
[Ca2+]i in primary podocytes (from 179 ± 36 to 121 ± 25 nM, P < 0.05) and
conditionally immortalized podocytes (from 95 ± 10 to 66 ± 8 nM, P < 0.02) in the absence of extracellular
calcium. The type III receptor tyrosine-kinase inhibitor
PTK787/ZK222584 abolished this reduction. VEGF increased podocyte
[3H]-thymidine incorporation (3,349 ± 283 cpm,
control 2,364 ± 301 cpm, P < 0.05) and cell
number (4.5 ± 0.7 × 104/ml, control 2.6 ± 0.5 × 104/ml, P < 0.05) and
decreased cytotoxicity (5.9 ± 0.7%, control 12 ± 3%,
P < 0.05), whereas a monoclonal antibody to VEGF
increased cytotoxicity. Electron microscopy of normal human glomeruli
demonstrated that the glomerular VEGF is mostly podocyte cell
membrane associated. These results indicate that one of the functions
of VEGF secreted from podocytes may be to act as an autocrine factor on
calcium homeostasis and cell survival.
intracellular calcium; apoptosis; glomerulus; cell survival; immunogold
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INTRODUCTION |
THE GLOMERULUS IS a
unique functional unit characterized by differential permeability, high
to water and electrolytes and low to protein. The podocytes (visceral
glomerular epithelial cells; GECs) are believed to play a crucial role
in the maintenance of this selective barrier. Podocyte dysfunction,
either genetic (20) or acquired in glomerular disease
(13), results in loss of the macromolecular selectivity of
the glomerular filtration barrier and proteinuria. How podocytes exert
their influence is poorly understood, but the available data suggest
that podocytes contribute structurally by the provision of slit
diaphragm and glomerular basement membrane (20, 22) and
functionally by production of molecules known to affect endothelial
permeability in other vascular beds. Examples of these molecules
include vascular endothelial growth factor (VEGF) (2),
known to increase microvascular permeability (5, 6), and
angiopoietin-1 (33), the only podocyte-secreted molecule
that has been shown to decrease macromolecular extravasation
(38).
Despite the production of VEGF by podocytes at high levels,
the detailed role of VEGF in normal glomerular physiology and its
potential contribution to glomerular macromolecular permeability remain
controversial. Normal VEGF biology is complex. Differential exon
splicing of the VEGF gene results in a number of mRNA species, which
code for a series of isoforms containing different numbers of amino
acids termed VEGF189 and VEGF165 (the most
widespread isoform and also that found predominantly in the renal
glomerulus) and VEGF121 (10). VEGF isoform
expression in glomeruli is heterogeneous. Individual human glomeruli
express one, two, or all three of these main isoforms at the mRNA level
(40). Minor VEGF mRNA splice variants
(VEGF206, VEGF183, VEGF148, and
VEGF145) have also been reported, but they are less well
characterized (18, 19, 29, 40). In addition, evidence for
a new set of almost identical sister molecules of
inhibitory VEGF isoforms in the renal cortex has recently been
described by this laboratory (4).
VEGF signals through two receptors. The primary targets of VEGF on
vascular endothelial cells are the class III receptor tyrosine-kinases, VEGFR-1 (flt-1) and VEGFR-2 (KDR), both of which are expressed by the
glomerular endothelium (9). The latter initiates
angiogenesis, cell migration, and permeability changes. VEGFR-1 also
exists in a soluble form, sVEGFR-1 (sFlt), which is inhibitory when
bound to free VEGF. In addition, the neuropilins have been shown to bind specific isoforms of VEGF, although their signaling properties remain unknown (14, 16, 35, 36). Neuropilin-1 (Np-1), for
example, facilitates the binding of VEGF165 to VEGFR-2
(12) enhancing VEGFR-2-mediated effects.
VEGF, one of the most potent mediators of angiogenesis and endothelial
permeability known, is produced at a high level by the podocytes
200-300 nm from its receptors on the glomerular endothelial cells.
A paracrine action for VEGF would therefore appear clear
(7). For this to occur, however, VEGF needs to act against
a significant filtration of fluid across the glomerular barrier. It has
therefore been suggested that VEGF might act on cells other than the
glomerular endothelium. This led us previously to investigate VEGF-R
expression by human podocytes themselves. Although we were unable to
detect tyrosine-kinase VEGFR-2 expression, we demonstrated the
expression of Np-1 by normal human podocytes in vitro and in vivo
(17). These results suggest that podocytes may have the
potential to bind the VEGF they secrete. We therefore hypothesized that
the potential VEGF-Np-1 interaction may be important in terms of an
autocrine loop or in VEGF sequestration in podocytes (17).
Although Np-1 has been considered as a nonsignaling VEGF coreceptor,
VEGF has more recently been identified as an autocrine survival
factor for Np-1-positive, VEGF tyrosine-kinase receptor-negative breast carcinoma cells (1). Because VEGF has been shown to stimulate increases in cytosolic calcium concentration
[Ca2+]i in endothelial cells, we were
prompted to investigate intracellular cytosolic calcium responses of
cultured human podocytes to exogenous VEGF to address the hypothesis
that VEGF may play a role as a podocyte autocrine factor. We studied
these potential functional responses in proliferating dedifferentiated
primary culture podocytes and nonproliferating differentiated podocytes
in vitro. In addition, we investigated the potential of exogenous VEGF
to act as a survival factor for dedifferentiated proliferating
podocytes. Finally, we determined the distribution of VEGF within the
region of the glomerular filtration barrier using transmission electron
microscopic (TEM) analysis of colloidal gold immunohistochemistry on
normal human glomeruli.
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MATERIALS AND METHODS |
VEGF used in these experiments was recombinant
VEGF165, a kind gift of N. Ferrara (Genentech). All
chemicals/solutions were from Sigma unless otherwise stated.
Primary culture podocytes.
Nephrectomy tissue was supplied by the Department of Urology, Southmead
Hospital, from patients undergoing nephrectomy for unipolar renal tumor
(age range 43-68 yr). All patients were nondiabetic, normotensive
with normal excretory renal function and no urinary sediment. Cells and
mRNA from human tissue were derived from material removed at surgery
and the excess to diagnostic requirements or postmortem. Informed
consent was obtained from patients or relatives as appropriate.
Podocytes were isolated from the nonmalignant histologically normal
pole of renal cell carcinoma nephrectomy specimens by sieving and
cultured under standard conditions as previously described (26). Cells grown by this method demonstrate a typical
polyhedral shape with a cobblestone appearance on confluence and have
been characterized as positive for cytokeratin and Wilms tumor
protein-1 (WT-1) by immunofluorescence; positive for VEGF, WT-1, and
synaptopodin by RT-PCR; and negative by RT-PCR for von Willebrand
factor, CD45, and smooth muscle myosin, excluding contamination by
endothelial cells, leukocytes, or mesangial cells, respectively, as
previously described by ourselves and co-workers (17, 26).
This phenotype was confirmed by regular sampling of cells studied.
Conditionally immortalized podocytes.
This cell line has been conditionally immortalized from normal human
podocytes with a temperature-sensitive mutant of immortalized SV40 T
antigen. These cells have been previously characterized in detail
elsewhere (32). At the "permissive" temperature of 33°C, the SV40 T antigen is active and allows the cells to
proliferate rapidly. Thermoswitching the cells to the
"nonpermissive" temperature of 37°C silences the transgene and
the cells become growth arrested and differentiated. Under these
conditions, they express antigens appropriate to in vivo arborized
podocytes. Cells were grown on coverslips for a period of 14 days to
ensure growth arrest and differentiation.
Intracellular calcium studies.
Podocytes were grown on coverslips to confluence. Cells were incubated
with fura 2-AM (10 µM) for 90 min in DMEM at room temperature, and
the coverslip was then placed in a holder. The holder was then mounted
on a rig consisting of an inverted fluorescence microscope (DM IRB,
Leica) equipped with a UV source (Cairn Instruments, World Precision
Instruments) with filters for excitation at 340 and 380 nm. Fast
switching was achieved using a rotary filter wheel at 50 Hz and a
spectrophotometer for photometric measurement (Cairn Instruments). The
spectrophotometer received emitted light via a 400-nm dichroic filter
and a 510- to 530-nm barrier filter in front of the photometer.
Powerlab software was used for analysis and graphic display.
Experiments were conducted in HBSS media containing 1.3 mM calcium
(i.e., normal extracellular calcium concentration,
[Ca2+]o) and in nominally calcium-free HBSS
(Gibco BRL). Test samples of 1 nM VEGF, 30 µM ATP, used as a positive
control, and HBSS, used as a negative control, were left to wash and
record for 5 min. To ensure that changes in
[Ca2+]i were effectively detected, 5 µM
ionomycin were added to stimulate Ca2+ entry into the
cells. One millimolar manganese chloride (MnCl2) in the
continued presence of 5 µM ionomycin was then used to quench the
calcium-sensitive fura to determine the background (Ca2+
independent) fluorescence signal. Three washes with appropriate HBSS
were used between stimuli, and cells were allowed to rest for 20 min.
VEGF was used at 1 nM, because this concentration has been shown to
produce physiological responses in our previous in vivo experiments
(3, 6, 27).
Emission fluorescent measurements (If) were taken 50 times
a second. The ratio of the If measured during 340-nm
excitation to that during 380-nm excitation (R), proportional to the
calcium concentration, was calculated from
where Rexp = (If340
B340)/(If380
B380).
If340 is the If measured during excitation at
340 nm, If380 is the If measured during
excitation at 380 nm, and B340 and B380 are the
background If values measured during excitations at 340 and
380 nm, respectively (measured as the If after
Mn2+ quenching). Rmin is the in vitro ratio for
zero [Ca2+][Ca2+]i was
calculated from the following formula
Where Kd
(the product of the fura dissociation
constant from bound-to-free calcium and the ratio of maximal-to-minimal
If380) was calculated from an in vitro calibration curve.
The order in which the test samples were added was varied between
experiments. Inhibition studies were conducted in which conditionally
immortalized cells were challenged with 1 nM VEGF after preincubation
for 10 min with the class III tyrosine-kinase receptor inhibitor
PTK787/ZK222584 (100 nM) (a kind gift from J. Wood, Novartis, Basle,
Switzerland), a response recorded, the cells washed three times with
HBSS (minimal calcium) and allowed to rest for 20 min.
[3H]-thymidine assays and cell count.
A 24-well plate was seeded with primary cultured podocytes, which were
incubated in RPMI media containing 1% penicillin/streptomycin, 1%
L-glutamine, 1% insulin transferrin selenite (all Life
Technologies), and 20% FBS. One well was set aside for 1 ml serum-free
media and one for FBS-free media plus VEGF. These were used as negative controls. Podocytes were left for 48 h until established, and then
media were removed and replaced with FBS-free media. One nanomolar
VEGF165 was added to half the wells and one-half were left
untreated. Twenty-four hours later, 37 kBq methyl-[3H]
thymidine (Amersham Pharmacia) were added to each well. Four hours
later, media were removed and 0.2 ml trypsin was added to each well and
left for 2 min. Two hundred microliters of RPMI were added and a
hemocytometer was used to determine cell number in each well (Weber).
Remaining cells were pipetted into 1.5-ml tubes (Eppendorf) and spun at
300 g for 10 min (Biofuge, Heraeus). The supernatant was
removed, and 0.2 ml NaOH was added and left at room temperature for 30 min. Cells were then pipetted into scintillation vials (Fisher), 5 ml
of biodegradable scintillation fluid (Amersham Pharmacia) were added,
and counts per minute were read by a scintillation counter (1217 Rachbeta, LKB Wallac).
Cytotoxicity assays.
Ninety-four wells of a 96-well plate (Costar) were seeded with
primary cultured podocytes and 100 µl 20% FBS-RPMI. Podocytes were
left for 48 h, and then media were removed and replaced with FBS-free media. After 24 h, 100 µl media were removed from each well and cytotoxicity was assayed using a lactate dehydrogenase (LDH)
cytotoxicity detection kit (Roche) and quantified using a Bichrometric
Multiscan plate reader (Labsystems). These samples were used for
background LDH measurement (Txmin). The media of half of
the wells were replaced with 100 µl FBS-free media, the other half
with FBS-free media containing 1 nM VEGF165. Twenty-four hours later, 100 µl media were again removed from each well and cytotoxicity was assayed and quantified. These samples were used to
determine the cytotoxicity (Txexp). Finally, 100 µl of
2% Triton X-100/1 × PBS (final concentration 1%) were added to
each well and left for 10 min to completely lyse the cells. One hundred microliters were removed and cytotoxicity was again assayed and quantified. This enabled determination of the maximum LDH from the well
(Txmax). Percent cytotoxicity (Tx) was
calculated for each well as
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The fold-increase in cytotoxicity was calculated as
[Tx(treatment)]/[Tx(control)].
The percent reduction in cytotoxicity was calculated as
[Tx(VEGF) × 100]/[Tx(No VEGF)].
RT-PCR.
Total RNA was extracted as previously described (8) using
the TRIzol method from the conditionally immortalized human podocyte cell line, human thyroid, brain, and kidney. Reverse transcription was
carried out using 1 µg RNA and 5 µM oligo dT (Promega) in 10 µl
RNAse-free water (Sigma). This mixture was incubated at 65°C for 5 min and immediately placed on ice. The reaction mixture was then
altered to 1× first-strand synthesis buffer (Roche), 10 mM DTT
(Roche), 2.5 mM dNTPs (Promega), 1 U RNA guard (Amersham), and 2.5 U
expand RT (Roche) in a total of 20 µl RNAse-free water (Sigma). This
was incubated at 42°C for 2 h. PCR was performed using the
primers as detailed in Table 1. The PCR
mixture consisted of 1× PCR buffer (Abgene), 1.25 mM MgCl2
(Abgene), 375 µM dNTPs, 10 µM forward primer, 10 µM reverse
primer (except for GAPDH where 5 µM of each primer were used), 1 µl
cDNA, and 1 U Taq (Abgene) in 20 µl RNAse-free water. A
standard PCR cycle was used, i.e., 55°C, 35 cycles (Hybaid). RT-PCR
products were run on 2% agarose (Roche) gels in the presence of 0.5 µg/ml ethidium bromide (Invitrogen). Gels were photographed under UV
transillumination (Gibco). A 100-bp ladder (Sigma) was used to
visualize bands from 100 to 1,000 bp.
Western blot analysis.
Confluent primary cultured podocytes from a T75 flask were left
untreated or treated with VEGF (1 nM) for 30 min. The cells were then
trypsinized, rinsed in PBS, pelleted, and the protein was extracted in
0.2% (vol/vol) SDS, 300 mM NaCl, 20 mM Tris, 10 mM ethyldiamine
tetraacetic acid, 2 mM Na3VO4, 1 mM
phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 20 µM E64, 2 µg/ml aprotinin, 62.5 mM
-glycerophosphate, and 1 µM pepstatin
A. Protein quantification was performed spectrophotometrically using
Bio-Rad dye. Equal amounts of protein were electrophoresed against a
prestained protein size marker using 10% SDS-polyacrylamide gel
electrophoresis. Proteins were electroblotted to polyvinylidene
difluoride membrane. Membranes were blocked in 10% (wt/vol) nonfat dry
milk (Marvel) in 1× PBS-Tween 20 0.1% (vol/vol) (PBST) for 1 h
and incubated with primary antibody (1:300 goat anti-VEGF-R1, SC-316,
Santa Cruz) for 1 h in 1× PBST plus 5% (wt/vol) Marvel. Unbound
primary antibody was removed by five washes in 1× PBST (5 min/wash).
The membrane was incubated for 1 h with 1× PBST plus 5% (wt/vol)
Marvel and secondary antibody (1:3,000 donkey anti-goat IgG). Washes were performed as previously described, and the protein was detected by
enhanced chemiluminescence.
TEM.
One-millimeter cubed pieces of renal cortex were taken from the normal
pole of nephrectomy samples taken for unipolar cancer and fixed in
0.2% glutaraldehyde (Agar Scientific) and 0.2 M phosphate buffer at pH
7.4 at room temperature for 30 min. The tissue was then stored in 0.2 M
phosphate buffer until processed. Specimens were partially dehydrated
using a 10-min wash in 50% IMS followed by three 10-min washes in 70%
methylated spirits (IMS). Specimens were infiltrated with LR white hard
grade resin (London Resin) in a 2:1 ratio with 70% IMS for 30 min.
Specimens were infiltrated with LR white resin for four 30-min periods.
The specimens were then embedded in LR white resin plus an accelerator
(London Resin) in size 00 gelatin capsules (Agar Scientific) and left
to polymerize via a cold catalytic process at 4°C for at least 2 h. The blocks were then transferred to a 50°C oven for 2 h. The
capsules were then exposed to the air and left to set. Sections were
cut at 0.5-0.9 µm on a Leica Reichert Ultracut S ultramicrotome
and placed on a glass slide and stained with 1% toluidine blue in 1%
borax to determine whether the tissue was suitable for further
investigation. Appropriate tissue was cut into 90-nm sections and
mounted onto 300 mesh hexagonal nickel grids (Agar Scientific) and left
to air dry. Grids were washed in 0.01 M PBS (pH 7.4) for 10 min and then incubated in polyclonal rabbit anti-VEGF antibody (A. Menarini) in
PBS (pH 7.4) and 0.6% BSA at 1:10 in Antibody diluent (A. Menarini) for 60 min at room temperature. Sections were then washed for 1 min in
PBS (pH 7.4) and PBS (pH 8.2). The secondary antibody was 15-nm
gold-conjugated goat anti-rabbit IgG in Tris (pH 8.2), sodium azide,
and 0.6% BSA (BioCell at Agar Scientific) 1:10 dilution, applied for
60 min. Grids were washed in PBS (pH 8.2) and deionized H2O
for 1 min and then stained with a saturated solution of uranyl acetate
for 20 min; sections were then washed in deionized water and stained
with lead citrate for 1 min. The grids were then rinsed with deionized
water and left to air dry. Grids were viewed under an electron
microscope (Philips CM10). Podocyte (intracellular or membrane
associated), glomerular basement membrane, and glomerular endothelial
cell-associated gold particles were enumerated in 16 random fields from
four different kidneys. Colloidal gold particles were considered
membrane associated if they were within two particle widths (i.e., 30 nm) of the membrane on either side.
Statistics.
Data are presented as means ± SE. Two-tailed, paired
t-tests were used to compare paired data on the same cells,
and unpaired t-tests were used to compare separate cell
populations treated differently. ANOVA was used to compare distribution
of gold particles on podocyte foot processes.
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RESULTS |
Primary cultured podocytes.
Figure 1 shows the effect of HBSS on
[Ca2+]i in primary cultured podocytes (Fig.
1A) and human tubular epithelial cells (HK2). HBSS did not
change calcium in either cell line or in human tubular epithelial cells
(Fig. 1B) in either the presence or absence (not shown) of
extracellular Ca2+. ATP, on the other hand, caused a
transient rapid increase in [Ca2+]i on all
occasions (Fig. 1C). [Ca2+]i
increased from 113.2 ± 22.1 to 209.5 ± 41.0 nM
(P < 0.01; Fig. 1D) peaking at 30 ± 10 s and returning to baseline after 3.2 ± 0.5 min.
Surprisingly, although VEGF did not alter
[Ca2+]i in the presence of extracellular
calcium (Fig. 2A), VEGF
produced a slow and sustained reduction in
[Ca2+]i, which was significantly
different from baseline in minimal extracellular calcium (Fig.
2B in primary cultured podocytes). There was a significant
reduction in the ratio (R) in minimal, but not normal extracellular
calcium (Fig. 2C), which corresponds to a change in
[Ca2+]i from 178.9 ± 35.6 to 121.1 ± 25.4 nM with VEGF (P < 0.05). A minimum was reached
after 5 ± 1 min.

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Fig. 1.
Effect of HBSS and ATP on podocyte and HK2 cell calcium.
A: example of no significant change of cytosolic
Ca2+ concentration ([Ca2+]i) in
primary culture human podocytes in response to 1 µl HBSS in the
presence of 1.3 mM extracellular Ca2+ concentration
([Ca2+]o). B: means ± SE of
[Ca2+]i change in podocytes and human tubular
epithelial cells in response to 1 µl HBSS in the presence of 1.3 mM
[Ca2+]o. C: transient increase in
[Ca2+]i in primary culture human podocytes in
response to 30 µM ATP. D: means ± SE of
ATP-stimulated response compared with baseline values
(n = 6, **P < 0.005, paired
t-test).
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Fig. 2.
Effect of vascular endothelial growth factor (VEGF) on
[Ca2+]i of primary cultured and conditionally
immortalized podocytes. A: example of
[Ca2+]i change in primary culture human
podocytes in response to 1 nM VEGF in the presence of 1.3 mM
[Ca2+]o. B: example of
[Ca2+]i change in primary culture podocytes
in response to 1 nM VEGF in the absence of
[Ca2+]o. C: means ± SE ratio
before (open bars) and after (filled bars) VEGF treatment.
D: example of [Ca2+]i change in
response to 1 nM VEGF in the presence of 1.3 mM
[Ca2+]o. E: example of effect of
VEGF on [Ca2+]i change in the presence of
minimal [Ca2+]o. F: means ± SE ratio before (open bars) and after (filled bars) VEGF treatment.
*P < 0.05, **P < 0.02 compared with
before treatment.
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Conditionally immortalized podocytes.
ATP administration stimulated a transient increase in
[Ca2+]i in conditionally immortalized cells
from 102.2 ± 34.5 to 142.1 ± 35.8 nM postexposure,
P < 0.05. In a similar pattern to that seen in primary
culture podocytes, VEGF did not alter [Ca2+]i
in differentiated podocytes in the presence of
[Ca2+]o (Fig. 2D) but again
produced a slow but sustained significant reduction in
[Ca2+]i when these differentiated podocytes
were incubated in minimal extracellular calcium (Fig. 2E).
There was a significant reduction in the ratio (Fig. 2F),
which corresponds to a reduction in [Ca2+]i
from 94.7 ± 9.8 to 66.1 ± 8.4 nM (P < 0.02).
To determine whether VEGF was acting on type III tyrosine kinases such
as VEGF-R2 or VEGF-R1, we performed the experiments after preincubating
the cells for 10 min with the type III tyrosine-kinase receptor
inhibitor PTK787/ZK222584. To our surprise, the addition of VEGF to
cells preincubated in PTK787/ZK222584 resulted in a small but
significant increase in [Ca2+]i (Fig.
3A), whereas addition of
PTK787/ZK222584 to cells did not result in any change in intracellular
calcium by itself (Fig. 3B). This VEGF-induced increase in
the presence of PTK787/ZK222584 was consistent in all six sets of
experiments, with a mean ± SE increase from 78.8 ± 35 to
115.2 ± 50.6 nM (P < 0.05, paired
t-test; Fig. 3C). Therefore, the reduction in
Ca2+ stimulated by VEGF (0.69 ± 0.06-fold) was
reversed by this inhibitor (1.47 ± 0.15-fold, P < 0.001; Fig. 3D), suggesting that the VEGF-dependent reduction in [Ca2+]i may be a constitutive
event in podocytes, mediated by one or more type III receptor tyrosine
kinases.

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Fig. 3.
Effect of class III receptor tyrosine-kinase inhibitor
PTK787/ZK222584 on VEGF-mediated [Ca2+]i
changes in transformed human podocytes. A: example of
[Ca2+]i change in response to 1 nM VEGF after
treatment with PTK787/ZK222584 incubated in minimal calcium.
B: means ± SE ratio before (open bars) and after (gray
bars) treatment with PTK787/ZK222584 and 1 nM VEGF.
C: example of [Ca2+]i
measurement in response to treatment with PTK787/ZK222584 alone.
D: comparison of the response of vGEC
[Ca2+]i to 1 nM VEGF in the absence and
presence of PTK787/ZK222584. Values are the relative change in R from
baseline (1 = no change). *P < 0.05, ***P < 0.001.
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Proliferation and cytotoxicity.
In primary cultured podocytes, addition of 1 nM VEGF to culture
medium resulted in a significant increase in
[3H]thymidine incorporation (from 2,364 ± 301 to
3,349 ± 283 cpm, P < 0.05; Fig.
4A). Assuming that the
[3H]thymidine incorporation is the same for each cell for
each division, then [3H]thymidine incorporation gives the
number of cells dividing within a defined time. If more cells are
surviving, then there will be more cells present to undergo the normal
rate of division. Therefore, to determine whether this increase in
[3H]thymidine incorporation was due to increased
proliferation rate or due to an increase in the survival of
VEGF-treated cells (and hence increased cell number), we measured the
number of cells in each well. The cell number also increased from
2.6 ± 0.5 to 4.5 ± 0.7 × 104/ml
(P < 0.05; Fig. 4B) with VEGF treatment.
[3H]thymidine incorporation calculated per cell was
therefore not affected by VEGF (untreated 0.1 ± 0.015 cpm/cell, treated 0.125 ± 0.029 cpm/cell, not significant; Fig.
4C), suggesting that VEGF was acting not by increasing
proliferation rate but by reducing cell death. To assess independently
whether VEGF could reduce cytotoxicity, the effect of VEGF on LDH
release into the media (which occurs when cells lyse) was carried out.
VEGF stimulated a reduction in cytotoxicity from 12.5 ± 3.0 to
5.9 ± 0.67% (P < 0.05; Fig. 4D).

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Fig. 4.
Effect of VEGF on primary cultured podocytes proliferation.
A: means ± SE 3H-thymidine incorporation
without (open bars) and with (filled bars) 1 nM VEGF. B:
means ± SE cell number without (open bars) and with (filled bars)
1 nM VEGF. *P < 0.05 compared with 1 nM VEGF.
C: means ± SE proliferation rate (measured as thymidine
incorporation per cell) without (open bars) and with (closed bars) 1 nM
VEGF. D: means ± SE cytotoxicity without (open bars) and
with (closed bars) 1 nM VEGF. * P < 0.05 compared with
or without VEGF.
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To determine whether this decrease in cytotoxicity was also brought
about by endogenous VEGF, proliferating primary cultured podocytes were
incubated with a neutralizing antibody to VEGF. This resulted in a
significant increase in cell death, which was abolished by addition of
VEGF. The effect of exogenous VEGF, furthermore, was abolished by the
addition of PTK787, although this concentration of PTK787 alone did not
significantly increase endogenous cytotoxicity (Fig.
5). The reduction in cytotoxicity
appeared to occur through phosphatidylinositol (PI3)-kinase activation,
because the reduction in cytotoxicity was blocked by treatment with the
PI3 kinase inhibitor Wortmannin (Fig. 6).

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Fig. 5.
Effect of VEGF on primary culture podocyte cytotoxicity. VEGF
significantly reduced the cytotoxicity of primary cultured
podocytes. A monoclonal antibody to VEGF increased cytotoxicity
(VEGFMab) and blocked the VEGF-mediated decrease (VEGF+VEGFMab). The
reduction in VEGF-mediated cytotoxicity was reversed by
inhibition with the type III receptor tyrosine kinase PTK787
(PTK787+ VEGF), although PTK787 alone did not stimulate an increase
in cytotoxicity. P < 0.001 ANOVA. * P < 0.05 compared with control, Student-Newman-Keuls post hoc test.
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Fig. 6.
VEGF-mediated reduction in cytotoxicity is PI3 kinase
dependent. Pretreatment of conditionally immortalized podocytes (CIP;
gray bars) and primary cultured podocytes (PCP; black bars) by the
phosphatidylinositol 3-kinase inhibitor Wortmannin abolished the
reduction in cytotoxicity induced by exposure to 1 nM VEGF
(P < 0.001, ANOVA). Cytotoxicity was reduced by
74 ± 3.9% in CIP and 74 ± 2.7% in PCP cells treated with
1 nM VEGF. This was inhibited by 25 nM (PCP) and 100 nM (CIP)
Wortmannin. *P < 0.05 compard with 100% Dunnet's
post hoc test.
|
|
VEGF receptor expression.
mRNA for VEGF-R1 (333 bp), R3 (381 bp), and Np-1 (504 bp) was detected
in the conditionally immortalized human podocyte cell line, but VEGF-R2
(332 bp) was not (Fig. 7A).
Brain, thyroid (not shown), and kidney cDNA (Fig. 7B) were
used as a positive control for the primers and GAPDH (364 bp) for
integrity of the cDNA. Each came up positive (results not shown). All
negative controls (water and RNA without reverse transcription) were
blank (results not shown). Furthermore, expression of VEGF-R1 but not
VEGF-R2 protein was detected in Western blot analysis of protein
extracted from conditionally immortalized podocytes (Fig.
7C).

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Fig. 7.
A: expression of VEGR-R1, VEGF-R3, and NP-1 mRNA in
human conditionally immortalized podocyte cell line. VEGF-R2 was not
detected. B: VEGF R2 and GAPDH mRNA expression in human
kidney tissue. C: Western blot for VEGF-R1 with and without
treatment with VEGF.
|
|
Transmission electron microscopy.
Immunogold transmission electron microscopy was carried out to detect
the subcellular localization of VEGF in isolated human glomeruli
derived from the normal pole of nephrectomy specimens. The protocol
described to detect VEGF by colloidal gold resulted from a compromise
between fixation, morphology, and antigen detection, optimized finally
for antigen detection. A short fixation with 0.2% glutaraldehyde was
the only fixation protocol to result in antigen detection.
Colloidal gold particles were seen throughout the glomerular
filtration barrier, within the podocyte foot processes (77.9 ± 1.81%), glomerular basement membrane (11.9 ± 1.2%), and what were taken to be glomerular endothelial cells (10.2 ± 1.6%)
(Figs. 8 and
9A). Unfortunately, the
endothelial morphology was poor with this technique, despite good
morphological preservation of podocytes and basement membrane, so we
were unable to determine whether the staining was predominantly luminal
or abluminal. Of the podocyte foot process-bound VEGF, 63.15 ± 3.29% was membrane associated in contrast to 36.85 ± 3.29%
(P < 0.03), which was intracellular (Fig.
9B). Particles were seen throughout the glomerular basement
membrane and on both luminal and abluminal surfaces of the glomerular
endothelial cells. The differential expression of VEGF at progressively
further distances from the membrane was highly significant
(P < 0.0001, ANOVA; Fig. 9C). Colloidal
gold particles were only identified within Bowman's space when
associated with podocyte cell debris. Negative controls (no primary
antibody included) revealed no gold particles at all within the
glomeruli, although occasional scattered particles were seen in tubular
cells (not shown).

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Fig. 8.
Identification of VEGF expression by immunogold TEM in a
normal human glomerulus. Gold particles can be clearly seen on the edge
of the podocyte foot processes. BS, Bowman's space; GBM, glomerular
basement membrane; PFP, podocyte foot process.
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|

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Fig. 9.
Distribution of VEGF expression by immunogold in a normal
human glomerulus. A: means ± SE% of gold particle
distribution within the 3 components of the glomerular filtration
barrier. B: gold particle distribution within podocyte foot
processes, either membrane associated or intracellular. C:
means ± SE% of gold particle distribution at 25-nm intervals
from the membrane, with results significanly different using ANOVA.
|
|
 |
DISCUSSION |
Characteristics of primary cultured and conditionally immortalized
podocytes.
Despite the extensive use of primary cultured podocytes for renal
research, two longstanding criticisms remain. First, there is the
observation that podocytes alter their phenotype in culture, becoming
dedifferentiated and proliferative. This contrasts with the
growth-arrested, differentiated podocytes in vivo. We therefore studied
both phenotypes. Second, there is the issue of purity. We did our
utmost to ensure podocyte purity of primary cultures. There has been
some debate over the origin of epithelial cells grown using the sieving
method particularly with regard to the effective removal of parietal
epithelial cells. These issues are fully covered by ourselves and
co-workers elsewhere (17, 26). We showed that the vast
majority of cells isolated using this technique have identical
expression characteristics of visceral GECs but cannot exclude a
minority of cells being parietal in origin. For both of these
reasons, we also chose to study a conditionally immortalized podocyte
cell line in addition to primary cultured podocytes. The conditionally
immortalized cell line has a differentiated phenotype and is a pure
population (32). Both primary cultured podocytes and
conditionally immortalized, differentiated podocytes demonstrated a
similar fall in [Ca2+]i in response to
VEGF165 when intra- and extracellular calcium concentrations are similar. Furthermore, in the event of significant mesangial or glomerular endothelial cell contamination of primary cell
cultures, an increase in [Ca2+]i in response
to VEGF165 would be expected rather than a decrease. Both
mesangial and glomerular endothelial cells have been shown to respond
to a variety of agents with increased [Ca2+]i
(reviewed in Ref. 24). Moreover, it is well characterized that the VEGF-mediated increase in permeability of systemic capillaries in vivo is mediated via an increase in
[Ca2+]i (6, 28).
What are the physiological roles for VEGF in the glomerulus?
The physiological role of podocyte-derived VEGF is still poorly
understood. It has, however, been hypothesized that glomerular VEGF may
have an important function in the maintenance of the glomerular
endothelium (including maintaining fenestration) and/or selective
permeability to macromolecules (7, 30). In fact, we
previously showed in vivo that VEGF can effectively increase hydraulic
conductivity (permeability to water) without reducing macromolecular
selectivity in the mesenteric microcirculation, exactly that scenario
present in the glomerular endothelial barrier (3).
Continued controversy over the role of VEGF in normal glomerular
physiology stems from a number of anomalies, however, not least of
which is the apparent minimal disruption that results from the in vivo
administration or inhibition of VEGF165 in normal animals
(25, 39). Although both of these reports only administered or inhibited VEGF165 (one species among many potential
isoforms), neither study demonstrated any abnormality save for
VEGF-associated hypotension (39).
In addition, despite a high level of podocyte VEGF production and
associated high permeability of the glomerular filtration barrier to
water, the glomerulus is not a site of new vessel formation in healthy
subjects. It is clear then that under normal circumstances, the
proangiogenic properties of VEGF must be modified by other factors.
Angiopoietin-1, VEGF165b, and other members of the
inhibitory family of isoforms are good candidate molecules (5,
33). It has therefore been suggested that VEGF may have no
physiological role in health but, in contrast, may only be important in
glomerular disease (stimulating endothelial cell proliferation).
Evidence for this, however, is only apparent from animal models rather than in human pathology (37), and it does not explain why
VEGF is so strongly expressed in normal human glomeruli. Although VEGF decreases cytotoxicity of podocytes grown in culture, the relevance of
this finding to healthy kidneys in vivo is still unclear. Future studies addressing the amounts of specific isoforms of VEGF including the inhibitory isoforms of VEGF (VEGF165b) produced by
podocytes in vivo will help clarify this issue.
Furthermore, the microanatomic positioning of glomerular VEGF
production and receptor expression suggests that VEGF has to diffuse
against a significant filtration gradient to bind to its target
molecules [receptor binding studies would suggest there are no VEGFRs
in the distal nephron (34)]. Therefore, the synthesis of
some isoforms of VEGF, for example VEGF121, which has
little or no heparin-binding properties (and therefore no ability to sequestrate into the glomerular basement membrane), would appear redundant since the glomerular filtration would tend to wash such molecules into Bowman's space. Our colloidal gold TEM finding of
significant localization to endothelial cells would suggest that at
least some VEGF isoforms are able to travel against the gradient of
glomerular filtration down a concentration gradient.
The above paradox, in conjunction with the identification of Np-1
podocyte expression, led us to study potential VEGF-podocyte autocrine
responses. In this report, we provide the first functional data to
support the notion that podocyte-derived VEGF may have autocrine
potential in addition to its other putative roles. Not only have we
shown that exogenous VEGF acts directly on cultured human podocytes,
but inhibition of endogenous VEGF, by a neutralizing monoclonal
antibody, increases cytotoxicity of podocytes, an effect that is
overcome by exogenous VEGF. Interestingly, however, this response is
not mimicked by VEGF receptor inhibitors, suggesting that the
endogenous effect may circumvent receptor inhibition [possibly by
activation of an internal autocrine loop (15)]. Furthermore, we provide the first TEM studies of VEGF expression in
human renal glomerulus. These indicate that most VEGF within the
glomerular filtration barrier is podocyte cell membrane associated. This phenomenon could be explained either by an accumulation of VEGF
protein before secretion or by the sequestration of VEGF onto the
podocyte cell surface, via chemical or receptor binding. Therefore, we
have evidence that supports the hypothesis that one of the roles of
VEGF in the glomerulus is to act as an autocrine survival factor for podocytes.
How does VEGF act on podocytes?
[Ca2+]i is an important second messenger in
most cells and, certainly in endothelial cells, plays an important role
in VEGF-mediated permeability, mitogenesis, and vasodilatation. The
nature of the [Ca2+]i response we
demonstrated in podocytes, however, is atypical. This is the first
evidence that VEGF can stimulate a reduction in
[Ca2+]i under any circumstances in any cells.
The conditions under which reductions in
[Ca2+]i are seen in podocytes in response to
VEGF are nonphysiological (i.e., minimal calcium). The fact that VEGF
can stimulate a reduction in calcium under low external calcium
conditions, however, suggests that VEGF is activating calcium extrusion
or sequestering mechanisms. It is possible that VEGF stimulates
sarcoendoplasmic reticulum calcium or plasmalemmal calcium ATPases
(SERCA or PMCA). Activation of either of these pumps would reduce
[Ca2+]i, but this effect would normally be
masked by normal calcium homeostasis. The functional significance of
this VEGF-mediated reduction in [Ca2+]i in
podocytes is not clear, but there are a number of possibilities. VEGF
is known to act as an autocrine survival factor in breast carcinoma
cells that express the same VEGF receptor profile as do podocytes.
Bachelder et al. (1) showed that VEGF inhibits the
apoptosis of tyrosine-kinase VEGF receptor-negative,
neuropilin-positive breast cancer cells via stimulation of PI3-kinase.
In addition, other studies showed that PI3-kinase activity mediates the
in vitro inhibition of cyclosporin A-induced podocyte apoptosis
via Bcl-X (11). The evidence described above is consistent
with a role of PI3-kinase in this VEGF-mediated reduction in
cytotoxicity. It is well recognized that intracellular subcellular
Ca2+ localization plays an important role in regulating
apoptosis (21). Our findings, that VEGF acts as a
survival factor for podocytes when dedifferentiated and proliferative,
support this hypothesis. Further details of the signaling mechanisms
that underlie this mechanism await further investigation.
Alternatively, the effect of VEGF on the calcium-handling properties of
podocytes may be to modify the response of podocytes to other agents.
Although many molecules (including bradykinin, thrombin, arginine,
vasopressin, and serotonin) have been shown to have no effect on
podocytes [Ca2+]i (24), other
studies have highlighted a number of agents that result in a
dose-dependent increase in podocyte [Ca2+]i.
These include polycations (in primary culture and conditionally modified mouse podocytes) (31) and angiotensin
(24). This latter effect is thought to be mediated via
angiotensin receptors because angiotensin receptors signal by
increasing [Ca2+]i and the response is
inhibited by the ANG II type 1 receptor blocker losartan. Continued
injury of podocytes participates in the progression of chronic renal
lesions. It is generally accepted that ANG II accelerates this process,
because inhibition of the renin-angiotensin system produces benefits in
renal survival in both animal models and humans. If the VEGF-induced
reduction in [Ca2+]i is functionally
important in vivo, then it may explain the mechanism by which VEGF acts
as a cytoprotective agent in such lesions by counteracting the ANG
II-driven increases in podocyte [Ca2+]i.
Our results prompt questions concerning which receptor(s) or
intracellular pathway(s) mediate the response we identified. We
demonstrated that human podocytes in vitro (primary culture) and in
vivo express Np-1. Np-1 has no signaling domain, however, nor does it
have a commercial inhibitor. Podocytes are not believed to express
tyrosine-kinase VEGF receptors (VEGF-R1 or R2) but we demonstrated that
although VEGF-R2 could not be identified, VEGF-R1 and VEGF-R3 mRNA and
VEGF-R1 protein are in fact expressed in the conditionally immortalized
human podocyte cell line. We therefore addressed the possibility that
VEGF may act through a type III tyrosine-kinase receptor (for example,
VEGF-R1). The podocyte response to exogenous VEGF was inhibited by
PTK787/ZK222584. In fact, the addition of this inhibitor produced a
significant increase in podocyte [Ca2+]i.
Because PTK787/ZK222584 is a class III receptor tyrosine-kinase inhibitor that has been shown to inhibit all such tyrosine kinases in
the submicromolar range, including PDGF-R, c-kit, VEGF-R2
(41), we cannot use our data to show that VEGF acts on
podocytes via VEGF-1 with or without neuropilin [the literature
suggests that NP-1 acts as a coreceptor for VEGF-R2 but not VEGF-R1
(23)]. The likelihood is, however, that in podocytes VEGF
either acts on VEGF-R1 and/or VEGF-R3 or via another unidentified
podocyte expressed class III tyrosine-kinase receptor. These data
suggest that these receptors may play a role in normal podocyte
function, and this possibility requires further investigation.
In conclusion, we showed that VEGF can act as an autocrine factor for
podocytes, acting via an alteration in calcium handling of the cells
and reducing cell death. This appears to be true for proliferating
primary cultured cells and growth-arrested, differentiated cells. The
details of the receptor and intracellular regulatory pathways involved
in this phenomenon and any potential effect on cell function or
survival in vivo remain to be determined. In vivo studies including
conditional knockouts of specific VEGF receptors expressed on the
podocytes will need to be done to clarify further the role of VEGF in
an adult glomerulus.
 |
ACKNOWLEDGEMENTS |
This work was supported by the Southmead Hospital Research
Foundation Grant RF157 and Wellcome Trust Grant 58083. S. J. Harper is supported by The Wellcome Trust (057936/Z/99), D. O. Bates by the British Heart Foundation (BB2000003), and S. C. Satchell by a South West NHS R and D grant.
 |
FOOTNOTES |
Address for reprint requests and other correspondence: D. O. Bates, Microvascular Research Laboratories, Dept. of Physiology, Univ. of Bristol, Preclinical Veterinary School, Southwell St., Bristol
BS2 8EJ, UK (E-mail: Dave.Bates{at}bristol.ac.uk).
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.
First published March 4, 2003;10.1152/ajprenal.00276.2002
Received 2 August 2002; accepted in final form 22 February
2003.
 |
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0363-6127/03 $5.00
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