Nephrol Dial Transplant (2003) 18: 1286–1292
DOI: 10.1093/ndt/gfg176
Original Article
Vascular endothelial growth factor expression and glomerular
endothelial cell loss in the remnant kidney model
Darren J. Kelly, Claire Hepper, Leonard L. Wu, Alison J. Cox and Richard E. Gilbert
Department of Medicine, University of Melbourne, St Vincent’s Hospital, Fitzroy, Australia
Abstract
Background. Vascular endothelial growth factor (VEGF)
is constitutively expressed in the glomerulus where it
may have a role in the maintenance of capillary endothelial cell integrity. The present study sought to
examine changes in VEGF expression in a model of
progressive renal disease and to assess the effects of
angiotensin converting enzyme (ACE) inhibition.
Methods. Subtotal nephrectomized (STNx) rats were
randomly assigned to receive vehicle (ns10) or the ACE
inhibitor perindopril (8 mgul drinking water) for 12 weeks
duration (ns10). Sham-operated rats were used as
controls (ns10). Glomerular capillary endothelial cell
density was evaluated by immunostaining for the panendothelial cell marker RECA-1 and VEGF expression
was assessed by quantitative in situ hybridization.
Results. In STNx rats glomerular capillary endothelial
cell density was reduced to 19% that of sham rats
(P-0.01) with a concomitant reduction in glomerular
VEGF expression, also to 19% of sham rats (P-0.01).
Perindopril treatment was associated with normalization of both capillary endothelial cell density and
glomerular VEGF mRNA.
Conclusions. Reduction in glomerular VEGF expression is a feature of the renal pathology that follows
subtotal nephrectomy. In the context of the known
functions of this growth factor, these findings suggest
that diminution in VEGF may contribute to the
demonstrated loss of glomerular endothelium that
develops in this model of progressive renal disease.
Keywords: angiotensin converting enzyme; endothelium; glomerulosclerosis; renin–angiotensin system;
vascular endothelial growth factor
Progressive capillary loss, with the obliteration of the
microvasculature, frequently accompanies fibrosis as
a characteristic feature of chronic renal disease [1].
Indeed, capillary loss correlates closely with declining
function in a range of kidney diseases [2] and may
represent the endothelial component of a common
adverse response to renal insult. However, while much
attention has been focussed on the mechanisms underlying the development of renal fibrosis, the pathogenesis of microvascular injury has only begun to be
unravelled with recent studies suggesting a central
role for vascular endothelial growth factor (VEGF) in
endothelial cell survival and repair [1,3].
Blockade of the renin–angiotensin system (RAS) has
repeatedly been shown to have renoprotective actions
in a range of human and experimental renal diseases
[4,5]. While these effects were initially viewed to result
from attenuation of angiotensin II-mediated effects on
intraglomerular pressure [6], studies conducted over
the past decade have highlighted that other mechanisms may also contribute. These include, among others,
angiotensin II-mediated stimulation of transforming
growth factor-b (TGF-b) expression and the induction of macrophage chemotaxis [7]. However, the effects
of angiotensin II on the expression of VEGF have
been more controversial [8,9].
The aims of the present studies were therefore 2-fold.
First, we sought to examine the effects of progressive
renal disease on VEGF expression and glomerular
endothelial cell integrity. Secondly, we also sought to
determine the effects of angiotensin converting enzyme
(ACE) inhibition on these parameters.
Subjects and methods
Animals
Introduction
The integrity of the glomerular capillary tuft is vital
to the kidney’s primary function of plasma filtration.
Correspondence and offprint requests to: Dr Darren J. Kelly,
Department of Medicine, University of Melbourne, St Vincent’s
Hospital, 29 Regent Street, Fitzroy, 3065 Victoria, Australia.
Email: [email protected]
#
Thirty male Sprague–Dawley rats weighing 200–250 g were
randomized to three groups of 10 animals each. Anaesthesia
was achieved by the i.p. administration of pentobarbital
(7 mgu100 g body weight, Boehringer Ingelheim, Artarmon,
NSW, Australia). The control group underwent sham surgery consisting of laparotomy and manipulation of both
kidneys before wound closure. The other 20 rats all underwent subtotal nephrectomy (STNx) performed by right
subcapsular nephrectomy and infarction of approximately
2003 European Renal Association–European Dialysis and Transplant Association
VEGF and endothelial cell loss
1287
two-thirds of the left kidney by selective ligation of two of
three to four extrarenal branches of the left renal artery.
Animals were then randomly assigned to two groups: STNx
alone or STNx with the ACE inhibitor perindopril (8 mgul
drinking water, Servier, Neuilly, France). Rats were housed
in a temperature (228C) controlled room with ad libitum
access to commercial standard rat chow (Norco Co-Operative
Ltd., Lismore, NSW, Australia) and water during the entire
study. Rats from each group were sacrificed at 12 weeks
post-surgery. At death the remnant (left) kidney was then
sliced sagitally and one half immersion fixed in 10%
neutral-buffered formalin for in situ hybridization and the
other half fixed in 4% paraformaldehyde (0.1 M phosphate
buffer, pH 7.4) for immunohistochemistry. All tissues were
subsequently embedded in paraffin. All experiments adhered
to the guidelines of the Animal Welfare and Ethics
Committee of St Vincent’s Hospital and the National
Health and Medical Research Foundation of Australia.
Renal function
Body weight was measured weekly. Plasma urea and creatinine were measured by autoanalyser (Beckman Instrumentals, Palo Alto, CA) at the beginning and end of the
study. Glomerular filtration rate (GFR) was measured prior
to death by a single shot Tc99m-DTPA clearance. Systolic
blood pressure was measured in conscious rats using an
occlusive tail-cuff plethysmograph attached to a pneumatic
pulse transducer (Narco Bio-system Inc., Houston, TX).
Before death, rats were housed in metabolic cages for 24 h
for subsequent measurement of urinary protein excretion
using Coomassie Brilliant Blue method.
Renal structure
The glomerulus was considered as the area internal to and
including Bowman’s capsule. In 3 mm kidney sections stained
with PAS, 50–80 glomeruli from rats were examined in a
masked protocol. The degree of sclerosis in each glomerulus was subjectively graded on a scale of 0 to 4; Grade 0,
normal; Grade 1, sclerotic area up to 25% (minimal);
Grade 2, sclerotic area 25–50% (moderate); Grade 3,
sclerotic area 50–75% (moderate to severe) and Grade 4,
sclerotic area 75–100% (severe). Glomerulosclerosis was
defined as glomerular basement membrane thickening,
mesangial hypertrophy and capillary occlusion. A glomerulosclerotic index (GSI) was then calculated using the
formula:
GSI~
4
X
Fi (i)
i~0
where Fi is the per cent of glomeruli in the rat with a given
score (i).
Immunohistochemistry
Glomerular capillary endothelial cell density was evaluated
by immunostaining with RECA-1, a pan-endothelial cellspecific monoclonal antibody. Three micron sections were
placed into histosol to remove the paraffin wax, hydrated in
graded ethanol and immersed in tap water before being
incubated for 20 min with normal goat serum (NGS) diluted
1:10 with 0.1 M PBS at pH 7.4. Sections were then incubated
for 18 h at 48C with RECA-1 (Serotec, Oxford, UK). Sections incubated with 1:10 NGS instead of the primary
antiserum served as the negative control. After thorough
washing with PBS (3 3 5 min changes), the sections were
flooded with a solution of 5% hydrogen peroxide, rinsed with
PBS (2 3 5 min) and incubated with biotinylated goat antimouse IgG (Dakopatts, Glostrup, Denmark) diluted 1:200
with PBS. Sections were rinsed with PBS (2 3 5 min) and
incubated with an avidin–biotin peroxidase complex (Vector,
Burlingame, CA) diluted 1:200 with PBS. Following rinsing
with PBS (2 3 5 min), sections were incubated with 0.05%
diaminobenzidine and 0.05% hydrogen peroxide (Pierce,
Rockford, IL) in PBS at pH 7.6 for 1–3 min, rinsed in tap
water for 5 min, counterstained in Mayer’s haematoxylin,
differentiated in Scott’s tap water, dehydrated, cleared and
mounted in Depex.
The magnitude of RECA-1 immunostaining was quantified using image analysis as described previously [10]. Briefly,
the glomerulus, as defined previously, was outlined by interactive tracing. Images were then captured using a BX50
Olympus microscope attached to a Fujix HC-2000 digital
camera and a Pentium III IBM computer. The colour range
for RECA-1 positive cells (brown on immunoperoxidase
labelled sections) were selected and image analysis was performed using a chromogen-separating technique [10]. The
proportional glomerular area showing positive RECA-1
immunostaining was measured from three sections per rat
(ns6ugroup), providing in excess of 50 glomeruliutreatment
group.
In situ hybridization
In situ hybridization was performed using a cDNA encoding
mouse VEGF164 (gift of Dr Steven Stacker, Ludwig Institute
for Cancer Research, Melbourne, Australia). The fragment
containing the entire open reading frame of VEGF was
cloned into pGEM 4Z (Promega, Maddison, WI) and linearized with HindIII to produce an antisense riboprobe using
T7 RNA polymerase. In situ hybridization was then performed on 4 mm paraffin sections using 33P-labelled antisense
riboprobe. In brief, tissue sections were dewaxed in histosol,
hydrated through graded ethanol and immersed in distilled
water. Sections were then washed in 0.1 M PBS, pH 7.4 and
hybridized with 33P-labelled antisense and sense-specific
probes (5 3 105 c.p.m.u25 ml hybridization buffer) which
were added to hybridization buffer (300 mM NaCl, 10 mM
Tris–HCl, pH 7.5, 10 mM Na2HPO4, pH 6.8, 5 mM EDTA,
pH 8.0, 1 3 Denhardt’s solution, 0.8 mg yeast RNAuml, 50%
deionized formamide and 10% dextran sulphate), heated to
858C and 25 ml added to the sections. Coverslips were placed
on the sections and the slides incubated in a humidified
chamber (50% formamide) at 608C for 14–16 h. Slides were
then washed in 2 3 SSC (0.3 M NaCl, 0.33 M
Na3C6H5O7.2H2O) containing 50% formamide at 508C to
remove the coverslips. The slides were again washed with 2 3
SSC, 50% formamide for a further 1 h at 558C. Sections were
then rinsed three times in RNase buffer (10 mM Tris–HCl,
pH 7.5, 1 mM EDTA, pH 8.0, 0.5 M NaCl) at 378C and
treated with 150 mg RNase Auml in RNase buffer for a
further 1 h at 378C, then washed with 2 3 SSC at 558C for
45 min. Finally, sections were dehydrated through graded
ethanol, air dried and exposed to Kodak Biomax MR
Autoradiography film for 4 days at room temperature. Slides
were coated with Ilford K5 emulsion (Ilford, 1:1 with distilled
water), stored with desiccant at room temperature for 21
days, developed in Ilford phenisol, fixed in Ilford Hypam and
stained with H&E.
1288
D. J. Kelly et al.
Quantitative autoradiography
Statistics
Quantitative in situ hybridization, which permits the assessment of gene expression equivalent to northern blot analysis
was used to determine the magnitude of gene expression.
Quantification was performed using two methods: autoradiographic film denistometry and grain counting of
emulsion-coated sections using established methods, as
reported previously by our group [11].
Film densitometry of autoradiographic images obtained
by in situ hybridization was performed by computer-assisted
image analysis as described previously [12] using a Micro
Computer Imaging Device (MCID, Imaging Research,
St Catherine’s, Ontario, Canada). With this method quantification of transcript is based on the changes in X-ray film
density that follows exposure to the radioactive emissions of
radiolabelled VEGF mRNA. In situ autoradiographic images
were placed on a uniformly illuminating fluorescent light box
(Northern Light Precision Luminator Model C60, Image
Research, Ontario, Canada) and captured using a video
camera (Sony Video Camera Module CCD, Japan) connected to an IBM AT computer with a 512 3 512 pixel array
imaging board with 256 grey levels. Following appropriate
calibration by constructing a curve of optical density vs
radioactivity quantification of digitalized autoradiographic
images was performed using MCID software. Data were
expressed as optical density per cm2 relative to control
kidneys (Relative Optical Density, ROD).
In addition to film densitometry, grain counting of emulsioncoated sections was also used to quantify gene expression
and to explore the cell-specific sources of transcript within
the kidney. With this method exposure of the photographic
emulsion to radiolabelled VEGF mRNA leads to the
formation of silver grains that can then be quantified. In
brief, light microscopic images viewed through a 20 3 objective lens were captured and digitized using a Fujix HC2000 digital camera (Fuji, Tokyo, Japan). The outline of 50
glomeruli as defined by interactive tracing was used to assess
glomerular gene expression in each kidney section. Regional
gene expression was quantitatively measured to determine
the proportion of each area occupied by autoradiographic
grains as described previously [12], using computerized image
analysis (Analytical Imaging Station, Imaging Research
Inc.).
All sections were cut in a uniform manner in the midsaggital plane, hybridized to their respective probes in the
same experiment and analysed in duplicate under identical
conditions. All analyses were performed with the observer
masked to the animal study group.
Data are expressed as means"SEM unless otherwise stated.
Statistical significance was determined by ANOVA with a
Fishers post-hoc comparison. Because of its skewed distribution, proteinuria was analysed using log transformed data
and are represented as geometric means 3 u% tolerance factors.
Data derived from the glomerulosclerosis index were not
normally distributed and were expressed as median (range)
and analysed using the Kruskal–Wallis test. Analyses were
performed using Statview II q Graphics package (Abacus
Concepts, Berkeley, CA) on an Apple Macintosh G4 computer (Apple Computer, Inc., Cupertino, CA). The correlation between VEGF and glomerulosclerosis was assessed
non-parametrically using Spearman’s rho. A P-value -0.05
was regarded as statistically significant.
Results
Renal functional and biochemical studies
Rats that underwent STNx surgery became hypertensive
and developed heavy proteinuria, elevated serum
creatinine and glomerulosclerosis. Each of these
parameters was reduced with perindopril treatment
(Table 1).
RECA-1 expression
In sham rats there was intense localization of RECA-1
to the endothelial cells of glomerular capillary loops.
RECA-1 immunolabelling was reduced to 19% of
control levels in the glomeruli of STNx rats (P-0.01,
Figures 1 and 2). This decrease in RECA-1 immunolabelling was attenuated by perindopril to levels similar
to that in sham animals (Figures 1 and 2). Sections
stained with normal IgG showed no staining (data not
shown).
VEGF expression
In situ hybridization autoradiography revealed punctate cortical expression of VEGF mRNA consistent
with localization of transcript to glomeruli (Figure 3).
This pattern of distribution was not detected in
Table 1. Clinical characteristics
Body weight (g)
Kidney weightubody weight
Systolic blood pressure (mmHg)
Proteinuria (mgud)
Plasma creat (mM)
GFR (mlumin)
Glomerulosclerosis
Control
STNx
STNx q perindopril
488"13
0.27"0.01
127"2
21 3 u%1.1
50"1
3.3"0.3
0.1 (0–0.5)
419"13*
0.42"0.08*
186"12{
328 3 u%1.2*
80"0.7*
0.7"0.1*
3.5 (1–4)*
428"24*
0.41"0.03*
124"11"
172 3 u%0.9*{
80"1{
1.5"0.2*{
1 (0–3){
Proteinuria expressed as geometric mean 3 u% tolerance factor.
Glomerulosclerosis index expressed as median (range).
*
P-0.05.
{
P-0.01 vs control.
{
P-0.05.
"
P-0.01 vs STNx.
VEGF and endothelial cell loss
1289
Fig. 1. Representative photomicrographs of glomeruli labelled with RECA-1 antibody showing endothelial cell immunostaining in sham
kidneys (A) and following subtotal nephrectomy in rats treated with either placebo (B) or perindopril (C). Sections show loss of microvascular
endothelium in STNx kidneys that is attenuated by perindopril treatment. Magnification 3 380.
rats. While detectable and still confined to podocytes,
the magnitude of its expression was substantially
decreased in STNx rats (Figures 4 and 5). Sections
labelled with sense probe (negative control) showed
no hybridization (data not shown).
Densitometric analysis of autoradiographic images
confirmed a significant reduction in renal VEGF
expression in STNx rats, which was attenuated by
perindopril (Figure 5). Similarly, quantification of
emulsion-dipped sections also showed that treatment
of STNx rats with perindopril was associated with
restoration of renal VEGF levels, similar to those
of sham animals (Figures 4 and 5). A close inverse
correlation between the degree of glomerulosclerosis
and the magnitude of VEGF expression was noted
(Figure 6).
Fig. 2. Quantification of proportional endothelial cell density as the
proportional area immunostained with RECA-1 antibody. Data are
shown as mean"SEM. *P-0.01 STNx vs control; {P-0.01
perindopril-treated vs untreated STNx rats.
Discussion
autoradiographs from kidney sections of STNx animals
but present in animals that were treated with perindopril following renal mass reduction. In emulsiondipped in situ hybridization, sections showed that
VEGF mRNA was localized to the glomerular visceral
epithelial cells of sham and perindopril-treated STNx
In the present study of progressive renal disease, renal
mass reduction was accompanied by a diminution in
glomerular VEGF expression and a decrease in glomerular endothelial cell density. In the context of the
known role of VEGF in the maintenance of capillary
endothelium these findings suggest that reduction in
1290
D. J. Kelly et al.
Fig. 3. In situ hybridization autoradiographs for VEGF mRNA in sham kidneys (A) and following subtotal nephrectomy in rats treated with
either placebo (B) or perindopril (C). Punctate cortical expression in sham and perindopril-treated rats, consistent with glomerular expression.
Fig. 4. Photomicrographs of glomeruli labelled in situ with antisense riboprobe to VEGF from sham kidneys (A) and following subtotal
nephrectomy in rats treated with either placebo (B) or perindopril (C). Sections show abundant VEGF mRNA in glomerular podoctyes
(arrow) from control and perindopril-treated STNx rats compared with untreated STNx. Magnification 570 3 .
VEGF and endothelial cell loss
Fig. 5. Quantification of VEGF gene expression by film densitometry (upper panel) and by grain counting of glomerular transcript
in emulsion-dipped sections (lower panel) from sham kidneys and
following subtotal nephrectomy in untreated rats (STNx) and those
treated with perindopril. Data are shown as mean"SEM of the
relative optical density (OD) as arbitrary units (AU) and as proportional area occupied by autoradiographic grains. *P-0.01 STNx
vs control, {P-0.01 perindopril-treated vs untreated STNx rats.
Fig. 6. Correlation between glomerular VEGF gene expression (as
assessed in emulsion-dipped sections) and the extent of glomerulosclerosis. There is a significant inverse correlation between glomerular
VEGF mRNA and the glomerulosclerotic index (rhos–0.48, P-0.01).
1291
this cytokine may contribute to the endothelial cell loss
that develops in progressive renal disease.
Subtotal nephrectomy provides a well-characterized
model of non-inflammatory proteinuric renal disease,
frequently used to examine the pathogenesis of progressive kidney disease. Following renal mass reduction, endothelial cell loss due to apoptotic cell death
occurs as an early and progressive feature, commensurate with the development of glomerulosclerosis [13].
Such endothelial cell loss has clear implications for the
maintenance of blood flow within the glomerulus.
However, in addition, the injured endothelium may
also initiate platelet activation and the coagulation
cascade, both also implicated in the pathogenesis of
glomerulosclerosis [14]. In the present study, substantial glomerular capillary loss was noted in animals that
had undergone subtotal nephrectomy. The cause of
this endothelial cell loss cannot be directly determined
from this study. However, given the important role of
VEGF in endothelial cell survival and repair [3,15]
these findings do infer that the demonstrated reduction
in VEGF may be contributory.
The interaction between the renin–angiotensin
system and VEGF expression has been a matter of
controversy. For instance, in cultured vascular smooth
muscle cells and in mesangial cells, angiotensin II
induces VEGF expression [8,16]. Furthermore, renin
gene transfer has also been shown to restore VEGF
expression to skeletal muscle [17]. However, the
opposite effect has been reported in tubular epithelial
cells where angiotensin II leads to a diminution in
VEGF expression [9]. Similarly, divergent effects have
also been reported with blockade of the RAS in the
in vivo setting. For instance, in the retina, ACE inhibition has been shown to reduce VEGF expression [18],
contrasting the findings of the present study in which
ACE inhibition was associated with an increase in
VEGF mRNA. Together, these in vitro and in vivo
studies, suggest that the effects of angiotensin II on
VEGF expression are cell type-specific. A similar cell
specificity may also apply to the relationship between
blood pressure and VEGF where hypertension is
associated with increased VEGF in the retina [19]
contrasting the decrease in its renal expression
demonstrated in the present study.
Like many other growth factors, VEGF is a multifunctional cytokine, initially noted for its permeability
enhancing actions, leading to its implication in the
pathogenesis of proteinuric renal disease [20].
However, in the present study, heavy proteinuria was
instead accompanied by a reduction in VEGF expression suggesting that it is unlikely to have a major role
in the pathogenesis of proteinuria in this model.
In addition to its angiogenic and permeability
enhancing properties, VEGF also induces collagen
synthesis in cultured mesangial cells [21], possibly
via transforming growth factor-b (TGF-b)-dependent
mechanisms [22]. However, in the present study, an
inverse correlation between VEGF expression and the
extent of glomerular matrix deposition was found.
Furthermore, previous reports of increased TGF-b
1292
following subtotal nephrectomy model and its attenuation with RAS blockade [4], suggest that VEGF is
unlikely to directly contribute to the matrix accumulation and consequent sclerosis that develop in this
animal model.
In summary, the present study indicates that
progressive glomerulosclerosis in the remnant kidney
model is associated with diminution in VEGF expression and reduced glomerular capillary endothelial cell
density. These findings were both attenuated by treatment with the ACE inhibitor, perindopril. Whether these
changes are due to a primary effect of ACE inhibition
on VEGF expression, or whether they are a consequence of other renoprotective effects of this class of
drug cannot be determined form the present study.
Acknowledgements. This project was supported by a grant from
the National Health and Medical Research Council of Australia.
The authors thank Miss Mariana Pacheco for and Miss Giao Tran
for technical assistance.
Conflict of interest statement. R.E.G. has received
honoraria for speaking engagements and travel
grants to attend scientific meeting from Servier
Pharmaceuticals, the manufacturers of perindopril.
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Received for publication: 19.5.02
Accepted in revised form: 12.2.03