of Texas. Health. Sciences. Center at San Antonio. San. Antonio,. TX. Correspondence ..... of the Medical. School of Marburg. University. Immunofluorescence and ... immunofluorescence and double-label immunohistochemistry tech- niques. .... San Diego, ... by auto- radiography with. Hyperfilm-3H. (Amersham-Buchler,.
Receptors of Vascular Endothelial Growth Factor/Vascular Permeability Factor (VEGF/VPF) in Fetal and Adult Human Kidney: Localization and [1251]VEGF Binding Sites MATTHIAS ELISABETH EBERHARD *I,istittjte
SIMON,* WOLFGANG ROCKL,t CARSTEN HORNIG,t F. GRONE,* HENNER THEIS,* HERBERT A. WEICH, FUCHS, AVNER YAYON, and HERMANN-JOSEF GRONE*
of Pathology,
Differentiation,
German
of Neurobiology, Weizmann
institute
Vascular
endothelial
important
function
in renal
expressed
growth
in podocytes
implications for mans, the cellular specific
receptors:
to localize
(1)
genesis;
(2)
kidney;
and
(3) by
and
aims
binding
into
only
of
binds
was
proteins
possesses a complex vascular and morphologic characteristics.
pressure vessel kines
(2).
The
system and
Vascular
and
their
receptors
endothelial
to
be
the have
and
integrity
regulated
by
known aim
placenta
of
lointerstitial
solely
to
fenestrated
Department
of Molecular
In developing
Cell
glomerubi,
network
(Kd)
Kd: gbomeruli
of
38.6
±
(fK, ii binding receptor cells.
(aK)
Biology,
VEGF
A
n
19.4
±
coexpression
shown,
with
receptor
binding
of
receptor
sites
2.6
(aK,
both
VEGF
togen
for
endothelial
cells
permeability
factor
(VPF)
kidneys
was:
± 7. 1 (fK, n
5),
=
binding
the
in the kidney.
1998)
(1K)
n
5);
=
1 1.6
± 7.0
growth factor-2 displaced VEGF by approximately 60%. VEGF found only in renal endothelial
a function for of angiogenesis.
1044,
developmental
fetal
5), 36.3
=
Flt- 1 demonstrating of
different
and
1 1 .2 (aK,
5) pmol. Placenta in all renal structures proteins thus were
=
in
adult
tubulointerstitium
microvaseubar
renal
blood
factors/cyto-
most
These
VEGF (J Am
is a potent
mi-
mice
an important
function
sites
could
abundant studies
be
VEGF
support
the
in adult kidney that is Soc Nephrol 9: 1032-
Four
and
[indicating
The
(9).
readily the
larger
associated.
This
homologous
chain
increase
brane
binding
studies gene
and
phenotypes
for VEGF
point
to
ontogenesis
a molecular
of VEGF
of amino
VEGF
NH, to
(PDGF)-A
with
and
also
exon
6
enriched
have
are
display
a typical
these because
of
two
to glycosaminoglycans
and sequence
are acid
growth on
that the
are
Although
leader
residues
i89,
detected
isoforms
of a 24-amino
basic
i65.
been
cells.
platelet-derived
with
of 34 to
secreted
of transfected
terminus,
is probably
weight
(VEGF121
acids])
isoforms
in medium forms
the
to increase
in vascular
isoforms
smaller
measurable
two
VEGF
protein
number
preceding
as vascular
ability
its
(3,7,8).
molecular
two
known
of
a functional
for
is a dimeric
46 kD.
It is also
because
(6). In situ
lacking
development
206
(4,5).
permeability
of transgenic kidney
Received June 20, 1997. Accepted November 26, 1997. Drs. Simon and Rock! contributed equally to this work. Dr. Simon’s current address: Division of Nephrology. Department of Medicine, University of Texas. Health Sciences Center at San Antonio. San Antonio, TX. Correspondence to Dr. Hermann-Josef GrOne. Department of Pathology, Klinikum Lahnberge. Universit#{228}tMarburg, Conradistrasse. D-35043 Marburg. Germany. 1046-6673/0906l032$03.00/0 Journal of the American Society of Nephrology Copyright 0 1998 by the American Society of Nephrology
and
Germany;
Departinent
capillary
Affinity
hypothesis independent
transcapillary
of this
(VEGF)
Braunschweig, and
vessels.
VEGF
factor
Regulation
protein appeared as soon as endothelial cells were positive for von Willebrand factor. Specific ‘25I-VEGF binding could be localized to renal arteries and veins, glomerubi, and the tubu-
(3).
growth
of Gene
and post-
are
growth
glomerular
cortical
system with distinct The endothelial to a high
Biotechnology,
were:
immunohis-
glomeruli,
Departtnent
Germany;
stages.
autoradiography
localized
for
In huto two
latter and
capillaries
are exposed
development
seems
vessels,
peritubular
capillaries
two
VEGF
were
The kidney functional
of glomerular
its
By double-label
receptor
of preglomerular
Glomerular
ability
study
The
by
cells
(I).
an
to Flt- I . Quantification
performed
endothelial
cells
The
ofthis
Flt- 1 receptors.
densitometry. VEGF
kidney.
has is consti-
and to increase activator may
binding
sites
computerized
tochemistry,
and
The
the
and
which
binding
(VEGF)
proteins in human renal ontobinding in human fetal and adult
competitive
factor-2,
‘25IVEGF
receptor VEGF
KDR
factor
Germany;
Israel.
and renal disease. depend on binding
KDR.
to dissect
the
achieved
growth
and
VEGF to quantify
components: was
Flt-l
Gottingen,
Rehovot,
of the adult
renal development actions of VEGF
Center
Center,
ontogenesis
for monocytes plasminogen
Marburg,
Research
Primate
of Science,
vascular
of VEGF to be chemotactic activity of collagenase and
University,
National
German
Abstract.
tutively
Philipps
cellregion factor
apparently
plasma
mem-
(10).
VEGF
mRNA
and protein
have
been
demonstrated
in meso-
VEGF/VPF
and metanephros in glomerular leeting duct epithelia by in situ tochemistry ney
cortex
leads
activity
by
ability
increase
the also
and
to development beads
tor)
selectively
receptors;
and
fik-
to mediate tions
are
have
cell-to-cell KDR
and
and
Flt-
mesangial
cells,
exert
effects
VEGF
may
of endothelial
cell
for
that
VEGF
For been
the
other
receptor
receptors
are
human
localized,
and
data
for
kidney.
Cultured
sources
have
binding
characteristics
VEGF activity
aspects an
been
analysis
cannot
always
VEGF/VPF
adult
rat,
conditioned cells
(27).
With we
between
of
rodents,
and receptor
and
been
receptor
that
highly
are
for
developed
in
in
mammals
the
from ovary
vasoactive
important
receptor
proteins
pobyclonal
and
binding the
by
binding
kidney
quantitative into
study
two
were:
in amino
is an endothelial acid
sequence
to localize using
antibodies; during
components,
cell growth to VEGF
(2)
ontogeny
radiographic one
to quantify and
method;
to Fbt- 1 . The third aim was achieved VEGF and placenta growth factor VEGF,
(1)
immunohistochemistry,
monocbonal
in human
an in situ
ofour
due
and to KDR
in the (3) and
g;
pling
humans
column
was
VEGF adult
by
to dissect due
by
1). VEGF
glycosylated
to sKDR
analysis
eluted
at least
with
0.1
M glycin,
dodecyl
purified serum
with
other
VEGF
mined by SDS-lO% polyacrylamide ing conditions. followed by Western proteins
were
membranes
transferred
(Millipore, was
then
completed
anti-rabbit Mouse
IgG
recombinant Freund’s intervals,
of
Antibodies BALB!c
extracellular
mouse
injection.
myeloma
sKDR
hypoxanthine.
cells
Hybridization
was
10% 4
X
the
gel elecblot
reactivity proteins
of the was
deter-
difluoride the Western
(3 1), using
affinity-purified
The
were
bands
alkaline
were
visualized
phosphatase-labeled
Booster spleen was
M aminopterin,
Soluble
FIt-I
with
FIt- I (sFlt-
25
Prog
of
I ) emulsified
in
injections were given at 3-wk cells of immunized mice and
performed
induced
heat-inactivated lO
and
immunized
or soluble
glycol (MW 1500). in Dulbecco’s modified with
in
with I was
Western
the transfer,
to sKDR
mice
complete adjuvant. and fusion between
NS-l
pH 2.4,
antibody.
Monoclonal Groups
p.g!ml. for
The
Bound
at once purification
cross
After
as described
substrate
NaCI,
polyvinylidene
MA).
of 0.55
buffer.
and
receptor
was
pH 8.0.
gel electrophoresis under reducblotting. Fractionated receptor
to Immobilon-P Bedford,
a chromogenic
The
a cou-
(r2l2)
same
staining
to KDR.
protein
with
NaCI.
150 mM
silver
in an
KDR
(SDS)-polyacrylamide
by
r212
mM
were neutralized of the antibody
sulfate
followed
bled.
to DNBr-activat-
antiserum
10 vol of the
after
was
dilution
Germany)
500
A total
the rabbit;
the animal
4 ml of rabbit
eluted proteins The efficiency
sodium
of the
Freiburg,
isolated
(r212). into
was coupled
Tris,
mo-
Germany). 5) was
Receptor
0.2 mg of protein,
Then,
puri-
an apparent
shown by N-terminal chemistry on an auto-
injected
domain)
overex-
were
1 through
was
of KDR
Weiterstadt,
(domains
was Gerinsect
and
with
Biosystems,
with
presence
supernatants
protein
protein
with
analysis
plemented
(30,3
washed were
checked
cell
domains Heidelberg. into Sf158
the
sKDR was degradation
in 100 mM
aliquots. The M Tris-HCI, pH 8.0.
factor
identity
Insect
Biotech,
column
S00-p.l
teins.
specific
one
boosts
of 50%.
to this
using
VEGF
for virus
protein
(Pharmacia
efficiency
to polyethylene were grown
a 53%
sepa-
KDR
extracellular (Clontech, transfection amplification
(Applied
sKDR
Soluble
for
1 10 kD extracellular
by competitive binding of (PLGF)-2. PLGF-2, like with
KDR
1 and
revealed a half-maximal titer at a I :7500 immunosorbent assay for sKDR. Purification of r212 Antiserum. Purified
applied
goat
the aims
These
Flt-
by screening (sKDR).
FIt-I
r21 2 at a concentration
(28,29).
Therefore,
two
The serum enzyme-linked Affinity
protocol
differences
and
(32).
to quantify
used
purified as described (34). Rabbit Polyclonal Antibody
affinity-purified
hamster
binding
there
affinity
Receptors. KDR was cloned cDNA library. A 2.3-kb BgIII IgG-like
1 10 kD with using Edman
soluble
trophoresis
studies
VEGFI#{212}Sobtained Chinese
in
situation.
performed
and
extracellular
it possible
the
of the purified
sequencer
antibodies
and
features
binding
to the in vito have
human
cells
functional
identity
ed-Sepharose
combined As
gas
were
KDR
mass of analysis,
human
(900
not
tissues
the
lecular sequence
The
VEGF placenta
obtained
of soluble
(33).
against
vector pBacPAK9 expression. After
clones
an additional
is part of
(26).
fled
were
These
muscle
characterized.
define
receptors
high
make
receptors
for the first seven
of 0.5 mg of purified
cells.
in
clones
pression
and
suggest
function
coding
matic
In situ
receptors
to the
Methods
of Soluble from a human
of pan-
have
different
generally
to
shown
been
from
transfected
regard
have
not
recombinant
medium
peptides,
kidney
VEGF
these
studies
human
in human
VEGF
be transferred
binding
using
(23-25).
VEGF
ofAntibodies Receptors
transferred into the many) for baculovirus
The
to modulation
integrity
proteins
culture can change their morphologic compared with the in vivo situation, vitro
smooth
receptor
of
to of
cells
on endothelial
cells
used
be expressed
beta
uterine
has
kiKDR/
interac-
found
of renal
endothelial
by
may
in addition
and
exclusively
their
1 also
with
1033
(VEGF-Rl)
binds
of PLGF
of the
and
message.
1 is essential
cell-to-matrix
and
mRNA
kidney,
of these
basic
sites
Preparation and Flt-J
cells,
kinase
that
Flt-
as monocytes.
proliferation
VEGF
Knowledge the
such
=
recep-
tyrosine
suggest
whereas
ereatic
studies
features
binding
fragment
characterized with
mice
vasculogenesis,
Because
domain
domains
cells
cells,
plasma
III tyrosine
are
knockout
on nonendothelial cells,
type
to Flt-l
PLGF
of Fit- I , but not to KDR,
Generation from cDNA
( I 9,20).
intracellular on
endothelial
islet
to two
insert
domains repeats
for
(2 1 ,22).
development
affinity
(kinase
receptors
Results
1 is important
high
or KDR
immunoglobulin-like
receptors
kidney
(15-18).
with
extracellular
activity.
activator
called Flt- 1 (fms-like tyrosine kinase1 I ) and fik- I (fetal liver kinase = VEGF
These
their
normal
disease
affinity
high
in contrast,
Kidney
rately.
and to
plasminogen
with
in Human
cap-
for monocytes
and
for
of renal
receptors receptor [RI
Both
lacking
bind
(VEGF-R2);
receptor-binding the
of VEGF
gbomeruli
of collagenase
implications
in humans.
nase
kid-
in organotypie
inhibition
in vito
to abnormal
[R] 2) in mouse
seven
embryonic
of capillaries
to be chemotactic
activity
binds
receptor
KDR
Materials
of VEGF
have
VEGF VEGF
to rabbit
(1 3); in contrast,
the pathogenesis
membrane
given
(14).
The may
protein
antibody
tufts
homodimers domain
VEGF
conditions
iblary
and in cobimmunohis-
(11,12).
Recombinant culture
visceral epithelia hybridization and
Receptors
3 d after
by exposure
After
fusion,
Eagle’s horse and
the third of the
mixed
medium serum
cells
cell cultures (DMEM)
containing
1.6 X l0
booster
mixed
suplO
M thymidine
M
1034
Journal
(HAT
of the American
medium).
Cultures
Society
were
refed
with
every 2 to 3 d. After 10 to 14 d, culture specific antibodies by enzyme-linked munoblotting,
using
were
KDR-l
(IgG-l
determined
using
DMEM-HAT
supernatants immunosorbent
were
) and
clone
the Sigma
190.1
1 for anti
Immunotype
TM
sFlt-4,
for im-
domain
Positive wells were isolated, The mouse immunoglobulin
sFlt-l , sFlt-4, or cross-react with
by incubation
with
SO p.1 of unspecific
for 30 mm supernatant
at RT and centrifuged was then incubated
(diluted
1 :20)
1 h at RT.
The pH
(extracellular
pellet
8.0),
buffered buffer
the
was
0. 1%
domain,
washed Triton,
gels.
rabbit
was
jugated
After used,
goat
twice
with
once
Human
control,
TSA
with
blotting,
an
followed
IgG
adult
the
with
removal frozen
(n
due to renal in isopentane,
The
fetal
formed tissue. for this be well
serum
2.5 tg
and
once
dissolved gel
affinity-purified
diluted
Tris,
to use
5) were
=
was
applied
(diluted
for S serum
body
for
the
of purified
10 mM
with
NaC1,
phosphate-
After
tions
for
monoclonal
dilutions
from
peroxidase-con-
surgical
autopsies
per-
was
structures could listed in Table I. obtained
of Marburg
and
Immunohistochemistry
from
the
University.
Immunohistochemistry
was
in acetone
immunofluorescence
at
and
carried -
out
10#{176}C for
double-label
on
S-p.m
10 mm,
frozen
using
by
Hamburg, goat Germa-
(FCS;
factor
10%)
antibody
at 37#{176}C followed
I :20;
Sigma,
by
Munich,
medium.
8.4,
body were
tissue
double-label
immunohistochemistry
tech-
or
antibody
mouse
of 20
antibody
was
in PBS,
then
pH
(Z 259,
7.6.
For
DAKO)
mM),
naphthol-AS-BI-phosphate and levamisole 146 mM
sections
new
(0.5
(S mM)
NaC1
at 22#{176}C for
staining,
(28
for
seemouse
incubated
nitrite
p.g
to the tissue
1 h; alkaline-phosphatase
of sodium
containing
anti-
the
fuchsin
mM),
in SO mM
dim-
TnsIHC1
15 mm.
were then washed in H2O and PBS, followed by an with FCS (10%). Anti-von Willebrand factor rabbit anti-
(DAKO), washed
diluted 1 :500, was applied for 18 h at 4#{176}C. Sections three times with PBS at 22#{176}C.A mouse anti-rabbit
two
diluted antibody
antibodies
R (Camon,
incubated
rabbit
IgG,
to produce
for the with
respectively,
without
antibody.
For competition
I h.
a blue
color.
1:40) Blue
Sections
medium.
Control experiments entailed immunohistology antigens,
followed by a 1:40. Each of
monoclonal antibody (diluted 1 h and developed with Vector
Germany)
in aqueous
applied, diluted
at 22#{176}C for
mouse at 22#{176}C for
Wiesbaden,
mounted
1 :50; DAKO) was (Z 259; DAKO)
was
Alkaline-phosphatase was then incubated were
monoclonal
IgG!ml
at a concentration
primary
I :40)
done
(2 1 mM),
mouse
p.g
anti-mouse
(diluted
(64 mM),
pH
the 15
at 22#{176}C for
were
fuchsin)
buffer,
of the
to a solution
these
Immunofluorescence fixed
antibody
exposed
(basic
with
190. 1 1 to flt-l
was applied
Sections incubation
specimens
serum
Willebrand
(diluted
of
2 h at 22#{176}C, a rabbit 1:40
during
School
(con-
Eching,
calf
in aqueous
incubated
application
obtained
of these
mounted
were
(n
Medical
were
antibody
IgG!ml.
1 h. All
abortion and were handled as described for adult of fetuses of I 7-wk gestation or older were taken
of the
fetal
for 45 mm
IgG
primary
at 37#{176}C,followed
37#{176}C; Biozol,
incubated
the dilution
(M 737, DAKO, by rhodamine-labeled
PBS,
at a concentration
monoclonal
mune
sections
was
anti-rabbit
antibody (M 737, rabbit anti-mouse
5) were
mm
at 22#{176}C.Von
sheep
parts were excised, and stored at -70#{176}C.
=
mm
with
DAKO)
KDR-l
ethylformamide
IgG
after
20
sections
anti-KDR
immediately
45
used
at a 1:10
Immunohistochemistry
Tissue
were
horseradish
washes
Sections
Double-Label
I :2500.
received
Germany).
for
1 :20, 45 mm,
three
for
1 :25;
incubated
(diluted
after
Tumor-free nitrogen,
sections
committee
Then,
in 30 j.d of sample electrophoresis with
study; in these, renal glomerular and tubular visualized. Gender and age of patients are
Approval
IgG
ny).
(r212)
monoclonal antibody mm at 37#{176}C, followed
cell carcinoma. cooled in liquid
kidneys
shortly after Only kidneys
IgG!ml)
mouse for 45
diluted
(10 mM
TSA,
by anti-rabbit
(Promega)
kidneys
j.g
with serum
Kidneys
The
35
experiments
antibody
over
I 10 kD).
saline (PBS). The pellet was and used for SDS-polyacrylamide
7.5%
ethics
For
serum
incubated bovine
in a microcentrifuge with rabbit anti-KDR
(50 pA) was preincubated
protein
rabbit
(35). The lysate was then slurry (saturated with
albumin) mm. The
KDR
taming
anti-KDR
FITC-labeled
1 h at room temperature (RT) SO pA of protein A-Sepharose
immunoprecipitation
immunofluorescence
polyclonal
anti-mouse
The
or PDGF-R.
over
rabbit
(IgG-1)
sFlt-l
Immunofluorescence
Double-label
anti-rabbit Germany)
Isolation of KDR from Porcine Endothelial Cells Overexpressing the KDR Receptor. Cell lysate from 1 X lO cells (800 pA) was precleared
Double-Label
and cells isotypes
Kit (150-1).
KDR-l monoclonal antibody did not cross-react with PDGF-R3. The Flt-l monoclonal antibody did not sKDR,
medium screened assay and
KDR extracellular
baculovirus-expressed
or sFIt-l protein, respectively. were cloned by limiting dilution. of the clone
of Nephrology
immunhistochemical nonimmune mouse and
primary
with
antibody,
sKDR
added
to the section,
together
higher
concentration
than
the primary
used
for
as competing
alkaline
the respective
with
that
or sFlt-1
or without
experiments,
demonstrations IgG or nonimphosphatase competitor
antibody,
was
at a SO-fold
the antibody.
niques.
VEGF/VPF
in Vitro
Binding Table
I.
Gender were
and
age
obtained
of patients for
from
‘25I-VEGF
binding
Adult Gender
kidneys studiesa
(yr)
Gender
bovine medium Age
(wk)
x
cells
were
washed
19
buffer
20
VEGF165,
Female
22
mately
46
Female
57
Female
65
Female
22
(31,36). PLGF-2
a
VEGF,
vascular
endothelial
growth
factor.
(bFGF)
lO
serum
cells
(36).
with
(Sigma)
Diego,
similar
pM
were
(Life
Technologies, plates.
of
VEGF amounts
of
PDGF-BB, were
used
human and
for
seeded Paisley,
competition
Scot-
3 to 4 d, the
Hepes,
and
1 mg/mI
recombinant activity
fibroblast experiments
human of
Bensheim,
recombinant basic
for
in EGM
for 3 h at 4#{176}C in binding
a specific
(Immundiagnostik,
pas-
described
After
25 mM
‘25I-labeled with
between
to those
cells
with
microvascular
CA)
in 12-well
DMEM
25I-VEGF,
j.tCi/ng
(3 1 ,33),
FCS
human
pH 7.4, and incubated 23
named Different
5%
using
San out
Assay on
Briefly,
per well
albumin,
containing 2000
Clonetics, carried
cells
Binding
performed
were
(Clonetics) at 2
Female
Male
8 and
land)
Male
Male
were
(MVEC;
endothelial
bovine
42
cells
6 and
I7
Female
63
endothelial sages
Fetal Age
Male
whom
Receptor
experiments
approxiGermany)
VEGF165 growth (see
(33), factor Figure
VEGF/VPF
5). Radioactivity
present
in the lysates
was
quantified
using
a gamma
the
counter.
human
with
KDR
purified
in Situ
Frozen
sections
thaw-mounted uum
Receptor
(10
p.m;
or
Control
periodic
pathologic sites
In short.
consecutive
DMEM
supplemented
4 .tg!ml
leupeptin,
then with
and
the
labeled
binding
was
of a 700-fold
excess
were
twice
washed
to remove
For
unspecific
the specificity
‘251-VEGF VEGF and
binding
incubated
The
standards
sections
Imaging,
tissue
using
The
sites generated
and Pad and
dry
(Bniax) with
After
San
Diego, more
incubation were
Rochester,
NY).
consecutive
(Figure that
von
covered
fixed
sections
with
were
densito-
of
Kodak
stored
Prism,
‘25I-VEGF
we used
the coverslip
for
tech-
‘251-VEGF.
the
(Kodak,
but
and
In
human
and
4c)
positive
more
label
for
4a) colocalized
the
latter
antigen
(Figure
ex-
2, e and also
Willebrand
were
in In
capillaries obtained
d,
had
factor.
a
Compa-
with
the
or Fit- 1 protein
KDR
as
appeared
factor.
VEGF-R
von
glomeruli for
depicted
be demonstrated
(Figure
Peritubular
was
as soon
factor
not
with
than
specific
which 1 ),
(FIt-
Willebrand
and KDR
4a).
fetal
(Figures
(Figure
3b
3c)
were
monoclonal also
in peritubular
cells
and
kidney,
mono-
(Figures
3a
cells
VEGF-R. endothelial (Figures
smooth
glomerubar
endotheliab
for only
capillaries
vascular
VEGF
adult and
positive
antibodies,
muscle
cells
capilof
As
shown
cells
were
3d
and
4b).
did
not
exhibit
arteries by
the
stained, Mesangial staining
receptors.
Characteristics
counterstained
with
VEGF To
binds
receptors,
the
MVEC.
The
specifically
specific
to
radioactive
binding PDGF
could
was
be
was
achieved bFGF
Fifty with
did
whether
cell
surface
incubated
with
human
in
percent 58 pM
not
iodinated
VEGF/VPF
competed
or PLGF-2. and
Receptors analyze
the
ligand
binding
VEGFI6S
PLGF-2.
eosin.
of
MVEC in Culture.
VEGF165
unlabeled
10 to 14 d. developed
and
could
factor, and
1
convolutes,
3a),
for
r2l2,
Willebrand
von
strongly
antibodies
Binding
binding
2 emulsion
at 4#{176}C for
Readymatic,
the
Scatchard
(GraphPad
NTB
antibody
von
for
of VEGF-R
results
laries
for
experiments.
SO pM
to
were
and S-structures
glomerular
Adult Kidney.
per mg
between with
belong
as PDGF-R
and 4a).
of the
quantification
3a
coexpression
system
values
of
more
Figures
Human
precisely,
in Kodak
were
using
second
proteins
1B).
VEGF-R
VEGF-R
Willebrand often
rable
d).
FIt- 1 (Figure
clonal
of ‘25I-VEGF
programs,
the
immunofluorescence
for
negative
stages
VEGF-R, pressed
[i25I]
bound
analysis
level,
Proteins
with
positive
were
developed with
kidney)
with
saturation
but
(Figure
in comma-shaped
2, a through
cells
CA).
of renal
Slides
D19,
air.
in fmol for
from
relationships
structures
sections
derived
regression
the anatomical
renal
in dis-
amount
method
to a
lymphatic
with
such
with the anti-KDR
cells
has been established (37). (Kd) and the maximal num-
curve-fitting
nonparametric
Software,
in Kodak
were
the
expressed
of this
were
nique.
was
sections constant
To define sites
to determine
which
proteins
endothelial
fetal
Grey
in
receptor
receptor
kinases
Double-label
in the pre-
analysis
Canada).
led 1 A).
at a moderate
Other
VEGF-Receptor
cross-reactive
M) of unlabeled
image
Ontario,
validity
analysis Graph
a computerized
of
protein
of cold
films
antibody
(Figure
reactivity
VEGF
antibody
Kidney.
VEGF-R
performed by autoBraunschweig,
The
the tyrosine
the
slightly
once
for 2 d, together
binding sites in tissue equilibrium dissociation
ber of binding Data
exposed
used
sections,
equivalent.
peptide The
with
for
to l0
i2
binding sites was (Amersham-Buchler,
were
were
to tissue
(lO
cross
noticed.
by
Fetal VEGF-R
the
and
recognized
same
incubation,
in detail,
a weak to
the
(I 10 kD)
is expressed
I) was
related
1035
of
KDR
detected, that
Also,
III receptor
Localization
in I .5 M NaCl
(adult
not
class
also
(FLT-
used.
were
a stream
binding 40 pM
doses
St. Catharines,
[1251I-microscales bound
under
Amersham-Buchler).
analyzed
(MCID
After
are
not
up to
experiments sections
that
of
immunobands
a receptor
cells.
receptor
and
(microscales,
metrically
dried
with
Quantification of ‘251-VEGF radiography with Hyperfilm-3H Germany).
in the
to proteoglycans,
then
increasing
at RT pM
endothelial
Preincubation
specific
antibody
protein,
Kidney
MgCI,.
and
once
in
Sections
experiments
5 to 250
in PBS,
of ‘251-VEGF
were
and with PLGF-2.
0.5 mM
polyclonal
same
‘25I-VEGF.
fluoride.
VEGF.
10 mm
of
taken.
(35).
in Human
domain
of the
FLT-4
VEGF
free
were
Hepes,
on alternate
of unlabeled for
at RT for 30 s, and
sections
tissues with
saturation from
determined
slides
To test
25 mM
ranging
vac-
at RT for 30 mm
experiments
ligand.
sence
kidney
only
by incubation
FCS.
under
the
by hematoxylin-
microscopy,
preincubated
competition
Nonspecific
water
labeled 10%
that
light
up to I 2 h for association
concentrations
15 mm
ensure
This
on a cryostat,
placed
S nM phenylmethylsulfonyl
‘25I-VEGF,
tilled
stained
were
with
saturation
buffer
were
and
incubated
3 h for
and
were
by
Kidney
cut
slides,
to
sections
were
glass
determined
for VEGF
in Human
fetal)
sections
acid-Schiff
changes,
Binding
were
and
on gelatin-coated
at 4#{176}C overnight.
eosin
Binding
adult
protein
extracellular
disappearance
VEGF/VPF
Receptors
the
excess
inhibition VEGFI#{212}S and
compete
for
of of the
VEGF
8 nM
binding
(Figure 5). Statistical Data
Human
Analyses are
determine
given
as means
statistical
regarded
± SD.
significance
as a significant
The
with
difference
unpaired
t test
a two-tailed
between
groups
was
used
to
adult
P value
of