Inhibits Advanced. Glycation. End Products ... advanced glycation end products. (AGE)- modified. /32-microglobubin. (AGE-j32M) ...... Seo H: Involvement of /32- ...
Aminoguanidine Inhibits Advanced Formation on 2-Microglobulin FAN FAN HOU,* JOSHUA BOYCE,t and WILLIAM F. OWEN, JR.* *Renal
Division
Women
‘s Hospital,
Hospital,
Because
modified
advanced
of amyloid
2M
may
prevents
end
products
is a dominant
amyloidosis
D-glucose
in the pathobiology
mellitus,
blocking
tissue
the
damage.
linked
and
amyloidosis
incapacitating
complication
nance
(1,2).
dialysis
and
(DRA)
for
Amyloid
the
deposits
In
thropathy, of DRA
and
tenosynovitis,
subchondral
is incompletely
can
is
be
and hypothetical been recommended: and
amyloid
end
(AGE),
a major
and
component
directly
and
of
of
participate
chemotaxis
amyloid
32M
fibrils
in the pathobiology of monocytes;
Received
June
30,
1997.
Accepted
to Dr.
William
Office,
and Women’s
(2)
tive
July
17,
F. Owen,
in
DRA
Nephrol
9: 277-283,
(6,7).
On
inhibited
of
manner.
by 30 to 70%.
the AGE formation If aminoguanidine of
At
I : 1 , fluorescent
I :8 to
necrosis factor-cr (TNF-a), (3) production of collagenase
the
basis
of
studies
of
consequences
Furthermore, on 32M bound is similarly ad-
clinical dialysis.
utility (I Am
for Soc
hydrazine in vitro
(8).
compound,
to collagen
and
can
complications,
the
if the
products
are
aminoguanidine,
inhibits
AGE
phar-
a nucleo-
formation
the cross-linking
on
of soluble
the cross-linking
maccells
be prevented
glycosylation
Because
by suppressing
and IL-6 from in synovial
diabetic
of AGE
of early
blocked
of collagen
collaplasma
to itself
(8,9), it has been advocated for in viva use. In animal models of diabetes mellitus, aminoguanidine minimizes the develop-
with
ment (10).
the
use
of
of glomerulopathy, vasculopathy, Based on the suggested efficacy
diabetes
mellitus,
beneficial
in other
an appropriate
glycation is may
a disorder
and
and neuropathology of aminoguanidine
in
with
be
associated
AGE-associated
diseases,
necessary
preliminary
AGE, such
it may as DRA.
investigation
nonenzymatic
glycosylation
of
As
before
the administration of this compound as a therapeutic agent DRA, we examined the in vitro effect of aminoguanidine
for on
2M.
interleukin-l3
and Methods
Unit
Administrative
of 2M-AGE
AGE-modified
f32M
1046-6673/0902-0277$03.00/0
or 100 mM D-glucose buffer (PB) containing
Street,
ratios
formation
1998)
intermediates
proteins
1997.
75 Francis
the
in a dose-dependent
and
Journal of the American Society of Nephrology Copyright 0 1998 by the American Society of Nephrology
Hospital,
examina-
inhibited
this compound may be in patients on maintenance
viva,
Preparation
Jr., Dialysis
2M
was
of 1 :8 to I : 1,
Fluorospectrometry also
suppressed collagen.
treating
gens
by stimulating: of
on
formation ratios
solutions have removal of
AGE-j32M
of DRA synthesis
occurred.
generation
(5,11,12). Biochem,
Brigham
Medical
aminoguanidine
molar
molar
aminoguanidine to AGE-modified
philic
(AGE-/32M)
(5).
product
antibody,
aminoguanidine
AGE
Materials Correspondence
Nanfring
Hitchcock
M-(carboxymethyl)lysine
aminoguanidine-glucose
macologically
studies
to advanced
AGE-modified
that
of fluorescent
reactive
mea-
of retrospective
is modified
hemocyanin
inhibition
showed
pathobiologic
pathogenesis
preventive
associated
to 53%
rophages;
spondyloar-
Two principal the intradialytic
limpet
(IL- 1 f3), tumor
often
mainteof fibrils
(1 ,2). The
basis
synthesis
tissue
and
anti-
in osteoarticular carpal tunnel
therefore,
on the
j32M
using
dialysis membranes (3,4). The longitudinal of these interventions are unknown. has demonstrated that j32M isolated from di-
alysis-related products
cysts
considerations. enhancing
minimizing
bioincompatible clinical sequelae Recent work
bone
only
of enzyme-
destructive
understood;
be proposed
basis
26 tion
of incremental
composed
flexor
(1)
At aminoguanidine-glucose
it
undergoing
syndrome,
/32M
on 32M.
may
is a progressive
patients
(2M) are localized mainly result in shoulder periarthritis,
sures
glucose-induced
study,
immunoblots
2-microglobulin tissue and may hand
Lahev
inhibited
of AGE
this
absence
On
assay
Dialysis-related
Brigham
of Nephro!ogv,
of Rheumatology,
AGE-keyhole
of DRA.
intervention
of aminoguanidine.
immunosorbent
Departtnent
Section
KAY,
of Medicine,
constitAGE-
formation In
pharmacologic
for 3 wk in the presence
concentrations
Department
Massachusetts; and
Products
JONATHAN
(AGE)-
(DRA),
involved
similar
Boston,
in DRA. Arninoguanidine, a nucleophilic hydrazine that prevents AGE formation on collagen, may have effect on the advanced glycation of 2M. To test this 2M was incubated in vitro with 50 or 100 mM
hypothesis,
-.-
glycation
in dialysis-related
that
M. CHERTOW,*
Rheumatology,
of China;
End
Massachusetts.
(AGE-j32M)
AGE-mediated
and
School,
‘s Republic
diabetes
postulated beneficial compound a similar
Medical
People
be directly
experimental
GLENN
of Immunology
Harvard
Burlington,
/32-microglobubin
uent
tDivision
Guangzhou,
Center,
Abstract.
and
Glycation
Boston.
MA
021 15.
was
in Solution prepared
in vitro
as described
previously
Briefly, 1.75 mg/ml purified normal human 32M (Corter San Leandro, CA) was incubated at 37#{176}C for 3 wk with 50
(Sigma, St. Louis, MO) in 100 mM phosphate 200 U/mb penicillin, 70 g/ml gentamicin. and
278
Journal
of the
American
1 .5 mM phenylmethylsulfonyl were also incubated under various
concentrations
Zee M. Look, of glucose
of Nephrology
fluoride (PMSF) identical conditions
of aminoguanidine
Alteon,
with
Society
Ramsey,
and
After incubation, all ered solution (PBS), tion was determined albumin (BSA) as a
hydrochloride
NI). Samples
without
(Sigma). Samples in the presence of
were
samples were dialyzed pH 7.4, and lyophilized. by the Bradford method standard.
430
(5). Fluorescence nm,
in the absence
used
Preparation
as controls.
of 2M-AGE
Enzyme-Linked
against phosphate-buffThe protein concentra(13) using bovine serum
immunosorbent
assay
anti-AGE-KLH
antibody
limpet
hemocyanin
of South reacted
vitro
ofAGE-Modifled
The
major
ford,
epitope
structure
recognized
as
has been
was
formed of each
examined
microtiter
plate
was
(concentration
incubated
of /32M,
University
antibody
specifically
of Maillard
to 20
at 1 :2500
sample
(14).
This
anti-
to the anti-AGE-KLH Kit (Pierce, RockEach well of a 96-well
Starter
0. 1 ml of sample buffer.
in coating
The
wells
60 mm
at room
temperature.
The wells were then washed with buffer A, incubated for 60 mm at room temperature with 0. 1 ml of goat anti-rabbit IgG-peroxidase at 1:5000 dilution (Pierce), and developed by the addition of the substrate
2,2 ‘ -azino-bis-3-ethylbenzthiazoline-6-sulfonic
bance
at
(Titertek rabbit
405
nm
Multiskan, antihuman
stead
on
Control
(Boehringer
anti-AGE-KLH
32M
In separate
measured
Mcc/340). IgG
of rabbit
unmodified ples
was
instead
acid. a micro-ELISA
studies
Mannheim,
antibody
The absorplate
reader
were performed Indianapolis,
and coating
using IN)
the plates
in-
experiments,
to evaluate 200
g
5 mM sodium borohydride (NaBH4) room temperature for 4 h ( 16). The
the reactivity
of AGE-f32M
was
of reduced
sam-
incubated
with
in 1 ml of 0.2 M PB, pH 8.5, at samples were dialyzed against 50
mM acetic acid to destroy excess NaBH4, followed by dialysis against PBS, pH 7.4. ELISA was performed as described. Immunobbotting. Immunoblots were performed as described (5). After incubation as described above, the samples were run on a 16% Tris-glycine gel (Novex, San Diego, CA). After sodium dodecyl sulfate-polyacrylamide
gel electrophoresis,
the gel was electrophoreti-
membrane.
The
transbbotted
soaked
in blocking solution and reacted with rabbit anti-AGE-KLH at 1 :3000 dilution for 60 mm. After washing, the membranes incubated with peroxidase-conjugated goat anti-rabbit IgG
membranes
were
were at 1 :3000 dilution (blotting grade, Bio-Rad, Hercules, CA), followed by the addition of enhanced chemibuminescence solution (Amersham Life Science, Arlington Heights, IL). The membranes were exposed to Kodak X-Omat films, which were developed by an automatic X-Omat processor (Kodak
cence
spectrometer
(Aminco
The fluorescence spectra of the samples concentration of 0.26 mg/mb in a fluoresBowman
Briefly,
0.45
was
mg/mI
purified
at 37#{176}C for 200 U/mI
Series
and
quantified
by
2), as described
ofAGE
prepared
type
ELISA
using
in
I collagen
30 d with penicillin,
100 mM 70 g/ml
sample was
was char-
anti-AGE-KLH
fluorospectrometry
was
Formation
previ-
and substrate
the ELISA
tions,
all
Starter
solutions
(Sarstedt,
of
j32M
0.05N acetic AGE-collagen
acid
filtered
NC).
above.
36.9
U/mg
Bound
to
through
Type
polystyrene
solution
was discarded,
and
buffer.
One
were
hundred
filter
solubilized
in
a coating buffer. was aliquoted for
rinsed
microliters
condi-
pore
was
incubated
and the wells
sterile
a 0.22-p.m
I collagen-AGE
microplates
were obtained
Under
and adjusted to 10 j.g/ml using solution (1 p.g of protein/well)
96-well
j.d of wash
used in these experiments
Kit as described
were
Newton,
The into
12 h at 4#{176}C. The
three
times
with
of D-glucose
100
solution
(final concentration of glucose, 100 mM in 100 mM PB) with or without f32M (75 g/ml) was then added and incubated at 37#{176}C for 3
wk in the presence incubation
or absence
solution
also
and 1 .5 mM by aspiration
replacement
with
fresh
of 32M, glucose, period, the wells control,
buffer
of 2M
(75
To
1 :2500)
I h. Each
well
was
at room
temperature
measured
using
To confirm
KLH
were
antibody
100 sl was
the
antihuman
or sheep
The
reader
of 2M
with
and absence
anti-AGE-KLH temperature
for
of goat-anti
added.
wells
rabbit
After
were
incuba-
washed
and
IgG
(dilution
antihuman
the
antihuman
Emeryville,CA)
studies
1 :2500)
32M
sheep
anti-sheep
( 12). Control
nm was
AGE-collagen,
(BiosPacific,
rabbit
at 405 above.
affinity-purified
dilution
of
absorbance
as described
to immobilized
with
addition
(Pierce)
IgG were
instead
and
peroxidase
performed
of rabbit
at using
anti-AGE-
antibody.
Analyses
All experiments
were
performed
expressed as mean ± SD, dative terms were included variables. pairwise
considered ducted
and
mm,
for 30 mm.
1:2500
the
dilution
evaluate
incubated
at room
(Pierce)
60
an ELISA
incubated
at
by
Statistical
for
was
(100 mM). of rabbit
j.d
times,
dilution
the binding
microplates
rabbit
incubated
three
solution and its
concentration
in the presence
added
and
jsg/ml
100 .d of 2,2’-azino-bis-3-ethylbenzthiazoline-6-sulfonic
then
1 :5000
( 1 jig/well)
both 100
rinsed
acid
,32M
The 70
the original
aminoguanidine
temperature
with
penicillin,
At the end of the incubation times with wash buffer. As a
formation,
at 1 :5000
at room
reacted
and
was
IgG peroxidase
containing
D-glucose,
AGE
(dilution
tion
solution
AGE-collagen
p.g/ml)
aminoguanidine.
U/mI
PMSF. Every 7 d, the incubation of the original incubation solution
without
quantify
200
and aminoguanidine. were rinsed three
immobilized
the same
of 100 mM
contained
gentamicin, was changed
mental
M35A).
Fluorospectrometry. were measured at a protein
content
developed
cally antibody
to a nitrocellubose
from
with
of AGE-f32M.
to the anti-AGE-KLH,
(collagen-AGE)
by fluorospectrometry
AGE
The buffers
has been
12 h at 4#{176}C with
for
(I 1,12).
Characterization
sg/ml)
dilution
(5).
AGE-Collagen
were washed three times with washing buffer containing 0.05% Tween 20 (buffer A) and blocked with 1% bovine serum albumin. After washing with buffer A, each well was reacted with 0. 1 ml of anti-AGE-KLH
360 and
Collagen
I collagen
(calf skin, Sigma) was incubated D-glucose in 100 mM PB containing
reaction.
by the antibody
(12,15).
for
0.01
keyhole
W. Baynes,
This
the ELISA
previously
were proteins
a polyclonal with
(CML)
prepared
maxima of AGE
of protein.
used in ELISA assays to measure /32M in vitro (12) and in human lens protein
using
as described
using
products
N-(carboxymethyl)lysine
antibody
IL),
(14).
the early
and collagen AGE ( I 5). The reactivity antibody
SC)
but not with
enzyme-linked
immunized
of Dr. Iohn
AGE
characterized AGE-KLH
Columbia,
An
performed
in rabbits
(a gift
type
as described
(12).
Assay.
was
raised
(KLH)
Carolina, with
(ELISA)
and emission
is characteristic
gentamicin, and 1 .5 mM PMSF. After incubation, the dialyzed against PBS, pH 7.4. The prepared collagen-AGE
in Solution
Immunosorbent
which
AGE-modified
acterized Characterization
excitation
respectively,
(a gift of Mr.
incubated
aminoguanidine
ously
The
SAS
Student-Newman-Keuls
comparisons.
statistically with
(The
in triplicate.
procedure
Two-tailed
significant. SAS
Continuous
variables,
were compared using ANOVA. Multiplito evaluate for interaction among experi-
Institute,
P values
Statistical Cary,
analyses NC).
was used 90%
by of the
to
attenuated
by
of aminoguanidine
on
relationship established
of by
quantity of immunoreacon the concentration
incubation
induced
not
bind
solution
(Figure
2A).
by aminoguanidine
was
of The
a func-
0.6
to
of the Maillard products
by this
anti-AGE-KLH
Maillard
of the
NaBH4,
reaction
are
is the ( 14),
not
D-
0.4
Mail-
(17),
in Figure
antibody
with
with
treatment
8). As shown
treatment
reac-
incubated
but 1 , the
was
not
I
al-
indicating
that
recognized
by
principal AGE antigen these data suggest that
0.2
0 0
A
6.25
12.5
Concentration
-
0.4
The dependent
modification
was
effect
279
because
An irrel-
did
f32M Early
are not (14,17,1
the antibody. Because CML recognized by anti-AGE-KLH
0.5
its
with
aminogua-
anti-AGE-KLH.
NaBH4.
to the
significantly
nonspecific
with
early in the
with
immunoreactivity
tered
or without
(antihurnan
are reduced
and AGE
with
in
process
formation of AGE on f32M, a dose-response aminoguanidine on AGE modification was ELISA,
less.
this
aminoguanidine. To further
aminoguanidine,
significantly
resulted
and
Formation
shown).
treated
reaction
CML
reflect
whether
recognized was
incubated
the anti-AGE-KLH incubated with
not react
antibody
was
2M
AGE,
AGE
with
1), suggesting
and
was
of glucose
not
did
2M
D-glucose
product
was not recognized by The interaction of 2M
anti-AGE-KLH
lard
of
of
in the absence
nidine shown).
by incubation (Figure
When
concentrations
immunoreactivity
modified
anti-AGE-KLH
of AGE-/32M.
equimolar
tion
2M
of
CML-containing
of 2M
Solution
On the
incubation
glucose
Effect
Inhibits
25
50
100
of Aminoguanidine
(mM)
80
-
60
0.3
-
0
4O
0.2
-
0
U)
0.1
20
-
0I
0.01
I
I
I
0.1
1
10
100
CONCENTRATION Figure products
1. The
effect
offl2M
of aminoguanidine
(AGE)-modified
/32-microglobulin
samples
were
incubated
(NaBH4) (A, 0). AGE-/32M munosorbent assay (ELISA) cyanin
(KLH).
pressed
as
mean
aminoguanidine, concentration,
P
interaction,
Data P
P
