between simvastatin- and vehicle-treated nephritic rats at day. 2 and day 4, which corresponded to the times when simvastatin treatment resulted in a reduction.
J Am Soc
Simvastatin Macrophage Proliferative
Suppresses Infiltration Nephritis
Abstract.
Inhibition
reductase
inhibits
shown
production
proliferation
a beneficial cell
glomerular
injury.
tin
and
was
and
was
maximal
at day
4 after
progressive
glomerular
membranoproliferative
phritis,
and
focal
angial cell extracellular opment of clearly
4
glomerulosclerosis
demonstrated
(3).
strategies
Products
Received Correspondence
Showa 227,
1046-6673/0901 Copyright
5, 1997.
pathway
May
Yoshimura,
University,
12,
Hospital,
IgA
©
1998
by the
Society American
nephrolupus
ne-
by mes-
and
future
an important
therrole
in
of a change
DNA
replication
into
and
pharmacologic
action
the
that
possibility
merular
disease.
mesangial growth
factor
report
of Medicine, Fujigaoka,
Division Aoba-ku,
proliferation.
they
may
(HMG
glomerular
nal
of Nephrology of Nephrology
cause
reductase
have
a beneficial
previous
of
3-hy-
reductase
These
may
suppress diseases
was designed
to examine
pathology
in a rat model
(anti-Thyl studies
.
mesCoA
cell in
HMG
proliferation sclerosis
addition
injury
the effect
to
by
of simvastatin
by anti-thymocyte
1 GN).
This
model
demonstrated
in their
lowering
of mesangial
induced have
in human that
of gbomerular in vivo,
of rat
platelet-derived
suggest
mesangial
development
in gb-
proliferation
synthesis
studies
raised
effect
inhibits
DNA
(13).
inhibitors
inhibits
simvastatin
glomerubonephritis antibody
indepen-
CoA)
CoA
lovastatin
(1 2) and
subsequent
study
were
).
of HMG
progressive
This
partly
monocyte/
Inhibition
A
(PDGF)-induced
on gbomerular
was
and
which
been
of sim-
of mevalonate and has been shown to in many cell types in vitro (4-1 1 This
and prevent
of
that
have effect
1 . 1 ON
potential beneficial effect on gbomerular serum lipids, as shown previously (14,15). 1-30
cell
to find
expansion protective
glomeruli,
when
lipids.
cell
Indeed,
cells
first
proliferation
coenzyme
the production proliferation
nephritic times
in mesangial
in anti-Thy
in circulating
There or triglyc-
to the
matrix The
cell
droxy-3-methylglutaryl
ative Society
and in vivo.
the reduction
cellularity.
vehicle-treated
is the
expansion
angial cells in vitro. reductase inhibitors
diseases
this
recruitment
dent
IV collagen
reflect
in a reduction
mesangial
disease
suppressed
cholesterol
and
matrix of
simply
corresponded
resulted
2 after type
mesangial
4, which
proliferation
inhibition
inhibits suppress
mesangial the develstudies
expansion in of mesangial
into
Japan.
American
for
of proliferation
1 -2027$03.00/0
of the
cell
and
in plasma
In conclusion,
mesangial
at day
simvastatin-
treatment
macrophage
muscle
1998.
Department
Fujigaoka
simvastatin
day
in the
induction,
glomerular play
between 2 and
by
insights
progressive
Accepted
to Dr. Ashio
Nephrology,
Journal
human
of mevalonate
August
Yokohama
for
levels at day
vastatin
provide
apeutic
and
disease
cell
useful
of
might
difference
a prominent
of glomerubar
also found that the plateletwas reduced in simvastatin-
which
significant
also
remarkably
expansion
proliferation
by simvastatin
cells in subsequent matrix Therefore, the regulation
may
rats, cell
proliferation.
7.0
a-smooth
role
matrix
blocked
are characterized
important
no
rats
of the ±
was eride
activity
and accumulation of processes may precede Recent experimental
the
of the between
(26.5
nephritic
in mesangial
pronounced
including
treatment simvastatin
cells/glomerulus); was suppressed
activation of mesangial the glomerulus (1 ,2). proliferation
degree
glomerulonephritis,
proliferation matrix. These glomerulosclerosis.
simvastatin
was
Japan.
in the number
treated
which is a marker of monocyte/macrophage
diseases,
decrease
proliferative
proliferative
after
by
Yokohama,
by simvastatin
a 30%
of simvasta-
suppression
decreased
glomerulus, Inhibition
mesangial
active
was
accumulation in glomeruli. We derived growth factor expression
most
induction
day
also
in the activation.
pathy,
the
The
At
administration
actin expression mesangial cell
was
disease
subsequent
because
injuries
The
antigen-positive 70% of proliferation
treatment.
simvastatin
rats.
proliferation.
Furthermore,
antibody
in the
initial
GN
cells
induction.
been
UDA,
Hospital,
gbomeruli
ED- 1
anti-thymocyte
no difference
GN
cell nuclear approximately
simvastatin
Many
by
control
cell
effect
into
inhibitors
in the
mesangial
complement-mediated
glomerular
proliferating however,
the
in a rat
of simvastatin-treated
early
by
examines
induced
There
simvastatin-treated feature
study
recruitment
Fujigaoka
Therefore,
disease, feature
A
University
There
has
reductase
in glomerular
pathology
1 GN).
types.
Showa
feature.
and
cell A
is a central
(ON) .
antibody
effect
This
glomerulonephritis
(anti-Thyl
in many
proliferation
on glomerular
coenzyme
of mevalonate
coenzyme
glomerular
of Nephrologv,
of 3-hydro-3-methylglutaryl the
to suppress have
Division
1998
and
INUI, TAKASHI NEMOTO, SUSUMU WATANABE, NAOKO YOKOTA, IWASAKI, and TERUKUNI IDEURA
SHIGEKI
of Medicine,
3-hydro-3-methylglutaryl
may
KIYOKO SUSUMU
TAIRA,
Department
9: 2027-2039,
Glomerular Cell Proliferation in Rats with Mesangial
ASHIO YOSHIMURA, YOUICHI SUGENOYA, TAKAYASU
Nephrol
was
prolifermonoclochosen
that
be-
it
is
2028
Journal
Table
of the
1. Score
for
B-chain
American
aSMA protein
Society
J Am
of Nephrology
staining, expression’
mesangiolysis,
mesangial
matrix
expansion,
type
Nephrol
Soc
IV collagen
staining,
9: 2027-2039,
and
1998
PDGF
Day Parameter 0
2
4
7
12
20
aSMA vehicle
0.1
± 0.1
0.5
± 0.3
3.5
± 0.3
3.1
± 0.7
2.3
± 0.8
0.9
± 0.2
simvastatin
0.1
± 0.1
0.5
± 0.2
2.1
± 0.3”
2.8
± 0.9
2.0
± 0.6
0.7
± 0.1
Mesangiolysis
vehicle
0
1.6
±
0.4
1.2
±
0.4
0.5
± 0.3
0.2
± 0.2
0.1
± 0.2
simvastatin
0
1.6
± 0.3
1.1
± 0.4
0.5
± 0.1
0.1
± 0.2
0.1
± 0.1
vehicle
0
0.3
± 0.1
1.2
± 0.4
2.0
± 0.5
1.3
± 0.3
0.7
± 0.2
simvastatin
0
0.2
± 0.2
0.7
±
03b
0.7
±
03b
0.5
±
0.2
± 01b
Mesangiab
Type
matrix
IV collagen
vehicle
0.2
± 0.3
0.7
± 0.3
1.7
± 0.2
3.0
± 0.5
1.9
± 0.2
1.2
± 0.4
simvastatin
0.2
± 0.2
0.8
± 0.2
0.8
± 0.2”
2.3
± 0.3k’
1.2
± 0.5’
0.4
±
vehicle
0.1
± 0.2
0.3
± 0.1
2.2
±
0.4
1.4
±
0.3
1.5
± 0.3
0.5
± 0.2
simvastatin
0. 1 ± 0. 1
0.2
± 0.2
1 .5 ±
02b
0.7
± 03b
0.6
±
0. 1 ±
PDGF
vehicle-treated
anti-Thyl
muscle
I nephritic
simvastatin:
versus
vehicle-treated
rats
on
the
same
experimental
day.
p < 0.05
versus
vehicle-treated
rats
on
the
same
experimental
day.
by and
macrophage
platelet-derived
infiltration,
subsequent
mesangial
mesangial
18). Although anti-Thy injury and is associated
1 1 ON with
growth
simvastatin-treated
h
liferation,
PDOF,
rats;
C
characterized
actin;
.
a-smooth p < 0.01
matrix
cell
expansion
best model for cell proliferation
anti-Thyl
nab antibody (2.5 mb/kg Limited, Hornby, Ontario,
pro-
(1,16-
In each group, and total
studying the and matrix
expansion.
RNA
and
Disease
The study was designed to determine whether HMO CoA reductase inhibitor) reduces gbomerular mental mesangial proliferative nephritis (anti-Thyl.l statin (Banyu vehicle (0.5%
Pharmaceutical, carboxymethyl
Katayama
rats
were
were
Kagaku,
prepared
divided
vehicle-treated twice
(Nippon
into
two
(n
a day
Ikagaku
disease
induction
tomy.
amount
of simvastatin
of
simvastatin-
disease
vehicle-treated 2
after
disease
rats,
0,
respectively),
and
by a single
(n
7
at day
8,
=
8,
(ii
20
intravenous
(n
to the
(2
(19).
with
respectively).
of anti-rat
at at
.
day
8, respectively).
=
B-chain
Northern
blots
were
each.
Kidneys
to histologic
performed
that
stained
the number proliferating
actin
previously
(22,23).
In addition,
evaluated
by periodic
acid-Schiff
evaluation
of mesangial
tive in
as well
scores the
for
extent
thermore,
the
staining
stained
the
the
the gbomerubar
expression (16).
All
for evaluation
sections
and
IV
the
cells cross
stained
for type
reflect matrix
B-chain coded
and
pathologic
IV
changes
( 1 ). Fur-
was studied then
changes
immunohistochemical
For the
semiquantita-
mainly
mesangial
were
was
(22).
because
of PDGF
of both
(PCNA),
as described
mesangiolysis
sections
of
cells,
per gbomerular
sections
collagen
slides
also
antigen
of
( I ,22),
intensity
sets
were
mesangial
nuclear
degree
used
of two
group
respectively,
expansion, were
of type
than
immunohistochemistry
to two doctors
cell
specific
For each
of proliferating
of cells
(aSMA),
using (2 1 ).
RNA
activated
(PAS)-stained
matrix
as for PAS
rather
number
and
for
RNA
all rats in each The
muscle
were removed,
analysis,
on gbomerular
from
by enumerating
Laboratories
the kidneys blot
(20) and 28S ribosomal
examination.
ED- I , or a-smooth
(Cedarlane
sacrificed,
monocytes/macrophages,
assessed
collagen
solution)
staining
by
presented
on PASas
shown
below.
weight
rats
anti-Thyl
02b
Six groups
and
respectively),
injection
of nephrec-
studied:
respectively), 8,
from
per rat per body were
and mg/kg
started
day
report
and
48)
was
simvastatin-treated
and rats
Japan)
simvastatin
continued
rats 8 of
Dawley
(n
control)
administered
a =
induction
Tokyo,
of
(for
to a previous
respectively)
at day
Doubutsu,
was
vehicle-treated (day
respectively), induced
and
according
or
induction
six Sprague
simvastatin-treated
or vehicle
2 d before
The
Ninety
Administration
tube)
per day was decided
Japan).
groups:
48).
by gastric
simvastatin (an injury in experimodel). Simva-
Tokyo, Japan) was diluted to 0.2% in cellubose/0. I M phosphate-buffered saOsaka,
(n
for Northern
of four kidneys
section
Model
the rats were
was extracted
group,
was
and Methods Protocol
rats
10% Canada).
for PDGF
infiltrating
Experimental
of
probes
subjected
Materials
.1 nephritic
02b
factor.
is not a model of progressive only a mild decline in renal
.
function, it is thought to be the effect of simvastatin on mesangial
line,
02b
B-chain
Vehicle:
a
SMA,
04b
8 of
-
1 ON 4
Renal
without n
at day
(ii
(n
day
12
The
disease
=
fixed
for
light
in methyl
Four-micrometer
8,
terstained
with
number
of nuclei
mesangiolysis
microscopy
Carnoy’s
8,
was
Thy I . I monoclo-
Morphology
Tissue
sections hematoxylin. was
and
solution were
stained In the
per gbomerular graded
immunoperoxidase
(24)
with
semiquantitatively
PAS
PAS-stained
cross
staining
and embedded
section
was
in paraffin.
reagent
and
sections,
coun-
the
was determined, on
a scale
of 0 to 4+
total
and as
J Am Soc Nephrol
9: 2027-2039,
1998
Effect
of Simvastatin
Immunohistochemical MonocvteslMacrophages, Type IV Collagen Kidney Carnoy’s
tissues
from
four
solution,
and
4-sm
l9A2
(Coulter
Immunology,
against
cbonal phages,
and
An IgO
fraction
Gbostrup,
PCNA+
zyme
cetts/glom
PCNA,
20
in
MA)
of polycbonal
rabbit
10
Bedford,
with
segments and
cross
day 0 1. Total
Figure
gbomerular
(PCNA)-positive
and
day 2
in
both
induction,
groups
in
normal
by
a rapid
from
ON
lower
day
The
Total
at day
day
20
in simvastatin-treated
of
was
ON
rats
cells
at days
was
2, 4, and =
(ii
7,
Isolation Glomerular
were
as follows:
(22).
0, no
matrix
of the glomerular the gbomerular gbomerular
tuft.
tuft; tuft;
tuft;
A minimum
to 47).
Furthermore,
4+,
3+,
mesangial
sections,
and each
each
MO)-treated
ON
rats.
and
experimental
1 +,
expansion;
2+,
matrix
matrix matrix
expansion
expansion expansion
of 20 gbomeruli
matrix
was
matrix
gbomerulus between
between over
examined
75%
25 and
irrelevant
proliferating performed
containing
22 to 36;
by
more
mean
of
for aSMA, graded
than
28.7
positive
±
8.3)
cells
per
B-chain,
as
or absent mesanof staining; 1 +, diffuse,
1 to 25%
of the glomerular
25
of the
2+,
or
weak,
increase
staining:
PDGF
semiquantitatively,
very
with
to 50%
3+, manner;
tuft
gbomerular
50 to 75% of and 4+, >75%
the of
strongly.
and
isolated
by
stability
Preparation differential
of the
than
and 20
Houston,
TX).
all groups as
saline,
as little
elapsed in
time
from
Total
RNA
with
reported
(27).
RNA, autoclaved
steps
St.
Louis, sieves,
for the preparation. to
dissolution
of
(Cinna/Biotecx
Laborato-
from
glomeruli
extracted
RNAz0IB#{174} following
previously
preserve
or baked
as possible
solution
was
To
all isolation (Sigma,
nephrectomy
RNAzo1B#{174}
of rats
sieving
diethylpyrocarbonate
using
mm
gbomeruli
of
glomerubar
phosphate-buffered
Northern
graded 25% 50%
50 and
75%
of
gbomerular
per biopsy
of was
number
was
localized
at 4#{176}C using
glassware
Less
below
the
of the
of an
isobated
the
the manufacturer’s
(2,23).
expansion
was
expansion
no
staining
were
and
instructions
on the same
(range, the
staining
of Glomeruli RNA
done
isolated
previously
IV (Col-
of substitution
all gbomeruli
0, diffuse,
increased
Gbomeruli
from
evaluated
and
tuft
the integrity
ries,
was
collagen
Quantification (ED-b cells)
glomerubus
matrix
focally
8 in
respectively.
reported
(Gen-
signifi-
previously,
PCNA
PDGF-BB
and
4. The
predicts the subsequent cells increased from day
number
activated
stain
concentration
each
(1,16):
staining
the gbomerular
disease
at day
(DAKO,
to
antihuman
tuft demonstrating focal, strong staining; gbomerular tuft staining strongly in a focal
glomerular
to aSMA
anti-mouse
of the staining
each
mesangial
showing
shown
consisted
enumerating
previously
matrix
weak
antigen
2 after
increase
compared with vehicle-treated GN rats, respectively group). El, vehicle-treated ON rats; U, simvastatin-treated *P < 0.01 versus vehicle-treated ON rats in the same day,
nuclear
As reported
accurately of PCNA
rats.
rats.
4 to
treatment.
the degree of PCNA expression cellular response, and the number
gial
day 20
in simvastatin-treated
below
cellularity
12
cell
(ON)
by simvastatin
2 in vehicle-treated
day
(cells/glom)
followed
gbomerular
7
and proliferating
decreased
suppressed
significantly
day
glomerulonephritis
respectively,
increase
cantly
cells
cells/glomerubus
vehicle-treated
cells
day 4
monomacro-
section.
IV collagen,
described
controls
manner,
For the evaluation type
to
during
MA).
antibody. macrophages
capillary
glomerular
protein
CA), a murine in monocytes.
(26);
an equivalent
in a blinded biopsy
indirect
monocbonal
auxiliary
antibody
rabbit
monocbonal cells and
each
the
in late G1, peaking of the cell cycle;
been
Cambridge,
negative
antibody
20 discrete
*
has
vito (25); of polycbonal
Research,
examining,
in methyl
by
in
fraction
murine (PCNA)
fixed
a murine
is an
monocbonal
which
For all staining, primary
Cells, and
cells;
of murine
Diagnostics,
laborative
2029
included:
FL),
which
that is expressed to the M phase
dendritic
cells fraction
An IgG
.
30
were stained
antibodies
Hialeah,
human
Denmark),
mesangial An IgG
.
group were
(Chemicon International, Temecula, IgG to a cytopbasmic antigen present
ED-I
.
in each
sections
Primary
DNA polymerase delta S phase, and extending S
rats
method.
antibody
Nephritis
Staining for Proliferating aSMA, PDGF B-Chain,
immunoperoxidase S
on Proliferative
of
of the
(range,
Total a
1%
20
agarose
membrane baked light tion
Blot
Analysis
gbomerular
RNA gel
(IS
(Hybond
was j.g
denatured of
N#{174},Amersham,
at 80#{176}C for 2 h. Examination in the presence and
integrity
and
RNAJlane),
of ethidium of
the
285
a
bands
nylon
IL),
under
demonstrated
ribosomal
to
Heights,
of the membrane 185
through
transferred
Arlington
bromide and
electrophoresed
and
ultraviolet good
(23).
resolu-
2030
Journal
of the
American
Society
of Nephrology
J Am
Soc
Nephrol
9: 2027-2039,
1998
“-s
. ‘
‘V.
#{149}s .#{149} 4.
‘
Figure
2. Immunostaining
PCNA
cells/glom
difference
in the number
a drastically D and
reduced
J: day
The DNA PDGF
I
for
began
probes
was
K:
day
used
response I 2; F and
PDGF
at day
site
(A
4, and
then
between at days
L: day
genomic of
the
both
2, 4, and
20
blot analysis
B-chain
the SinaI
into
in vehicle-treated
cells/glom
for Northern
Murine cloned
cells
2, peaked
of PCNA
proliferative
7; E and
B-chain.
fragment)
PCNA
at day
(after
through
slowly groups
at day
285
ribosomal
detect 285 and Helene Probes with
RNA.
A bovine
ribosomal RNA Sage, University
of PDGF
[a-32P]-deoxycytidine
B-chain
280-bp
cDNA
285
5-triphosphate
ribosomal (3000
during
0 (A and 0);
induction,
(0
the course however,
X600.
through
L)
of the disease simvastatin
A and
0:
day
ON
0;
B and
DNA
(a 326-bp
transcription
vector
RNA Ci/mmol,
used
to
Nuclear
Research
aminetetra-acetic dodecyl
acid
sulfate
liter.
Hybridization
2 X
I 06
cpm/ml
and
was of
were
OMAT
New
En-
-
drying,
film
70#{176}C. The
membranes
(Kodak, obtained
Tokyo,
H:
There
was
associated
day
2; C and
sg
performed
Japan)
in the
was
no with
I: day
4;
of
with eDNA
by random
primer
in a solution containing phosphate-ethylenedi-
Denhardt’s
SX
100
32P-labeled
Iruela-Arispe
After
Products,
(SSPE),
(SDS),
Blots were then washed each at room temperature
labeled
increase
respectively).
gland
was
The
model.
treatment
extension. Membranes were prehybridized 50% (vollvol) formamide, 5 X saline-sodium
probe
rats.
in this
were as follows:
(provided by Drs. Luisa of Washington) (21). and
simvastatin-treated
7. Immunoperoxidase,
disease
pGEM I at a site between the SP6 and T7 promoters (provided by Dr. Daniel F. Bowen-Pope, University of Washington, Seattle, WA) (20). .
F) and
decreased
salmon
solution, sperm
the same was
added
0.1% DNA
solution, for
24
sodium per
milli-
to which h at 42#{176}C.
three times in 2X SSPE, 0.1% SDS for 5 mm and then in 0. 1 % SSPE, 0. 1% SDS at 50#{176}C. were
exposed
to nonpreflashed
Kodak
X-
Tokyo,
Japan)
with
screens
at
autoradiograms
were
enhancing read
by linear
densitom-
J Am Soc Nephrol
9: 2027-2039,
1998
Effect
of Simvastatin
on Proliferative
Nephritis
2031
macrophages/glom
15
10
5
*
0 day 0 Figure 3. The vehicle-treated decrease
day 2
day 7
number of glomerular and simvastatin-treated
in gbomerular
vehicle-treated
GN
day 1 2
macrophages ON rats.
macrophage
induction by simvastatin n 8, and 17.5 ± versus
day 4
infiltration
day 20
(ED-i cells) in There was a 30%
at day
2 after
disease
treatment (12.0 ± 1.7 in simvastatin-treated, 2.6 in vehicle-treated ON rats, n = 8).
rats;
vehicle-treated
U,
ON
simvastatin-treated rats
ON
in the same
rats.
experimental
LI,
< 0.01
*P
day,
respec-
tively. etry
(Electrophoresis
TX)
at 600 nm. Some
probes
Data
Center,
(up to a maximum
glomerular
RNA
amounts
pressed
of 285
as optical
observed
were
these
obtained
from
were
ribosomal
density
units
in gbomerular
Laboratories,
RNA
Beaumont,
rehybridized
of 3 times);
preparations
Obtained autoradiograms above) at 600 nm. Densitometry
alent
Helena
membranes
were
with additional repeated
different
twice
sets
with
of animals.
read readings
by linear densitometry (see were normalized for equiv-
RNA relative
per lane, and values are exto the specific mRNA level
from
normal
rats
as described
(2,23).
4. Immunostaining in vehicle-treated (A) Figure
after
disease
induction.
macrophage
Administration We
of Simvastatin
also
effects
performed
toxic
At first,
nephrectomy
Administration
tube)
was
biopsy.
on
respectively;
renal
4 and
12 rats was
biopsy
was
tail
vein
Beckman
Instruments,
by enzyme
assay,
Measurement
each
to the
2 and Renal
samples
CA),
day
were
7 on six rats tissues
Serum
were
Protein
assay
6,
=
and
in
Immunohisto-
from
creatinine
Excretion
8
individually
urine housed
collections
were
in metabolic
cages.
obtained Rats
were
from fasted
using
a whole
Puerto
Rico).
Statistical
lightly
Japan).
in rats
serum
but were measured standard
allowed free access to water. by a sulfosalicylic acid method (Lab
Trol,
Dade
Diagnostics,
Urine (27),
Aquado,
Analyses
test
or one-way
that
during
ANOVA
± SD 0.05)
modified
unless stated was evaluated
Simvastatin treatment (2 mg/kg erated in normal rats (the number
twice a day) was well of PCNA cells/gbomerubus
tol-
[cells/glomi
t
test
otherwise. using the the
correction
with
