to both test media because. NO production was negligible in the absence of the cytokine combi- nation. A hyperosmolality control medium consisted of DMEM. +.
High
Glucose
Inhibits
Rat Mesangial HOWARD for
accumulation.
high
messenger
directly
stable external
glucose
reduced
nitrite
manner
in this
modulates
and
study
levels
(5.6
high
glucose
in cell
media
was
monophosphate
in rat
The
Hyperglycemia
is a major
complications glucose
plasma
flow
cells
(3).
(ECM)
(2),
oxide
dioxygen
the
by at least
synthase
(NOS)
arteniolar
handling
(6).
tone (10).
possess
response
different participates
and
cells
cytokines
are conflicting
and
(iNOS)
cyclic was
no
on NO
(1).
matrix
L-arginine
renal
yet
April
Correspondence Division
to
of Nephrology.
l()46-6673/0808-
Journal
18,
1996. Dr.
Accepted Howard 269-01
March Trachtman. 76th
Avenue,
17,
(7),
effect
diabetes
increased
I 276$03.00/0
and high
glucose
and
a role
causes
compromises
precursor
reduce
NO
and
other,
production
by
ambient
in the
(J Am Soc
glucose
development
Nephrol
(13,14),
NO
of
8: 1276-1282,
guanosine
meruli
isolated
of NO
in the pathogenesis
and
NO
nitrite,
the effect
diabetic
of high
cells
the
and
monophosphate from
rats
glucose
docu-
NO-dependent generation
by
gb-
(15-17).
To
the
role
clarify
nephropathy, production
measuring
metabolite
indicate
have
(cGMP)
on NO
by
reports
others
decreased
of diabetic
(RMC)
stable
Some
whereas
production
cyclic
mesangial
NO production.
synthesis
reduced
med
cell
in cultured
the
of NO,
we examrat
accumulation
in the
of
incubation
media.
NO
Materials
of experi-
and
Hospital, NY
I 1040.
Methods
Cells
epithelial
Children’s
of the American Society of Nephrology Copyright U 1997 by the American Society of Nephrology
high
of an elevated
play
on renal
NO
in
RMC
were
obtained
passages
identity
from
6 and
of mesangial appearance:
puromycin
aminonucleoside
plated
75-cm2
flasks
analysis ified
index for
of iNOS atmosphere
modified streptomycin,
Eagle’s 100
was
there
was
plates,
25
NO
synthesis of
protein.
Cells
of 10%
C02-90%
medium
were
(DMEM) penicillin,
I0
growth
cells/mb,
10%
and
nitrite
grown Western
fetal
in Dulbecco’s with bovine
of
Cells
at 37#{176}C in a humid-
maintained
supplemented and
were
content
incubated
effect (18).
to assay
RMC
L-arginine
air and
stellate,
no inhibitory
( 19).
the
elongated,
on cell
X
and used
microscopy,
by their
or d-valine of
explants
phase-contrast
confirmed
determination
pg/ml
glomerular
Using
in addition,
in 96-well an
primary
10.
cells
or fusiform
production, Park,
may
mesangial
in normal
cells
on
manner.
1997)
that
1997. Hyde
that
media
glucose
increased
that
to
presence
gbomerulosclerosis.
between
New
indicate
act
high
levels
in the metabolic
in the
a change
mM)
(20
factors
con-
to external
of
in mesangial
Limitation
caused
L-arginine
a dose-dependent
to comparable results
glucose
cell
effect in
(vitamin
anti-transforming
ambient
to 20 mM)
inhibitory
These
by inhibition
tubular
mesangial
tubular
Schneider
(10
of L-arginine
unidentified,
level,
modified of antioxidants
in mesangial
production
content
cells
not
or a pan-specific
of L-arginine
diabetic
were Received
dose
synthesis.
as
was
of affer-
synthesize
the
NO
media.
mented
to mesangial
of the enzyme
and
concerning
University
An elevated
the
cell
glucose
mental
An
nephron
regulation
and
reversed
mesangial
NO
(I 1,12).
data
Washington
addition
of L-arginine
highest
NO in
molecule
(8,9),
of Molecular
the
reduction
Addition
depletion
of target
circulation
balance
NOS
h
(4,5).
isoforms
a time-dependent
mesangial
glucose
single
of
antibody.
partially
synthesis
injury
dismutase),
tent.
to 72
nephropathy
in the
island
Department
cells
C (H-7),
factor-a
cell L-arginine
of extracellular
glomerular salt
Mesangial
inducible
to various
There
pathogenesis
nitrogen
NO
Long
Center,
and
Laboratory,
growth
The
glucose
cell-signaling
three in the
of sodium
proliferation
in the
guanidino
York;
E or superoxide
the
inducible
of high
cells
L. CRIMMINS
Medical
by mesangial
the
there
of
oxidative
New
kinase
nitrite,
24
however,
production
DAN
Jewish
Chemistry
Raising
decreased
effect
is a volatile,
from
by
enhances
by renal (NO)
synthesized
for
in NO
including
causes
the
components
Nitric
cells
GFR
enhances
matrix
in normal
decline
concentration
and
and
factor
of diabetes
in
whether
of
media.
expression
suppressive
oxide
in a time-dependent
generation;
cell
of protein
tested
mM
paralleled
mesangial
protein.
elevated
The
production
by rat mesangial
of 14 ± 3% of the amount
alteration
organ
33.3
Park,
Acid
concenpromotes
mesangial
it was
supernatants
(P < 0.01).
mM)
guanosine synthase
to
Island
Hyde
Nucleic
participates
NO synthesis
concentration
to a nadir
media
ent
GFR,
New
develop-
Nitric
that
in vitro by measuring the accumulation metabolite of NO, in the incubation
cells
is
flow,
the
kidney.
molecule
blood
Therefore,
glucose
to
glucose and
in the
and
and
Long
‘s Hospital,
of Medicine, Protein
contributes
matrix
of renal
in Cultured
Missouri.
A high-serum hemodynamics
of extracellular
the regulation
and
St. Louis,
directly
is a short-lived
Production
FUTTERWEIT,
Children
College
Pharmacology.
Hyperglycemia
(NO)
Einstein
of Medicine,
ment of diabetic nephropathy. tration alters intraglomerular deposition
STEPHEN
Schneider
Albert
and
School
Abstract.
TRACHTMAN, of Nephrology,
Biology
Oxide
Cells
Division
Campus
Nitric
1(X) g/ml serum.
in
High
Experimental
Conditions
At 24 h after reached
plating
confluence
randomly (1)
tions:
glucose:
to one
Control: DMEM
concentration
Western RMC
into 96-well
in 75-cm2
assigned
flasks
of the
plates
(usually following
DMEM containing supplemented with
or when
within two
5.6 27.8
of 33.3 mM. Lipopolysacchanide (IFN)-y (50 U/mI) were added
interferon
I wk),
the cells cells
experimental
mM mM
were
(2) High to a final
of the cytokine combiconsisted of DMEM
netetra-acetic
of 125
tl
was added
and
Nitrite
production
was of
naphthylethylene
measured
a solution
diamine
cGMP
10 mm,
and
the
containing
1%
dihydnochloride, was
assay
Samples
were
measured
0.1%
phosphoric
acid
incubated
at 550
nm.
at
and say
glucose
concentration.
RMC
cGMP in the supennatants after acetylation. using
Chemical
Co.,
Ann
were
grown
was measured a commercially
Arbor,
1% Triton
0.05%
Lexington,
sham,
Arlington
in 25-cm2
ex-
Protein a
flasks,
protein
RMC fate
in these was
used
to determine
the number
Twenty-five
of viable
A total of 100 jd of phenazine
microliters
in 96-well
plates. 37#{176}C in the 10% 490
freeze-
dodecyl for 45 mm,
sulfate gel. Gels and protein was
20, the membrane
was
to iNOS
followed
exposed
to a
(Transduction
by a secondary
Labo-
antibody
(horse-
of the iNOS (Amen-
IL).
of the cell
blue
reagent
homogenates
(BioRad,
were
Richmond,
determined
CA).
polyclonal
(TGF)-
antibody,
and TGF-5, MN).
methosul-
with
anti-transforming
reagents
were
growth
I , TGF-2,
TGF-
was purchased
All other
Co. (St. Louis,
neutralizing
reactive
from
TGFI .2, Systems (Minne-
R&D
purchased
from
Sigma
Chemical
MO).
to 2 ml of 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxy-
methoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium.
dark.
was
(25 pg of protein)
Reagents
apolis,
experiments.
added
suspension lysates
anti-mouse IgG). Immunoblots with enhanced chemibuminescence
contents
a Coomassie
TGF-/33,
was
ether)phenylmethylsul-
by enzyme immunoasavailable kit (Cayman
MI).
method
buffer
ethylenediami-
Assay
The using
Assay
A colonimetnic
The ofthe
antibody
Heights.
A pan-specific.
Proliferation
of lysis
mM
a nitroceblulose membrane electromembrane with buffer containing
Tween
KY)
peroxidase-binked were visualized
2
I mM
X-l00). aliquots
monoclonal
radish protein
factor
Cell
NaCl,
Nitrite
production was normalized to the number of viable cells and pressed as a percentage of the value in control media containing normal
mM
at 1000 rpm for 5 mm in 100 l
dithiothreitol,
and equal
and
murine
ratonies,
(19).
sulfanilamide,
and 2.5%
media.
absorbance
Griess
1277
ethyleneglycol-bis(3-aminoethyI
1 mM
and times,
gelatin
primary
Production
using
to I 25 j.tl of conditioned
25#{176}C for
2 mM
dissolved
loaded onto a 7.5% acrylamide-sodium run at 200 V and 70 mA (milliamps)
0.25%
Briefly,
was 100
then transferred from the gel onto phonetically. After blocking the
Measurement
RMC
in Cultured
in PBS and centrifuged
acid,
three
were were
mM mannitol.
Nitrite
acid,
fluoride,
thawed
+
Production
The pellet pH 7.6,
N,N’-tetra-acetic fonyl
NO
were harvested
to sediment cells. (50 mM Tnis/HC1,
condi-
glucose; glucose
Inhibits
Analysis
RMC
(LPS) ( 10 pg/ml) and to both test media because
NO production was negligible in the absence nation. A hyperosmolality control medium 27.8
Glucose
in the
Statistical
to each well
Results
inner
of this mixture
salt
was added
Plates were wrapped in foil and incubated for I h at CO,-90% air atmosphere. Absorbance was read at
nm in samples
and
solution
Analyses are
experimental group
presented groups
comparisons
as mean were
were
± SEM.
compared made
The
using
using
means
ANOVA;
between post
the Bonferroni
hoc
the inter-
correction.
blanks.
Results Production
NO
HPLC
Assay
for
After contain
incubation LPS and
L-Argimne in normal IFN-y, cell
phosphate-buffered pension were
was
saline spun
Vacuum-dried hydroxysuccinimidyl
and high monolayers
(PBS).
at 10(X) rpm
resuspended
methanol was and the cleared
There
in 500
l
RMC
glucose were were
for 5 mm of
PBS,
media rinsed
removed,
and
to sediment and
then
derivatized with using the Waters
the
sus-
the cells. 1000
added. Samples were spun at 10,000 supernatants were analyzed by HPLC. extracts were carbamate,
that did not twice with
X
l
They
of
g for
100% 10 mm,
6-aminoquinolyl-Nkit (Millipore,
Mil-
by
IFN-y exposure inhibition
exposure
media
10 pJ of 20 mM
maximum
HPLC
of 20 M1 of the denivatized
column
without
obliterating
the chromatogram. Therefore, the sample to be analyzed. were
HCI,
30 l
of borate
adapted
Calibration was (Pierce Chemical
from
Liu
this Mobile
(2 1) for
performed with Co., Rockford.
the internal standard. ible to
U, -J
w
neuronal activity
75
L
> U U I-
I
25
z 0
E
%E.o 24H Figure
1. Effect
levels
of high
(nmol/unit
percentage point). versus
(50
normal
glucose
paired
that
synthesis
per
control
cell
Results
media
(RMC)
nitrite
are expressed
(n
10 for
=
glucose
RMC
media,
Escher#{252}hia
two The
media
before
media.
values represents ratio of NO,/NO,
high
glucose
with
20 mM
The
as a
each