of antioxidant enzymes. (J Am. Soc Nephrol. 8: 1722-1731,. 1997). There is indirect evidence that reactive oxygen species. (ROS) could mediate the glomerubar.
Reactive Puromycin
Oxygen Species Aminonucleoside WILFRIED
GWINNER,*
BIRGIT
and
Abstract.
Division Hannover,
from
changes
nine
days
oxygen
species
1 5 mm
after
from
were
increased
puromycin
of
related
of
in glomeruli
aminonucleoside
injection,
increased
radical
There
is indirect
could
mediate
puromycin
Further
generation,
using
evidence the
reaction
minimal
of
nephrosis
ROS
with
scavengers
reduced
phobogical
alterations
not
several
been
of increased available
time of
(2,8
induction times
lipid
directly ROS
-14),
ROS
to interpret,
tabolism
in PAN determined
generation
were
was
because
nephrosis which
Furthermore,
a correlate the
are
of
kinetics
unknown.
radical
of
radical ROS and
disease,
Copyright
© 1997
by the
American
true
ROS it has the
are
PAN
me-
with
not
patho-
oxygen were
not
catalase puro-
indicate
that
a
oxygen
species
is
injury
observed
in
necessarily
respond
of antioxidant 1997)
enzymes.
knowledge
luminol,
which
peroxide,
cence
and
with
radical
anion
scavengers
were
a more
assess
the
glomerular
were
of increased
in PAN
radicals
examined,
that
(i5,16).
of increased
the
defense, observed
of mRNA
of
specific specific
for
radical
measurements
radical enzyme
and
hydro-
chemilumines-
is relatively
chemibuminescence
catalase,
as a
course
radicals,
and
In addition,
in the
and
could
in the
anion
analysis
by the determination
ROS
nephrosis
chemiluminescence
(15),
an enhancer
therapy of renal
strategies
ROS using
superoxide
dismutases,
or are
glomeruli.
gbomerular
therapeutic
determined
antioxidant
alone
of the
in most instances onset of symptoms
antioxidant
detects
used
a protec-
generation defense
defense
radicals
detailed
superoxide
further
course
hydroxyl
lucigenin,
superoxide
different
demonstrated
measure. levels of glomerular were
hydrogen
with
anion scavengers only. A third, elevated gbomerular ROS levels
to the antioxidant specific
investigators
radical,
protective
et a!. (9)
antioxidant
of the
some
anion
were
of an increased
nephrosis
dase of Nephrology
Diamond
to an impaired
to establish
gen
for
radical
superoxide is whether
interventional Therefore, the
the
Society
not
Whereas
the clinical setting, where be implemented after the
help of
of lipoperoxide
of Nephrology
may
induc-
9 d after
results
gbomerular
gbomerulus
hydroxyl
result
in relation
Received December 9, 1996. Accepted June 4, 1997. Correspondence to Dr. Wilfried Gwinner, Division of Nephrology, Medical School Hannover, Carl-Neuberg Strasse 1. 30625 Hannover, Germany. 1046-6673/0801 1- 1 722$03.00/0 Journal of the American Society
declined
of superoxide
(8, 10),
due
In the initiated
and
the
also In would
for various
in
a
hydroxyl
contrary,
reactive
gbomerubopathy.
tive effect with unsolved question
measurements
is involved
the
after and
reactive
the
The
gbomerular
scavengers
efficiencies
course
conclusion
Second,
activities
the oxidative
that
days
enzymes
On
injection. in
the
that
are
pathomor-
increased
of
found peroxide,
of the
time
maintained
of nephrosis.
peroxidase
and
genesis of
a model
was
no clear-cut
increased.
as
yet
and
allowing
suggested
anion
antioxidant
to oxidative stress with an induction (J Am Soc Nephrol 8: 1722-1731,
(ROS)
is unknown. treatment
model
Nine to increased
stress,
course
glutathione
this
gb-
nephrosis, the
in the
to induce
course
the
First,
induced
sufficient
to
scavengers,
superoxide
to contribute
oxidative
increase
gbomerular PAN
antioxidant
of nephrosis
peroxidation
in
unsolved.
found
Despite
transient
oxygen
mitigated
were
species.
isolated
phase
in the
However,
remain
thereafter,
difficult been
and
of
en-
acute
various
radical
aminonucleoside
Metabobites
increased
with
(2,8-14).
glomerular
intervals
which
were
determined
studies
before
lipids
KOCH,* Department
aminonucleoside.
mycin
rose again by chemilu-
in rats,
nephropathy.
and
radical
primarily
and
species
the
nephrosis
treatment
issues
oxygen in
(PAN)
proteinuria
important
of
Medicine,
different
puromycin nephrosis,
reactive
chemiluminescence
injury
change
and
( 1-7),
the
reactive
glomerubar
aminonucleoside
of the human
have
that
analysis
and
changes
returned
minescence
luminob.
and gross
typical
Levels
eightfold
to
Five
of internal
lucigenin
after
and
merular
KARL-MARTIN
predominant involvement of hydroxyl radical and hydrogen peroxide in the initial increase in reactive oxygen species 15 tion
and
and
hancer
mm
injection,
present.
Department
was
baseline bevels on days 1 and 5 after injection, to 14-fold on day 9 after injection, as determined with
P. BRANDES,t
EBERHARD,*
in the acute
defense.
clearances,
were
in
species
rats
aminonucleoside
creatinine
mdi-
gbomerubopa-
antioxidant
morphology
in
Germany.
species
oxygen
nephrosis
gbomerular
reduced
studies
oxygen
reactive
isolated
puromycin
gbomerubar
Hannover,
aminonucleoside of
aminonucleoside
after
RALF
OLIVER
of Gardiology,
scavenger
of reactive
in gbomeruli
in the
proteinuria,
radical
generation
of puromycin
the
several
of puromycin
directly
phase
School
involvement
study,
Defense
J. OLBRICHT*
Medical
an
this
examined
of
CHRISTOPH
LANDMESSER,*
PLASGER,*
Anatomy,
the pathogenesis In
ULF
JENS
of Nephrology,
suggested
thy.
KUBAT,
*Divisiopi
Results
rectly
and Antioxidant Glomerulopathy
levels.
To
activities
of
glutathione
changes
and protein
peroxi-
were
analyzed
levels
of the
ROS and Antioxidant
enzymes.
In addition,
number
of macrophages
to identify
potential
were
sources
examined
of ROS,
the
(Sigma)
with
PAN
and
Basic
Sprague
(Sigma,
mg/lOO
rats (8- to lO-wk
St. Louis,
MO)
wt via
of0.9%
the
NaC1.
dissolved tail
ROS
1 , 5, and 9 d after
and
in 0.9%
vein.
were
old) were
Control
NaCl rats
determined
PAN
injected
received
urine
collections
excretion
were
and creatinine.
directly
after
PAN
of these
measured
in a sample
isolated
and enzymes
All
were
Munich,
was determined
on animals
of Health
heart
were
Guide
Serum
performed
using
Protein
according
to Lowry
conducted
for the
Care
Use
x-
protein
an
in accord and
each
was the
et a!. (17).
with
National Ani-
mals.
Isolation
were
by cervical
20 ml of ice-cold sieves
(300,
dislocation.
Kidneys
Krebs-Henseleit-saline
Minced
catheter.
tive
cortices
150,
and
were
buffer
passed
75 tim)
were
using
(KHS)
through
2000
flushed via
three
an
consecu-
ml of ice-cold
gbomerular
suspension
suspension
by light
was
more
microscopy.
than
95%
The
purity
activity
Generation
Generation
of ROS
SOD cording
(Biobumat ments
LB 9505,
of the gbomer-
loss
were
performed
Corp.,
Wildbad,
1 mm.
corded
Subsequently,
over
enhanced (0.1
30
of 0.23
mm.
superoxide
added anion
scavenging case, and
showed
the
antioxidants
and
hydroxyl
had
were
obtained
ROS
was
expressed
mM
from
to the
nary
experiments
able to respond
Chemicals.
plus
at
the
protein
I 2.000
X g at
at -70#{176}Cuntil assay with
isolation
different
was
resulted
no
not used even
of
Triton
procedure
leaving
(>50%)
measurable
because
at very
this bed
bow energy
by the cytochrome c assay, activity, i.e., copper/zinc-SOD
manganese
SOD
potassium
cyanide
confirmed
to inhibit
Catabase phosphate,
(MnSOD),
was
to avoid
assayed
interference
were
‘,
assayed
10 mM
0. 1 mM and
(20).
I mM
activity
ac-
in the by cyto-
separately after cyanide. Prelim-
potassium
cyanide
was
in gbomerular
samples
by more
suf-
unit of SOD was defined as the amount of SOD to inhibit the reduction of cytochrome c by 50%.
of
‘cm
that
CuZnSOD
activities rate
spectrophotometricabby
hydrogen
EDTA,
peroxide
in
pH 7.0. Calculation
coefficient
of hydrogen
the change
in absorbance
Measurements
were
by the con-
50
mM
potassium
of activities
peroxide during
performed
was
based
at 240 nm, 0.0425 the first
only
60
the
s of
in the linear
range
sumption
the
60 mm
CL
hydroperoxide
the
reaction
2 mM
and
0.5
Activities
buminol-
NADPH
at 340 nm, 6.22 mM
thiourea
bance
during
was
de-
the
only
reductase.
first
of
the
range
con-
that
reductase.
the extinction
(21),
‘
the
enzyme
re-
Reactions were performed mM EDTA, 20 mM gbuta-
using
‘cm
2 mm
in the linear
for GSH-Px, by
the
U of gbutathione
calculated
by
modifications.
as a substrate
oxidized by GSH-Px. 0.1 M Tris-Cl, 0.5 were
determined
minor
spectrophotometricabby
by glutathione
NADPH,
were
with
served
followed
(Sigma).
performed
(21),
(70 ,.M)
was
of NADPH
thione,
activities
by Beutler
duces the glutathione in a buffer containing re-
(GSH-Px)
described
and
the
reaction.
pH
7.7
coefficient change
of
in absor-
Measurements
were
of the reaction.
(200
say
the
was
U) ( I 8). In each added
thus
after
10 mm
confirming
dosage.
Gbomerular
that
All antioxigeneration
minus
concentration
that
tert-Butyl
contain-
of
background
(Bio-Rad
activity
of fresh
and
gbomeruli
that
l2-O-tetradecanoylphorbol
protein
glomerubi are still
13-acetate
variation.
Analysis
homogenates
Lowry’s
of
containing
method
glomeruli
procedure, Bio-Rad Laboratories, Muin the gbomerular suspensions. Prelimithan
peroxidase
method
and
specifically
peroxide
counts/mm
confirmed
to the stimulus
From
of erythrocyte CuZnSOD, GSH-Px, and included in each assay to control interas-
activity,
over
more
Sonication
7.8.
Bovine enzyme standards liver catalase (Sigma) were
by catabase
protein
this
buffer
(1 U) and subsequent
in a sufficient
Sigma
based on the Bradford Germany) determined
Hydrogen
in CL
or
was
with
to detect
scavenger
as the average
centrifuged
activities,
determined Total SOD
experiments
medium
to equilibrate
30 U) or dimethyl
peroxidase
added
pH
c oxidase. MnSOD activity was measured of CuZnSOD activity by I mM potassium
version
the
buminol
activity
measurements
radical.
decrease
assay nich,
constant
allowed (CL)
thereafter
signal
been
related
and
(SOD;
of the radical
no further
dants
remains
the
of horseradish
dose
and
of
dismutase
of the obtained
a second
activity
In some
to the gbomeruli
by addition
for
added
were
that
were (19).
activities to Crapo
Glutathione
Measure-
Mannheim, Germany). 10 mm. freshly prepared
chemiluminescence
CL, superoxide
M) was
tected
gbomerubi
10,000
to determine
experiments
activity
than 90%. One activity necessary
luminometer
Germany).
at 37#{176}C in the presence
of 1000
of
of the reaction. in a multichannel
ing 50 mM Hepes (Boehringer-Mannheim, After recording the background activity suspensions
was
of catalase
of 10 M
mM
bucigenin (Sigma) in 500 pl of Dubbecco’s minimal essential for chemiluminescence without phenol red, and bicarbonate,
for
Also, to 2500
ethybenediamine
X-bOO,
drawn
was stored
enzymatic
debris.
pellets
0. 1 mM
Triton
remainder
of
activities,
was
confirmed
on the extinction
in all preparations.
determined
Berthold
1%
aliquot
Preliminary
release
(CuZn-SOD)
KHS.
of ROS
was
the
in the cellular
reaction
Glomerular
and
The supernatant
100 concentrations
ficient
Gbomeruli were washed twice with KHS and resuspended in 15 ml of KHS. Quantitation of gbomerubi was performed in aliquots of the ular
time.
of 500
I h in 1 ml of hypo-osmobar phosphate,
an
activity.
in a maximal
mary
killed
(EDTA), and
enzyme
chrome inhibition
of Glomeruli
Animals with
this
levels.
concentra-
of Laboratory
acid
potassium
homogenate,
to a severe
autoana-
at 4#{176}C for
mM
4#{176}C for 20 mm.
killing
Germany).
50
concentration,
protein
creatinine after
containing
of enzymatic
lysed
were
presence
aortal
after
in the range
Enzymes
determination
resulting
were obtained
puncture
Antioxidant
the
tetra-acetic
injection, of
the individual
injection. by
Instruments,
procedures
Institutes
with
15
injection.
determination
For gbomerubi
and histology
6-h urine samples
before
obtained
samples
the
and compared
measurements
(Beckman
tion in urinary
for
In addition,
animals
Creatinine
lyzer
made
injection
excretion animals.
generation
to be linear,
1723
vial.
per
Glomerular
of 15
a similar
in glomeruli
injection,
with
at a dose
were examined in glomeruli isolated 1, 5, and 9 d after PAN Before the isolation of gbomeruli I , 5, and 9 d after PAN 24-h
Nephrosis
Procedures
Dawley
g body
volume mm,
ROS
shown
PAN
in
and Methods
Animals Male
increased
were
gbomeruli
Materials
an
measurements
in the gbomeruli.
Defense
( I 7). Protein
revealed
protein
Triton-X
concentration
concentrations
no differences
between
Detection
12% detected rocyte
of antioxidant
enzymes
polyacrylamide with catalase
gel
a polycbonal (Calbiochem,
and
blotted
antibody San
Diego.
of and
relative
blotting
was
to per-
of 80 g of gbomerubar sodium dodecyl sulfate/
to nitrocellulose. from
animals
expressed
Western
as described previously (22). Samples were separated on a 1.5-mm-thick
a modified
to the number
PAN-treated were
by
gbomerular
with
relative
controls. Therefore, all enzyme activities the glomerular protein concentration. formed protein
in
100 was performed
Catalase
rabbit
against
human
CA).
GSH-Px
was
was eryth-
detected
1724
with
Journal
a polyclonal
GSH-Px,
kindly
primary
of the American
Society
of Nephrology
antibody
rabbit
against
antibody
was
AR,
and
NY).
The
by densitometry,
and
expressed
as percentage
of the
different
loaded
amounts
on
the
established,
gel,
an anti-IgG
exposure
Rochester,
the
performed
rabbit
antibody
from
(Amersham Corp., Paisdetected by using a CL kit
of blots
to x-ray
film
(Kodak
X-
of the detected
bands
was
readings
of PAN-treated
controls.
linear
and creatinine
bound
(23).
intensity
protein
range
animals
In preliminary
of gbomerubar
enabling
erythrocyte The
human
Avissar
peroxidase bands were
analyzed with
Nelly
with
horseradish Protein
Corp.)
OMAT
by Dr.
reacted
donkey coupled with ley, United Kingdom). (Amersham
from
provided
and
enzyme
evaluation
of
standards
mRNA
levels
according
of antioxidant
enzymes
were
to established
protocols
described
readings
was
antioxidant
en-
quantitatively
6-h
individual Wilcoxon’s ation
test.
mRNA
levels
of -actin
Morphological After from
(22).
of the
cortex,
cacodylate
fixed
in Epon
copy,
l-m
812
to
2.5%
(Serva,
sections
antigen methyl
was
number
gbomeruli
of was
Statistical All
was
in 0. 1 M tetroxide.
osmium Germany).
sodium and
For light
toluidine
blue.
Protein
Kingdom),
electron acetate anti-
a cytoplasmic
and macrophages (27). was fixed and embedded in paraffin. Staining
performed
using
cells
per
an
immunoperoxidase
glomerubar
in of
system.
cross-section
of
30
are
expressed
as
activities
means
and
±
mRNA
SD.
For
levels;
the
results
protein
of
excretion;
Proteinuria
Protein
*
600
compared
tested
analysis
Creatinine
excretion
not
in the
different
from
whether
protein
with
no of
with
the
Dunn’s
the
nonpara-
multiple
and CL radical
com-
scavenger
A P value
U test.
related
to creatinine
in urine creatinine;
172
± 44
p.g/p.mol
creatinine).
after
5 and
