Reactive Oxygen Species and Antioxidant Defense in Puromycin ...

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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