Expression and Localization of Cyclooxygenase Isoforms and ...

Expression Cytosolic

and Localization of Cyclooxygenase Isoforms Phospholipase A2 in Anti-Thy-l Glomerulonephritis SHINTARO KATSUTOSHI CURTIS

HIROSE,t TADASHI YAMAMOTO,* LILI KAWASAKI,* SHIN GOTO,t HIDEHIKO

B. WILSON,

*De/,art,iie,it School

MASAAKI

of Pathology,

of Medicine,

Institute

Niigata,

ARAKAWAt

of Nephrologv,

Japan;

and

and ITARU

and

Departtnent

FENG, EISHIN FUJINAKA,*

Department

YAOITA,*

KIHARA*

of Medicine

of immunology,

and

The

(II),

Scripps

Niigata

University

Research

Institute,

La Jolla, California.

Gbomerular

Abstract.

enzymes

for

expression

of

the

rate-limiting

negligible

isoforms

(COX-l and COX-2) and cytosolic phospholipase was investigated in anti-Thy1 nephritis in rats.

A2 (cPLA2), Ribonuclease

sion

synthesis,

major

cycbooxygenase

protection

prostanoid

assay

demonstrated

in gbomeruli

of control

of expression monocbonal ruRNA

minimal

rat kidneys

from day I to day anti-rat Thy-b antibody.

expression,

also

COX-l

minimal

mRNA

and

in the normal

gborueruli,

in a pattern

similar

Immunofluorescence COX

the

showed

are

gbomerular

injury

presumed

to

of

in the

GFR

(COX;

synthase;

EC

the

model

known

as

COX-l

2.8-kD cDNA

mRNA library

fibrobbasts cDNA

and was

COX-2

first isolated COX-2 was

(5).

transformed is 45%

differences

by

homologous in the

(5-8).

Rous

sarcoma

to COX-2

enzymatic

virus

cDNA,

activity

to

and

generate

is

there

greater

known

the

These generation and

especially

tion,

was

localized

epithelial

cells

University

School

of Medicine.

757

Asahimachi-dori

1 . Niigata

1046-6673/0903-()408$03.00/0 Journal

Copyright

of

the American ©

1998

by the

Society American

of

Nephrology

Society

of Nephrology

between

cells.

the

COX-l

(60

95 1 , Japan.

that

(I

Am

Soc

the A2

cell

kidney

cells),

types has

activation

dence

also

signal

transduction

mediates

matory

reactions.

1

,

COX-2,

that

cell Here,

by

and

cytosolic is more

its

of Henle

densa

acts

and

in

in

releasing

molecular

in the

weight

cytoplasm

neutrophibs,

PLA2 findings

during

the

expression

the

localization

of and

(cPLA2) and

as a second

processes report

enzyme

PLA2

(22). These

in

cells

system.

role

prompt of

addi-

adjacent

of gbomerular

ruonocytes,

cPLA2

cPLA2

limb

A high

form

we

In

in macuba

mainly

activation

dif-

a rate-limiting

another

pathways

role

growth,

feedback

cascade

termed

a major

and

regulation

is also

(macrophages,

indicates

cPLA2

in the

of cPLA2

cells

ascending

generation.

PLA2,

plays

usu-

of COX-l

processes.

of COX-2

identified

been

that

regulate

densa

thick

(PLA2)

expression

COX-2

tubulogbomerubar

PLA2,

cytokines,

than

inflammatory

of prostanoid

to 1 10 kD)

expression

mRNA

in ruacula

acid

mRNA factors,

or lipopolysac-

rapid which

of prostanoids

arachidonic

several

COX-

indicate

cortical

via

total

more

presence

a role

precursors

Niigata

of COX-2

The

heruodynaruics

COX-2

intermediates,

and

of the

(15).

suggests

vesicle embryo

prostanoids

Nephrology.

ruesangiab

interaction

growth

of prostanoids,

Phosphobipase

1, 1997. of

response

ferentiation, COX-2

and

several

oxygen

results

that of secretory Institute

cell expres-

selective

epithelial

COX-b by

in magnitude

the kidney

no

that

relative

10-14).

1 8). The Received July 25. 1996. Accepted October Correspondence to Dr. Tadashi Yamamoto.

of COX-2

the

and

ruesangial

1998)

regulated

in the

(

H

are

It is now variably

encoding

(8,9).

gboruerubar

9: 408-416,

with

for generation

from a sheep seminal isolated from chicken

and

in

prostan-

COX-2,

1,

of the

an intercellular

the

the course that

COX-

upregubation cells

suggests

cells

charides,

cell damage

cDNA

(7).

ally

prostaglandin

COX-l

the

cells, in

10 during

mediation

epithelial

model

in

of prostanoids in the cycbooxygenase pathway of the arachidonic acid cascade. Recently, two isoforms of COX have been identified:

in the

capillary

endothelial

indicate of

was

enhanced

findings

induction

rat

COX-l

gbomerular

and/or

at 1 h and day

In particular,

ester,

(3,4).

1) is a key enzyme

injury

the

exclusively

These

implicated

phorbol

an involve-

in gloruerular

cells

in gbomerular

cell

along

was

through

of

cell injury have been demand leukotrienes were shown

also

1 . 14.99.

in and

synthesis and

model.

of control for

were

induction

of prostanoid

gboruerubonephritis

Cyclooxygenase [PGH]

to

injury

glomerubi

epithelial

nephritis.

are

10

staining

generated

cPLA2

in the

immunofluorescence

4 and

epithelial

Nephrol

demonstrated

gbomerubonephritis

and to be involved

anti-Thy-b

COX-2

gborueruboncphritis,

contribute

( 1 ). An elevation

of anti-Thy-b

1 and

against

days

anti-Thy-l

oids

sion

the

COX-2

ruesangial

expression.

antibodies

has been

models

ment of prostanoids in mesangial onstrated (2-4). Thromboxanes to decrease

COX-

generation

in several

prostanoids

both

was

of

detectable

in gbomerular

gbomerular

at 1 h and day undetectable in increased grad-

I ruRNA

using

that

of prostanoid

glomeruli

the gboruerubi

of COX-

microscopy,

isoforms,

An increase

to that

on

probably

whereas

10 after administration of On the other hand, COX-2

enhanced in a biphasic pattern with two peaks 10. Expression of cPLA2 ruRNA, which was normal glomerubi, was induced on day 1 and ually

walls

increase

faintly

In contrast,

intensified

expres-

a gradual

or

kidneys.

(16-

striking

than

(19-21).

Evi-

messenger suggest

the

in that

inflam-

profile

of

of

COX

COX

isoforms

in

glomeruli

in

the

anti-Thy-b

placed

gbomerubonephritis

gels

model.

Materials

of Anti-Thy-i

Anti-Thy-I rats weighing

gloruerubonephritis was induced in female inbred 160 to 220 g (Charles River, Atsugi, Kanagawa,

by

intravenous

a single

against

rat Thy-b

Porton

Down,

injection

(OX7;

The

Salisbury,

of

mouse

European

United

Collection

Kingdom)

ulin fraction per 100 g body wt (23). sacrificed, and the kidneys were removed after cause

antibody glomerular

1 h, lysis ative

administration. accumulation

of mesangial

lesion

glomerular kidneys

recognizable

lesions

were

were

Preparation

was

from

apparent

of COX-1,

Rat COX-l,

and cPLA2

lysaccharide-stimubated

as reported

eDNA), vector

then blunted and (Promega, Madison,

cDNA BamHI

was prepared and EcoRI

rophage

COX-2

A cPLA2 site

COX DNA

isoforms sequencer

cDNA cloned

and cPLA2 cDNA (ABI 373A, Perkin

of

from COX-l

(Promega). 719

subcloned

to

into

sequences

KpnI

of the

were

sieving

guanidinium-thiocyanate

followed

isolated

buffer

(Isogen,

by sonication

[32P]-Labeled

and

the

cRNA linearized

of each

rat

in a modified Gene

total cellular

sectioned

-70#{176}C and cryostat

Co.,

a goat

with

rat

templates

A (0.3

j.g/ml, Grand

ribonucleases were digested with at 37#{176}C.After phenol/chloroform

ethanol

precipitation,

the hybrids

Sigma) Island,

NY)

that were Co., St.

17

COX-l.

TI

(90

for 1 h at 30#{176}C. Then,

the

ribonuclease

proteinase K (0.5 mg/mb) for 30 mm extraction and sodium acetate-

were denatured

for COX

for

10 ruin

Biotechnology,

against

COX-2

normal

(EDI

3 h at 37#{176}C, and

Detection

Systems,

for COX-l

was

Santa

murine

(Kirkegaard

Teknica,

antibody

for

To localize

shown

West

with

1:50

Chemical

Seiyaku

Co.,

To-

anti-goat

IgG

I : 100 dilution),

FITC-

Kogyo Co., anti-mouse

Gaithersburg,

anti-mouse

IgG

1: 100 dilution)

MD; antibody

as the second-

temperature.

cells

in the gbomeruli,

sections

were

isothiocyanate-conjugated

glomerular basement membrane antibody prepared after the COX- 1 staining. The COX-2 localization immunostaining anti-rabbit

to a glomerular

Dr. P Mundel) goat

PA;

to react

(RP3, 1gM, a School of Mcdi-

donkey

Laboratories,

tetramethylrhodamine

examined by double and FITC-conjugated antibody

Chester,

1 h at room

4%

CA;

(Cayman

PA;

rabbit

COX-b-positive

stained

rabbit anti-rat our laboratory

& Perry

or FITC-conjugated

and

with

rabbit serum as a control

Cy3”’

Pittsburgh,

then

Cruz,

Dainippon

,

with

The

After washing in were incubated

which

,

at

in a cryostat.

for 5 ruin and

COX-l

and leuko-

in n-hexane

thickness

acetone

tissues

isoforms

quick-frozen

acetone

human

Cruz

antibody

1 : 100 dilution),

epithelial

IgG

with and

the anti-COX-2 then murine

cell

antigen,

IgG

(Cooper

Biomedical,

and monocytes/macrophages under a fluorescence

antibody monocbonal

pp44 (a kind gift from isothiocyanate-conju-

(27), and tetramethylrhodamine

anti-mouse

in was

Malvern,

infiltrating microscope over

in a section of each rat kidney. The data of these cells per glomerular cross-section

PA). the gbomeruli more than 30

were expressed as (mean ± SD).

Results Light

by in vitro (for

of each labeled antisense probes were digested and

with with

against (Santa

for

1gM antibody

Microscopic

The antibody

injection

Life Technologies,

fixed

forma-

and hematoxylin.

monocbonal antibodies against neutrophils from Dr. F. Sendo, Yamagata University

(Biological

glomeruli was hybridized with 1 X I O cpm cRNA probes for 16 h at 50#{176}C.Unhybridized ribonuclease

acid-Schiff

were

or monocytes/macrophages

gbomeruli numbers

The

protec-

in buffered

for 10 mm for COX-2 staining. saline three times, the sections

a rabbit

Japan)

normal

U/mI,

tissues

or fixed

antibody

Induced

were synthesized

eDNA

were

COX-l

or mouse kind gift

ary

later.

paraffin-embedded

periodic

at 2- to 4-pm

staining,

with

were fixed The

examination

sectioned

paraformabdehyde phosphate-buffered

kyo,

days

by ribonuclease

Tokyo,

sacrifice.

probes

with

of kidney

sections

leukocyte

COX-2, and cPLA2) or Sp6 (for GAPDH) RNA pobymerase and a-[32P]UTP (24). The total cellular RNA (20 g) isolated from the

with

parts

tissues

in paraffin.

and stained

other

cine),

a few

The

Japan)

samples.

kidney

For immunohistochemical cytes,

times

Tokyo,

RNA (26). As a

prepared from lungs of rats (4 mg/kg, Sigma Chemical

6 h before

antisense using

cortex

homogenized Nippon

to isolate

intraperitoneably

transcription,

from renal and

RNA

embedded

Neutrophils were counted

method

total cellular RNA was with bipopolysaccharide MO)

eDNA,

Assay

glomeruli

by a standard

were

then

doubly

by an automated Japan). A l23-bp

for COX-2

different

16 h and

three

gels.

(Fujiphoto,

Examination

and

gated

Protection

At sacrifice,

lin

and

subcloned

eDNA.

Ribonuclease

using

(Organon

1002,

dehydrogenase (GAPDH) an equal loading of sample linearized with either BamHI for EcoRI

assay,

polyacrylamide

film

developed

repeated

409

conjugated goat anti-rabbit IgG antibody (Seikagaku Tokyo, Japan; 1 : 100 dilution), FITC-conjugated rabbit

for rat glyceraldehyde-3-phosphate in pGEM4Z was used to evaluate

for cPLA2

Louis,

was

from

were analyzed Elmer, Tokyo,

HindIII

control, injected

vector

encoding

These

The plasmids were cDNA and GAPDH eDNA,

Japan)

fragment

COX-2 cDNA with by to rat peritoneal mac-

in pGEM4Z

cDNA,

vector.

(25).

kidney

by reverse

A 34l-bp

were was

in Thy- I Nephritis

on 6% to x-ray

Co., Ann Arbor, MI; 1:50 dilution),

from a lipopo-

library

ofthe cloned corresponding

COX-l

RNA

cDNA

(24).

fragment

cPLA2

of pGEM3Z

films

expression

dilution),

were cloned

macrophage

and subcboned

of 284-bp to rat

BamHI

and

subcboned into the SinaI site of pGEM3Z WI). A 242-bp fragment of rat COX-2

by digestion to 650,

eDNA)

the

As a control,

by BstXI digestion (encoding to rat peritoneal macrophage

(409

eDNA

corresponding

cDNA

previously

the COX-l cDNA was excised 1350 to 1690, corresponding

prolifer-

rats.

cPL42,

rat peritoneal

PCR,

at 28 d (23).

COX-2,

beat

at 10 d, and

Dehydrogenase

COX-2,

transcription

remarkable

WKY

Giyceraidehvde-3-Phosphate

of five rats was I , 4, 10, and 28 d

at 1 d, mesangial

normalized normal

Cells,

points were selected became conspicuous

exposed

For light microscopy,

of I mg y-gbob-

Each group at 1 h, and

at 4 d and

almost

obtained

at -70#{176}C.The

Histologic

antibody

of Animal

at a dose

These time of neutrophils

cells

was

monocbonal

WKY Japan)

electrophoresed

and

tion

Giomeruionephritis

and

dried

mRNA

and Methods

Induction

on ice, were

and cPLA2

at 85#{176}C for 3 ruin,

and

gbomerubar

gbomeruli (Figure

glomerubar

(mesangiobysis)

quently, number matrix,

of the

lesions

at various are

(Figure was lB).

Giomerular

Lesions

Antibody

administration

gbomeruli

in the

Findings

by Anti-Thy-i

shown

time

points

1A), an accumulation

observed Marked

1 h after expansion

hypoceblubarity

with

were

at

apparent

a glomerular proliferative of gbomerular cells and was noted on days 4 and

day

the

after

1 . Compared

in Figure

with

of neutrophils

anti-Thyof the

loose

OX7

I antibody

gbomerular

ruesangiab

I (Figure

lC).

tuft matrix

Subse-

lesion, with increases in the expansion of the mesangial 10 (Figure 1, D and E). The

410

Journal

. - . . .

of

\

the American

of Nephrology

?

,2

(

A

Society

‘

.

.

.

,

\‘

.

jb

:r:-

‘DI

y:’:a

0

::iff&I*;; ;‘

;(

.p

I

*jYi,

.

,

11

microscopic

in a gbomerulus

proliferative segmental

findings.

on day

lesion mesangiab

was

“

,F--

C

-‘

(A) Normal

glomerulus.

(B) Neutrophils

‘i

D

(arrows)

28 (F).

almost

sclerotic

Magnification,

normalized lesion

(Figure

Ribonuclease COX-I mRNA

of mRNA for Glomeruli protection expression

COX-i,

COX-2,

.

‘

-

are observed

in glomerulartufts

a gloruerulus

at day

sclerotic

lesion

I

.

1 h after

Proliferative

(arrow)

administration

gloruerular

is observed

lesion

at a segmental

X500.

by day

28,

leaving

normal

1F).

rat kidneys.

increased antibody

Expression the Nephritic

‘I

,

of anti-Thy-b antibody. (C) A decrease in the number of glomerular cells is shown in with an increase in mesangial cell number is present on days 4 (D) and 10 (E). Glomerular area

q\\

?

..

1. Light

.

t9rr

;,

,

:

,L>a-

,..

--

.

plp

Figure

.

cv’

and

cPL42

in

and

expression

COX-2 assay showed a minimal level of in gbomerubi isolated from control

slightly

glomeruli, day I and ,

The at

markedly

on day

of COX-b

ruRNA

gboruerubar

1 h after ruRNA

was

then

increased

1 and

thereafter

was

apparently again

mRNA

administration

decreased

expression

but

COX-l

the

also

enhanced on day

expression

of anti-Thy-b (Figure

somewhat

minimal

2A).

on day

The 28.

in the normal

at 1 h, decreased 10, revealing

a biphasic

on

coxI probe cox

pattern.

C0X1mRNA

-----

2 probe

than

that

The cPLA2

COX2mRNA

-

but

reach

cPLA2

Immunofluorescence In normal

7

medullary

probe,. 4cPLA2mRNA

-

ubi (Figure

3, A and

along

and

-S

..

-

I 0 after

with

the

(Figure

ing

was

ducts

the

7

No

along

JA

cPLA2

.

I m

mRNA

day

the

found

probe

4A).

4E).

Double

and

anti-pp44

7

28

solic

phospholipase

A2

expression is detected slightly at I h, markedly able

in normal

biphasic

(GAPDH) exposure

rat gbomeruli,

pattern

(A).

expression

of a gel

to x-ray

in the

normal

gbomeruli

with

anti-Thy-l

increases

ruRNA

expression

ruRNA showing gbomeruli;

glorueruli

similar lane

nephritis.

gradually

to x-ray films. the gloruerular

and ruRNA

at I h and

with

In another expression

mRNA

on day

of cPLA2

is apparent

on

Glomerular

a peak

at day

in a gel by ribonuclease

injection; rats.

lane

day

is

1 in the

ruRNA

10 (B).

using

different

1 , COX-2,

protection

7, lungs

associimmu-

of

collect-

anti-Thy-

I

obtained

the COX-2

4G).

These

of the faintly

rat

kidneys

became

intense

(Figure 4C),

4B).

The

recovered

at

10 (Figure

anti-COX-2

staining

antibody

of COX-2 cells,

tended

in the

nearly

ruRNA

protection

found

epitheliab

changes

were

isoform

was

cobocalization

gbomerubar

in

course

at day

the

rat

the

COX-2

increased using

normal intensity

control

1 (Figure

markedly

for

Then,

in

injection

at day

marker

and at day

to decrease

staining

at

for

comparable

expression

COX

to the

pro-

demonstrated

by

assay.

of neutrophils

glorueruli

COX-2

for

showed

in

staining

walls

staining

antibody,

cells

was

and

numerically

microscopy.

The

per

ruonocytes/ruacrophages

examined

numbers

of

gbomerular

by

immunofluo-

neutrophils

and

cross-section

at 1 to 4 d, respectively

(Figure

mono-

were

highest

5).

Discussion

cx-

GAPDH

The

RNA

and cPLA2 assay (C),

from

trace

normal

COXoids

li-

expression

glomeruli

now

known

that by

intermediates, our previous regulated, kidneys

several

1 mRNA

present

These

regulation

growth

and

of

(28).

in

suggests

glomerular

On

COX-2

factors,

demonstrated

which

that

generation of prostanare presumed to play

prostanoids

previously

COX-l

was

study,

in the constitutive

physiological

as speculated

regulated

the

gbomeruli.

in the

naruics,

of COXin

I participates in the

a role

results as demonstrated in A and B. Lane 1, normal 2, 1 h; lane 3, 1 d; lane 4, 4 d; lane 5, 10 d; lane 6, 28 d

after anti-Thy-b antibody popolysaccharide-stimulated

3, C

10 in a

mRNA

cPLA2

gbomeruli,

in the gbomeruli

at 1 h and

3 d (a) and 16 h (b) after the gel experiment of COX-

on days

in the

during

I antibody

and

cytes/macrophages

is enhanced is undetect-

observed

immunostaining,

COX

rescence

3 d (a) and 16 h (b) after

Expression and

cyto-

dehydrogenase

is examined films.

and

COX-l

expression.

but is intense

was also examined

is analyzed

COX-2,

Glyceraldehyde-3-phosphate

mRNA

pression

mRNA

1 became

in close

course

COX-2

capillary

4D),

Accumulation

in the normal gbomeruli on days 1 to 10. COX-2

minimal

exposure samples.

(cPLA2)

in the

(Figure

1 staining

densa

was

transiently

(Figure

of

in the (COX-l),

the

gbomer-

by double

the

ruacula

Gbomerubar

ribonuclease

cycbooxygenase-l

COX-

observed

COX-

of

the anti-Thy-

isoforms

2. Gbomerular

for

membrane

F).

in

cells

declined

files

Figure

present in the

injection

1 was

change In the

4 (Figure

day

123456

of

in the gbomeruli

during

gbomerubar

pp44, a specific 10 (Figure 4F).

GAPDH

C

gbomerular

obscure

staining

basement

unchanged

densa

intensity

COX2mRNA

-

of

profiles

intensely

1 antibody

of COX-

apparent

ruacula

1 h after

-

set

was

walls

anti-Thy-

3, D and

was

kidneys.

(Figure probeu.. cPLA2 probe. cox 2 probe.

but

However,

gbomerular

nostaining

cells,

B).

gbomerubonephritis.

coxi

to

Findings 1 was

the capillary

E). Localization

COX-2

123456

gradually

gbomerubonephritis. GAPDH mRNA

B

different

rat

1 antibody

representative

same or similar (Figure 2C).

COX-

duct

conspicuous

ation GAPDH, probe

in the normal

anti-Thy-

Another

Microscopic rat kidneys,

collecting

4 and *

was

observed.

mRNA(a)

GAPDH ‘mRNA(b)

123456

a

mRNA

points

increased

2B).

with

41 1

COX-l

1 after

expression

RNA samples showed the expression for these mRNA

A Y

of

was not found

10 (Figure

obtained

Nephritis

at all time

on day

cPLA2

on day

in Thy-I

ruRNA

detected

The

a peak

autoradiograph

A

expression

expression

was

administration.

,

the

of COX-2

mRNA

glomeruli,

GAPDH probe

and cPLA2

Interestingly,

greater

-

COX

the

mRNA

cytokines,

hemody-

other

hand,

it is

expression

is

relative

oxygen

or lipopolysaccharides ( 10-13). We showed in study that COX- I mRNA expression was downwhereas

after

COX-2

lipopolysaccharide

expression

was

stimulation

upregulated ( 14).

in rat This

result

412

Journal

Figure

and

rat kidney. wall

C through

suggests

the American

3. Irumunofluorescence

in a normal capillary

of

that

of Nephrology

microscopic COX-

in gbomeruli E; X200

Society

I staining

findings in kidneys

of rat kidneys

of COX-l obtained

delineated

.

COX-l

4 (C) and

by anti-gbomerular

basement

induced

COX-2

and

lipopobysaccharide

in pathological

cPLA2

in a glomerulus

anti-Thy-

(A)

1 antibody

membrane

antibody

and

intense

administration (D and

in collecting

ducts

is apparent

F). Magnification:

(B)

along X500

the in A

in B.

influences

ruRNA

duced

expression

of COX isoforms differently. A gradual increase in COX-l ruRNA during the course of anti-ThyI glomerulonephritis observed in the present study indicates that this COX isoform is also

is negligible 10 (E) d after

mRNA

or inflammatory

were

previously

conditions,

shown

(10-14). which

glomerubar

induction

in a similar

COX-2

as

to be in-

The

expression,

ubi,

but

of the to that

of COX-

increase of COX-

1 and

cPLA2

of cPLA2 1 , indicates

may

ruRNA that

the

be regulated

manner.

mRNA was

profile

is similar

expression

upregulated

was at

both

minimal

in normal

I h and

10 d in

gbomera biphasic

COX

Figure 4. Immunofluorescence for COX-2 increases along I (C). epithelial

The

staining cells

glomerulus

normal

to increase

by combining

at day

rabbit

turns

microscopic the capillary

serum

28

(G).

No

is used

findings of COX-2. walls in a glomerulus

again

double

staining

specific

staining

instead

of the

on day

4 (D).

for pp44, is observed

anti-COX-2

The

In a normal at I h after COX-2

a specific antibody

staining

(H).

in Thy-I

Nephritis

413

rat gloruerulus, COX-2 is imruunostained faintly (A). The staining anti-Thy1 antibody injection (B), but declines to be weak on day

gboruerular

in a gbomerulus

and cPLA2

ofa

is most

remarkable

epithelial

cell

kidney

obtained

Magnification.

X500.

at day

antigen

(F).

10 d after

10 (E) and The

COX-2

anti-Thy-I

is localized intensity antibody

in glomerular decreases injection

in a when

414

Journal

of the

American

Society

of Nephrology

and

cPLA2

COX-2

mRNA

ruRNA

regulation

0

of TxB,

phases

Cl

of

anes Cl)

Cl) 0 Cl

and

are

1 nephritis

in glomerular

by amelioration

receptor

antagonist

cell

of GFR

lytic

thromboxane

rats

increment

of

sustained

up-

and

The

role

proliferative of

nephritis

with

or the

in the

of this

nephritis-induced in

second

model.

dysfunction

onstrated

prostanoids

the

involved

at the mesangiab

anti-Thy-

anti-Thy-l E

expression

expression

been

administration

of

inhibitor

However,

the

dem-

of TxA,

synthetase

(2,4).

pathogenesis

thrombox-

has

the

lesion

in

role

of

to

be

remains

clarified.

0

The

=

at

enhancement

1 h after

trophils ruerubi,

C)

lh

0

iday

4

10

28days

evidence

indicate

leukocytes

or

5. Changes

(#{149}) observed

macrophages course

in the numbers

of anti-Thy-b

(El)

of neutrophils

in a glomerular

nephritis.

Results

and monocytes/

cross-section

are

mean

the

± SD.

The

present

ubar basement

from

that

that

of COX-

the

from

regulation

that

have

suggesting

was

induced

is different

cells

or

have

been

synthesize

speculated, has

shown

gbomerular

cells

that

reasonable

model glomeruli. prostanoid synthesis

of prostanMesangial

not

test

in

vivo,

has

been

whether as

shown

examined

cell

in

duced

ruesangiab

injury

showed slightly

that synthesis of PGE,, impaired at a time point

to

vivo

cell cells

cells.

The

injury

in mesangial

or in mesangial

It

rats

injury

has

disrupt

in normal

cells

might

synthesize

with

(29),

prostanoid

ing

an

anti-Thy-

(2-4).

I

the

studies

cytokines cells

(LT)B4,

strated

acid

(HETE)

early

time

synthesis synthesis lyric

and

gbomerular

was points

shown

and

to be upregulated

(1 and 2 h) in the model

returned

to

control

remained

increased

proliferative

synthesis

levels,

but

in the glomerubi (4). Thereafter, TxB,

in association

lesions

(days

of prostanoids

and

be reasonable

of TxB,

at the early

to speculate

time

mesangial

14).

The

in this

nephritis

that the sustained

Furthermore, increase

cell

has

incidence

with

logic

changes

it may 1

were

wild-type

expression

showed

reported

tissue

may cell

interfere

cells,

result-

for

proteins.

in prostanoid the

syn-

release

gloruerular

of

epithelial

isoforrus

was mice

in anti-Thy-b

(35).

recently

(35,36).

to develop

in tubules

in the

had

ulceration

and

acid

no significant organs

COX-

but

to arachidonic

Although

in systemic

demonThe

normally

gastric

responses

found

indicates of renal study.

present

in

injury

may

through

kidney

mutant

mice

were

the

and

epithelial

of indomethacin-induced

constitutive

capillary

in humans

epithebial

study

of COX

in the

abnormalities

This result velopment

decrements expression to the upof COX-

pared

rela-

(33,34).

of inflammatory

occasional

been

expression

homozygous

mice

decrement

enhanced

stimulate

to

epithebiab

cell

be involved

that

at

and

intercellular

permeability cells

of prostanoids

in COX

1-deficient less

12-HETE

with

points.

at LTB4

4 and

noted to mediate the anti-Thy1 antibody-induced of GFR (2,4). The first increase of COX-2 mRNA demonstrated in the present study may contribute regulation

A role

l2-hydroxyeicosatetraenoic

the

as predicted

activate

expression

cell-gboruerular

may

mediators

to enhance

nephritis

(Tx)B,,

or

may

the

I gbomerubonephritis

present

epithebial

the

that

gloruerular

of gbomerular

leukocytes

during suggest

mesangial

gbomerubar

pp44, COX-2

in mesangial

gborucrular or

anti-Thy-

the

role of

gbomerular

and

however,

in

with

gbomerubonephritis

mesangiab

disturbance in

suggesting a synthesis of these prostanoids in mesangiab cells in a physiologic condition. In contrast, synthesis of leukotriene thromboxane

proteinuria

a physiological in

thesis

PGF,a, and 6-keto PGF1a was of marked mesangiab cell lysis,

of

possible

findings

a possible cells

predicted

Alternatively,

antibody-in-

These

predicting

other

that not

1 in

the

glomeruli

ruRNA

(30glomer-

of COXOn

proliferation

mesangiab

clarified;

but

the

COX

proliferative been

interaction

in culture

model

not

and

proliferative

the

indicating

These

injury

mechanism

I along

cells in

to enhance

prostanoids, between

in

clarified.

gbomerubar

mesangiab

was

synthesize

epitheliab

cell

cells

or platelets

cobocalized

(27),

of

largely

cells.

selectively

1 nephritis.

initiation

the presence

marker

than

prostanoid

synthesis COX-

leukocytes

mesangiab epithelial

tionship

and

in

been

the prostanoid-synthesizing

To

selective

the type

enzymes

have

a specific

COX-2

however,

were

in gbomerular

in neu-

other

of leukocytes

endothebial

cell

of anti-Thy-

least

of origin 1 and

these

conditions

to use

to elucidate

COX-

of phys-

of pathologic

of debate.

(10,12,13);

express

or pathologic

seemed

an issue

in culture

regulation

mediation

the cell

been

to express

prostanoids

physiologic

in the and

proteins

infiltrating

course

of prostanoid

been

cells

of

expression, expression

after

suggesting

epithelial

mRNA

hemodynamics

in the glomeruli

COX-2

a gbomerular

mRNA

the robes

oids

hand,

of COX-2 or cPLA2.

gbomerular

processes

profile of different

isolated

prostanoid

and/or

cells enhanced

depletion

membrane,

expression occur

for

demonstrated

epithebiab

1 or cPLA2

of COX-l

Although iobogic

gbomerubi. The was apparently

glomerubi

study

may

intrinsic

account

the gboruerubar

glomerular

pattern in the anti-Thy1 nephritic glomerular COX-2 mRNA expression

glomerubar

in which

ruRNA

injection

accumulating in the gb(4). In contrast, several lines of

may in

injuries,

did not affect 32).

that

platelets

observed

immune

during

COX-2

1 antibody

or monocytes/macrophages as predicted previously

synthesis Figure

of gbomerubar

anti-Thy-

com-

patho-

in these

mice,

were

noted.

kidneys

a physiologic robe of COX- I in the tubules, which may correlate with of COXOn

the

abnormalities

other

dethe

1 in collecting

ducts

hand,

knockout

mice

kidneys,

heart,

restricted

COX-2

to the

shown

in

and cPLA2

COX

and

ovaries,

renal

and

altered

abnormalities

immature

were

glomerubi

COX-2

in the

findings

also

development and

limiting

cPLA2,

for

are

in the

These ated

strongly

through

the

gbomerular and

between

mesangial

induction

a role

during

and

1 and

the

and

14.

gener-

COX-2,

intercellular

gbomerubar

in

the

K. Yoshida

(University clonal in-Aid tion This ogy,

for technical

communication

of Freiburg,

Freiburg,

assistance

15.

Germany)

antibody, pp44. This study was for Science, Health, and Culture and

by Grant

DK-20043

is publication The

from

Scripps

Research

for

providing

U.S.

from

Public

a mono-

Health

the Department

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of Immunol-

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