Glomerular Epithelial Cells Produce

Glomerular Andrey

V. Cybulsky,2

A.V. Cybulsky. Royal Victoria

of

J. Stewart,

D.J. Stewart. Department Hospital. McGill University,

Cybulsky.

Vascular

Pathology.

Brigham

yard

Duncan

Cells

Produce

and

Myron

Medicine, Montreal,

of

Canada

Quebec

Ml.

Epithelial

Medical

(J. Am.

Research

and

School.

Soc.

Boston,

Nephrol.

Division,

Women’s

Department

Hospital

and

Har-

MA

1993;

I. Cybulsky

dose of phorbol myristate acetate. Thus, glomerular epithelial cells may be an important source of endothelin-1, which might influence glomerular vasoconstriction or proliferation of target cells, particularly in the presence of proinflammatory molecules in the glomerulus. Key Words; forming

3:1398-1404)

E vascular

a vasoactive peptide originally isolated from vascular endothelial cell culture supernatants, has constricting or mitogenic effects on smooth muscle and glomerular mesangial cells. Whether or not cultured rat glomerular epithelial cells synthesize endothelin-1 was assessed. Under basal culture conditions, the synthesis and release Endothelin-

I

,

peptide

endothelin-1

cells

by

glomerular

epithelial

was time

dependent, reaching 0.231 ± 0.0 17 pg/1,000 cells at 24 h. For comparison, unstimulated bovine pulmonary artery endothelial cells and rat mesangial cells produced 0.982 ± 0.237 and 0.004 ± 0.002 pg of endothelin-1 peptide/1,000 cells per 24 h, respectively.

In addition

to endothelin-1

pep-

tide, unstimulated glomerular epithelial cells expressed preproendothelinI mRNA. Transforming growth factor-f, complement C5b-9, thrombin, and phorbol myristate acetate significantly enhanced peptide

endothelin-1 hal

cells

(45,

15,

synthesis

and

55,

24 h, respectively),

in glomerular

25% above

whereas

basal

epidermal

levels

growth

at

factor

had no effect. Thrombin and phorbol myristate acetate appeared to stimulate endothelin-1 peptide by activating protein kinase C, because the protein kinase inhibitor 1-(5-isoquinolinyl-sulfonyl)-3-methylpiperazine abolished the thrombinand phorbol myristate

acetate-induced

rise in endothelin-1

but

had

no effect on basal production. The stimulatory effect of thrombin was also markedly diminished in glomerular epithelial cells that had been depleted of protein kinase C by prolonged preincubation with a high I 2

Received June Correspondence

Hospital,

687 Pine

1046-6673/0307-

3, 1992. Accepted October 2, 1992. to Dr. A. V. Cybulsky. Division of Nephrology, Avenue

West.

Montreal,

Quebec.

Canada

1398$03.00/0

Journal of the American Society of Nephrology Copyright © 1993 by the American Society of Nephrology

I398

Royal H3A

IAI.

Victoria

cell

from

an

cabled

in a reduction

protein

kinase

a vasoactive

also

culture

big properties

ET

release,

In the peptide

perfusion

of ET

suggesting

that

hemodynamics 3). Recent also

by endothebiab

be

in

produced

by

addition

nonendothebial ET production

an

the result

filtration,

activity,

vasopressin-in-

can

Na excretion, (2.3). The synbe

stimulated

by

substances, including intenbeukin-1 , vasopressin, growth factor-fl (TGF-fl), gbomerulus.

ET

may

conditions of inflammation have demonstrated that

studies

dude kidney-derived as primary cultures duct cells (5) and in

the

under

cells

kidney, can

ATPase

antagonize

of proinfbammatory II, endotoxin, and transforming

via

and

Na/K

1 ). ET

(

preproET,

(1 .2). of ET

21

porcine

supennatants

duced water permeability, enhance and be mitogenic for mesangiab cells a variety angiotensin thrombin,

of

from

--200-residue

antagonize

renin

thesis

C. trans-

peptide

isolated

in gbomerular

ET can

diminish

is

originally

endothebial

intermediate vasoconstnicting

Thus,

epithe-

(ET) acids,

is produced

but

thrombin.

factor-1

ndothelin-1 amino

ABSTRACT

of

Complement,

growth

nonendothelial

cells.

alter (1 can

ET

These

-

in-

epithelial cell lines (2,4), as well of rat inner medublary collecting gbomerubar mesangial cells (6,7). to gbomerubar

renal under

endotheliab

cells

(8),

cells might also participate in normal or inflammatory condi-

tions.

Among the intrinsic cells of the renal gbomerulus are visceral and parietal gbomerulan epithebial cells (GEC). Both cell types are of common embryologic origin.

In normal

kidneys.

visceral

GEC

play

a robe

in

the maintenance of gbomenular capillary wall permselectivity and there appears to be little turnover of GEC (9). Injury to visceral GEC may lead to abnormal gbomerular permeability ( 1 0). ProlIferation of parietab and possibly visceral GEC may occur in other forms of gbomerular injury, e.g. , focal segmental sclerosis and crescentic glomerubonephnitis ( 1 1 ). Moreover, expenimentab models of gbomerubopathy that manifest GEC injury/proliferation are often associated with perturbations in gbomerular hemodynamics ( 1 2, 13). In this study, we examined whether rat GEC can synthesize ET and whether inflammatory mediators

Volume

3

‘

Number

7

‘

1993

GEC Produce

can

El-I

alter

in

ET

culture

under

production.

We

synthesize

basal

further

conditions

enhanced

pbement.

demonstrate

barge

and

after

thrombin,

that

of

ET

GEC peptide

ET synthesis

stimulation

and

that

amounts with

phorbol

can

TGF-9,

mynistate

be

com-

acetate

(PMA).

METHODS

Tissue

culture

ICN

media

and

(Mississauga,

Chemical

Co.

(St.

reagents

were

Ontario,

Louis.

MO),

obtained

Canada),

and

Sigma

Collaborative

(Mississauga,

Ontario,

was

from

tanio,

Canada),

from

Peninsula

dium

and

virus netic

rabbit was

from

purchased

transcriptase (Tampa,

from

was

CA).

from avian

Guani-

Fluka

was

(Cleveland.

(La

Cetus

Jolla,

from

United

OH).

Thrombin,

bromo-cGMP,

and

Bio-

myeboblastosis

was from Molecular GeFL), Taq DNA polymerase

Elmer

Stratagene

Ci/ On-

antiserum

(Belmont, NY),

Perkin

(2,000

(Norwalk,

CT),

Klenow fragment of DNA polymerase I and nucleotide kinase were from New England Inc. (Beverly. MA). the plasmid vector pBS erase

CA),

and

States

DNA

polym-

Biochemical

PMA,

the

T7

the

T4 polyBiobabs, +1was Corp.

dibutyryl-cAMP,

protein

kinase

8inhibitor

1 -(5-isoquinobinyb-sulfonyb)-3-methybpiperazine, were purchased from Sigma. The latter compound. which we call isoH-7. had been originally sold by the manufacturer as 1 -(5-isoquinolinybsubfonyb)-2-methylpiperazine Iments

(H-7). with

this

manufacturer icab

(1 4).

that

The

an error

we

completed

we

were

had

Nevertheless,

hibitor

KT5823

the Co.

factor

at a H-7

inhibitor

kinase

from

in-

“

Oaks, CA). Epidermab TGF-f3 were from Collabo-

Ontario,

Canada,

and

Boston,

in rabbits,

cultures gbomeruli,

of the American

of rat as

GEC

were

detailed

Society

established previously

of Nephrology

of aminonucbeoside

of puromycin,

whether

from

17).

GEC

in

for

cytokeraby electron to determine

culture

originate

from

visceral or panietal epithebium. Primary cultures of rat mesangiab cells were estabbished from explanted gbomerubi and were characterized by published methods ( 1 8). Mesangiab cells were grown on plastic substratum in Dulbecco’s modified Eagle medium with 20% fetal bovine serum. Studies were done with cells between passages 2 and 10.

of ET

Measurement

GEC were Incubated with or culture media (37#{176}C)in 35-mm for 4- to 24-h periods. To evaluate ment,

cells

were

first

incubated

without agonists in tissue culture wells the role of complewith

GEC

antiserum

(diluted 1 : 10; 40 mm; 22#{176}C),followed by a concentration of normal human serum ( 1 .5% 37#{176}C: 24 h) or heat-inactivated human serum trobs ( 1 6). Preliminary studies demonstrated der these conditions, assembly of the C5b-9 on GEC membranes (measured by [‘25bJC9 reached a maximum at 6 h and assembled persisted

until

at

beast

24

sublytic vol/vol: in conthat, uncomplex binding) C5b-9

h.

At the end of incubations, culture supernatants were extracted on Sep-Pak C18 cartridges and were eluted with methanol. ET was measured by RIA, as detailed previously (19, 20). Briefly. samples and ET standards were reconstituted in assay buffer and incubated with rabbit anti-ET antiserum for 24 h at 4#{176}C. The addition of -4,000 cpm of [‘25I)ET was followed by a second 24-h incubation, and bound and free radioligand were separated by the second-antibody

method.

Bound

after

Total

strand action

radioactivity

bogit-bog

data

were

then

transformation.

of ET mRNA

cellular

guanidinium

described as

( 1 6.

doses

Demonstration

(2 Ci/mmol) purchased from

produced

to bow

evaluated

Kamiya

Cultures

Primary

Journal

shown

but than

kinase

(methyb-3HThymidine (500 Ci/mmol) were

(Mississauga.

by the

been

protein

purchased

(Thousand (EGF) and

exper-

in its chem-

has

protein

MA). Antiserum to GEC was described previously (15).

explanted

occurred

cGMP-dependent were

native Research. and cx-[35SJdATP

Cell

the

informed

isoH-7

cAMP-dependent

and

Dupont

after

protein kinase C (PKC) effectively to fourfold higher concentration

KT5720 Biomedical growth

but compound.

synthesis.

to inhibit threefold

mod5.0% NuSerum and hormone supplements (“K 1 medium) ( 1 6). Studies were done with cells between passages 20 and 60. Characterization of GEC was published previously (1 6). According to established criteria (18), the cells demonstrated a polygonal shape and cobblestone appearance at confluency, cytotoxic suscepti-

specifically

Re-

(Oakvible,

anti-ET

(Ronkonkoma.

reverse Resources

was

[‘25IJET Canada

Laboratories

thiocyanate

Chemika

Canada).

Amersham

grown on collagen gels in Dulbecco’s medium/Ham F 1 0 (1 : 1 ), containing

positive immunofluorescence staining tin. and presence ofjunctional complexes microscopy. Presently. it is not possible

search (Bedford, MA). Pepsin-solubibized bovine dermal collagen (Vitrogen) was from Cobbagen Corp. (Palo Alto. CA). Sep-Pak C18 cartridges were from Milbipore mmol)

were Eagle

bility

Materials from

GEC ified

RNA

was

isolated

from

GEC

thiocyanate-phenob-chboroform

by cDNA volume)

Chomczynski was synthesized with 2 g

and of

by the method

Sacchi

(21).

(42#{176}C:2 h: 25-jL RNA as template,

Firstre150

pmol of oligonucleotide primer complementary sequence found in the 3’ untranslated region preproET mRNA (AGCCATGACTTACATAGAG) and 27 U of avian myebobbastosls virus reverse

to the of rat (22). tran-

scriptase.

(95#{176}C

After

the

reaction

was

inactivated

I 399

GEC

for

Produce

El-I

5 mm)

erase

and

chilled

chain

nested

on

reaction

primers.

ice,

two

(PCR)

rounds

were

First-strand

cDNA

(6

template for the first round of PCR. the reverse primer was carried over transcription

reaction

complementary

and

to

the

(GTTCCATTTGCAACCGAG) second round of PCR. was

used

sisted

as

only

with

ML)

was

forward

and

untranslated

the

the

cycles

complementary (22). Both

of denaturation

29 s, annealing 72#{176}Cfor 60

primers

reaction

Taq

at

volumes

DNA

were

50

pobymerase,

concentrations products

were

of 2.5 and ML) were analyzed

(8

contained

Rat

The product of second-round mM IMgCl2I reactions) was

35

U of

at MgC12

electrophoresis product

and were

DNA polymerase nucleotide kinase, into the standard

modified

T7

Ml3-20

by

viousby growth

ends

PCR

DNA

pobymerase.

primers

(23).

of Cell of GEC

fragment

fetal

of

counter. counting placed

as

DNA detailed

cells serum-poor serum)

1 7).

phosphate-buffered 0.25 N NaOH. The fluid and counted

and

suspended

hemacytometer. incubations

was

Calculations The concentration was normalized

1400

cells

mean

-5

x iO

for

synthe-

48

saline bysate in a

were

then

number

pre-

2).

ET

per

35-mm

cells,

K!

with medium

period)

by

in

This

(pg/bOO

GEC

was

unstimulated cells by

comparison

“-25%

of

bovine

but was rat mesangial

between

pub-

-60-fold cells

cell

types

is

cells)

0.10

4

12

8

ET

(pg/bOO

16

20

24

(hours)

basal ET peptide production collagen gels, were cultured measured by RIA in culture consists of six measurements.

by rat in KI super-

cells)

h (17).

and bysed was added /3-scintillation

counted

of cells

of

artery endotheliab than basal synthesis

were first medium

in to

in

in control

webb.

Statistics for

a 24-h

produced

Figure 1 . Time course of GEC. GEC, adherent to medium. ET peptide was natants. Each time point

was determined by visual end of incubations. Cells were as described previously (16,

The

and

absence

and

linearly

0.00-

T3

Proliferation

bovine

Cell number of cells at the into suspension.

the

production

increased

didewith

and

as

The

conditions, culture me-

0.20

Growth factors were added for 24 h, and [3H)thymidine (0.25 MCi/well) was added for an additional 24 h. At the end of Incubations, cells were washed four times with situ with scintillation

(over

amount

0.30

In

peptide.

medium

Time

some experiments. by culture in

0.5%

the

a-[35SJdATP,

1).

the

culture in Ki

by

pBS +/sequences

by procedure

measured

by the

or the collagen gels (alone) contained no deET. When compared with other cells grown their standard culture conditions, basal ET

synthesis

the

standard matrices

ET

(Figure

(alone) tectable under

0

was

exper-

determined

of

with T4 polywas subcboned

incorporation.

(17). In arrested

of the

Klenow

were determined termination

I3Hjthymidine

(containing

and gel

of the plasmid vector (23). Nucbeotide

DNA chain

Proliferation

The

the

time

(Figure

(pool of 2.5 by agarose

I and phosphorylated and the product

Measurement sis

beads. with

HincIl site procedures

purified plasmid oxynucleotide

and

glass

repaired

between

was

test.

grown under to collagen

of ET into

monary higher

mM. PCR reaction by 1 .0% agarose gel

PCR purified

controls

synthesized

release

ebectrophoresis.

5.0

sum

GEC

dium,

at PCR

0.25

performed

5.0

rank

i.e. , adherent

at 92#{176}Cfor

30 s. and extension 8-mm extension. ML,

and

1 s and

and

RESULTS

and

94#{176}Cfor

number of cells or as cells above controls.

of differences

groups

Wibcoxon

con-

to CCGAGCACATTGACrounds of PCR were

at 55#{176}C for s. with a final

± SE of ET per in stimulated

significance

imentab

For the reaction

of CTTCTTCGGATCCCTTTGCAGAATGG

a sequence TACAGAGCCC

Statistical

region

nested

as mean increase

primer

(22) was added. 1 pL of the first-round

template

sented percent

pobym-

In this reaction, from the reverse

the

5’

of

performed

of ET in culture supennatants cell number. Values are

pne-

o.1

o.o1: li a

0.001

Endo

GEC

Mes

Figure 2. Basal ET peptide production by rat GEC, rat gbmerular mesangial cells (Mes), and bovine pulmonary artery endothelial cells (Endo). ET was measured in culture supernatants after 24 h of incubation. The value for endothelial cells has been reported previously (20).

Volume

3

.

Number

7

‘

1993

Cybulsky

[MgCI2]

2.5

5.0

2.5

5.0

et al

mM TGF-

(13)

C5b-9

IIIIj*

(9)

Thrombin

L-

(19)

PMA

*

(12)

EGF (6) isoH-7

PKC-

(6)

depleted

Thrombin Figure

3. Detection of preproET mRNA in rat GEC. mRNA was converted to single-stranded cDNA by reverse transcriptase and was amplified by two rounds of PCR (at (MgCI2) of 2.5 and 5.0 mM) with nested oligonucleotide primers based on the sequence of rat preproET (22). PCR products were electrophoreSed in I .0% agarose gels, containing ethidium bromide, and were visualized under ultraviolet light. The positions of nucleic acid Size standards, obtained from A DNA digested with Hindlll (shown on the left), correspond to

2,322,

2,027,

and

564

base

pairs

(top

to bottom).

The first

round of PCR (left panel) yielded a faint band at 5.0 mM (MgCI2), and in the second round (right panel), intense bands were visualized. The products of both PCR were consistent with the expected lengths of 727 and 643 base pairs, respectively (22), indicating that GEC synthesize preproET mRNA. approximate mab for each To

because cell type,

provide

further

GEC produce we examined Our

preproET

mRNA to

(22)

(see

the

primers.

then product

and

of on

yield

first 727

preproET was

mRNA.

obtained

tide sequence product was cDNA

then by

rat

be present Compared

Journal

of

the

of the identical

cDNA

to the

643

base

confirmation that

second-round to the sequence

GEC

could

whether be

altered

in the gbomerulus with basal bevels,

of

cDNA round

pairs.

The

predicted express

4. Regulation

Figure culture

of the American

Society

_____ _____

#{149}*

.

25

#{176} ;:

above

media

ET was

PMA

measured

GEC

or media

(5 U/mL),

#{176}:

Control)

of collagen-adherent

in control

ng/mL), thrombin or isoH-7 (100

containing

(300

nM),

in

after

24-h

TGF-l (10

EGF (10

ng/mL),

The effect of C5b-9 was determined by the incubation of antibody-sensitized GEC with normal human serum or heat-inactivated serum in controls (see Methods). Inset. ET production in PKC-depleted GEC (24-h preincubation with 2 MM PMA) that were stimulated for 24 h with

The

MM).

ng/mL)

or thrombin

The

number

P< stimulatory

(5 U/mL)

or were

of measurements

0.001; effect

unstimulated

is indicated

P< 0.05; stimulated of thrombin (but

PKC-replete GEC was significantly GEC (P< 0.05). hanced brane

after attack

reduced

in

versus connot TGF-(1) in

in PKC-depleted

PKC

isoH-7

by

of

identity

(Previously.

the

nucbeo-

preincubation deplete PKC independent

of ET

pep-

by

that

may

during inflammation. ET synthesis was

24 h of incubation with complex of complement

en-

however, ± 7% of

the C5b-9 and with

had control).

memTGF-

no stimubatory ET production

was also enhanced with thrombin (Figure 4). Because thrombin is known to increase 1 ,2-diacylgbycerob and PKC activity secondary to stimulation of phospholipase C (24), we examined the effect of thrombin on ET production in the presence of the protein kinase inhibitor

production

of Nephrology

(4)

of ET production.

supernatants

incubations

of

subcboned PCR of rat preproET

factors

Thronn

ET (% Increase

7 inhIbited

examined

(4)

TGF-$(4)

0

(Figure 4). TGF-/3, effect after 4 h (79

a

of GEC

25

/3

in GEC,

yield second

products that

demonstration

by

design

present

of PCR would pairs and the yielded indicating

GEC

obigonucleotide preproET cDNA

was

Unstimul.

of

of

(22).

We tide

according mRNA

Further

by

presence of

nested of rat

product

PCR 3),

the conversion

-

trol.

for

.j

+

parentheses.

that

conditions, mRNA

-HJ

PMA (6) isoH-7

TGF-t (10 (Unstimul.).

opti-

observation

amplification

Thus,

a cDNA

two rounds of length (Figure

detect

PCR with the sequence

round base

the

the

the

if preproET of

would

to

involved

Methods).

the

for

while

standard culture GEC contained

approach

cDNA

two rounds primers based

conditions, not identical.

support

ET under whether

preproET. mRNA

culture were

(6)

+

isoH-7

and

a 24-h we

in

that

demonstrated

had

been

with that

depleted

2 MM PMA.

isoH-7

and

24-h

with 2 MM PMA functionally inhibit/ activity in GEC 1 71). isoH-7 had no effect on basal ET synthesis, and isoHthe

stimulatory

4). Basal ET production depleted GEC, but the was reduced by more results

GEC

preincubation

indicate

that

effect

of thrombin

(Figure

was also not altered in PKCstimubatory effect of thrombin than 70% (Figure 4). These the

action

of

thrombin

was

1401

GEC Produce

El-I

through PKC. Further support for PKC in the stimubation of ET release was obtained by demonstrating that synthesis of ET was enhanced after 24 h of Incubation

(300

with

nM)

and

abolished

the

that

in

exogenous

the

the

PKC

stimulatory

presence

activator

effect

of

isoH-7

cantly with

different in PKC-depleted that in PKC-replete cells

ecubes

that

might

including

be

EGF.

significant addition.

the ,

24

ET

of PMA

factors

medium) as

1 mM)

not

with

of the

An

and guanybate 1 mM), as well

In

culture

affect

ET

cyclase

cyclase as the

in GEC.

Although they

TGF-/3 both

and

PMA

decreased

enhanced

pro-

incorpo-

ration (Table 1 ). EGF and thrombin potently stimubated 3HJthymidine incorporation (Table 1) and produced parallel increases in cell number (data not shown). but only thrombin stimulated ET. Furthermore. isoH-7 did not inhibit thrombin-stimubated 13H1 thymidine thrombin-stimulated pendently sis

incorporation.

produced

when

added

that

herent rices

GEC

to collagen but

not

presence

of

it inhibited isoH-7 inde-

increase

In DNA

containinggrowth

synthefactors,

(Table 1 ), but had no effect in serum(data not shown). Finally, we reported

medium

previously

a small to medium

i.e. , K 1 medium

poor

although production.

ET

to

will

gels plastic

extracelbular

proliferate

or to other substrata

matrix

only

when

ad-

extracelbular ( 1 7): however. did not affect

matthe ET

production.

DISCUSSION We demonstrate preproET mRNA

1402

that (Figure

cultured 3) and

14

±

34

EGF (10 ng/mL) Thrombin (5 U/mL) Thrombin (5 U/mL)

±

lsoH-7

+

11

1,089 1,010

±

488 478

1,795

±

901

MM)

0

Factors

were

(3H)thymidine in serum-poor

added

to GEC

in KI medium

performed in triplicate. trol (P< 0.05).

or serum-poor

medium.

i.e., cells in KI medium or ± 1.800 and 2.939 ± 1.01 1 cpm/ Values are from three to five experiments

incorporation in control medium was 24.155 All values

GEC.

are

significantly

different

from

con-

pathcAMP-

ET

[3Hjthymidine

164

well per 24 h. respectively.

medium

adenybate

±

37±

no

4).

K!

67

PMA(300nM) isoH-7 (100 MM) Serum-Poor Medium

and cGMP-dependent protein kinase inhibitors. KT5720 (4 MM) and KT5823 (4 MM), respectively (25). had no effect on basal ET production at 24 h (data not shown). We also examined whether, in addition to soluble mediators, ET synthesis might be regulated by extracelbubar matrix. However. we observed that the synthesis of ET was not significantly different between GEC adherent to collagen gels and cells adherent to plastic substrata (0. 1 5 ± 0.03 versus 0.17 ± 0.05 pg/i .000 cells per 24 h, respectively: N = 6). Some of the factors that were tested for their ability to stimulate ET production in GEC are also known to stimulate or inhibit cell proliferation. Thus, we examined the effects of these factors on DNA synthesis duction,

KI Medium TGF-/t (10 ng/mL)

(100

had

in the

did

compared

Activators

(dibutyryl-cAMP. ways (8-bromo-cGMP.

(17), (Figure

Incorporation

(% Control)

gbomerubi.

mitogen

in GEC#{176} (3H)Thymidine

as compared 4). Other mob-

production

DNA synthesis

.

was

4).

noted after ± 7% above stimulatory not signifi-

inflamed

of growth h,

shown).

in

GEC

on

serum-poor

at

not

active

major

absence

(i.e.

medium production

(data

a

effect

GEC (Figure

I

PMA

(Figure

upward trend in ET production was also a 4-h incubation with PMA (300 nM: 1 1 control). In contrast to thrombin, the effect of TGF-/3 on ET production was

TABLE

rat GEC synthesize ET peptide. ET pep-

tide was released in a time-dependent 1), and the amount produced under by

GEC

was

approximately

comparable

duced by bovine pulmonary artery and greatly exceeded production cells

(Figure

manner (Figure basal conditions

2) when

similar

to

that

pro-

endotheliab cells by rat mesangial

culture

conditions

and

the same RIA for ET were used. Although the amount of ET production that we measured in unstimulated rat mesangial cells (Figure 2) was approximately 5fold

to

1 0-fold

less

than

that

reported

for

mesangial cells (7). our results, nevertheless, that GEC produce significantly more mesangial cells. Our study is in keeping a recent report, which also demonstrated thesis

of

ET

preproET therefore, thesis

by

rat

mRNA confirm is not

GEC

in

culture,

expression the emerging

restricted

to

as

(26). These view that

endothebial

cells.

on the basis of this and previous studies now be concluded that all of the intrinsic cell types can synthesize ET. In GEC, the production of ET peptide bated by TGF-/3. as well as by thrombin both of which increased ET production activation (Figure 4). veabs several potential

phorbob sites), nuclear

The

structure regulatory

ester-responsive where factor-

acts

1 (28),

where

collagen gest that expression

gene transcription TGF-/3. thrombin, of the gene

fashion

similar

to

that

(27),

and TGF-/3

well

Second.

(6-8). it can gbomerular was stimuand PMA, through PKC re-

(AP- 1 -binding binding may

sites act

endothelial

cells

for

to induce

(29). Thus, our data and PMA augmented for the ET precursor for

as

studies, ET syn-

of preproET sites, including

elements

PKC

human

indicate ET than do with that of basal syn-

sugthe in a

and

the

renal epithebial cell line LLCPK1 (4). C5b-9 also stimulated the release of ET, but to a lesser extent as compared with the other agonists. Possibly, C5b-9

Volume

3

‘

Number

7

1993

#{149}

-

would

,

have

tions

been

more

there

activate

cells

over,

in

going

cellular

limits nism

basal

kinases

not

appear was

the

there

ET

but

through mitogenic

1 ). TGF-/3

results notjust Although

the

in vivo reports.

and

mesangiab

to the

was ET

of ET

be relatively preproET

and

aminonucleoside athy results

Institutes

The

in preliminary have been

cells

hammed

example.

by

mRNA

a rat

to

preliminary

is transiently

GEC

injury

of

(passive

( 1 0) might

Heymann

be associated

elevated

ET

experimental

might

with

enhanced

contribute

7.

our

the

American

Society

of Nephrology

altering

Research from

Blais,

A.V.

technical

A.

Medical

Canada.

Cybulsky of

Canadian

Cyr.

the

of

Council

the

M.D.

from

and holds

a

Canada.

Heart

D.J.

Foundation.

McTavlsh.

and

F.

Mo-

assistance.

the

M, Kurihara vasoconstrictor

endothelial

cells.

KF:

S. et at. : A produced by

Nature

Biological

(Lond)

1988;

and

patho-

actions

significance of endothebin Int 1991:40:1-12.

Simonson A possible

MS. Dunn MJ: role in gbomerular

Invest 1991:64:1-4. Ohta K, Hirata Y,

Imai

in the

kid-

Endothelin peptides: inflammation. Lab et at. : Cytokine-in1 from porcine renal Biophys Res Com-

T,

1990:169:578-584.

Kohan

DE,

Fiedorek

FT:

Endothebin

synthesis

rat inner medublary collecting Soc Nephrol 1991:2:150-155.

duct

cells.

J

Sakamoto H, Sasaki S. Hirata Y, et at. : Production of endothelin1 by rat cultured mesangial cells. Biochem Biophys Res Commun 1990:169: 462-468. Zoja C, expression mesanglal forming

Orisio 5, Pericon et at. : Constitutive of endothebin gene in cultured human cells and its modulation by transgrowth factor-fl, thrombin and a throm-

A2

Marsden

analogue.

PA,

BM,

Orkin

Doriman

SH,

expression

lary rat)

H, Kimura peptide

Lab

Invest

1991:64:

16-20. 8.

of

endothelial

DM,

Collins

Ballermann

endothelin

cells.

T,

BJ: 1 in

Am

Brenner

Regulated

gbomerular

J Physiol

capil-

1991:261:

Fl 17-F125.

9.

enhanced

renal vascular resistance, gbomerular hypertension. decreased gbomerular ultrafiltration coefficient, and the portion of proteinunia that appears to be due to changes in gbomerubar capillary pressure (13). Simi-

of the

Medical

expert

boxane

in

ET synthesis. to

Grants

(HL45563).

332:411-415. Kon V, Bath

by Am

membranous in

their

mun

5. 6.

of nephrop-

nephnitis

Besides

Foundation

duced release of endothebinepithebiab cell line. Biochem

study

increased

model

for

Research

Health

D.

vascular

an

receptors have (33), although

analogy

thank

physiologic ney. Kidney

vas-

wall permselectivET functions in

a recent

the

1 . Yanagisawa novel potent

ad-

gbomerubar

ET GEC

(3).

Kidney

a Scholarship

authors

cells

muscle

holds

by

Canada, of

from

Stewart

is

specific binding of [‘251JET observations). The synthesis become more prominent in

nephrosis,

nephropathy

Journal

National

involving GEC injury (34). Furthermore, our in cultured GEC suggest that the C5b-9-me-

diated

This

GEC

gbomerular

smooth

capillary that

GEC, ET

of

2. by

to modulate

For that

by the by

GEC

REFERENCES

These

ET

supported

Council

4.

we could not demonstrate to rat GEC (unpublished and effects of ET might

shown

of

work

3.

and possible

in cultured

inhibited

from

the

production.

low, mRNA

autocrine manner in GEC because been found in cultured human

findings

not

was

Research Scholarship

signaling pathbe mediated

because

release

ACKNOWLEDGMENTS This

de-

cells

gbomerubopathies.

stimconsist-

Conversely, EGF. no effect on ET both ET and DNA to

ET

vivo.

rat gbomerubi, the latter by ET synthesized by GEC in vivo may act in a paracnine fash-

gbomerubus

cular tone, function, ity (2,3). It is also

of

while

in GEC.

the synthesis of cell growth.

tected in microdissected PCR (31,32). Thus, under basal conditions

has

PMA,

of PKC,

unlike

production

appears to ET and/or

producpresence

stimulation

synthesis

of thrombin

indicate that a reflection

ebuci-

culture medium, between GEC

by different appeared

isoH-7,

ET

bar changes in gbomeruban hemodynamics may occur in inflammatory nephropathies (12), where inflammatory mediators derived from platelets, infiltrating cells, or intravascular coagulation, such as thrombin or TGF-/3, may be present in gbomeruli and might stimulate

protein

on the

the

independent

effect

inhibitor

to be

et al

gbomerubar hemodynamics and permsebectivity. ET could also promote proliferation of mesangial cells, because it is known to be a mesangial cell growth factor (2, 35). Further studies are required to document a pathologic robe for ET in gbomerubar injury in

mecha-

and to plastic. It should of ET enhancement in

DNA

a pathway

on-

which

The

basal

from

probably synthesis

that

is also

remains

in the different

with More-

complexes,

dependent

matrices pattern

(Table

DNA

on

doses (16).

previous reports ( 1 7.30). GEC mitogen ( 1 7), had Thrombin stimulated

synthesis ways.

jacent

between cytolytic

to mediate

inhibited

but

cGMP-dependent

significantly

ET,

ent with a potent production.

of

and not

concentnaused,

of C5b-9.

growth factors release was not

synthesis

PKC

effects

to collagen noted that

ubating

are

of C5b-9

synthesis

differed

DNA

activation,

cell

release

of exogenous and basal

GEC

to

cAMP-

did basal

adherent also be

that

and

been

range

doses

metabolism

PKC

if higher

have

is a narrow and

stimubatory

for

tion,

could

addition

the

dated.

ion

effective

of complement

C5b-9,

Cybulsky

.

1 0.

Pabst R, Sterzel RB: cell types in normal 626-641. Cybulsky

DJ: cell

AV,

Rennke

Complement-induced injury.

in rat

Role

membranous

of the

Cell rats.

renewal Kidney

HO,

of gbomerubar Int 1983:24:

Feintzeig

ID,

gbomerubar membrane

nephropathy.

Salant

epithelial

attack

J Clin

complex

Invest

1986:77:1096-1107.

1403

GEC

Produce

El-I

1 1 . Morita T, development Pathol

1 2.

Susuki Y, Churg of the gbomerular

1 4.

1 5.

1 6.

1 7.

EA,

Yoshioka Ichikawa

GA,

and serum

thromboxane nephnitis.

Muller

K,

Hilz

tion of Biochem 981-987.

protein kinase Biophys

Quigg RJ. alamenti

Salant

DJ:

in rat 1983:

DJ, Deen WM, high transmural

promotion

and

C in epidermab Res Commun

PA,

cells.

Robinson

Karnovsky

MJ:

Am JM,

in

Physiob

Hoover

RL,

methods

release

2 1 . Chomczynski

Sakurai cloning, tion of Biophys

T, Yanagisawa

J, Fritsch A

Laboratory

.

M, Inoue

EF,

Maniatis Manual.

28.

Anal

29.

Ed.

Cold

J A,

Pathol

1992:141:

Takuwa

Y,

Mitsui

T: The

Complete

J Car13-17. Mitsui Y,

human

preproen-

nucleotide

of expression.

Y,

preproenby endothe-

turnover signalling. 1 989: 1 3(suppl 5):S M, Takuwa Y,

M, Masaki

Sporn

MB,

Roberts

Crombrugghe chemistry

30.

factor-beta. MacKay Agodoa

sequence

J Biol

and

K,

AB,

B: Some biology

Chem

1989:

3 1 . Uchida Detection

LY,

Striker

Murine

LM,

GE:

gbomerubar

microdissected J Am

Doi

T,

growth

receptors

gbomenubar

de-

in the growth

Transforming cells.

and J Clin

5, Takemoto F, Kurokawa of endothelin-1 mRNA

stracti.

32.

Wakefield

recent advances of transforming

J Cell Biob 1987:105:1039-1045. Striker U, Stauffer JW,

sponses of isolated 1989:83:1160-1167.

re-

Invest

K, Ogata RT-PCR

by

nephron segments from Soc Nephrol 1991:2:420.

E: in

rats

lAb-

Ujiie K, Terada Y, Nonoguchi H. Shinohara Tomita K, Marumo F: Endothelin-1 mRNA endothebin-1

segments

production

along

lAbstracti.

J Am

the

Soc

rat

M, and

nephron

Nephrol

1991:2:

420.

33.

34.

Rebibou R, He CJ, JD: Characterization thelin stractJ. Osada

1 receptors J Am Soc 5, Nakamura

Tomino Y, Koide in gbomerubi and

Biochem

T: Molecular

Pharmacol

264:14954-14959.

vascular

distnibuBiochem

of pro-

Rev

M, Masaki T: The human 1 gene: possible regulation

regulation

Invest

Davalath 5, Marsden cells synthesize endo-

Am Inoue

in

Lab

Annu

peptides.

1 gene.

Labora-

proteases

differentiation.

Fried TA, epithebial

M,

Harbor

R: Pharmacology

hal phosphoinositide diovasc Pharmacob Inoue A, Yanagisawa

and

sclerosis 412.

A, et at. : cDNA

2nd

thebin 279-283. Yanagisawa

Kobayashi

cul-

method

Spring

of extracellular

and

Kasinath BS, PA: Gbomerular

dothebin-

1990: Wright

robe

H, Kobayashi

Kobayashi dothelin-

thiocyanate-

sequence analysis and tissue rat preproendothelin-1 mRNA. Res Commun 1991:175:44-47.

Sambrook Cloning.

of

N: Single-step

by guanidium extraction

27.

by coclu-

cells with cells 1990:259:H1928-H1932.

P. Sacchi

of RNA isolation phenol-chloroform 1987: 162:156-159.

is inhibited

Hidaka

factor-/3.

turing rat gbomerular cells. Kidney Int 1984:26: 875-80. Cernacek P, Stewart DJ: Immunoneactive endothebin in human plasma: Marked elevations in patients in cardiogenic shock. Biochem Biophys Res Commun 1989:161:562-567. Stewart DJ, Langleben D, Cernacek P. Cian-

K: Endothelin

26.

gb-

for

W: The

Cold

tein kinase inhibitors. Toxicol 1992:32:377-397.

regulates

J

Improved

25.

deple-

turnover

Scher

cell proliferation 1987:57:607-633.

JB6 cells. 1989:165:

matrix

phospholipid

ture of endothelial media. Am J Physiol

1404

synthesis J Clin Invest

Extraceblular

and

Harper

flone

23.

Gbomerular

with antibodies to cultured rat gbomerular epithelial cells. Subepithebial immune deposit formation after in vivo injection. Am J Pathol 1989: 134:1125-1133. Quigg RJ, Cybulsky AV, Jacobs JB, Salant DJ: Anti-Fx1A produces complement-dependent cytotoxicity of gbomerular epitheliab cells. Kidney mt 1988:34:43-52. Cybulsky AV, Bonventre JV, Quigg RJ, Wolfe

TC,

22.

MJ:

DR. Cybuisky AV. BadAWM, Salant DJ: Studies

merular epitheliab 259:F326-F337.

20.

Dunn

H: Tumor

Abrahamson J, Minto

Spring Harbor, NY: tory Press: 1989.

24.

Andres

T, Rennke HO, Salant I: Role of abnormally

proliferation

1 9.

and Am J

pressure in the permselectivity defect of gbomerular capillary wall: A study in early passive Heymann nephritis. Circ Res 1987:61:531-538. Kischel T, Harbors M, Stabe 5, Borowski P.

LS,

1 8.

Structure crescent.

1973:72:349-368.

Lianos prostaglandin nephrotoxic 72:1439-1448.

1 3.

J:

35.

Simonson

Meulders and in Nephrob

MS,

Delarue

J

Wann

F, Sraer

of

endo-

human gbomerubi 1990:1:425. T, Ebihara I, Suzuki

H: Endothebin medulla from

lAbstract].

bin stimulates change, c-fos rat mesangiab 708-7 12.

Q,

localization

Am

Soc

5, Mene

phospholipase expression, cells. J

gene focal

and Clin

Volume

lAb-

5,

expression gbomerubo-

Nephrol

1991:2:

P. et at. : EndotheC. Na7H exmitogenesis in Invest 1989:83:

3

‘

Number

7

-

1993