Renal endothelin system in polycystic kidney disease

J Am Soc Nephrol

Renal

Endothelin

System

BERTHOLD

HOCHER,*t

CLAUDE

BRAUN,t

HANS-HELLMUT

and PETER

Free

Heidelberg;

and

interstitial

fibrosis

human

endothelin-

fore,

analyzes

centrations

renal

formation

conscious,

neys

of affected

cyst

were

also

This

study,

seen

of ET

of PKD:

(cy/+)

and

homozygous Han:SPRD

in

therecon-

receptor

Han:SPRD

(cy/+) Sprague

rats.

(cy/cy), and

rats

Dawley

rats rats,

instrumented

showed

with P

dominant

0.01;

polycystic

accounts

for

up to

therapy. areas

Han:SPRD

to that seen

disease

membrane, to end-stage

as

(ADPKD),

of Berlin;

University

tubular

renal

segments

enlarge

with

October

10.

similar

ADPKD by

thickening

of

5, a quantitative and plasma urea

1997.

Accepted

Berthold Humboldt Universit#{228}tzu Berlin, 20-21, 10098 Berlin, Germany. to

1046-6673/0907Journal

Copyright

of

age

of progressive in many

respects

in humans structural

the

as well

alterations

tubular

basement

of cysts, gene was

trait locus concentration.

Dr.

leading mapped

controlling The ho-

January 23, 1998. Hocher, Universit#{228}tsklinikum Charit#{233} der Klinik f#{252}r Nephrologie, Schumannstrasse

0.01;

<

American

C) 1998 by

tlnstitute

of Molecular

Klinikum

Mannheim,

Benjamin

typical

Biology

and

University

Franklin,

the

American

of

Nephrology Society

of

Nephrology

0.4-fold

±

autosomal

of

Free

in old cy/+

dominant

expression

within

Scatchard

analysis

ETB receptor blockade

the

University

rats, P

of

due

0.001)

<

of both

endothelin

receptor

after

bosentan,

bosentan

and RBF in age-matched the paracrine renal endothelin in the regulation

contributes

to renal

gene

region

cyst

PKD I gene,

localized

16 encoding a high 500,000 kD named on human rats,

little

is

mechanisms chemically

induced

composition implicated

and

endothelin such

cyst

1 (ET-l) system

as lupus

cell

models

with

7).

acute

Thus,

play

analyzed measured

expression

Scatchard

analysis

compared

with

in the age-matched

also

(4). a major renal

failure

an activated

therefore the

were

impaired

an important

in

and

and

underlying

in genetically renal

cysts.

or

Altered

matrix (3) is thought to be Interstitial fibrosis, glomeru-

mice

renal

advances

in humans

biology formation

to play

nephritis,

and

tem may We

seems

the

chromosome

recent

of PKD

to cyst

formation

transgenic

these

basis

the

animal

8. The

rat is neither

of human

Despite

of the extracellular in cystopathogenesis.

losclerosis,

thelin-

about

that and

and possibly

chromosome cy/+

arm

genetic

contribute

on MAP,

weight protein of approximately nor the PKD2 gene, localized

4 (2).

known that

on human

molecular polycystin, of the

ml/min,

RBF,

<

flow P <

fibrosis.

in Han:SPRD

on the short

chromosome

determination

P

blood

0.5

±

GFR, and

to reside

for PKD

acute

2. 1 mmHg,

Renal

had no effect

of MAP, formation

is likely

responsible

and

The

controls. These data show system is activated in PKD

GFR,

participates

well.

ETA

rats.

±

0.005). (+2.1

<

ET-l

as

decreased

(- 19.7

BP (MAP)

whereas

cysts

subtypes using bosentan led to a significant de-

rats

± 5%, P increased

cells with

a high

the

of affected

PKD

arterial

of

a markedly

heterozygous

in mean

demonstrated cells

in all groups

0.05) and GFR (-41 (RBF) was significantly 0.05)

PKD

epithelial

revealed

density

in 6-mo-old

analyzed

Society

6.2

histochemistry,

1 169$03.00/0 the

BAUER,t

to a highly increased ET-1 synthesis within the epithelial of the cysts. Analyzing tissue sections from patients

the

a form

appears

P

rats,

through Correspondence

VOGT,

BRAUN,t

Hospital

phrectomy, Received

VOLKER

CHRISTIAN

of Nephrology,

mology

of origin.

interstitial fibrosis, and formation kidney disease (I). The rat PKD

on the rat chromosome PKD, kidney mass,

cy/+

requiring

renal

is characterized

such

± 0.3-fold

kidney disease in live births. This

progressively

in humans.

rats

kidneys

disease

kidlevels

in young

patients

develops

that

in ADPKD

as in Han:SPRD

all

from

the nephron

rat strain

gender-dependent

of

The

tissue

(3.5

0.2-fold

kidney

arise They

from

±

hereditary 1 in 1000

I 0%

Cysts

of dilation.

separate

controls

1.4

rats.

elevated

age-matched <

disease

replacement

SCHWARZ,*t NICOLE

crease

littermates

(cy/+)

significantly

to be the most common affects approximately

the

by

Interestingly,

expression

of a rat model

thought humans,

of

Medicine,

of ET- 1 , the tissue

as the

chronically

cy/cy

Autosomal

The

of Nuclear

cysts.

mice.

distribution

age-matched

of ET- 1 compared

and may

Department

were analyzed. Furthermore, the acute effects of (A/B) endothelin receptor antagonist bosentan on and renal function were investigated in 6-mo-

old,

1998

Disease

KOPPENHAGEN, University

of Berlin;

is characterized

renal

transgenic

heterozygous

corresponding

as focal

(PKD)

and

heterozygous

in young

Department

disease

1 (ET-l)

as 6-mo-old

(SD) (+1+) the mixed hemodynamic

Charit#{233}, Humboldt

University

of

in the kidneys

as well

(IAUS

formation

ET- 1 , as well

Six-week-old

THONEREINEKE,t

and

the tissue of

subtypes

the

kidney

fibrosis

CHRISTA

ANJA

177,

Germany.

Polycystic

interstitial

ZART,*t

NEUMAYER,* of Nephrology,

Biochemistry,

Abstract.

RUDIGER

Kidney

1 169-1

ROHMEISS

*Department

Berlin,

in Polycystic

9:

in human

tissue

function renal

in references endothelin

of endothelin controls.

S sys-

of PKD.

of ET- I by immuno-

concentrations

kidneys

renal

disorders after 5/6 ne-

in the pathogenesis

the expression

endo-

the

role in renal

(reviewed

paracrine

role

seen

In addition,

of

receptor of

male

ET- 1 , and subtypes

Han:SPRD

In addition,

the

by rats acute

Journal of the American

1 170

effect (8),

of bosentan, on renal

blood

was analyzed Furthermore, typical expression

Nephrology

a combined

ETAIETB GFR,

and

within

of

flow

(RBF),

in conscious we analyzed

ADPKD

Society

receptor

heart

rate

antagonist

(HR),

and

BP

chronically instrumented PKD tissue sections from patients

could

demonstrate

the epithelial

cells

a very

rats. with

high

ET-l

of the cysts.

ET- 1 and cross-reacts I 89% ET-3 was less than 0.001.

frozen

further analysis. Male well

and 6-wk-old

as 6-mo-old

corresponding analyzed. The

Methods

enized

heterozygous

(cy/+

) and homozygous

heterozygous

(cy/+

) Han:SPRD

age-matched Sprague Dawley animals (a generous gift from

(cy/cy),

rats

(9)

as

and

the

rats (SD) (+1+) were Dr. N. Gretz, Klinikum

Mannheim, Mannheim. Germany) were fed a commercial diet tromin#{174},Altromin, Lange. Germany) and given water ad lihitum.

(AlAll

animal experiments were conducted in accordance with local institutional guidelines for the care and use of laboratory animals. [I25I] ET-l (2000 Ci/mmol) was obtained from DuPont (Hannover, Germany).

Unlabeled

ET- 1 was

from

Peninsula

Laboratories

phenoxy)-2,2’-bispyrimidine-4-yl}-benzenesulfonamide)

erous gift from Dr. Martine Clozel Roche Ltd., Basel, Switzerland). ligands

(BQ

(Napa,

CA).

Peninsula analytical ny),

123 and BQ The

3020)

polyclonal

from

rabbit

California

anti-ET-l

Laboratories. Unless otherwise grade and were purchased from

Boehringer-Mannheim

nich,

was

a gen-

(Mannheim,

Inc.

was

from

antibody

stated, Merck

all reagents (Darmstadt,

Germany),

were of Germa-

or Sigma

(Mu-

Germany).

membranes,

formed

The

as described performed

in the

buffer

as recently

described

by Biho-

Tissue

ET-l

at 40,000

levels

were

kidney

samples

Kidney

measured

as

saturation”).

studies

recently

described

wet

thawing,

0.5 M NaCl. disrupted using a polytron and subsequently The homogenate was centrifuged at 4#{176}C for 15 mm

at

25,000

The supernatant

was retained

g

After

homogenized. x g.

in 2 ml

(10).

M

was

suspended

endothelin weight

for ET- I RIA and the pellet

discarded.

Radioimmunoassay. tissue

supernatant

One-milliliter centrifuged

using

AmprepTM

ET- 1 was extracted

500 mg C2 columns

samples were acidified with at 10,000 X g for 5 mm at room

onto the columns. 0. 1 % trifluoroacetic collected

from

(Amersham).

0.25 ml of 2 M HC1, temperature, and loaded

The columns were washed with 5 ml of water + acid, and the adsorbed peptide was eluted with 2

ml of 80% methanol were

Immunoreactive

in water

in polypropylene

+ 0. 1% trifluoroacetic tubes

and

dried

acid. under

The eluents a stream

of

at 4#{176}C for

15

bacitracin,

100

the expression

in the kidney,

contained

binding

of the

(3 tM)

I mg

-

whereas

from

crude

BQ3020

ml

(5).

The

bacitracin,

the concentration

plasma

of assays

subtype-specific

and/or

from 0 to 25 nM (competition

Samples

100

in a total was kept

of unlabeled

studies

membranes

with “cold

were

concentration of 0.53 mg of protein . mlt. Binding performed at room temperature for I 20 mm. Nonspecific

used

studies binding

at a were was

assessed in the presence of excess ET- 1 (5 p.M). After adding 1 ml of cold binding buffer, free and receptor-bound radioactivity was sepaobtained

at 30,000

were

buffer.

washed

efficiency

for

(3)

and

g (4#{176}C) for I S mm,

X

two

additional

[12511 was counted

times

in a Packard

and the pellets

with

counter

I % BSA.

biotinylated

and

Ramasubbu

(13),

using

Buehler,

Detection

of the bound antibody

Braunschweig,

instructions. antibody

a polyclonal

I antibody. Briefly, for antibody incubation, sections were mounted on poly-L-lysine-coated

second

antibody according

experiments

were

phosphate-buffered

For immunohistochemistry, and homozygous

(cy/cy),

was performed

male as well

6-wk-old

as 6-mo-old

using

to the manufacturer’s omitting

the first

instead.

heterozygous

(cy/+)

hetetozygous

(cy/+)

Han:SPRD rats and the corresponding age-matched Sprague Dawley rats (SD) (+1+) were analyzed. In a second set of experiments, we analyzed with

kidney

normal

sections

human

from

patients

with

typical

PKD

compared

kidney.

nitrogen.

The probes were reconstituted in 250 tl of assay buffer (0.02 M borate buffer, pH 7.4, containing 0. 1% sodium azide), and 2 X 100 l were taken for analysis in a commercial ET-l [125I1 RIA kit (ET-l,2 (high sensitivity) [ ‘ 251]assay system, Amersham). Separation of the antibody-bound

fraction

the Amerlex-M

Separator

was

effected

(Amersham).

by magnetic

separation

This assay reacts

using

100% with

a

red (Amersham

performed saline

5-j.tmglass

Laboratories) saline contain-

and streptavidin-Texas

Germany).

Control and using

(78%

for the detection of ET- I in the kidney was modifications, as recently described by Schafer

slides. Polyclonal rabbit anti-ET- 1 antibody (Peninsula was applied at dilutions of I :50 in phosphate-buffered ing

I ml of cold

gamma

[125I]).

Bachmann

rabbit anti-ETthick cryostat

of 0.2

CH3COOH,

were

tissue-immunoreactive

was homog-

a motor-driven

( 1 mg/mI

or absence

BQI23

cpmltube,

was increased

et al.

Preparation.

using

To analyze

ETB)

presence

Immunohistochemistry performed with minor

ET- 1 Radioiinmunoassay

at -80#{176}C until

tissue

mM Tris-HCI, S mM MgCl2, and 0.1 g% BSA, pH 7.4, volume of 150 tl. The [‘251]-ET-I tracer concentration

Im,nunohistochemistrv

concentrations

stored

centrifuged

buffer

(12). (ETA,

ligands

for binding

serum

urea

was

in assay

subtypes

receptor

counting

urine

and

NaHCO3,

homogenate

previously

receptor

The carrier status of each animal was established by determination of the kidney weight/body weight ratio, typical kidney histology, and and

nitrogen

150 mg of kidney

was resuspended

endothelin

binding

reau et al. (2).

liquid

mM Tris-HCI, 5 mM MgCl2, and 0. 1 g% bovine serum albumin [BSA], pH 7.4) at a protein concentration of 200 jg/ml. Binding Assay for ETA and ET8. Binding studies were per-

thus Determination

with

g. The

x

rated by centrifugation Phenotypic

177. 1998

supernatant was decanted and centrifuged at 4#{176}C for 30 mm at 40,000 x g. The pellet, consisting of crude plasma at 1000

constant

Peptides,

with

Approximately

homogenizer.

mm

assay

(Pharma Division, F. Hoffmann-La The selective endothelin receptor were

pestle

endothelin

bosentan

9: 1 169-1

Big ET- 1 . Cross-reactivity

at 4#{176}C in 10 ml of 20 mmollL

were

(Frankfurt,

Germany). The mixed (A/B) endothelin receptor antagonist (Ro 47-0203, 4-tert-butyl-N-[6-(2-hydroxy-ethoxy)-5-(2-methoxy-

with

Soc Nephrol

Scatchard Analysis Membrane Preparation. Membranes were prepared according to Nambi et al. ( 1 1 ). The rats were sacrificed and the kidneys were immediately

Materials

Am

I

Measurement

of GFR,

in Conscious,

Chronically

Surgical the rats catheters

before

Procedures. were anesthetized were implanted,

starting

Mean

Arterial Instrumented

One

week

with ether, as recently

the experiments,

flow

before

BP,

HR,

and

RBF

Rats the acute

experiments,

and femoral artery and vein described ( 14). Three days

probes

( 1RB with implantable

J Am

Soc Nephrol

connector,

Transsonic

implanted

around

probe

had

been

small

access.

was

then

envelope

All

neck

and

at the point

conductance,

the envelope cables

were

Transsonic

RBF

system

were

(Tl06,

displaying

a two-compartment

(16).

concentrations

Blood

samples

drawn

at 0, 15, 30, by

two

bolus 90,

of

the

system RBF

in 6-mo-old

nous

analyzed. injections

rats were n

3 (SD; injections

in PKD

rats,

we

tested

6-mo-old

heterozygous

(cy/+)

sponding

age-matched

Sprague

divided

into

(PKD,

n

group

3 (SD.

injection

6) received

=

n

=

Effects

,

to

the

effect

ex-

MAP,

injection

followed

ET-l

n

Group

I

n

Applied renal

rats and

mates.

To avoid

a possible

ET- 1 , the rats received ET-l.

appears heterozygous

study

bosentan.

for

RBF.

In

analyzing in PKD

rats,

tested

the hemodynamic

of ET-l

(20,

50,

in independent

100, (cyl+)

Sprague

experiments

are due

cells

tissue

to ET- I ex-

of the cysts within

(Figure

the interstitial

of homozygous

Han:SPRD

rats.

differences

with

cysts

is stronger

in homozygous

PKD

6-mo-old

increased

(cy/

respect

to the

of ET- 1 in 6-wk-old homozygous Han:SPRD rats. ET- 1 staining

of the

stronger

in 6-mo-old

rats.

Control

PDK rats

of het-

homozygous on the other rats

compared

(6-wk-old

as well

as

2C).

the

a similar

kidney

ADPKD

was

(Figure

from

four

one woman)

pattern

seen 3).

due to the size

of

ET- I immunoreactivity

in kidney Kidneys

sections were

of the polycystic

different

with

classic

patients

ADPKD

from

patients

removed

kidneys. (38

to

in

these

We analyzed

56

yr

compared

old;

with

three

normal

I 60 z

140

:-

120

.

100

and

..e:

r

80 60

200

the

20

the

0

ng of

intravenous

of bolus

0.005

E’.

old

Sprague-Dawley PKD heterozygous PKD homozygous

6 months

1. Bar graph of renal tissue endothelinin 6-wk-old and 6-mo-old rats with polycystic

and the corresponding ± SEM.

age-matched

old

littermates

Figure

means

-

I-’

EI

litter-

<

77/..

6 weeks

Han:SPRD injections

‘

40

of exog-

Dawley

repeated

S

catheter

We

after

above

and glomeruli

some

and

the

of inulin,

arterial

system

age-matched

6-wk-old that

180

2 and

RBF.

tachyphylaxis

heterozygous

in

a single

on BP and

heterozygous

in ho-

with

was obtained

(cy/+) are

effect,

higher

littermates) had a very low renal ET- I tissue immunear the detection limit of the method used in this

(Figure

within

of

the epithelial

vessels,

litter-

1).

revealed

signal

the tissue

a gene-dose

rats compared with 6-wk-old staining of the interstitial tissue,

Interestingly,

rats

the effect

in 6-mo-old

PKD ET-1

with

men,

group

to analyze

on BP and

corresponding

The

injection

experiments

endothelin

performed

doses

of

within

cells

hand,

sections

of 150 mg of inulin

ET-1

set

(SD). 6) and

=

and 180 mm after the

(Figure

PKD,

much

rats compared

PKD

with

age-matched

were

as described

blood

epithelial

with

=

GFR

7) received

=

by an injection 135,

were

to increasing intravenously)

n

of 100 mg/kg

4 (PKD.

of

rats and the corre-

littermates I (SD,

with

there

6-mo-old noreactivity

(PKD)

on

if appro-

elevated

with

analysis

specific

tissue,

patients

of bosentan

rats

mainly

erozygous PKD rats.

littermates

groups:

rats

differ-

used

at a value

significantly

rats, we observed

ET- 1 fluorescence pattern and 6-mo-old heterozygous the

Antagonist

Han:SPRD

(PKD)

Dawley

above-described

experiments

of

the

a single

of Exogenously

(PKD)

injections

is

6) and group 4 (PKD;

=

(200 .d) were taken from of serum inulin concentrations.

added

responses

was

of vehicle.

Group

of the endogenous

enously

GFR

Dawley

in 6-mo-old

Han:SPRD

2)

However,

were

Inulin

on resting

into four

n

groups:

At 0, 15, 30, 90,

blood samples determination

following

(cy/+) Sprague

Han:SPRD

7) and group

of placebo,

mm later.

ET-l

four

(cy/cy)

(Figure

renal

rats

the endogenous

of bosentan

significant

showed

PKD

cy) and heterozygous

of the Mixed (A/B) Endothelin Receptor Antagonist on GFR. To analyze the endogenous renal endothelin

system

impact

by

7) received cumulative intrave(10 mg/kg) every 15 mm up to a

bolus

contrast

inulin

The

Receptor

was

statistically

ET- I concentrations

2). A weaker

(Inutest#{174}).

(17).

of statistical

by t test

ET- I compared

ET- 1 concentrations

=

Group

Effects

of

tissue

as

rats

Han:SPRD

rats

of the plasma

injection.

method

divided

of bosentan

intravenous

the

well

Immunohistochemical

blood

functions.

after

heterozygous

6) received

Bosentan

as

In 6-wk-old

(cy/+)

(15).

evaluated

resolution

To examine

total load of 100 mg/kg.

were

using

Endothelin

6) and group 2 (PKD:

bolus

flow

absolute

was

considered

affected

mozygous

wt.

age-matched

The

The

measured

mm

rats, the effect

rats and the corresponding

recorder.

of 150 mg of inulin

and RBF.

in PKD

Inc.)

clearance with

180

(A/B)

HR,

flow

of serum inulin concentrations and

mates.

Brush

Systems, volume

monoexponential

100 g body

Mixed

endothelin

was

f3-fructosidase

per

on MAP,

=

of

pression

injection

135,

a modified

Bosentan

(SD; n

were

In 6-wk-old

because

implanted

2600

and measured

single-shot

for determination

as ml/min

was

Results

ANOVA

0.05.

<

kidneys

transducer

to a Gould

absolute

model

into

pressure

Brush

determines

The

an intravenous

and

means.

1 171

Results

and HR

Transsonic

a Gould

precalibrated,

method

Effects

P

with ultra-

BP (MAP)

arterial

via the chronically

on

single-shot

measured

followed

of group

in PKD

CN)

attachment. to the

coupled

flowmeter

recorded

probes

received

for determination

ence priate. a

subcutaneously

processor

flow with an accuracy of ±2%. Measurement of GFR. GFR

(SD)

signal using

Mystic,

was filled

a Statham

was measured time

flowmeter

The flow

HR,

Corp.,

line with

pressure

a transit

continuously

pressed

the best

position

of reflector

led

Mean

via the arterial a Gould

with

inulin

after

The

System

Statistical Analyses The unpaired t test was used

concentrations

recorder.

probe

and,

by

dissected.

in proper

Op-Wipe#{174} (Merocel

Measurements.

and

2600

fixed

chronically

was exposed

was carefully

the artery

was

and

were

(14).

measured

P23Db

NY)

and the artery

catheters

Circulatory were

Ithaca,

The left kidney

artery

around

probe

of Merocel

signal

gel.

rat’s

the

the probe

To improve

Inc., artery.

The renal

placed

achieved,

covering sound

Systems,

the left renal

a retroperitoneal flow

Renal Endothelin

9: 1 169-1 177, 1998

littermates

I (ET- 1) concentrations kidney disease (PKD) are shown.

Data

are

1 172

Journal of the American

Society

of Nephrology

J Am Soc Nephrol

9: 1 169-1

177, 1998

-, %

a -

; r

.

.

.

,

)

Figure

2. (A)

Kidney

histochemical

section

staining

an

kidneys of a 6-mo-old male only a very weak fluorescence

PKD rat (cy/cy) the epithelial epithelial staining

of renal

cysts

of the cysts

of the interstitial

cells

of

the

antibody

male

heterozygous

showing

PKD

a highly

increased

staining

but also of the interstitial

in the kidneys

tissue

renal

in 6-mo-old

appears

nephrectomies high ET-l cysts

of

stronger

littermate (D) Kidney

using an ET- I antibody of a 6-wk-old

PKD

rats (B) compared

in homozygous

PDK

rats (E and F) compared

patients.

with

also stained with an ET-1 antibody showed section from a 6-wk-old male homozygous showing

male

heterozygous

due to kidney immunoreactivity ADPKD

tissue

4J(.P4.

rat (cy/+) (Hematoxylin and eosin IH&E] staining). (B) ImmunoET-l expression within the epithelial cells of renal cysts in the

(E and F) Immunohistochemical

is stronger

human kidney tissue from We always detect a very epithelial

a 6-mo-old

ET-l

heterozygous PKD rat. (C) The corresponding age-matched signal within the tubules, blood vessels, and glomeruli.

(H&E staining).

cells cells

from

using

-

-.1’

,

an increased

homozygous

6-wk-old with

homozygous heterozygous

cancer. in the

interstitial tissue also signal. Normal kidney

showed tissue

Again,

kidney

only

cancer

showed

PKD

ET- I expression rat. ET- 1 staining PKD PKD

rats (E),

within of the whereas

rats (B).

a specific ET- I fluorescence from the nephrectomies due a very

weak

signal.

to

J Am

Soc Nephrol

9: 1 169-1

Renal

177. 1998

Endothelin

System

PKD

in

I 173

1000

800

600 400 a,

200

0

0. a)

0

E 0

E

800

C

600 400 200 0 6 weeks

old

6 months

Sprague-Dawley PKD

old

littermates

heterozygous

PKD homozygous Figure

4. Bar graphs

the kidneys corresponding

SPRD

rats compared

(Figure The

binding

(Table

(cy/cy)

chemical

staining

using

ET- 1 expression kidneys

section PKD

an ET-l

within

of this patient.

polycystic

from a 39-yr-old

(H&E

staining). antibody

showing

the epithelial Kidneys

were

patient

with typical

(B and C) Immunohisto-

cells removed

a highly

of renal

increased

cysts

due to the size

sponding

(cy/+)

(cy/+)

subtypes analysis,

decreased

6-wk-old case, density

ETA,

and

as

well

6-mo-old

an inverse was

on

much

gene-dose more

the

other

as

ETB

affected effect reduced

hand,

revealed

receptor

PKD was

a markedly

density

observed.

in homozygous

fl

(Bmax),

rats (Figure

4). The (cy/cy)

In this receptor Han:

such was

using

and

were

RBF

and

acute bosentan

the (10

mg/kg

6-mo-

of

the functional

completed 90 mm after basal MAP before the

was

of

in the

are not suitable

before

1 18.4

corresponding

blockade

of

rats. Measurement

90 mm

begun

hoage

in conscious,

only

in our study

as 6-wk-old

started

because at the

disease.

performed

used

PKD

homozygous

died

renal

HR,

was

was control

compared with and the expres-

± 9.3

rats and 109.8 ± 8.7 mmHg The basal MAP differences

rats The

usually

performed and was of bosentan. Mean

littermates.

significant. Scatchard

animals

and RBF

experiments

Han:SPRD SPRD

rats

in 6-mo-old

be determined,

MAP, rats

the methods

experiments were the last injection

of the

not

to end-stage

of GFR,

small

functional

with

in 6-mo-old

rats

10 wk due

HR.

in the

kidneys.

and the

SEM.

receptor

compared

affinity

subtypes

could

instrumented

for very

rats

binding

Han:SPRD

old rats, because

Kidney

the

rats

(cy/cy)

Measurement

dominant

±

and the ETB

PKD

receptor

Han:SPRD

chronically

3. (A)

PKD

) Han:SPRD

reduced for the ETB receptor 1 ). ET- 1 tissue concentrations

approximately

Figure

rats with are means

(cy/+

of the ETA

1), whereas

mozygous

autosomal

of ETA and ETH receptors

heterozygous

in 6-wk-old

of endothelin

MAP,

with

affinity

reduced

rats was only controls (Table sion

the density

as well as 6-mo-old littermates. Data

4).

slightly rats

showing

of 6-wk-old age-matched

in

both

mmHg

in the

corre-

between

controls

Han:

were

endothelin

intravenously

in

not

receptor every

15 mm

for 2.5 h up to a total load of 100 mg/kg) erozygous PKD rats led to a significant

in 6-mo-old hetdecrease in MAP

(Figure

of bosentan

on BP

littermates,

and

5).

No

age-matched

bosentan

significant Sprague

was

seen

effect Dawley

in the

control

group

(Figure

was no

seen effect

5).

in of

RBF,

I 174

Journal of the American

Table

I. ETA and ETB receptor corresponding

Society

of

binding

control

J Am Soc Nephrol

Nephrology

affinities

(Kd)

in the

kidneys

of 6-wk-old

and

6-mo-old

Han:SPRD

177. 1998

and

rats’

6-wk-old Category

Control Rats

ETA-receptor

9: 1 169-1

(n

=

6-mo-old

Heterozygous PKD Rats

SD 6)

(P1

=

Homozygous PKD Rats

6)

(ii

Control Rats

S to 6)

=

Heterozygous PKD Rats

SD

(ii

=

6)

(ii

6)

=

binding

affinity

0.24

±

0.08

0.60

±

001b

oso

±

005b

o.so

±

0.34

0.70

±

0.32

binding

affinity

0.3 1

±

0.0 I

0.68

±

002b

0.66

±

0.03k’

0.48

±

0.05

1 .50

±

009L

(nmol/L)

ETB-receptor (nmol/L) U

b

Values are mean ± p < 0.01 compared

however,

SD, Sprague Dawley; PKD, polycystic kidney disease. with age-matched control rats (Sprague Dawley littermates).

SEM.

was significantly

PKD

rats. HR Sprague Dawley

increased

in 6-mo-old

heterozygous

was not affected by bosentan rats (data not shown).

in PKD

or in

GFR was markedly reduced in 6-mo-old rats compared with control rats. A single

heterozygous PKD bolus injection of

bosentan

0.005)

GFR

led to a further

in PKD

in PKD

rats

rats

treated

100 g body

(0.42

with

wt in PKD

Intravenous ET- 1 produced sponse:

only

significant ± 0.06

placebo

and

per

0.29

with

g body

100

± 0.06

per

(Figure

5).

depressor

effect

The

blood

changes

wt

mI/mm

bosentan)

100, and 200

BP elevation.

of

decrease

of 20, 50, biphasic

short-lasting

by a long-lasting

<

ml/min

rats treated

bolus injections a dose-dependent

An initial,

(P

pressor was

sponding

controls.

response

pattern

to

a similar

130

-

-

E

.

110

-

100

-

0 <

ng of re-

followed

in BP

were

ac-

90

-

80

-

25

-

20

-

to exogenously in

controls

applied

the

ET- 1 . RBF

Han:SPRD

(data

not

(cy/+)

.g

15-

E

10-

decreased

rats

and

the 0-

shown).

3-

Discussion The kidneys

paracrine endothelin of Han:SPRD-PKD

I

system is highly activated in the rats. Renal tissue concentrations

probably

due

E

epithelial

cells

of the cysts

epithelial Endothelin

Models Renal

Patients

by a highly

cells

increased

of the kidney System

in PKD

of Chronic

Renal

Endothelin

It is important

ET- 1 synthesis

in Han:SPRD-PKD

immunohistochemistry.

characterized

the

increased

to note

with

within

the

rats, as shown ADPKD

ET- 1 synthesis

are

also

within

the

U-

0-

cysts. Compared

Failure

with

with

Sprague-Dawley

Other

Involvement

of

System that the increase

in renal

tissue

ET-1

littermates

PKD

(cy/+)

5. Effects of intravenous administration of 100 mg/kg bosentan or placebo (El) on mean arterial BP (MAP; mmHg), renal blood

Figure

()

immunoreactivity is much higher in rats with PKD than in any other animal model of chronic kidney disease reported thus far (4-7,10,12). ET-l transgenic mice and ET-2 transgenic rats,

Bosentan

-

C E

to a highly

Vehicle

I

I:1

a)

of ET- 1 were 3.5 ± 0.3-fold increased in young cy/cy rats and even more in old cy/+ rats (6.2 ± 0.4-fold). This finding is

by

*

C

similar in 6-mo-old rats with PKD and coneRBF showed a dose-dependent monophasic

extent

corresponding

-

x

companied by reciprocal alterations in HR. The observed effects of ET- I -induced alterations of the pressor as well as the depressor response were dose-dependent, and qualitatively and quantitatively

140

flow

(RBF;

6-mo-old

mL/min),

conscious,

and corresponding means

±

SEM.

and

GFR

chronically

control *JJ <

0.05;

(ml/min

per

instrumented

rats (Sprague **

<

0.01.

Dawley

100 (cy/+)

g body

wt)

Han:SPRD

littermates).

in rats

Data are

J Am

Soc Nephrol

for example, logic of

are characterized

renal

phenotype

only

slightly

Impaired

renal

associated

elevated

because

the

rhotic data

of

with

strongly

that

the

of

conditions-in

PKD

of an increased

ET- 1 tissue

with

report

ET- 1 mRNA (cpk

The

between cyst

ET-

fluid

in the

in the kidneys

role

is supported

role

a recent

expression

mice).

PKD

a major

of the

renal

by the

I concentrations

of patients

with

cirwater

Thus,

our

the

renal

of

of PKD.

system

ADPKD

(19).

Endothelin

Development

System of

male

ET-

(4).

of

in the

1 transgenic

NMRI

Nontransgenic

of small netic in

cysts.

alterations humans cause

receptors

renal

The

cysts.

cofactor

in the

primary

genetic

the

finding

ing

nontransgenic

tion

for

Furthermore, tion

typical

strategies

of BP and low Kidney focal

(Figures

1 and

renal

by:

(1)

predisposi-

cysts

(B.

of the

of

arise

(Figure

the

renal

because

in pathe

(e.g.

cells

ther-

, lowering

and the ETB receptor as demonstrated may

reduce

cyst

by Ong formation

effects

of

ETA

animal

models

renal

endothelin

Of

system

that

might is

a

prove major

the hypothesis progression

production

in

of ET-

that factor

endothelin

are,

PKD

with

I

rats,

re-

the

excep-

approximately

is of pathophysiologic

receptor

subtypes

a yet

and

unknown

of the

ETB

(Figures

1 and

system

using

4),

of endothelin

significantly

thelin

receptors

in PKD

Dawley

significantly

modified.

using

I and

the

the

expression

of

of a simul-

downregulation

of

(cy/+

endogenous

) rats

endothelin

demonstrate

because

MAP,

acute

blockade

after rats

only

(Figure

and

none

In addition,

blockade

bosentan

effects

with

increases

in Han:SPRD

littermates,

exogenous/v

rats. the

and

experiments

receptors,

altered

ing Sprague of

domain

In cpk mice

that

the

system in Han:SPRD (cyl+ ) rats is involved of BP, GFR, and RBF despite the downregu-

were

control Both

findings.

cell

consequences ET-

blocked These

outer

(18).

subtypes

we

bosentan.

renal endothelin in the regulation lation

1 mRNA

tissue

receptor

receptors

these

(23,24).

the pathophysiologic

endothelin

explain in the

mRNA

of

relevance.

endothelin

factor/mechanism

ET-

receptor

of may

identified

upregulation

both

alterations

were

expression

ETA

the

ET-1

of

endogenous

the

results

rats

does

not

of the

high

endogenous

reduce

applied are

endothelin

or

ET-l

obviously

similar

system

abolish

of

receptors

the

biological

ET- I concentrations

in PKD

in rats

and

application

of endothelin

rats.

counteracting

receptors

in PKD

endothelin

even

was

to increasing

exogenous

renal

RBF

endo-

parameters

response

was

after

and

of both

5). In the correspond-

applied

the

GFR,

of these

that the downregulation

of exogenously

Effects

of Bosentan

A major

Thus,

and

postreceptor

the downregulation

with

endogenous

in

the reduction

the of

on BP,

finding

et

have been no studies analyzing long-term ETB receptor blockade in humans or in

of PKD

sites

however,

dose there

affinity

endothelin

the

the

in

PKD. However,

recent endothelin

of

PKD.

as

express

is involved

recently

of

rats

in 6-mo-old

or glycosylation)

mechanisms

segments mainly

endothelin

with

of both

of PKD

(phosphorylation

in PKD

system

tubular

epithelial

blockade

receptor

binding

ET- 1 indicate

2).

endothelin are limited

from

ETA

constants

structural

doses

Hocher,

of the cysts

highly

in a reac-

of both

autocrine

kidney

Posttranslational

the

Tubular cells,

ETB

of

diet).

in PKD

of tubular

renal

correspond-

genetic

relevance,

available

(10, 12,20),

(2 1 ). Thus,

a!.

protein

(2),

additional

their

renal

cells

is of clinical

of dilation.

ETB receptors the growth

the epithelial

rats

rat

to be an important

and

without

develop

the

requiring

(10)

gene on

formation

is supported

density

a downregulation

binding

in the

To analyze

and (2) the finding that patients with characterized by a highly increased

currently

cysts

areas

rats

of an activated

ADPKD

cysts

the

resulted

are in agreement

to increased

the

subtypes

taneous

ge-

in rats of

and

stimuli (rats

not

are also

in PKD

with

apeutic

did

within

finding

gene

ET- 1 seems

transgenic

cysts)

or PKD2

growth

of renal

observation);

as seen

tients

pathogenesis

ADPKD

The

that

littermates

renal

ET- I synthesis 3),

hypothesis

ET-2

PKD1

that

rats

two times higher compared with Sprague Dawley littermates. Thus far there are no reports showing that such a slight alter-

growth number

unknown activation

promoting

of

that primary

of PKD

secondary

thus

or environmental

that

unpublished

in the

cyst

a small

propose

a yet

kidneys

renal only

in

suggest

receptor

results

showing

in response

PKD,

in the

to the development

a downstream system,

we

within

5, leading

gene

develop

as mutations

mutations

endothelin

in the

promoted

mice

Therefore,

such

or

chromosome might

mice

NMRI

renal

ET-l

These

experiments

study

in Han:SPRD

of the

subtypes.

vitro

both

human

study

(4).

Receptors

in this

synthesis

N-glycosylation

Cysts the

presented

downregulation

receptor

ation

relationship

concentration

as a Cofactor

Renal

Overexpression

of Endothelin

ET-1

of both Renal

by the present

mice

Rats

data

ceptor

in human

of an inverse sodium

as suggested

transgenic

I I 75

in PKD

(22).

concentration in PKD ( 1 8) showing increased of a mouse PKD model

and

tive

rats as well

pathogenesis

endothelin

finding

The

above-mentioned

(Han:SPRD

The

is in agreement

(12).

the

may

in PKD,

in ET-l

Downregulation

increased

these

ascites activation

as humans)

finding

play

in

a decreased

with

failure

Han:SPRD

tissue

relevance

in

stronger

is also

demonstrated,

system

resulted

system-compared

pathophysiologic

(12) in renal

clearly

endothelin

formation

suggest

endothelin

the

is

renal

and the findings

System

concentrations.

cirrhosis

increase

rats

chronic

in spite

the pathophysiologic

bosentan

increased

liver

mild

cirrhotic

blockade

and

but

of a patho-

developed

or ET-2

with

However, in

animals

excretion

ET-1

in rats

a significant

findings

development

pathology

tissue

function

with

these

by the

(4, 1 0). This

ET- I concentrations. of

Renal Endothelin

9: 1 169-1 177. 1998

after

endothelin

of BP (-

of bosentan

RBF,

and

acute

blockade

system

in PKD

I 9.7

±

rats using

2. 1 mmHg),

had no significant

effect

Dawley

littermates

(Figure

5).

paracrine

endothelin

system

in Han:SPRD

utes

substantially

BP-lowering

to the

effect

GFR of the highly

These

regulation

of the combined

whereas

BP

ETA/ETB

in

same

in Sprague

indicate

(cy/+)

was

the

on MAP data

of

activated

bosentan

rats PKD

receptor

that

the

contribrats.

The

antag-

Journal of the American

1 176

onist in activated

Han:SPRD endothelin

structural

(e.g.,

(cy/+ system

alterations

BP

as

transgenic

seen

rats

tions

in PKD

genie

animal

explain

in

(10).

system

ET- 1 transgenic

mice

much

with

of

demonstrate

higher

the

that the major

tial

tissue,

blood

the regulation GFR

RBF

GFR

after

manner

significantly system

in

after

bosentan

a single

injection

BP

in PKD

rats

reduction the

involved finding highly

the present study system is activated

to renal

cyst

acute

10.

ceptor

antagonist

protein

cysts

suggests

within

that

in human

the

ADPKD

the epithelial

renal

The by a

cells

endothelin

1 3.

Remuzzi

G, Benigni renal

system

Gretz

by grants

from

the Deutsche

Forschungs-

I S.

severity

Hocher

B, Liefeldt

J Kidney

Bihoreau

pathogenesis, and treatment of autokidney disease: Recent advances.

Dis 28: 788-803,

MT.

Ceccherini

Schafer

K, Bader

overexpression thelial changes 190-195,

4.

Hocher ski

M, Gretz

I, Kranzlin

B, Romeo

G,

a new

receptor

antagonist.

B, Kloting

kidney

disease

of rats.

HD, Distler

of the renal phenotype

the human

endothelin-2

gene.

of

Hyper-

1996

M, Contino

LC: Upregulation

cyclosporine

187:

of renal

A-induced

endothe-

nephrotoxicity.

1 1 3-1 16, 1990

R, Diekmann

mayer

HH, Bauer

C, Gross

in rats with

liver

Bachmann

5, Ramasubbu

F, Rohmeiss P: Paracrine

P, Distler

renal

Br J Pharmacol

cirrhosis.

A,

endothelin

Neusystem

I 18: 220-227,

1996

K: Immunohistochemical

alpha-subunit

of

neutrophil

in kidney

and

NADPH

liver.

oxidase

mt

Kidney

and

S 1 : 479-

1997

Hocher

Drost

M, Rohm-

L, Th#{246}ne-Reineke C, Orzekowski

in rats with

the

ac-

G: Gender-dependent

polycystic

B, Zart

of

orally

J Pharmacol

1995

196-201,

P, Pullen

potent

I, Devoto

R, Romeo

in autosomal

Hocher

B, Rohmeiss

P. Zart R, Diekmann

M, Koppenhagen

K, Gretz

F, Vogt

N, Bauer

A: Function

and expression

of endothelin

in the kidneys

of spontaneously

hypertensive

Res 31: 499-510, 1996 C: Vessel-diameter independent using

ultrasound.

Proc

San Diego

V. Metz

C, Neumayer receptor rats.

D, HH, sub-

C’ardio-

volume

flow

measure-

Biomed

Symp

17: 299-

1978

Gretz N, Ecker-Tschirner KH, KUhnle HF, Dahl KV, Kirschfink M, Drescher P, Lasser ii, Strauch M: Practicability of the inulin single-shot

clearance.

N, Oberb#{228}umer I, Bachmann

18.

V.

C, Rohmeiss

Siegmund

Bauer C, Schleuning

P, Schmager

F, Quertermous

Contrib

Nephrol

8 1 : 220-228,

WD, Theuring

F, Slowin-

F: Endothelin-l

HH,

transgenic

volume

104-107,

1992

samples

FH: Fully enzymatic

without

determination

deproteinization.

Nephron

62:

Nakamura T, Ebihara I, Fukui M, Osada 5, Tomino Y, Masaki T, Goto K, Furuichi Y, Koide H: Increased endothelin and endoreceptor

mRNA

expression

in polycystic

kidneys

of cpk

J Am Soc Nephrol 4: 1064-1072, 1993 Munemura C, Uemasu J, Kawasaki H: Epidermal growth factor and endothelin in cyst fluid from autosomal dominant polycystic kidney disease cases: Possible evidence of heterogeneity in cystogenesis. Am J Kidney Dis 24: 561-568, 1994 mice.

19.

5: Focal

T, Neumayer

small

thelin

20.

B, Thdne-Reineke

HF, Dahl D, Schmidt

Ktihnle in

of collagen 4 characterizes the initiation of epiin polycystic kidney disease. Exp Nephrol 2:

1994

T, Burst

17.

1996

I, Browne

Lathrop GM, James MR. Gretz N: Location of the first genetic locus, PKDrI, controlling autosomal dominant polycystic kidney disease in Han:SPRD cy/+ rat. Hum Mol Genet 6: 609-613, 1997 3.

and

endothelin.

1994

rats expressing 28:

Nambi

302,

16.

diabetec

for renal

I 990

Grantham ii: The etiology, somal dominant polycystic Am

role

of bosentan,

Waldherr

mt 48: 496-500,

ments

References

2.

B,

Kidney

vase

Industrie (to Dr. Mannheim (to Dr. and S. Schiller is

of proteinuric 1997

I, Kranzlin

disease

plasma

1.

228-235,

P. Hocher

types

(Ho 1665/2-I), Fonds der Chemischen Hocher), and Zentrum f#{252}r Medizinische Forschung, Rohmeiss). The technical assistance of 0. Chung greatly appreciated.

re-

4: 349-

of sustained 1995

73: 461-468,

A possible

endothelin

N, Ceccherini

Distler was supported

Invest

A: Progression

characterization

non-peptide

482,

Acknowledgments

Hypertens

M, Breu V, Gray GA, Kalna B, L#{246}ffier BM, Burn K, iM, Hirth G, Muller M, Neidhart W, Ramuz H: Pharma-

Fakhury

study

Nephrol

pathophys-

endothelin

G: The renal toxicity Lab

disease:

ecto-S’-nucleotidase 14.

This

Opin

In! Suppl 58: S66-S68,

Clozel Cassal

tion

of

as well.

gemeinschaft

traffic.

Eur J Pharmacol 12.

and is also

of BP, GFR, and RBF in PKD. ADPKD are also characterized

ET- 1 synthesis

be involved

shows that the paracrine in rats with PKD, may

Curr

A, Zoja C, Remuzzi

glomerular

tension

1 1.

177. 1998

1995

Benigni

transgenic

of GFR, of

studies.

in renal

unselective

A, Bauer C, Paul M: Characterization

in

setpoint

and renal fibrosis,

formation

in the regulation that patients with

kidney

and

lin receptors

increased

might

of endothelins

Exp Ther 270:

autoregulation.

contribute

the role of selective

eiss

9: 1 169-1

interstitial fibrosis and glomeruloscleJ Cli,z invest 99: 1380-1389, 1997

A: Defining

cological.

in PKD

cysts,

on the basis

Kidney 8.

to

of bosentan

below

Benigni

renal

Soc Nephrol

iology

tive

contributing

altered using

develop

but not hypertension.

nondiabetic

to the intersti-

thus

the bosentan-induced

by reducing

7.

of the endothelin techniques, we

9.

also

of MAP

In conclusion, renal endothelin

the

may

6.

cells of the renal cysts. in the epithelial cells of

glomeruli,

endothelin

rats may explain

probably

trans(4, 10)

mice rosis

353,

concentra-

these alterations were less pronounced cornBP-lowering effect of bosentan in PKD rats.

reduction

PKD

were

of the renal

rats. However, pared with the The

ET-2

of BP and GFR.

and

blockade

and

not

of ET- 1 expression

in a passive

vessels,

and

system

sites

S.

tissue

the above-mentioned

endothelin

an to

but does

tissue

hemodynamic effects Using immunohistologic

migrates/diffuses

kidney

(4)

ET-l

6-mo-old PKD rats are the epithelial We propose that the ET-l synthesized the cysts

because in general

and

fibrosis),

rats compared models

(25)

Am

I

is remarkable, or contributes

and interstitial The

the additional in PKD rats.

could

) rats causes

of Nephrology

in cardiovascular

glomerulosclerosis

affect

Society

Hocher

B, Rohmeiss

penhagen

K, Bauer

endothelin

receptor

haemodynamic

effects

P, Zart

R, Diekmann

C, Distler subtypes

A, Gretz in

of the mixed

the

rat

(A/B)

F, Braun

C, Kop-

N: Distribution kidney: endothelin

Renal

of and

receptor

I

Am

Soc Nephrol

antagonist 21 .

Ong

Eur

bosentan.

463-472,

Renal

9: 1 169-1 177. 1998

J Clin

Che,n

Clin

Biochem

expression

33:

1995

ACM,

Jowett

348:

TP, Firth

JD, Burton

5, Karet

FE,

Fine

LG:

24.

An endothelin-1 mediated autocrine growth loop involved in human renal tubular regeneration. Kidney Ira’ 48: 390-401, 1995 22.

Clozel

M, Ldffler

23.

of endothelin-1 . Am J Physiol 265: Cl88-C192, Arai H, Hori 5, Aramori L, Ohkubo H, Nakanishi

Downregulation

BM,

Breu

V, Hilfiger

of endothelin

receptors

L, Maire

JP, Butscha

by autocrine

B:

production

5: Cloning

Haendler human

and

encoding

System

in PKD

an endothelin

1 177

receptor.

Nature

1990

B, Hechler endothelin

Pharmacol 25.

20[Suppl

U, Schleuning (ET)

WD:

receptors

ETA

12]: 51-54,

1992

Molecular

and ETB.

cloning

of

J Cardiovasc

Hocher B, Thdne-Reineke C, Bauer C, Raschack M, Neumayer HH: The paracrine endothelin system: Pathophysiology and implications

1993

of a eDNA

730-732,

Endothelin

in clinical

35: 175-189,

1997

medicine.

Eur J Clin

Chem

Clin

Biochem