PQtnQtQIMqturQtiQnQIthe KaIIiKrein-Kinin3ystem in the - JASN

PQtnQtQIMqturQtiQnQIthe KaIIiKrein-Kinin3ystem in the Rat Kidney: From Enzyme Gene Expression 1

Activity

Jean-Loup

Castano,

Bascands,

Mireille

Gaucher,

Maria and

Encarna

Jean-Pierre

Mann

to Receptor Guy

Bompart,

Christiane

Pecher,

Girolami2 of the regulatory systems that control solute excretion. Kalllkrein is a serine protease, and one of its actions Is to generate potent vasodilator and

RBF,

knowledge

J.-L. Bascands, ME. Mann Castano, G. Bompart, Pecher, M. Gaucher, J.-P. Girolami, INSERM U388, tut Louis Bugnard, Toulouse, FRANCE

C. Insti-

GFR.

and

uretic

(J. Am.

Soc.

Nephrol.

1996;

7:81-89)

substances,

which circulation. esis role

ABSTRACT During

the

intrarenol changes

course

of

aging,

the

balance

between

reniri-ariglotensin tion on the

system, but comparable informarenal kallikrein-klnin system Is not yet The status of the kallikrein-kinin system was by (1) kalllkreln activity, measured by RIA;

available. assessed

(2) maximum

binding site density (Bmax) and affinity (Kd) of nonapeptide bradykinin (BK)-2 receptor, estimated by binding assays; (3) expression of BK2-receptor mRNA, detected by reverse transcription-polymerase chain reaction (RT-PCR) using specific BK2oligonucleotide primers. These parameters were determinated on renal glomerull of 3-, 5-, 8-, 2- and 38-wk-old normotensive rats. Kallikreln activity increased from 3.2 to 7.7 ng BK/mm per mg protein. The density

of BK2 binding

sites

also

rose

from

12 to 40

femtomoles/mg protein with no difference in affinity. There was no change in specificity, which remained that expected of a BK2 receptor. The increase In the density of BK2 binding sites was associated with an augmented mRNA expression, whereas p-actmn mRNA used as a control remained unchanged. The ratio of BK2 mRNA to p-actin mRNA Indicated maximum steady expression after 8 wk of age. The data provide

evidence that the renal ops postnotally. Key Words:

N tions

ormal (1). in

March

2Corsepondence 13-RDC,

8K2 receptor, of

aging

progressive

changes

1 ReceIved

Glomerulus,

kallikrein-kinin

the

kidney and

functional

Our understanding kidney functions

28. 1995. Accepted to Dr. J.- P. GirolamI,

CHU Ranguoll,

31054

Toulouse

system

ontogeny, Is

of the American

mRNA,

associated

structural

PGE2 with

deterlora-

of the age-related may be improved

by

July 27, 1995. Insfitut Cedex,

Louis Bugnard, Erance.

1046-6673/07010081 $0300/0 Journal of the American Society of NephrOlogy CopyrightS 1996 by the American Society of Nephrology

Journal

devel-

Society

of Nephrology

the the

lines

from

and

lIver

arises

Into

favor

the

system functions,

and electrolyte balance (2,3). One of the primary

kalllkrein-klnln system made by many investigators potential antihypertensive might be involved in the

kininogen,

released

of evidence

renal kalllkrein-klnln regulation of renal

the

hypoth-

could play primarily

a

and blood pressure interests in the renal

from that thIs system.

the system and

suggestion acts as on failure

a

development of hypertension number of investigators have reported a significant decrease in urinary kallikrein excretion in humans with essential hypertension (5), genetIcally hypertensive rats (6), and several animal models for secondary forms of experimental hypertension, such as the two kidney-one clip (2K- 1C) renal hypertensive rats (7). The physiological activity of the renal kallikrein(3,4).

A large

kinin The role pain,

system is carried out by the generated nonapeptlde bradykinln (BK) plays an in a number of biological processes, inflammation, vascular permeability,

muscle contraction, of blood pressure mediated macological of at least

BK2 the

cell proliferatIon, (8-10). These

and effects

by

cell-surface BK receptors. studies have demonstrated two types of BK receptors,

(8.9). The endogenous

BK1 BK

receptor metabolite

has

kinins. important including

smooth regulation

of

BK

are

Several pharthe existence termed BK1 and

a greater affinity des-Arg#{176}-BK. The

for BK2

receptor, which Is the more prevalent receptor type, has a greater affinity for the native BK. Cloning of the BK2 receptor from human and rat tissues (11-14) indicated that this receptor is a member of the superfamily of the seven transmembrane domain G-protein-coupled receptors. In the kidney, specific binding studies have reported the presence of BK2 receptor in cortical epithelial membrane (15), renomedullary interstltial cells (16). and in cortical tubules (17). We recently

Identified

the

glomerular

membranes

membranes

located

Micropuncture INSERM U388. Bet.

kinins.

in the

Several

that in

water control

hormones is disturbed. These age-related are well documented for the vasoconstrictor

named

is synthesized

major natri-

that

demonstrated BK2 receptor

(18), on

the

studies

BK perfused

glomerular

presence

into

ultrafiltratIon

that was

of

more mesangial

(20)

the

BK2

in

renal

rat

artery

(19). have

shown

decreased (Ks).

with

in

on those

cells the

coefficient

the activation associated

receptors

precisely,

of the an

Our

lab

glomerular increase

the then

in

81

Ontogeny

of Glomerulus

BK2 Receptor

intracellular calcium generation (22),

(21) as leading

cAMP

cells (23). glomerulus

mesanglal

sites

in the

dating certain BK. Although

and

renal

renal a role

The

presence

could

of BK2

be

functions

is generally

there

is,

as

in

effects

induced of water,

concerning

the

postnatal

well

yet,

accepted.

very

little

in of the

binding

important

hemodynamic in the regulation

knowledge kalllkrein-kinin reported that sites showed renal kalllkrein

well as an inhibition to a contraction

by salt.

postnatal

in the

development

rat

kidney

of

are

the

described

appeared

for mRNA

and efferent diethylpyrocarbonate

added

in

RNase.

the

buffer

4 rats

glomerular

during

Each

glomerulus

age group

were

for

Glomerular

4 rats

of adherent

measurements

not

determined

were

a light

fitted on perfectly

circular,

as

follows: xr2).

major

site

mRNA

performed

the

(largest

at

(Leitz and samples. mean

x 100

granular

for

Because

diameter

+

estimated

as

cells,

by

its

described synthesis which

kininogenase

previously and secretion cannot

be

(25). are

isolated,

homogenized, heated citrate dog plasma was used as a kininogen source, BK (Bachem. Switzerland) was used as a standard, and [12511 BK as a tracer. The BK generated was measured by RIA by using BK which Kininogenase

only

per

recognize activity was

minute

was

performed assessed uli in the

per

whole BK expressed

mg protein

but not BK in nanograms

(ng BK/mm

glomeruli 300 mated

measured

were

itL.

used

The amount after a 5-mm

fragof

per

mg

by enzyme

of PGE2 immunoassay

as currently

for

each

incubation

of POE2 was isolated glomer1500 to 2000

final

in a

volume

of prostaglandins secreted was incubation at 37#{176}Cin the presence

of estiof 10

riM BK. The incubation medium was centrifuged at 4,000 x g and the supernatant was stored frozen until assayed for determination of prostaglandins. The amount of PGE2 secreted in the medium was assayed directly by specific enzyme Ml).

Inimunoassays Briefly,

(Cayman

this

assay

was

coated

onto

Strasbourg. body. The

96-well

France) enzymatic

Chemical

is done

on

and

between free prostaglandin linked prostaglandin used rabbit-prostaglandin antisera.

as

a The

plates

via a tracer

the

basis

Corp,

Ann

of the

competition

(NUNC

Certified,

mouse-monoclonal reacts with

Binding

Studies

Poly-Lab. rabbit

the

Ellman

the well. and the amount of colored is inversely proportional to the amount present in the well. The data for POE2 POE2

Arbor,

the acetylcholmnesterasetracer for the respective prostaglandin antiserum

to

in pg of of glomerular

4 rats

Production

in the laboratory (25). The production by the amount released by freshly incubation medium. Approximately

expressed milligram

secreted per protein (pg/mm

mm per

antiReagent

substance of prostagrelease are

incubation mg prot).

per

for E2

extraction

magnifica-

Paris, were

binding

studies

(19.21).

Saturation

renal glomerulus membranes and 125I as described previously and used in as currently performed in the laboratory studies were conducted at 37#{176}Cfor 30 mlns

in the presence 0.2 to 10 nM.

of an Dissociation

site

(Bmax)

density

amount

of ‘251-[Tyr#{176})BK increasing constant (K.) and maximum

values

were

calculated

from

analysis of the data by using the GAND program (Elsevier-Biosoft, Cambridge. and were expressed as femtomoles of iodinated

milligram

was diame-

determined we estab± 0.9% (N

of crude obtained

assisted

glomeruli

diameter smallest

theoretical surface area was In preliminary experiments, variation coefficient of 3.3

was

RIA kallikrein

connecting

to inactiorganized as

SM-Lux, Leitz, all measurements

glomerular

02-5%

aliquots of tissue cortex were

Preparation [Tyr#{176}]BKwere

microscope

with a micrometer. 100-glomerulus

ter)/2. Glomerular by surface (SI = lished an intra-assay

82

the

added

An aliquot of the glomerular suspension was taken and the glomerull were counted by using a counting cell (Sedgewick Rafter, from Graticules Limited, Tonbridge. England). Gb-

are

Since

a kinin

released landin

prostaglandin

for

for

a 95%

prot).

Characterization

by using

of kallikrein

preparations was directly

activity;

of the

and

studies;

In (0.1%)

kallikrein

secretion

binding studies.

free

preparation 12 rats

contained

used

morphometry.

(PGE2), and and RT-PCR

and

arterioles.

unchanged with

during

of Animals

to be decapsulated

afferent

studies.

vate

France) performed

activity

activity by using the

remained oxygenated

Assays

BK generated

quickly removed and decapsulated. and glomeruli were obtained as described previously (18). The cortex suspension Is briefly passed twice through a 21-gauge needle to dissociate the tubule from the glomeruli. The glomeruli are then isolated by graded sieving through three consecutive steel sieves with decreasing pore sizes (180-. 125- and 75-j.tml. and the glomeruli are collected on the 75-nm sieve. In these conditions, we obtained approximately 15,000 glomeruli from one kidney, and under a light microscope, > 95% of the and

same periodically

In Vitro Glomerular

Preparation

glomeruli

of the when

(N

coefficient of 4.4 ± 1.2% certain that the glomerular

mixture.

antibodies, ments.

Male Sprague Dawley rats. 3, 5, 8. 12, and 38 wk of age. were housed in climate-controlled conditions with a 12-h light and dark cycle, and were fed with standard rat chow (UAR IA-041. IFA-Credo. Lyon, France: 104 mrnol Nat/kg of food) and water ad Ubitum. The animals were killed by decapitation to allow exsanguination, the kidneys were

tion

CO2

POE2

Glomerular

merular

3 h

The

renal

METHODS

follows:

preparation

to

Kallikrein

to our

development.

tubules

diameters

information

system in the rat. Moreover, we also the density of glomerular BK2 binding opposite variations to acute changes of activity induced by physiological con-

expression

variation to make

up

eluci-

ditIons such as dIsturbances in sodium balance (24) and renovascular hypertension (25). In the report presented here, we examine the relationships among the three indexes of the kallikrein-kinin system under strictly physiological conditions. Changes in the renal kallikrein activity, BK2 receptor binding sites, and gene

20) and an inter-assay =20). We also checked

fixed

amounts

of protein (finol/mg of membrane

at 37#{176}Cin triplicate, with BK or antagonist (from 10 =

protein). extract an 12

from binding

computer-

kinetic-EBDA-LIUnited Kingdom). BK bound per

For competition were incubated

for

studies, 30 mm

increasing amount of unlabeled l0 M) in the presence of 2 nM

of ‘25I-[’Fyr#{176}]BK.

Volume

7

‘

Number

1

1996

Bascands

Protein

Determination

In each

experiment,

Optimization samples

of renal

cortex

or glomerulus

suspension were used for determination of the protein content. After solubilization for 15 mmn at 100#{176}Cwith 1 M NaOH, proteins were measured by using the method of Lowry et a!. (26) with serum albumin as a standard.

Statistical Results separate variance parisons,

RNA

Analysis are expressed as mean ± SE of at least experiments. An unpaired t test or was performed when appropriate, and differences were considered significant

Isolation

and

RT-PCR

Total RNA was extracted (approximately 100 to

according Sacchi at the Promega

to the

method

three to five analysis of in all comat P < 0.05.

Procedure

from frozen 200 mg) or

described

by Chomczynski

and

(27). To study the expression of BK2 receptor mRNA different ages. polyA+ RNA was isolated using the reagent kit (PolyATtract mRNA Isolation System IV;

Promega.

Charbonni#{232}res.

Chain

Reaction

Five pA of each cDNA preparation were amplified. PCR amplification was performed by adding 140 pmol of each primer, 2 U Taq polymerase (Appligene, Illkirch, France), 0.5 mM dNTPs in a final volume of 100 pA PCR buffer containing either 3% formamide for BK2-receptor mRNA amplification, or 10% dimethyl sulfoxide for (3-actmn mRNA amplification. The samples were denaturated for 3 min at 95#{176}C,then PCR was performed for 30 cycles (1 mm at 94#{176}C,1 mlii 30 s at 58#{176}C.1 min at 72#{176}C).PCR was carried out using a Perkin Elmer DNA Thermal Cycler (Sigma. Saint Quentin Fallavier. France). In order to evaluate the PCR products comparatively and confirm the mntegrmty of the RNA. we amplified a housekeeping gene. that of f3-actin. The PCR products for BK2 receptor or (3-actin were amplified at the same time from the same cDNA and then electrophoresed. After analysis, the ratio of BK2 to j3-actin band densmty was determined. Amplification in the absence of cDNA did not yield any bands other than those of the primers for BK2-receptor mRNA and /3-actin mRNA at the bottom of the gel (not shown). The BK2-receptor primers were designed in the 3’ untranslated region of the cDNA from the sequence of the rat gene (11). Primer 1 was defined by bases 2955-2975 5’ACCAGAGATAGGATAGCCTCC-3’(sense) and Primer 2 was defined by bases 3451-3473 5’-ACAGTGTGTFAGCCTCAGAAGC-3’ (antisense). The cDNA amplification product was predicted to be 518 base pairs in length. The sequences of sense and antisense primers for /3-actmn (28) were defined by bases (2846-2865, exon 5) 5’-CCTAGCACCATGAAGATCAA-3’ (sense) and by bases (3171-3196 exon 6) 5’lTl’CTGCGCAAGTTAGGTITFGTCAA-3’ (antisense). The expected size of the PCR product for 13-actin was 227 base pairs.

Journal

of the American

Relative

Quantification

After amplificatIon. phoresed through ethidmum bromide. 665

The gel was

PCR product gel

and

photographed

was electrostained

with

with

Polaroid

(Poly-Lab, Strasbourg. at the same exposure and developing time. The bands on the negative ifim were scanned by using an image-analysis system (Elecphor#{176}#{176}. CRIS, Toulouse, France), which calculated the densitometric values of each band. Then, for relative quantification, arbitrary values were assigned as described in the figure legends.

France)

ifim

of PCR Products

20 pA of each a 2% agarose (Positive/Negatmve)

over ultraviolet

(UV) light

France),

First-strand cDNA was synthesized by using 1 ig of total RNA or PolyA+RNA, oligonucleotides (pd(T)15) for priming. RNase inhibitor (32 U). l,4-dithiothreitol (DT’F) (25 mM), dNTPs (5 mM). 5 X RT buffer, and 200 U RT (M-MLV from GBCO-BRL, France Life Technologies. Cergy Pontoise, France) were used. After RNA denaturatmon (10 min at 70#{176}C). the reaction was carried out at 42#{176}C for 50 min with a Perkmn Elmer TC-480 (Toulouse, France) heated to 95#{176}C for 5 mm and chilled on ice.

Polymerase

of PCR Conditions

To achieve optimal conditions for PCR, we first synthesized cDNA from 1 ag of total RNA obtained from normal SpragueDawley rat renal cortex and amplified It, mncreasing the cycle number from 15 to 50 for BK receptor and from 10 to 40 for -actin. We next studied the PCR product for BK receptor and (3-actin as a function of increasing amount of RT solution (cDNA). PCR amplification was performed for 35 and 25 cycles for BK2 receptor and $3-actmn respectively.

Type

powdered renal cortex from frozen glomerull

et al

Society

of Nephrology

Southern In order

to authentic

Blot to verify

Analysis that

BK2-receptor

the

amplified

fragment

transcript,

and

corresponded

to obtain

a fuller

definition of the variations observed on the agarose gel electrophoresis of the PCR products stained with ethidmum bromide, we hybridized Southern blots of electrophoresed PCR products with the rat BK2-receptor mRNA probe obtained by amplification of 200 ng of genomic rat DNA with our specific BK2-receptor primers and labeled wIth [a32P)dCTP by random priming (Amersham megaprime kit, Les Ulis. France), After amplification. 40 pA of each PCR product was electrophoresed through a 1.8 to 2% agarose gel. then the gel was denatured, neutralized, and transfered overnight onto a standard nylon membrane (HybondTM-N, Amersham) with 20 x SSC (sodium chloride/sodium citrate) as the transfer buffer (29). The DNA was fixed to the nylon under UV radiation (254 nm) in an UV-crosslinker (Stratagene, La Jolla, CA). After hybridization in stringent conditions with the 32P probe (42#{176}C overnight). the nylon was washed once wIth SSC 2X/0.l% sodium dodecyl sulfate (SDS) at room temperature and twice at 65#{176}C for 30 min. Autoradiography was performed for 1 h at -80#{176}Cby using Amersham hyperfflm-MP film with mntensmfymng screens, developed and analyzed as described above. To confirm that the PCR products were really BK2-receptor cDNA. they were sequenced using the CircumVentTM thermal cycle dideoxy DNA sequencing kit from Biolabs (Ozyme, Montigny Le Bretonneux, France).

MATERIALS Male Sprague-Dawley rats were from the IFA-Credo farm (Lyon, France). The commercial orIgin of the materials used was as follows: radioactive iodine as sodium salt was from Amersham (67.5 TBq/mmol). BK-related peptmdes were purchased from Sigma (Saint Quentmn Fallavier. France). HOE140 (D-Arg-(Hyp3,Thm5,D-Tic7,Oic8]-BK) was a gift from P.B. Schdlkens (Hoechst. Frankfurt. Germany). The enzyme immunoassay (ELA) used for PGE2 measurement was from Cayman Chemical (Ann Arbor, MI). dNTPs was from Bioprobe#{174}Systems (Montreuil-Sous-Bois, France) and pd(T)15 from Gibco-BRL. Oligonucleotides for primers were synthesized by Institut Pasteur (Paris. France). [a32Pl dCTP

83

Ontogeny

of Glomerulus

BK2 Receptor

TABLE 1. Glomerular

panameters#{176}

Age

Body

(wk)

(N

Wt (g)

Kidney (N

12)

=

Glomerular Content (ng/Glomerulus) (N 20)

Wt (g) 12)

=

Mean Glomerular Diameter (,Lm) (N 100)

Protein

=

=

3

40±4

5 8 12

110±7 210±5 350 ± 10 600±25

38 #{176}Body and

kidney

determinations.

weight,

values

The value

are

(3000Cm/nimol) and [a35S1 ICN (Costa Mesa, CA).

dATP

48±2

0.43±0.05 1.15±0.1

54±2.6 83±4.3 97 ± 5.2

1.39 1.46± mean

for the mean

0.25

±

0.5

CI/mmol)

were

As

for each age is the mean value ± SE of lifi glomeruli.

of Age

on

1. the

Parameters glomem-ular

diameter

protein

increased

.5:

30

o

shown

in

Figure

in Renal 1,

the

20

mg prot at Week

38.

The

at Week 8, and

slight from

3 to 6.6 ± 1.2 then remained

increase the

at Week

values

Cortex

obtained

is not at

8 or

of Age

on

the

Number

of BK Binding

Because the ular diameter

glomerular protein increased with age, the number of BK binding sites in protein and in fmol/j.tm2 Whatever sion used for the results, the same was observed. Over the age range of BK

postnatal

binding

life

sites

increased

(Figure

the

decreased

first

5

8

AGE

(weeks)

12

38

12

38

-

‘C

38.

IC

4-

1

Sites

content and glomerwe also expressed fmol/mg glomerular the type of exprespattern of evolution studied, the number

during

2). It then

(Weeks)

.5 .5

significantly

Weeks

8

AGE

5

ng BK/min per mg stable until Week

12

5

E

IC

Effect

SE of 20

10

continuously

kallIkrein activity occurred during the 3.7 ± 0.5 ng BK/min

renal

with age. The increase of postnatal life, from

per prot

different

±

I-

content

3

Age-Related Variations Kallikrein Activity As

value

40

Glomerular

in Table

the glomerular age.

increased first weeks

±4

a 9

shown

and with

228

from

RESULTS Effect

132±2 172±4 176 ± 4

123±7.5

SE of 12 rats. The glomerular protein content diameter for each age represents the mean

±

glomerular

(>1000

94±2

0.23±0.03

8 wk

3

of

to a lower

Figure 2. Age-related changes in the specific binding of 125i(Tyr#{176})BK. Each point represents the mean Bmax value of four independent glomerular membrane preparations: each of them was performed in triplicate. Values are expressed as means ± SE. Bmax values were deduced from Scatchard analysis of data and are expressed either as femtomoies per mg of glomerular protein (Panel A) or femtomoles per m2 (glomeruiar area, Panel B). P < 0.01, compared with the 3-week value. *

8 6 4

steady level, up to 38 wk. No variations in affinity were observed, and the mean Kd value for each age (3. 5. 8, 12, 38 wk of age) remained in the nanomolar range

1-

and 2.5 3

5

8 AGE

12

Specificity different

38

(weeks)

Figure 1. Age-related changes In the renal kininogenase activlly of Sprague Dawley male rats. Vertical bars indicate standard error (SE). Each point depicts the mean of triplicate determinations of 4 Independent experiments. P < 0.01 compared with the 3 week value. *

84

was respectively ± 0.25; and 2.85

competition antagonists BK binding

2.1 ± 0.5; ± 0.3 nM

of BK2 binding age groups was

2.4

(N

=

± 0.2; 2.7 ± 0.3; 4) for each age.

sites of glomeruli assessed on the

from basis

experiments and the K1 of different are given in Table 2. The specificity sites remained that of BK2 binding

since the specific was without effect.

BK1 antagonist Furthermore,

Volume

the of BK of the sites.

des-Arg9-Leu8-BK no significant varla-

7

Number

1 ‘1996

Bascands

TABLE

2.

et a)

K, valuesa K, Values

Age (wk)

Drugs

BK (nM)

Comparison calculated

5

± 0.12 >i0 1.16 ± 0.08

0.28 ± 0.15 >i0 1.27 ± 0.06

0.16

0.13

0.35

des-Arg9Leu8 BK (M) (D-ArgHyp3-D-Phe7)BK HOE-140 (nM)

(nM)

from

0.02

±

of BK antagonists equation: K, = EC/(1 from competition studies, and (L) is the concentration of three independent experiments.

#{176}

8

3

of the potency the following

for

±

0.38

1.25 ± 0.1 0.12 ± 0.03

resed

bromide, scribed

S

tionship

S

the

‘a C.

0

C,

12

(weeks)

the specificity among the

of the different

BK ages.

Effect of Age on Glomerular Stimulated by BK uli

functional

was

sites

were

merull the

BK2

by

BK-induced

receptor

To our

evaluate scanned

analyzed

DNA

Quantitation the

PGE2

to were at

release.

provided kit Number

those obtained for gbobtained. Interestingly. the 38th week corre-

of the

and

analysis

products

as

PCR tion of the PCR Each diluted

Journal

sensitivity

808-008, product. aliquot

American

linear

in

Society

d)

was

then

of Nephrology

relative

value

of

for

PCR,

we

first

1 p.g of total RNA obtained rat renal cortex, and cycle

receptor

(Figure

4B.

4C

number

and

from

and

4D).

from

10 to 40 The

PCR

15

PCR prod-

the

PCR

products

for

BK2

and

-actin

as a function of the increasing amount of product used for PCR amplification. Taken these results show that under the conditions above, PCR can be used in a semiquantitative to assess

mRNA Aging

mRNA of the

Expression

PCR

variation

in rat

product

confirmed

base 2955 previously

and base published

of the

BK2 Receptor

renal

cortex. complete

3473, with (11).

the

When

we

first

lamda Elmer

dilu-

electropho-

compared

to the

sion found in 8-wk-old rats 5C and 5D). The specificity firmed by the expression

of

During

As shown in Figure 5. BK2-receptor mRNA expression exhibited the same patterns of evolution as those obtained for the variations in the density of BK2 binding sites. We can notice a specific increase in BK2-receptor mRNA expression in 8 and 1 2-wk-old

band

and

which does not as a consequence,

intensity

12th

was

weeks

3-wk-old rats, the was 2.5-fold higher of the increase of a housekeeping

show any variations the ratio of BK

significantly

of age

(Figure

increased

expres(Figure was

congene

(Figure to f3-actin at

the

8th

SB).

DISCUSSION It has initial

(20

BK

f3-actin

Figure

rats.

binding funcrat.

response

bacteriophage template in Perkin using serial twofold

conditions

Sprague-Dawley it with an increasing

for

the

band.

ucts for BK2 receptor and (3-actin increased in a linear manner from 25 to 40 cycles for BK2 and from 15 to 35 cycles for /3-actin. We then chose 35 cycles for BK2mRNA receptors and 25 cycles for (3-actin. As shown

((3-actin), 5A). and

system,

from

a control

K, values were were calculated determinations

The

of PCR Products

densitometric

PCR

for

and

of each

from

identity, between rat cDNA sequence

on glomer-

sponded to a decrease in the number of BK2 sites and thus probably indicates the stabilized tional status of the BK2 receptor of the adult

Relative

cycles

dilution

optimal

cDNA

Sequencing

of the

and values similar from the 5th week diminished response

cycles

defined manner

results are given in Figure 3. A progressive increase in PGE2 secretion in response to 10 nM of BK was observed from Week 3 to Week 12. This rise in BKinduced PGE2 secretion was parallel to those observed for the density of glomerular BK2 receptors. At the 38th week of postnatal life, a small decline was observed

to 50

increased the RT together,

PGE2 Release

effect

assessed

binding

the

data

achieve

amplified

3. Effect of 10 nM BK on PGE2 production by 1500 to freshly isolated glomeruli. Values are expressed as ± SE. Triplicate determinations of four separate gbsuspensions obtained from four rats were performed. 0.01, compared with the 3-week value.

The

between

synthesized from normal

C’l

tions in observed

0.06

±

through a 2% agarose gel, stained with ethidium and then quantitatively analyzed as dein “Methods.” Figure 4A shows a good rela-

densitometric To

55

*

1.34 ± 0.09

0.15

In ‘25l-(Tyr#{176})BK binding to glomerular membranes. The mean where K0 is the dissociation constant for ‘25l-(Tyr#{176})BK.The EC values of 1 5l-(Iyr#{176})BK used in the assay (2 nM). Values are means ± SE of triplicate

C.

Figure 2000 means meruli P
iO 1.37 ± 0.08 0.22 ± 0.05

0.42

competition

I-

8

0.37 ± 0.14 >10

+ (L)/K9),

C

AGE

0.12

±

>10

0.04

38

12

maturation

been 9 wk

known, for a long time, that during the of postnatal life in the rat, the functional of the nephrons progress from the jux-

85

Ontogeny

of Glomerulus

BK2 Receptor

100

C

75

C

C.-,

C

50

50

#{149} C I-

25 C

S 0

0.2

0.4

20

15

0.8

0.6

25

L

100

product

35

40

45

50

number

100

10

5

RI

30

Cycle

Dilution

15

20

Cycle

(tI)

25 30 number

35

40

Figure 4. Optimization of PCR conditions, (A) Comparison of densitometric signal against dilution of a PCR product (bacteriophage lambda DNA). The densitometric value (arbItrary units) is plotted against dilution. (B) The figure represents the relative band intensity of PCR product for BK-receptor and j3-ACTIN as a function of increasing amount of RT solution (0.5 to 8 ML). PCR amplification for the BK receptor and for f3-actln was performed, respectively, for 35 and 25 cycles. An arbitrary value of 100 was assigned to the sample containing 8 L of RT product. Each point is the mean ± SE of four independent experiments. (C) and (D) PCR amplification for, respectively, BK-receptor and J3-actIn mRNA. The data represents the relative band intensity as an increasing

function

of cycle

number.

For the BK receptor, an arbitrary value of 100 was assigned to the sample

50 cycles. For 3-actin, an arbitrary value ± SE of four independent experiments. tamedullary ontogeny reported ceptors mine-i

course, report postnatal

region

towards

the

of some renal in the rat, such (31), receptor

renal (33).

natriuretic oxytocin

of

cortex

BK-binding capacity ular PGE2 release. in cortical kallikremn an enhancement

thereby

of B2-

likrein activity homogenate

renal

vasoactive

intriguing.

The

expression with an

activation. been

not

in

primary

measured isolated

explanation

sys-

characterized of the gbomerincrease in the

The

fact

gbomerincrease with

and kal-

that

in whole-cortex gbomeruli can

is that

the

be

major

sites for kallikrein synthesis and secretion are the granular connecting cells and, to our knowledge, it is not possible to obtain such isolated cells: the cortical homogenate appears to be the best compromise available for assessment of the renal activity of the system. The relationships between kallikrein activity primarily

located

86

in the

distal

tubule

and

glomerular

B2-recep-

tor

assigned

amplified

over 40 cycles.

activation

cussed data

have

been

(2,3.24). It is well and in vitro studies

kallikrein

investigated of the renal

and a rise in BK-induced addition, we found an activity, which is consistent of in viva kinin generation

has and

a2-adrenodopa-

(32). (34),

In

receptor

been

receptors and, of system (35-37). The the knowledge of the

these

in the mRNA associated

The

has

of a1 and

tems to the renal kallikrein-kinin system at the cortical level. The maturation kallikrein-kinin system was essentially by an increase ular B2 receptor

(30).

systems peptide

the renin-angiotensin presented here extends maturation

outer

vasoactive as that

of 100 was

present

in

the

to the sample

Each

previously admitted support

distal

amplified

point

over

is the mean

extensively that the

dis-

the anatomical possibility that

tubule

may

act

up-

stream to its synthesis and secretion site through the release of kinin in the renal circulation and/or in the interstitial space. Only a few studies have reported age-related changes in the kalhikrein-klnin system; one in humans (38) and one in female rats (39). Both studies reported a decrease in urinary kallikrein excretion. One study in fetal and newborn lambs (40) reported an increase in urinary kallikrein excretion during the last trimester birth. plasma lation urinary occurs

of gestation and an even further These increases correlate well with aldosterone concentration, but a was also observed between the kallikrein excretion and the rise in during fetal and postnatal life. Two

rise after the rise in good correincrease in RBF, which

more studies focused on kallikrein mRNA expression nephron maturation. With Northern blotting, ents et al. (41) detected kallikrein mRNA in the

recent during Clemkidney

of 1-day-old rats, reaching a maximum expression at 60 days old. Using an in situ hybridization technique, El-Dahr and Chao (42) detected mRNA kallikrein expression in the upper limb of S-shaped bodies of 1-day-old rats. Moreover, the transition from newborn to adult

life

was

associated

with

Volume

a fourfold

7

‘

increase

Number

in

1 ‘1996

Bascands

A3

38

et a)

Weeks

10.5

*

*

0. C 4-I

3

5 AGE

8 12 (weeks)

38

3

5 8 12 AGE (weeks)

38

227bp

U

CV

.5 r ‘CIC

C.,

SI

.C SIC

Figure 5. Age-related changes In giomerular BK2-receptor mRNA expression. (A) Panel A represents typical ethidium bromide-stained agarose gels of PCR amplification for BK2-receptor mRNA and (3-actin mRNA. cDNA was synthesized from I of polyA+RNA derived for glomeruli suspensions obtained at each age. 5 l of the some cDNA preparation obtained for each age were amplified at the some time either for BK2 receptor or for (3-actin. (B) Panel B shows the ratio of BK to /3-actin of the densitometric values. Each point is the mean ± SE of four independent experiments. (C) Panel C represents the Southern blot analysis of RT-PCR products of BK2 mRNA after transfer and hybridization with a 32P-labeled probe. (D) Panel D represents the relative bond-Intensity values of the Southern blot analysis as a function of age. An arbitrary value of 1 was assigned to the sample representing the age of 3 wk. Each point Is the mean ± SE of four Independent experiments. P < 0.05, compared with the 3- week value. *

renal mRNA showed that tally regulated. crease in the

accumulation. Both of these studies the renal kallikrein gene is developmenSince we observed a progressive inrenal kallikrein activity during the first 8

wk of postnatal life, reaching a steady level up to the 38th week, our data are consistent with these observations and with the hypothesis that kallikrein plays a role in the maturation of renal functions. It is well known

that

(2-5) that can tion coefficient

BK

is

a natriuretic

reduce (Kf).

(20) Direct

ment of the B2-receptor glomerular hemodynaniics kinin-receptor antagonists,

and

the evidence activation

diuretic

peptide

glomerular ultrafiltrafor a tonic involve-

came which

in the control of from the effect of demonstrate that

BK increases RBF with minor changes in GFR (43). In addition, we recently showed (23) that BK induces in vitro contraction via B2 receptor activation, a possible explanation consistent with reduction of Kf previously reported (20). On the other hand, the early postnatal period is accompanied by an increase in both K (44) and single-nephron GFR (30). Although we do not demonstrate any direct uration and maturation report presented here,

link between of glomerular the increase

B2-receptor expression may be tional development of glomerular

B2-receptor matfunctions in the in the gbomerular

relevant with ultrafiltration

maturain the

rat. To our knowledge, no previous mented the maturation of the system regarding the BK-receptor

Journal

of the American

Society

work has yet docurenal kallikrein-kinmn mENA

of Nephrology

expression,

the BK binding on its receptor, and the functionality the BK receptor altogether. It is therefore difficult compare our results with those of others. However, is well-known that the investigated in parallel system. Many authors the renin-angiotensin

of angiotensin maturation reported that the plasma

system is often renin-angiotensin have studied the maturation of system and especially the role II (All) in nephrogenesis and postnatal of the kidney. Pohlova and Jelinek (35)

in rats

kallikrein-klnin

with

between

the

20

and

40

days

of age,

angiotensinogen concentration increases Wallace et al. (36) also reported an increase renin content throughout the fIrst 6 wk of

with age. in renal postnatal life. They plasma renin activity and at values.

of to it

3 wk of age, The maximum

also and

describe concentration

followed plasma

by

an

a decrease concentration

increase during

in birth

to

adult of All a). (45)

was not attained until 5 wk of age. Grima et found an increase in angiotensin-converting enzyme activities during 3 to 8 wk of age in rat lungs and aorta, but they noted a very large decrease in the renal cortex. Furthermore, a recent study (46) that used quantification of the ‘251-ISarl IAJI binding (autoradiography) reported a significant rise in All binding between Day 3 and the 8th week of postnatal life of Sprague-Dawley rats, with a spike at 2 wk. Taken together, these studies indicate a very early postnatal maturation in the glomerular All receptors, occurring almost in the first 2 wk of postnatal life, followed by a slight de-

87

Ontogeny

crease ter.

of Glomerulus

at the

BK2 Receptor

week,

8th

and

remaining

stable

thereaf16.

Because it is well-known that and the surface area of gbomeruli

the

protein

content

we

17.

normalized our results by expressing the number of BK2-binding sites either in fmol/mg gbomerular protein or in fmol / m2. Both types of expression gave the

18.

same

pattern

of evolution.

increase

Moreover,

with

such

are consistent with BK2-receptor mRNA and with functional studies showing the increase in PGE2 release by glomeruli in

age.

variations expression age-related response

19. to

BK stimulation. the postnatal

Taken maturation

together, our data indicate that of gbomerular BK2 receptors

20.

occurs during the postnatal

the first maturation

8 wk of life, previously

21.

renal

receptors

receptors

(34),

such and

as a-adrenoceptors All

which is similar to reported for other (31), oxytocin

(46).

22.

ACKNOWLEDGMENTS The Miss

authors E.

thank

Mann

Dr P. Wlnterton Casta#{241}o

is

supported

for

revising by a

the grant

English

from

version.

23.

ERASMUS

program.

REFERENCES 1. Anderson S. Brenner BM: Effects of aging on the renal glomerulus. Am J Med 1986:80:435-442. 2. Margolius HS: The kallikrein-kinin systems and the kidney. Annu Rev Physiol 1984:46:309-326. 3. Scicli AG, Carretero OA: Renal kallikrein kinin system. Kidney Int 1986:29:120-130. 4. Margolius HS: Tissue kallikreins and kinins: Regulation and roles in hypertensive and diabetic diseases. Annu Rev Pharmacol Toxicol 1989:29:343-364. 5. Margolius HS. Horivitz D, Pisano JJ, Keiser HR: Urinary kallikrein excretion in hypertensive man. Relationship to sodium intake and sodium-retaining steroids. Circ Res 1974;35:820-825. 6. Ader JL, Tran-Van T, Praddaude F: Reduced urinary kallikrein activity in rats developing spontaneous hypertension. Am J Physiol 1987:252 (Renal Fluid Electrolyte Physiol 21 ):F964-F969. 7. Girolansi JP, Praddaude F, Ader ,JL, Tran-Van T, Echer JP, Sue JM: Bilateral urinary kallikrein excretion in the Goldblatt hypertensive. Eur Heart J 1983:4:67-72. 8. Regoli D. Barabe J: Pharmacology of bradykinin and related kinins. Pharmacol Rev 1980:32:1-46, 9. Farmer 5, Burch RM: Biochemical and molecular pharmacology of kinin receptors. Annu Rev Pharmacol Toxicol 1992:32:5 11-536. 10. Bhoola KD, Figueroa CD, Worthy K: Bioregulation of kinins: Kallikreins, kininogens and kininases. Pharmacol Rev 1992;44:l-80. 11. Mc Eachern AE, Smelton ER, Bhakta S. et at.: Expression cloning of a rat B2 bradykinin receptor. Proc Nail Acad Sd USA 1991:88:7724-7728. 12. Hess JF, Borkowski JA, Young GS, Strader CD, Ransom RW: Cloning and pharmacological characterization of a human bradykinin (BK-2) receptor. Biochem Blophys Res Commun 1992:184:260-268. 13. Eggerickx D, Raspe E. Bertrand D, Vassart G, Parmentier M: Molecular cioning. functional expression and pharmacological characterization of a human bradykinm B2 receptor gene. Biochem Biophys Res Commun 1992:187:1306-1313. 14. Powell SJ, Slynn G, Thomas C. Hopkins B, Briggs I, Graham A: Human bradykinin B2-receptor: Nucleotide sequence analysis and assignment to chromosome 14. Genomics 1993:15:435-438. 15. Cox HM, Munday KA, Peat JA: Location of angiotensin and bradykinmn receptors in rat renal cortex epitheliai membranes lAbstracti. Chin Exp Hypertens 1984:A6:

88

24.

25.

26.

27. 28. 29.

30.

31.

32.

33.

34.

35.

36.

1893A. Fredrick MJ, Abel FC, Righsel WA, Muirhead EE, Odya CE: B2-bradykinin receptor-like binding in rat renomedullary interstitial cells. Life Sci 1985:37:331-338. Tomita K. Pisano JJ: Binding of l3Hl-bradykinin in isolated nephron segments of the rabbit, Am J Physiol 1984:246 (Renal Fluid Electrolyte Physiol 15):F732F737. Bascands JL, Pecher C, Cabos G. Girolami JP: B2-kinin receptor like binding in rat glomerular membranes. Biochem Biophys Res Commun 1989:158:99-104. Emond C, Bascands JL, Pecher C, et at.: Characterization of a B2 bradykinmn receptor in rat renal mesangial cells. Eur J Pharmacol 1990:190:381-392. Baylis C, Deen WM, Myers BD. Brenner BM: Effects of some vasodilator drugs on transcapillary fluid exchange in renal cortex. Am J Physiol 1976:230:1148-1158. Bascands JL, Pecher C, Rouaud S, et at.: Evidence for the existence of two distinct BK-receptors on rat mesangial cells. Am J Physiol (Renal Fluid Electrolyte Physiol 33) 1993;264:F548-F556. Bascands JL, Pecher C, Girolami JP: Indirect inhibition by bradykinin of cyclic AMP generation in isolated rat gbomeruli and mesangial cells. Mol Pharmacol 1993:44: 818-826. Bascands JL, Pecher C. Bompart G. Rakotoanvony J. Leung-Tack J, Girolanii JP: Bradykinin-induced in vitro contraction of rat mesangial cells via a B2 receptor type. Am J Physiol 1994;267 (Renal Fluid Electrolyte Physiol 36):F871-F878. Emond

C,

Bascands

JL,

Cabos-Boutot

G.

Pecher

C.

Girolami JP: Effect of changes in sodium or water intake on gbomerular B2-kinin-binding sites. Am J Physiol 1989:257 (Renal Fluid Electrolyte Physiol 26):F353F358. Emond C, Bascands JL, Rakotoarivony J. et at.: Gbmerular B2-kinin-binding sites in two-kidney, one clip hypertensive rats. Am J Physiol 1991:260 (Renal Fluid Electrolyte Physiol 29):F626-F634. Lowry OM, Rosebrough NJ. FaarAL, Randall RJ: Protein measurements with the Folin phenol reagent. J Blob Chem 193:1951:265-275. Chomczynski P. Sacchi N: Single step method of RNA isolation by acid guanidium thiocianate phenol chloroform extraction. Anal Biochem 1987:162:156-159. Makino R. Sekiya T, Hayashi K: Evaluation of quantitative detection of mRNA by the reverse transcriptionpolymerase chain reaction. Technique 1990;2:295-301. Saxnbrook J, Fritsch EF, Maniatis T. In: Ford N. Nolan C, Ferguson M, Eds. Molecular Cloning. A Laboratory Manual. 2nd Ed. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press: 1989. De Rouffignac C, Momnens L: Functional and morphologic maturation of superficial and juxtamedullary nephron in the rat. J Physiol (London) 1976:262:119129. Mc Caughran JA, Juno CJ, O’Malley E, Rosenthal M: The ontogeny of renal alpha 1 and alpha 2-adrenoceptors in the Dahl rat model of experimental hypertension. J Auton Nerv Syst 1986;17:1-20. Semmekrot B, Roseau S. Vassent R. Butlen D: Developmental patterns of renal atrial natriuretic peptide receptors: [12511-alpha-rat atrial natriuretic peptide binding in glomeruli and inner medullary collecting tubules microdissected from kidneys of young rats. Mob Cell Endocrinol l990;68:35-43. Kinoshita S. Felder RA: Ontogeny of DA1 receptoradenylate cyclase coupling in proximal convoluted tubules. Am J Physiol 1990:259 (Renal Fluid Electrolyte Physiol 28):F97l-F976. Schmidt A. Jard 5, Dreifuss JJ, Tribollet E: Oxytocin receptors in rat kidney during development. Am J Physiob 1990:259 (Renal Fluid Electrolyte Physiol 28): F872-F88 1. Pohbova I. Jeinek J: Components of the renin angiotensin system in the rat during development. PflUgers Arch 1974:351:259-270. Wallace KB, Hook JB, Baible MD: Post-natal develop-

Volume

7

Number

1 ‘1996

Bascands

ment

of the renin-angiotensin system in rats. Am J 1980:238 (Regulatory integrative Comp Physiol 7):R432-R437. Wilkes BM: increased angiotensin II receptor binding with age in isolated rat glomerubi. Kidney mt 1988:34:

kallikrein J Physiol F705-F71

Physiol

37.

43.

241-247.

38.

Ogimara T, Hata T, et at.: The effect urinary kallikrein excretion in normotensive and in patients with essential hypertension. Naka

T,

docrinob 39. 40.

41.

42.

Metab

of aging on subjects J Clin En-

of the American

Society

of Nephrology

45.

in response

Physiol

1988:258

F408-F414, Ichikawa

WH.

(Renal

during nephron maturation. Fluid Electrolyte Physiob

Carretero

Scicli

OA.

to a kinin

AG: Renal

analogue

Fluid

Electrolyte

DA,

Brenner

BM:

I, Maddox

of rat gbomerular

hemody-

antagonist.

(Renal

Am 31):

Am J

Physiol

Maturationab

ultrafiltration

in

31): de-

the rat. Am

J Physiob 1979:236 (Renal Fluid Electrolyte Physiob 31): F465-F471. Grima M. Websch C. Glesen-Crouse EM. Coquard C. Barthelmeb SM Imbs JL: Age-related variations in tissue

angiotensin 46.

1992:262 1.

Belrwaltes namics

velopment

1981:52:1023-1026.

Girolami JP. Corman B: Urinary kallikrein excretion in normotensive aging female rats. Proc Soc Exp Biol Med 1991:193:149-154. Robillard JE, Lawton WL, Weismann DN, Sessions C: Developmental aspects of the renal kallikrein like activity in fetal and newborn lamps. Kidney mt 1982:22:594601. Clements JA, Matheson BA, Funder JW: Tissue-specific developmental expression of the kallikrein gene family in the rat. J Blob Chem 1990:265:1077-1081. EL-Dahr SS. Chao J: Spatial and temporal expression of

Journal

44.

and its mRNA

et al

converting

enzyme

activities:

between spontaneously hypertensive and rats. J Hypertens 1990:8:697-702. Ciuffo GM, Viswanathan M, Seltzer AM, Saavedra JM: Gbomerular angiotensin II types during development of rat kidney. 1993:265 (Renal Fluid Electrolyte Physiol F271.

Comparison Wistar-Kyoto Tsutsuml

K.

receptor

subAm J Physiob 34):F264-

89