JONATHAN. LYTTON, and BARRY. M. BRENNER. Renal. Division,. Brigham and. Women .... kidney. (20-25), and. ANP, BNP, and CNP gene expression has also been identified ... of Nephrology. Copyright .... and the scar-free remnant.
J Am
Upregulation
of Atrial
Remnant
Kidney
Natriuretic
of Rats
with
Peptide
Gene
Reduced
Renal
Abstract.
Atrial
kidney
but
lated,
natriuretic
its
determine
(n
ANP
mRNA
natriuretic (BNP),
a family
of
(n
and
mainly
CNP
peptide C-type
circulating
in
heart
is present
peptides. (1-3)
and
in the
brain
ANP are
are
into
the
adrenal
and
vasoconstrictive system
natriuretic
present
reabsorption
(13-16).
Previous
glands
play
an
receptor
antagonist
important
role
peptides,
the several
of
NP
Mg2,
and
is a principal reports
(10,20-32).
CNP each has been detected ANP, BNP, and CNP gene
also
inner
have water
medulla
implicate
Immunoreactive
was
markedly
sham)
the
in rats
other
with
peptides
have
in the
adaptive
shown
that in
for
natriuretic kidney
ANP,
BNP,
as a and
in the human kidney (20-25), and expression has also been identified
dietary
Massachusetts.
diminished
to 7.6
maintained
hand,
no
shown
of
remnant
responses
sodium
diet.
At
downregulation
restriction.
been
(n
± 2. 1 -fold
on a low
significant
sodium
previously
was
Because
natriuretic
by us to play
a major
nephrons
role
to renal
mass
ablation, contribute
these data suggest that ANP of renal origin may to the overall mechanism for enhancing sodium
excretion
in the face
of declining
some
ways
within
the
ologic
roles
unclear.
analogous
thesis
was
local
However,
of locally
the
recent
reports
and
confirm
that
To
test
ablation,
the
Materials
and
are
renal
still
ANP
acetate
hypothesis
a manipulation
sodium
pathophysi-
syn-
(DOCA)
to be established whether in the kidney is responsive that
expression is regulated, in the remnant kidney
in fractional
in
system
peptides
in deoxycorticosterone
stimuli.
observations may exist,
renin-angiotensin natriuretic
rats (32), it remains of natriuretic peptides peptide expression
number.
physiologic
synthesized
upregulated
salt-treated expression
mass
to the
kidney.
Although
nephron
and rats (26-32). These an intrarenal NP system
intrarenal
na-
we examined after extensive
associated
the to
with
large
ANP renal increases
excretion.
Methods
Animals Animal Institutes
experiments were performed in accordance with of Health guide for the Care and Use of Laboratory
and
were
approved
ical
School.
by the Animal
Male
Received February 21, 1997. Accepted March 9, 1998. Correspondence to Dr. Kazuhito Totsune, Second Department Medicine, Tohoku University School of Medicine, I-I Seiryo-machi, Sendai 980-8574. Japan.
rats
Committee
to regular
rat chow
(5/6NPX)
groups,
formed
and
tap
surgical
in four
groups
two-thirds
rats (groups
L4s,
laparotomy
and
of rats
normal low
sodium sodium
closure
and
and
n
N4s, NaCI groups
deionized
5/6
renal
anesthesia as normal
control
groups
(50
was and mg/kg) tissue,
Animals
water
L14s,
L4x,
(corresponding
and
by and
underwent were
N14s, N4x, and N14x received drinking solution (corresponding L4s,
perNl4x;
of sham-operated
6, all groups)
only.
±
nephrectomy mass
N4x,
=
Med-
g (303
All rats were with access
L14x,
Four
N14s;
For
total
L4x,
of the abdomen
Groups 0.4%
intake); diet
(groups
to 365
were used. conditions
of 5/6
methohexital which served
N4s,
253
ad libitum.
of the left kidney. Ll4s,
into eight groups. sodium diet and
water ablation
National Animals
of Harvard
weighing
4 g, mean ± SEM) at the time of surgery housed under temperatureand light-controlled
infarcting of Internal Aoba-ku.
Care
Munich-Wistar
n = 7, all groups) under removing the right kidney,
1046-6673/090916 13$03.00/0 Journal of the American Society of Nephrology Copyright © 1998 by the American Society of Nephrology
Boston,
water
homeostasis
the
School,
a highly
by sodium and water moderate hyperglycetarget
on
triuretic mRNA
recep-
and using
response
versus
7,
physiologic
kidney,
to increase renal and to inhibit the PO
(HS-l42-1)
renin-
to NP
and
laboratory
characterized mellitus with failure (17-19).
kidney recent
this
in sodium
pathophysiologic settings retention, such as diabetes mia and congestive heart Although
Ca2,
from
the
to the
access
has been shown concentrations),
of Nat, studies
of
respect
gain
in glomeruli
tubular
NP
With
(NP)
predominantly
ANP, for example, flow, GFR (at higher
specific
(9).
peptides
actions
=
Mass
in the kidneys of humans raise the possibility that
BNP
secreted
(4,5),
latter
in
HIROKO TOTSUNE, M. BRENNER
Medical
observed
In norin the
natriuretic comprise
and
Harvard
4 d,
in remnant
(ANP), brain or B-type natriuretic peptide (CNP)
(10-12). plasma
source
assessed
is regu-
in Munich-Wistar rats. ANP mRNA expression
related
angiotensin-aldosterone
NP
gene
To
adrenal tumors (6), and is also produced by vascular cells (7,8). ANP has multiple effects, such as natriuresis (I), reduction of BP, and the inhibition of
salt-retaining
tors
was
in the
is unclear.
of the ANP
and
6, all sham-operated groups) (*P < and by 28.3 ± 5. 1 -fold at 14 d. This
=
structurally
distributed
the
‘s Hospital
is synthesized there
expression
test)
circulation. including endothelial promoting
Women
was significantly increased by 5.0 ± 0.8-fold ± SEM) at 4 d when compared with sham-
operated controls 0.001 by Scheffe’s
Atrial peptide
(ANP)
expression
5/6 nephrectomy intake groups,
kidney mean
7,
peptide
and
significance
renal
renal
Brigham
physiologic
whether
kidneys after mal sodium remnant
Division,
Expression
Renal
KAZUHITO TOTSUNE, HARALD S. MACKENZIE, JULIA L. TROY, JONATHAN LYTTON, and BARRY
Soc Nephrol 9: 1613-1619, 1998
Ll4x
divided a low to
received
to low
sodium
a
1614
Journal
intake). and
Systolic
24-h
of the American
BP was
urine
cages
1 d before
N4s, were
and N4x) anesthetized
was
maintained
cDNA
measured
of Nephrology
by the
indirect
samples
were
collected
using
kidneys
were
harvested.
After
or
14 d (groups (methohexital.
harvested. In 5/6NPX leaving a wide margin remnant
Society
L14s, L14x, 50 mg/kg)
groups. the scarred of healthy-appearing
snap-frozen
in liquid
at -80#{176}Cuntil
nitrogen.
RNA
J Am
tail-cuff
method,
individual
above.
metabolic
4 d (groups
ANP
published
primers
fragment.
used
L4x,
stained
rats were
transluminator,
and
ing
scanner
Primers
primer
designed
to ensure
that only
Primers
used
amplification
for
(G3PDH)
respectively
All PCR 2 mM
(34).
mixes
MgCl-,,
RNA
method.
Then,
volume this
analysis
using
(Figure
1). Due
kidney
tissues
were
0.001%
according were
designed
to amplify
spanned
of
of ANP rat
corresponded
to nucleotides gave
primers
gelatin,
of
was amplified.
cycles and
extracted
10 tg
mM
pH 8.3, of
each
by the guanidinium
of total
murine Gaithersburg,
strand
eDNA
at 95#{176}C for then
ments reverse
RNA
leukemia MD),
was
1 mm.
72#{176}C for 5 mm
were performed transcriptase.
was
One
Packard)
of
size
Health
by Menzo
(35). at high
with
70#{176}C for 3 mm
For ANP
cDNA
and
Image
1 .54
software
compared
of ethidium densities were
with bromide adjusted
et a!. (37).
2
3
4
5
6
7
8
9
10
11 12 13
-+
Negative
CRS-DNA
control
-+
CRS-DNA concentration
0.2
(xl 0 molecules tube)
30
0.78
3.12
0.39
I
12.5
1 .56
50
6.25
25
100
B
extension): experi-
100
eDNA or omitting ( 1:320) 1 St strand
z 0 0
a) 0.
>
0
of PCR Products normal rat right
Sequencing RNA
of total
ci
from
atrium
and
ci
respectively, the respective
z
Applied
ligated into pGEM-T by an autosequencer
1
0 (/)
products were size-fractionated by gel electrophoresis and specific ethidium bromide-stained bands visualized
under ultraviolet light were excised, (Promega. Madison, WI), and sequenced
a: 0
r=0.95
0
vector (model
ci ci
Biosystems). 10
PCR competitive
tamed fortuitously used as an internal shorter
than
the
reference
objective
PCR
PCR using this clone
as template.
agarose gel, and the competitive quantitative
purified PCR
amounts scheme)
eDNA
was
standard
when the 357-bp PCR control. The CRS-DNA product.
added
of CRS-DNA were added,
to
was
The
nucleotide
DNA was as follows. tubes.
ob-
sequence
corresponding to nucleotides was deleted (data not shown). the PCR
PCR
(CRS)-DNA
product was subcloned and measured 186 bp, 171 bp
product
was
separated
used as CRS-DNA Constant amount To
each
tube,
of 153 After on
in the of wildincreasing
(0.2 to 100 X l0 molecules in a 1:2 dilution and all tubes were subjected to PCR as described
1. A typical
Figure
example
of a competitive
Electrophoresis
of a competitive
ing
amounts
increasing
of
DNA. Lane 1 , Standard products using 800 ng CRS-DNA/
tube
omitted
PCR.
in RT
(B)
plotted test equal
on the (i.e.
X-axis:
to standard. ,
ratio
12,
the Y-axis point
I ) is shown
standard
Reverse was
used
of the method
The
(A) us-
(CR5)-
Lanes 2 through I I , PCR and increasing amounts of
13. Water
samples.
PCR. (RT)-PCR
reference
DNA ladder. total RNA Lane
quantitative
transcription
competitive
Lane
in the test
DNA
reverse
as templates.
reaction.
An example
concentration
of
100
(xl o molecules/tube)
CRS-DNA
this clone revealed that a fragment to 324 of the rat ANP eDNA (36)
type
the
4 M1 of
as follows:
(annealing
extension).
1000
(Life in a total
PCR,
stringency
by substituting water for For G3PDH, 4 p.1 of diluted
and
Quantitative For ANP,
us-
and
computer. The densitometric
of the CRS-DNA
decreased incorporation and CRS-DNA band
digitized
KCI,
isothiocyanate-CsC1
kidney for ANP, or normal kidney tissue for G3PDH, was reverse-transcribed and subjected to PCR using
373A,
Hewlett
10
microgram
primers. PCR (2% agarose),
IIcx,
Institutes
established
1
extension).
Subcloning
was
deoxynucleotide
reverse-transcribed
previously
(final
an ultraviolet
photograph
(Hewlett Packard) on a desktop and enhanced before performing
to the smaller
Lane
product.
50 mM
virus reverse transcriptase using an oligo(dT) primer
amplified
with
The
A
cDNA was amplified at high stringency as follows: 25 cycles at 94#{176}C for 15 5, 64#{176}C for 0.5 mm. 72#{176}C for I mm: and then 72#{176}C for 5 mm (final
(Scanlet
visualized
PCR
5 to 30 and 964
rise to a 983-bp
Tris-HCI,
0.2
bromide,
the
electrophoresis,
glyceraldehyde-3-phosphate
These
10 mM
point, gel
a
the junction
mRNA
concentration
polyacrylamide
photographed.
National
by the equation
to sequences
ANP DNA, and the antisense primer of exons 2 and 3. The primers were
of 50 p1, as described
first
flatbed
The
(28-mer)
the eDNA
contained
was
U of Moloney Technologies,
a
equivalent a 5%
I .3 p.M ethidium
DeskScan 11 software image was inverted
triphosphate. 0.25 j.M of each primer, and 0.4 U Taq DNA polymerase (Pharmacia, Piscataway. NJ) in a total volume of 20 p.1. Total cellular
with
portions were excised, tissue, and the scar-free
synthesized
(33).
sense
exons 1 and 2 of rat genomic (33-mer) spanned the junction
to 987,
on
L4s,
extraction.
were
ci’ al.
The
dehydrogenase
the
run
the wild-type eDNA, was taken into account,
by Chen
357-bp
determine were
Nl4s, and Nl4x), and the kidneys
Amplification
The
To
products
Soc Nephrol 9: 1613-1619, 1998
used Concentration represents
at which
to the
densities
by the arrows.
transcriptase
was
as template
in the
determine of standard ratio
eDNA DNA is
of densities were
considered
of
J Am Soc Nephrol
The
results
(X-axis) the
9: 1613-1619.
are
versus
wild-type
chart.
plotted
the ratio band
The
point
ANP Gene
998
as
the
concentration
of the corrected
densities
where
(Y-axis)
the ratio
of
CRS-DNA in a full
was
equal
the
fe’s
CRS-DNA
band
densities
logarithmic
to
concentration
in each
sample
to correct
the initial amounts of the total RNA of G3PDH, a competitive reverse formed
using
CRS-DNA.
983-bp
PCR
product
which was
possible
obtained
subcloned
and
variations
fortuitously
measured
in
when
720
bp,
insert
and wild-type
in G3PDH gel
of the
and
greatest for
bp
stress
and
263
i.e.
subjected
with
ANP
mRNA
mean
value
G3PDH mole
aliquots
for each
of ANP
were
from
each
RT-PCR mRNA
tnRNA
reaction. levels
was
sample
was
I 7%
(ii
which
reported
=
were
assayed
Intra-assay
was
concentrations,
concentrations,
RNA
G3PDH
variation
of the
normalized
in micromoles
were
method
mRNA
before
performed data
analysis
Profiles
1.
SEM.
for multiple
concentration
distribution
±
to
per
were
used
Na
at day
ANOVA,
in the present
I
of basic followed
appropriate.
by ANP
to obtain
normal
followed
by Schef-
of
rats
among
the
groups.
0.05.
this
rates
This
30
eDNA
sequence
