Proc. Natl. Acad. Sci. USA
Vol. 96, pp. 7403–7408, June 1999
The natriuretic peptide clearance receptor locally modulates the
physiological effects of the natriuretic peptide system
(gene targeting?gene ‘‘knock out’’?guanylyl cyclase activity?urine osmolality?bone metabolism)
NAOMICHI MATSUKAWA*†, WOJCIECH J. GRZESIK‡, NOBUYUKI TAKAHASHI*, KAILASH N. PANDEY§, STEPHEN PANG¶,
MITSUO YAMAUCHI‡, AND OLIVER SMITHIES*?
*Department of Pathology and Laboratory Medicine and‡Dental Research Center, University of North Carolina, Chapel Hill, NC 27599-7525;§Department
of Physiology, Tulane University School of Medicine, New Orleans, LA 70112; and¶Department Anatomy and Cell Biology, Queen’s University, Kingston, ON,
Canada K7L 3N6
Contributed by Oliver Smithies, April 26, 1999
in heart [atrial (ANP) and B-type (BNP)], brain (CNP), and
kidney (urodilatin), decrease blood pressure and increase salt
excretion. These functions are mediated by natriuretic peptide
receptors A and B (NPRA and NPRB) having cytoplasmic
guanylyl cyclase domains that are stimulated when the recep-
tors bind ligand. A more abundantly expressed receptor
(NPRC or C-type) has a short cytoplasmic domain without
receptor, although it may have additional functions. To test
how NPRC affects the cardiovascular and renal systems, we
inactivated its gene (Npr3) in mice by homologous recombi-
nation. The half life of [125I]ANP in the circulation of ho-
mozygotes lacking NPRC is two-thirds longer than in the wild
type, although plasma levels of ANP and BNP in heterozygotes
and homozygotes are close to the wild type. Heterozygotes and
homozygotes have a progressively reduced ability to concen-
trate urine, exhibit mild diuresis, and tend to be blood volume
depleted. Blood pressure in the homozygotes is 8 mmHg (1
mmHg ? 133 Pa) below normal. These results are consistent
with the sole cardiovascular?renal function of NPRC being to
clear natriuretic peptides, thereby modulating local effects of
the natriuretic peptide system. Unexpectedly, Npr3 ???
homozygotes have skeletal deformities associated with a con-
siderable increase in bone turnover. The phenotype is consis-
tent with the bone function of NPRC being to clear locally
synthesized CNP and modulate its effects. We conclude that
NPRC modulates the availability of the natriuretic peptides at
their target organs, thereby allowing the activity of the
natriuretic peptide system to be tailored to specific local
Natriuretic peptides (NPs), mainly produced
The natriuretic peptides (NPs) play important roles in cardio-
vascular homeostasis. Three isoforms, atrial natriuretic pep-
tide (ANP), B-type natriuretic peptide (BNP), and C-type
natriuretic peptide (CNP), constitute the natriuretic peptide
family (reviewed in ref. 1). ANP and BNP are mainly produced
in the cardiac atria and ventricles, respectively; both are
present in the circulation; and they directly influence blood
pressure and body fluid homeostasis (reviewed in ref. 2). CNP
is most strongly expressed in the brain but also is produced in
vascular endothelial cells and in other tissues; its normal level
in the circulation is very low; and it may have a paracrine?
autocrine role. The biological functions of the natriuretic
peptides are mediated by two receptors, natriuretic peptide
receptor A (NPRA) [also known as guanylyl cyclase (GC) A]
(3) and NPRB (GC-B) (4), which have cytoplasmic GC
domains that are stimulated when the receptors bind ligand.
NPRA responds to ANP and, to a 10-fold lesser degree, to
BNP; NPRB responds primarily to CNP. NPRA is strongly
expressed in the vasculature, kidneys, and adrenal glands, and
its stimulation mediates vasorelaxant and natriuretic functions
and decreases aldosterone synthesis. NPRB is strongly ex-
pressed in the brain, including the pituitary gland, and may
have a role in neuroendocrine regulation. A third natriuretic
peptide receptor (NPRC) has only a short cytoplasmic domain
with no GC activity; it is generally thought to act as a clearance
receptor and remove natriuretic peptides from the circulation
(5), although several reports have suggested roles in addition
to this clearance function (reviewed in ref. 6). NPRC interacts
with all three natriuretic peptides in the order ANP ? CNP ?
BNP (7). NPRC is the most widely and abundantly expressed
natriuretic peptide receptor with a tissue distribution that
includes many but not all tissues that express a guanylyl cyclase
receptor; for example, kidney glomeruli (8) strongly express
both NPRA and NPRC whereas Leydig cells in the testis
strongly express NPRA but not NPRC (9). To gain a better
understanding of the relationship between the receptors and
their ligands and to test how NPRC affects the cardiovascular
and renal systems, we have inactivated its gene (Npr3) in mice
by homologous recombination. We find strong evidence that
NPRC, in addition to being involved in the systemic clearance
of circulating natriuretic peptides, plays an important role in
controlling local effects of the natriuretic peptide system. Its
complete absence produces unexpected skeletal abnormali-
MATERIALS AND METHODS
Gene Targeting. Portions of Npr3 (the mouse gene coding
for NPRC) were cloned from mouse strain 129 genomic DNA
fragments by using a probe based on the mouse Npr3 exon 1
sequence (D. G. Lowe, personal communication). The 5?
region of homology in the targeting construct (Fig. 1a) was a
5.9-kilobase (kb) XbaI-SpeI fragment from upstream of exon
1. The 3? homology region was a 0.7-kb XhoI-HindIII fragment
that includes parts of exon 1 and intron 1. Four electropora-
tions of strain 129 embryonic stem cells were carried out as
described (10). Candidate targeted clones were identified by
PCR amplification using primer C (5?-ACGCGTCACCTTA-
ATATGCG-3?) and primer D (5?-TCGGCTCCTTCCTC-
TATCTA-3?) (Fig. 1a). Targeting was confirmed by the pres-
ence of a 6.3-kb hybridizing band in addition to an 8-kb
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PNAS is available online at www.pnas.org.
Abbreviations: ALP, alkaline phosphatase; ANP, atrial natriuretic
peptide; BNP, B-type natriuretic peptide; GC, guanylyl cyclase;
NPRA, natriuretic peptide receptor A; kb, kilobase.
†Present address, Department of Geriatric Medicine, Osaka Univer-
sity Medical School, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan.
?To whom reprint requests should be addressed at: Department of
Pathology, CB 7525, Brinkhous-Bullitt Building, University of North
Carolina, Chapel Hill, NC 27599-7525. e-mail: email@example.com.
endogenous band in Southern blots of DNA digested with
HindIII and hybridized to probe A (a 0.7-kb BglII-SpeI frag-
ment) (Fig. 1b). Chimeric mice carrying the nonfunctional
allele were generated and mated to C57BL?6 females to yield
F1 heterozygotes, which were intercrossed to obtain Npr3
???, ???, and ??? F2 animals for use in the present studies.
Animals were handled under University of North Carolina-
Photoaffinity Labeling of ANP Receptors. Plasma mem-
branes (200 pg) were incubated with 4-azidobenzoyl [125I]ANP
as described (11). After photolysis, samples were washed twice
and were subjected to SDS?PAGE. The receptor bands were
localized by autoradiography.
ANP Clearance Measurements. [125I]ANP(rat; 1–28) was
rapidly injected (0.2 ?Ci?mouse) into the jugular vein; blood
was collected from the carotid artery as published (12) 0.5, 1.5,
Plasma (25 ?l) separated from the blood samples (60 ?l) was
precipitated with 125 ?l of ice-cold 10% trichloracetic acid.
125I-radioactivity in the pellets was determined by ?-scintilla-
tion. The residual counts at 16 min represent trichloroacetic
acid-soluble radioactivity trapped in the pellet and were
subtracted from the other values before calculations. The data
were normalized to 106cpm injected per animal. The P value
of ??? versus ??? was calculated by analysis of covariance.
Radioimmunoassay for ANP and BNP. The ANP assay was
with a published protocol (13) using a rabbit anti-ANP anti-
serum (Phoenix Laboratories, Belmont, CA). The BNP assay
was developed for this study. Synthetic mouse BNP conjugated
to bovine thyroglobulin was used to immunize sheep. The
resulting anti-mBNP antiserum cross-reacts 0.01% with rat
BNP and 0% with rat ANP[1–28], CNP-22, arginine vasopres-
sin, and angiotensin II.
Blood Pressure Measurements. Blood pressures, measured
in conscious young adult male mice aged from 3 to 4 months
by a noninvasive computerized tail-cuff method (14), were the
means of at least six measurement sessions on each of 5 days.
Animals were fed regular chow.
Biochemical Examination of Peripheral Blood and Urine.
Blood samples were drawn from the retroorbital sinus from
aged-matched anesthetized (Avertin, 2.5%, 0.3 ml?25 g body
wt) male mice of the three genotypes and were analyzed with
an Ektachem DT60II Analyzer (Johnson & Johnson, Roch-
ester, NY). Hematological determinations were with a Cobas
Micro Hematology Analyzer (Roche Diagnostics).
For urine studies, mice were maintained on a 12-hour
light?dark cycle in metabolic cages with free access to water
and food. On day 3, 24-hour water intake, urine excretion, and
urinary electrolytes were measured. Urinary cGMP was mea-
sured as described (15). All values were normalized to a body
weight of 30 g. To test ability to dilute urine, each mouse was
gavaged with a volume of water equal to 4% of its body weight.
Body weight and ensuing urine osmolalities were measured at
60 and 120 min. To test ability to concentrate urine, animals
were placed in cages without food or water for 12 hours, and
urine osmolalities were measured.
X-Ray and Histological Examination. Radiographs of mice
were taken with soft x-rays. For histology, mice aged 10 days
and 2 months were killed, were fixed in 4% paraformaldehyde
in PBS, and were decalcified in 10% EDTA, and samples were
embedded in paraffin. Five-micrometer sections were stained
for tartrate-resistant acid phosphatase as published (16) and
with hematoxylin. Some sections of bone were rehydrated and
stained with 0.1% Sirus Red (Sigma-Aldrich, Milwaukee, WI)
in saturated aqueous picric acid (pH 2.0) for 30 min. After a
brief wash with 0.01 M HCl, sections were dehydrated, were
mounted in synthetic resin, and were photographed by using
polarized light (17).
In Situ Hybridization. A 522-bp fragment from exon 1 of
Npr3 was amplified by PCR with the primers 5?-GCG-
TAGCGTGGAGGGCAAT-3? and 5?-CTGCCTTGGATG-
TAGCGCACTAT-3? and was inserted into pBluescript II
KS(???) plasmid (Strategene) for transcription of either a
sense or antisense
bones from 10-day-old mice were cut into 10-?m sections.
Hybridization was overnight at 50°C, and washes were at 65°C.
Slides were dipped in emulsion, were exposed for 2 weeks, and
were counterstained with hematoxylin and eosin.
Bone Marrow Cell Culture. Bone marrow stromal cell
cultures (18) were established with bone marrow from long
bones of 2-month-old ??? and wild-type mice. After 8 days in
culture, cells were evaluated for alkaline phosphatase (ALP)
activity by using a commercial kit (Sigma-Aldrich). ALP-
positive colonies were counted under low power magnification
on 20 randomly chosen fields, and the numbers of cells in 100
consecutive colonies were counted. Osteoclast formation in
vitro was assayed as described (19) after staining cultures for
tartrate-resistant acid phosphatase. Positively staining cells
35S-labeled riboprobe. Frozen vertebra
strategy. (Top line) The region of the Npr3 gene that includes exon 1
(black bar). (Middle line) The targeting construct. (Bottom line) The
targeted locus in which the gene is disrupted and from which 215
amino acids of the ligand-binding domain have been deleted. neo,
neomycin resistance gene; tk, Herpes simplex thymidine kinase gene.
Restriction sites are H, HindIII; N, NotI; S, SpeI; Xb, XbaI; Xh, XhoI.
The positions of two probes, A and B, and of two primers, C and D,
are indicated. (b) Southern blots of tail DNA from wild-type (???),
heterozygous (???), and homozygous mutant (???) mice digested
with HindIII and hybridized to probe A. The sizes of the hybridizing
bands in kb are shown. (c) An autoradiogram showing the presence or
absence of the 70-kDa band corresponding to NPRC after SDS gel
electrophoresis under reducing conditions of 4-azidobenzoyl
[125I]ANP photoaffinity-labeled lung plasma membranes from ???
and ??? mice. The 135-kDa radio-labeled band corresponding to
NPRA is indicated.
Targeted inactivation of the Npr3 gene. (a) The targeting
7404Genetics: Matsukawa et al.Proc. Natl. Acad. Sci. USA 96 (1999)
with three or more nuclei (osteoclasts) were counted under
high power magnification on 20 randomly chosen visual fields.
The number of nuclei in 100 consecutive cells was counted.
Bone Resorption. To assess bone resorption, urinary excre-
as described (20). Values were normalized by the urinary
Statistics. Except when indicated, analysis of variance and
pairwise comparisons were by the Bonferroni method. In some
comparisons, the data were analyzed by P-stat, a correlation?
permutation test in which the null distribution is approximated
by Monte Carlo sampling. Information about P-stat and a
program for its execution are available from its originator,
W. R. Engels (Genetics Department, University of Wisconsin,
Madison, WI 53706; http:??www.wisc.edu?genetics?CATG?
RESULTS AND DISCUSSION
Generation of Mutants. We used homologous recombina-
tion in embryonic stem cells to generate animals in which the
Npr3 gene was disrupted and partially deleted (Fig. 1 a and b).
The resulting heterozygotes (???) survived and reproduced
normally. The homozygous (???) mutants survived to birth,
but about half died before weaning; about one-third of the
survivors reproduced at least once. Ligand-binding analysis on
lung membrane extracts demonstrated the complete absence
of functional NPRC in Npr3 ??? animals but the continuing
presence of NPRA (Fig. 1c). The amounts of the two guanylyl
cyclase receptors (NPRA and NPRB) assessed by semiquan-
titative binding studies with isolated lung and kidney mem-
indistinguishable in the three genotypes (data not shown).
Clearance of ANP. The effect of absence of NPRC on
clearance of natriuretic peptides from the circulation was
assessed by measuring the disappearance of injected
[125I]ANP. The results (Fig. 2a and Table 1) show that ANP
half-life in Npr3 ??? mice is two-thirds longer than in
wild-type mice (P ? 0.0001), demonstrating that NPRC plays
a significant role in its clearance from the circulation. This
of [125I]ANP was reduced when NPRC was blockaded with a
truncated ANP (12). When ANP clearance decreases, its
steady state plasma level should increase unless ANP produc-
tion changes. However, in our Npr3 ??? and ??? mice, the
steady state levels of ANP and BNP were not higher than in the
wild type (Table 1); in fact, they tended to be lower than
normal, although the difference did not reach significance.
Two important inferences can be drawn from these measure-
ments: first, that some factor(s) in the ??? and ??? mice
have probably induced a homeostatic decrease in the cardiac
secretion of the two peptides; and second, that any changes in
the Npr3 ??? and ??? animals that we observe are not
caused by increased levels of ANP or BNP in the circulation.
Circulatory and Renal Effects. Blood pressures in the
homozygous mutants were significantly lower by 8 mmHg (1
mmHg ? 133 Pa) than in the wild type; blood pressures in the
heterozygotes were not significantly lower (Table 1). No
significant effects of genotype were observed on plasma Na?,
K?, Ca2?, Cl?, creatinine, and urea nitrogen concentrations or
on daily urinary excretion of Na?, K?, Cl?, creatinine, or
protein (data not shown). However, a substantial and progres-
sive increase in the ??? and ??? mice relative to the wild
type was seen in their total daily urine output, and their water
intakes tended to increase (Table 1), suggesting alterations in
renal function. Visual inspection of the Npr3 ??? mice shortly
after birth suggested that they were dehydrated. Hematocrits,
hemoglobin levels, and red blood cell counts of adults (Table
1) also increased progressively and highly significantly in the
??? and ??? mice relative to the wild type, indicating that
a reduced amount of NPRC in the heterozygotes or its absence
in the homozygotes decreases intravascular volume. As indi-
cated above, this would be expected to induce a decrease in the
cardiac secretion of ANP and BNP in the Npr3 ??? and ???
Because both NPRA and NPRC are strongly expressed in
renal glomeruli (8), if NPRC is acting in the kidney as a
clearance receptor, reduced amounts or absence of glomerular
NPRC should expose the glomerular NPRA to progressively
greater local concentrations of natriuretic peptides, thereby
enhancing its guanylyl cyclase activity and increasing cGMP in
the glomerular filtrate and in urine. To test this expectation,
we determined the total daily excretion of cGMP in the urine
of Npr3 ???, ???, and ??? mice. We found that a markedly
increased cGMP excretion does indeed occur in the Npr3 ???
and ??? mice (Table 1), being respectively ?200 and ?300%
normal. Because the levels of ANP and BNP in the circulation
of the ??? and ??? mice were not greater than in wild-type
trichloroacetic acid precipitable radioactivity after injection of [125I]
rat ANP (1–28) into wild-type (n ? 5, filled circles) and homozygous
mutant mice (n ? 4, open circles). The plotted points (log cpm) are
means ? SE of logarithms of trichloroacetic acid-precipitable counts
normalized as described in Materials and Methods. Where not shown,
error bars are smaller than the symbols. ?, P versus wild type ? 0.0001.
(b) Ability to dilute and concentrate urine assessed by loading and
depriving water; wild-type (n ? 7, filled circles), heterozygous (n ? 6,
open triangles), and homozygous mutants (n ? 4, open circles).
Urinary osmolalities (Uosm) at time 0, and 1, and 2 hours after 4%
body weight water loading by gavage, and after 12 hours of water
deprivation, are shown as means ? SE in mOsm?kgH2O.†, P versus
wild type ? 0.05;‡, P versus wild type ? 0.02.
Clearance of ANP and water handling. (a) Decline in
Table 1.Physiological and biochemical data
1.44 ? 0.05
114.5 ? 5.9
21.6 ? 3.8
118.7 ? 1.9
49.9 ? 1.0
15.6 ? 0.2
9.7 ? 0.1
3.0 ? 0.2
1.1 ? 0.1
ANP half-life, min
Plasma ANP, pg?ml
Plasma BNP, pg?ml
Water intake, ml?day
Urine output, ml?day
Data are means ? standard error using at least four males. N.D., not
determined; BP, blood pressure; Hct, hematocrit; HGB, hemoglobin;
RBC, red blood cells.
*P versus ??? ?0.0001.
†P versus ??? ? 0.8.
‡P versus ??? ?0.05.
§P ? 0.01 by a correlation?permutation test (P-stat, see Materials and
Methods) for effect of genotype on variable.
¶P ? 0.0001, also by P-stat.
2.40 ? 0.08*
88.5 ? 9.0†
22.1 ? 1.9†
110.4 ? 2.3‡
53.6 ? 0.9§
16.7 ? 0.2§
10.4 ? 0.1§
3.9 ? 0.3
2.0 ? 0.1¶
89.5 ? 13.4
23.4 ? 4.9
118.0 ? 1.8
51.8 ? 0.9§
16.2 ? 0.2§
10.1 ? 0.3§
3.2 ? 0.4
1.4 ? 0.1¶
3.2 ? 0.3 6.5 ? 0.6‡
10.9 ? 1.4*
Genetics: Matsukawa et al. Proc. Natl. Acad. Sci. USA 96 (1999)7405
mice, this result clearly demonstrates that decreasing NPRC
substantially increases the renal effects of systemically deliv-
ered and?or locally synthesized natriuretic peptides. This in
turn would be expected to alter the ability of the mice to
concentrate their urine. We tested this expectation by com-
paring the urine osmolalities of the ???, ???, and ??? mice
(Fig. 2b) and found that all genotypes can excrete dilute urine
after water loading but that the ??? and ??? mice were
progressively and significantly less able than wild-type mice to
Interpretation of Renal and Hypotensive Effects. In the
kidney, ANP and BNP are derived from the circulation
whereas urodilatin is derived from cells of distal tubules.
NPRA is expressed strongly in the renal vasculature and
glomerular podocytes and in medullary structures of the
kidney. NPRC is expressed in endothelial and vascular smooth
muscle cells throughout the circulation and is strongly ex-
pressed in glomerular podocytes, to a lesser degree in glomer-
ular mesangial cells, and minimally in medullary interstitial
cells (2, 21–23). In interpreting the effects of a decreased level
or absence of NPRC, it is important to recollect that the levels
of the natriuretic peptides in the systemic circulation and of the
guanylyl cyclase receptors in the kidney are essentially normal
in the Npr3 ??? and ??? mice. We therefore infer that the
observed renal effects of decreasing NPRC are not caused by
changes in the systemic levels of the natriuretic peptides.
Rather, they are caused by local increases in the concentration
of the peptides and possibly also by the effects of increased
cGMP in the glomerular filtrate (24). We presume, but at
present cannot prove, that the concentration of natriuretic
peptides in the circulation in and downstream of the glomeruli
will be less decreased than normally (i.e., increased relative to
normal). As a result, the known effects of ANP on the
structures exposed to glomerular and post-glomerular blood
flow are likely to occur: namely, an increase in filtered volume
and a decrease in water reabsorption (2, 25). In contrast, the
effects of urodilatin produced in the distal tubules are unlikely
to be affected because NPRC is not normally present in
significant amounts in the distal nephron.
Our finding that the Npr3 ??? and ??? mice maintain
their ability to dilute urine indicates that their defective ability
to concentrate urine is unlikely to be caused by impairment of
salt transport in the thick ascending limb. Anatomical inspec-
tion of the Npr3 ??? and ??? mice also indicates that the
deficiency in urine concentration is not caused by a shortening
of the renal papilla in these genotypes. Accordingly, the
of higher than normal post-glomerular concentrations of
natriuretic peptides in the blood and of cGMP in the ultrafil-
We interpret the hypotensive effect of the absence of NPRC
as being caused by a decreased blood volume resulting from
the observed diuresis or from a locally increased concentration
of the natriuretic peptides in endothelial and vascular smooth
muscle cells in the systemic vasculature. [The latter would
likely lead to vasorelaxation and?or to a shift of fluid from the
intravascular to the interstitial compartment, with a resulting
decrease in plasma volume (2).]
We conclude that NPRC in the kidney and other tissues
normally decreases in a ‘‘dose-dependent’’ manner the local
concentration of systemically delivered or locally synthesized
natriuretic peptides, thereby modulating and helping to com-
partmentalize their physiological effects. We suggest that
human genetic variations (if they exist) that modestly decrease
but do not abolish NPRC formation could have beneficial
cardiovascular effects under some circumstances whereas ge-
netic variations that increase NPRC formation could be det-
rimental. For example, hypertensive tendencies might be ame-
liorated by decreases in intravascular volume that accompany
decreases in NPRC or might be exaggerated by increases in
NPRC expression. Likewise, a genetic decrease (or increase)
lead to cardiac hypertrophy because absence of NPRA in mice
causes exaggerated and pathological cardiac hypertrophy (26).
A search in humans for genetic variations affecting the ex-
pression of the gene coding for NPRC therefore appears to be
Skeletal Effects. Unexpectedly, the ??? mice completely
lacking NPRC exhibit striking skeletal abnormalities, recog-
nizable 1 week after birth, including hunched backs, dome-
shaped skulls, and elongated tails that, in some mice, were
initially wavy (Fig. 3a). They had elongated femurs, tibias,
metatarsal, and digital bones, longer vertebral bodies, in-
creased body length, and decreased weight (Fig. 3 b–f). The
thoracic cages of the ??? mice were smaller than the wild type
and were constricted. Development of secondary ossification
centers in long bones was delayed in the ??? mice (Fig. 4 a
and b). In the cartilage growth plates of 10-day-old ??? mice,
cellular expansion was apparent in the zone of hypertrophic
chondrocytes although not in the zones with resting or prolif-
erating chondrocytes (Fig. 4 c and d). This difference was no
longer apparent in 3-month-old mice. The amino acid com-
positions of the organic bone matrix collagen in the wild-type
and ??? mice were virtually indistinguishable (data not
Wild-type (left) and homozygous mutant mice (right) at 2 months of
age. (b–e) Soft x-ray analysis of phalanges [wild type (b) and homozy-
gous mutant (c)] and lower bodies [wild type (d) and homozygous
mutant (e)] of mice at 3 months of age. (f) Body and bone dimensions
of homozygous mutants at 3 months of age as percent difference from
the wild type. ?, P versus wild type ? 0.001.
Skeletal phenotypes of Npr3 ??? and ??? mice. (a)
7406Genetics: Matsukawa et al. Proc. Natl. Acad. Sci. USA 96 (1999)
shown). The bony trabeculae in young ??? mice were thicker
and longer than in the wild type (Fig. 4 e and f). In situ
hybridization with an exon 1 probe showed strong expression
of NPRC mRNA in the osteoblastic cells lining the bony
trabeculae of developing wild-type bone (Fig. 4g). No signal
was seen in the same cells of the ??? mice (Fig. 4h). The
number and size of osteoclasts in cultures of bone marrow cells
from 2-month-old ??? mice were nearly twice that of the wild
type (Fig. 4i). The number of osteoblastic precursors from the
??? mice was ?3? that of the wild type, and they proliferated
almost twice as rapidly (Fig. 4j). These findings indicate a more
active bone metabolism in the mutants.
Bone Metabolism. We therefore compared the levels of
several relevant blood and urine components in 2-month-old
???, ???, and ??? mice. The data (Table 2) strongly
indicate that the homozygous ??? mice, but not the ???
ALP, an indicator of bone formation, is 150% of the wild-type
level, and urinary pyridonoline and deoxypyridonoline, indi-
cators of bone resorption, are all significantly increased
(?200% that of the wild type).
Interpretation of Bone Effects. Various components of the
natriuretic peptide system are known to be present in cells or
tissues involved in bone formation. For example, CNP and
NPRB (the guanylyl cyclase receptor essentially specific for
CNP) and the corresponding mRNAs have been demonstrated
in cultured fetal mouse tibia (27), and mRNA for NPRC is
readily detected in osteoblasts (ref. 28 and our present data).
A simple hypothesis consistent with previous and our present
observations is that NPRC in growing bone modulates the
autocrine?paracrine effects of locally produced natriuretic
peptides (mainly CNP). In favor of this hypothesis are obser-
vations that CNP, much more than ANP, increases cGMP
production in the fetal mouse tibia organ cultures and causes
an increase in longitudinal bone length, that this bone growth
stimulation is mimicked by 8-bromo-cGMP, and that it is
inhibited by HS-142-1, a nonpeptide GC-coupled natriuretic
peptide receptor antagonist (27). The growth stimulation in
the organ cultures was accompanied by an increase in the
height of the proliferative and hypertrophic chondrocyte zones
in the growth plate that also was seen in our Npr3 ??? mice
(Fig. 4d). Additionally 1,25 dihydroxyvitamin D3, a key reg-
ulator of mineral metabolism, stabilizes NPRC mRNA (28),
indicating a role for NPRC in this regulation.
Other Mutant Animals. Spontaneous and N-ethyl-N-
nitrosourea-induced mutations in mice causing skeletal ab-
normalities identical to those described here have recently
been identified in the Npr3 gene (J. Jaubert, personal com-
munication). Similar abnormalities also have been seen in
transgenic mice grossly over-expressing BNP (29), which could
be due partly to cross-stimulation of NPRB by the high levels
(200 times normal) of circulating BNP and partly to chronic
blockade of NPRC by this BNP.
Conclusions. We do not expect the effects of changes in
NPRC levels to be restricted to those that we have described
here in the kidney and bone. Effects can be expected in any
tissues or organs in which at least one of the natriuretic
peptides, an active receptor, and the clearance receptor are all
present in significant amounts. The interplay of these elements
is complex and is likely affected by the precise anatomical
location of the cells synthesizing each component of the
tartrate-resistant acid phosphatase staining (dark brown) in develop-
ing femurs of 10-day-old Npr3 ??? (a) and ??? mice (b); original
magnification 40?. The arrow indicates a secondary ossification
center. (c and d) Cartilagenous growth plates of femurs from 10-day-
old ??? (c) and ??? (d) mice; hematoxylin and eosin stain; original
magnification 200?. (e and f) Picro-Sirus Red staining of bony
trabeculae in femurs from 10-day-old Npr3 ??? (e) and ??? mice (f);
original magnification 400?. The amounts of unresorbed mineralized
cartilage (MC) and newly deposited bone matrix (arrows) are both
increased in the mutant. (g and h) In situ hybridization for mouse
NPRC mRNA in trabecular bones of 10-day-old wild-type (g) and
homozygous mutant mice (h); original magnification 400?. The signal
is specifically localized to the osteoblastic cells lining the bony tra-
beculae (BT). (i) In vitro osteoclast formation assessed by osteoclast
wild type; filled bars, homozygous mutant. (j) In vitro osteoblast
formation assessed by number of ALP-positive colonies and the
average cell number per colony in bone marrow stromal cell cultures
from wild-type (open bars) and homozygous mutant mice (filled bars).
?, P versus ??? ? 0.05;†, P versus ??? ? 0.01;‡, P versus ???
(a and b) Histochemical localization of osteoclasts by
Table 2.Bone-related variables
430 ? 120
510 ? 90
67 ? 7
620 ? 80
570 ? 70
106 ? 10§
1040 ? 200§
1090 ? 80¶
Data are means ? standard error for five males.
*Plasma alkaline phosphatase (units?liter).
†Urine pyridonoline (mmol)?urine creatinine (mmol).
‡Urine deoxypyridonoline (mmol)?urine creatinine (mmol).
§P versus ??? ? 0.01.
¶P versus ??? ? 0.001.
Genetics: Matsukawa et al.Proc. Natl. Acad. Sci. USA 96 (1999)7407
system, which may differ in different organs and will need to Download full-text
be more carefully delineated. In several different situations,
expression of NPRC is controlled differently from the expres-
sion of the two biologically active natriuretic peptide receptors
and of the natriuretic peptides themselves. For example,
chronic high salt intake leads to a decrease in expression of
NPRC in kidney glomeruli and papilla without affecting the
expression of the biologically active receptors (30). In the
heart, progressive hypertrophy induced by an aortovenocaval
fistula in the rat is accompanied by a striking gradual disap-
pearance of NPRC transcripts whereas expression of ANP,
BNP, and NPRA increases (31). In bone, 1,25 dihydroxyvita-
min D3 affects the expression of CNP and NPRC but not that
of NPRB (28). We conclude from these and our present results
that NPRC provides a biologically effective variable allowing
the physiological effects of the natriuretic peptide system to be
tailored to local needs because the several elements of the
system (ligands, active receptors, and clearance receptor) can
be changed independently. The potential flexibility of the
natriuretic peptide system and its possible compartmentaliza-
tion are thereby substantially enhanced.
We thank C. F. Best, T. M. Coffman, M. F. Goy, S. Hiller, H.-S. Kim,
J. Knowles, T. Maack, N. Maeda, F. Matsukawa, Y. Miyamoto, S.
Pornprasertsuk, H. Sagawa, K. Sakaguchi, Y. Tse, K. Uzawa, H. Ueno,
J. Vanhorne, and J. Vorobiov for help and discussions. cGMP deter-
minations were by the University of North Carolina Center for
Gastrointestinal Biology and Disease (National Institutes of Health
Center Grant DK34987). Our work was supported by the Heart and
Stroke Foundation of Ontario (Grant NA-3479), the W.M. Keck
Foundation, and the National Institutes of Health (Grants HL49277,
HL62145, and GM20069).
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