(Hypertension. 1997;30:177-183.)
© 1997 American Heart Association, Inc.
Articles |
Role of Natriuretic Peptide Receptor Type C in Dahl Salt-Sensitive Hypertensive Rats
From the Fourth Department of Internal Medicine, University of Tokyo School of Medicine (M.N., K.A., T.F.), and Department of Biological Sciences, Tokyo Institute of Technology, Yokohama, Japan (T.K., A.K., S.H.).
Correspondence to Toshiro Fujita, MD, Fourth Department of Internal Medicine, University of Tokyo School of Medicine, 3-28-6 Mejirodai, Bunkyo-ku, Tokyo 112, Japan. E-mail fujita-dis{at}h.u-tokyo.ac.jp
 |
Abstract |
|---|
 Top Abstract IntroductionMethodsResultsDiscussionReferences |
|---|
Abstract The natriuretic peptide system is suggested to be involved in the pathogenesis of salt-sensitive hypertension; a recent report indicated that disruption of the atrial natriuretic peptide precursor gene caused salt-sensitive hypertension. However, natriuretic peptide receptor (NPR)-A knockout mice did not show enhanced salt sensitivity of blood pressure. The aim of the present study was to investigate the role of NPR-C, the other receptor for atrial natriuretic peptide, in increased salt sensitivity of blood pressure. Dahl salt-sensitive (DS) and salt-resistant (DR) rats were placed on a 0.3% or 8% NaCl diet for 4 weeks. Blood pressure was elevated by salt loading only in DS rats. RNase protection assay demonstrated that NPR-C transcript level in the kidney was reduced by chronic salt loading in both DR and DS rats, whereas expression of NPR-A and NPR-B was not altered. The reduction of NPR-C mRNA in response to salt loading was enhanced in DS compared with DR rats. In situ hybridization indicated that the salt-induced NPR-C change was attributed mainly to suppressed expression of NPR-C in the podocytes. NPR-C gene expression was regulated by salt loading in a tissue-specific manner; the marked decrease in NPR-C mRNA by salt loading was seen only in the kidney. These data suggest that the exaggerated salt-induced reduction of NPR-C in the kidney of DS rats may play an important role in the pathogenesis of salt hypertension in this animal, possibly related to impaired renal sodium excretion.
Key Words: sodium • rats, Dahl salt-sensitive • receptors, atrial natriuretic factor • gene expression • in situ hybridization
|
Introduction |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
The natriuretic peptide family in mammals consists of three related peptides, ANP, BNP, and C-type natriuretic peptide (CNP), and is implicated in the blood pressure regulation, fluid and electrolyte balance, and cardiovascular homeostasis.1 2 3 4 ANP and BNP are cardiac hormones secreted mainly from the atrium and ventricle, respectively, whereas CNP is presumed to be a neuropeptide or local factor acting in an autocrine or paracrine manner. Three subtypes of receptors for natriuretic peptides have been identified so far5 6 7 : NPR-A, with high affinity for ANP and BNP8 ; NPR-B, which is selective for CNP9 ; and NPR-C, with less stringent ligand specificity.10 NPR-A and NPR-B possess a particulate guanylyl cyclase domain and are thought to mediate the biological actions of the peptides through the intracellular accumulation of cGMP. The third member, NPR-C, lacks guanylyl cyclase activity and has been proposed to function as a "clearance" receptor to eliminate the ligands from the circulation.11 Recent reports, however, suggest that NPR-C also mediates some biological functions via other intracellular signaling pathways.12 13 14
The natriuretic peptide system has been suggested to be involved in the etiology of salt-sensitive hypertension because of its biological actions, such as potent natriuresis, diuresis, and vasorelaxation. The significance of this system was confirmed by a recent report demonstrating that the gene targeting of proANP, the precursor of ANP, caused salt-dependent hypertension.15 Another more recent study, however, reported that the gene disruption of its receptor, NPR-A, yielded an elevation of blood pressure, which unexpectedly remained unchanged in response to either low or high salt diet (ie, non–salt-sensitive hypertension).16 These results suggest the possibility that NPR-C, the other receptor for ANP, may play an important role in salt regulation. Indeed, our previous work using in vitro cultured vascular endothelial cells demonstrated that NPR-C, but not NPR-A, was very sensitive to changes in the salt concentrations of the culture medium.17
DS rats have been extensively used as an animal model for salt-sensitive hypertension. Renal cross-transplantation studies between the DS rat and its control DR rat indicated that hypertension follows the kidney, suggesting that the abnormality responsible for salt-sensitive hypertension may be located in the kidney.18 In the present study, therefore, we performed an in vivo chronic salt-loading study in DS and DR rats and investigated the effect of salt loading on the gene expression of NPR subtypes in the kidney. We show that the modulation of NPR-C gene expression by salt loading is enhanced in the kidney of DS rats, which may be related to the impaired ability of renal sodium excretion and salt-induced hypertension in this animal model.
|
Methods |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Animals
Four-week-old male DS and age-matched DR rats (both n=28) were purchased from Shizuoka Laboratory Animal Center (SLC) (Shizuoka, Japan). The rats were housed in a room maintained at a constant humidity (60±5%), temperature (23±1°C), and light cycle (6 AM to 6 PM). The rats were fed either a 0.3% or 8% NaCl diet (n=12 for each group). Food and tap water were available ad libitum throughout the study. After 4 weeks of salt loading, the rats were analyzed as below. Body weight was measured on the experimental day.
Tissue and RNA Preparation
The rats (n=7 for each group; DR 0.3%, DR 8%, DS 0.3%, DS
8%) were euthanized by decapitation. Organs were quickly excised,
immediately snap-frozen in liquid nitrogen, and stored at -80°C
until use.
Tissues were homogenized, and total RNA was extracted by the acid guanidinium thiocyanate/phenol/chloroform method.19 Ten micrograms of total RNA was fractionated by electrophoresis on a formaldehyde-degenerated 1% agarose gel, stained with ethidium bromide, and photographed under ultraviolet light. The fluorescence of 28S and 18S rRNA bands was compared to verify the quality and quantity of the isolated RNA from each tissue.
Preparation of cDNA Probes
The cDNA probes for rat ANP (bases
216-443),20 BNP (bases 10-477),21 NPR-A
(bases 1067-1389),8 and NPR-B (bases
648-889)22 were cloned by reverse
transcription–polymerase chain reaction (RT-PCR), as described
previously. Poly(A)+ RNA was isolated from rat atrium (for
ANP), ventricle (for BNP), and vascular smooth muscle cells (for NPR-A
and NPR-B) using an mRNA purification kit (Pharmacia). One microgram of
poly(A)+ RNA was reverse-transcribed to cDNA with Moloney
murine leukemia virus reverse transcriptase (Bethesda Research
Laboratories) and oligo(dT)17 primer. Each cDNA (1/20 of
total) was subjected to PCR with Takara Taq DNA polymerase
and the following primers: The sense and antisense primers,
respectively, for ANP were 5'-ACTTAGCTCCCTCTCTGAGGT-3' and
5'-AAGCTGTTGCAGCCTAGTCC-3'; for BNP were 5'-GAGAGAGCAGGACACCAT-3'
and 5'-AAAGAAGAGCCGCAGGCA-3'; for NPR-A were
5'-GGGCCTTGTTCCCCAGAAAC-3' and 5'-CACCTTGGAAGCTTCGGTTCC-3'; and for
NPR-B were 5'-GGGCAGCAACCTCAGTGTGCA-3' and
5'-GCCGGCCTGTCGCACGTGTG-3'. PCR was carried out for 30 cycles by
repeating 94°C for 1 minute (denaturation), 55°C for 1 minute
(annealing), and 72°C for 2 minutes (extension). Rat NPR-C cDNA
(bases 1059-1231)23 was cloned by screening a rat cerebrum
cDNA library using bovine NPR-C cDNA fragment as a probe. The obtained
cDNA fragments were subcloned into pBluescript II SK- and
sequenced by the dideoxynucleotide chain termination method
using a Sequenase version 2.0 7-deaza-dGTP kit (United States
Biochemical) to confirm their authenticity.
RNase Protection Assay
RNase protection assay was performed according to the method
described previously.24 Briefly, radiolabeled antisense
cRNA probes were prepared with [
-32P]UTP (Amersham)
and T3 (for NPR-A and NPR-C) or T7 (for NPR-B) RNA polymerase using an
RNA in vitro transcription kit (Stratagene). The plasmids containing
cDNA fragments for NPR-A, NPR-B, and NPR-C were linearized with
Sal I, Xba I, and EcoRI, respectively,
and used as templates. After the transcription, template DNA was
degraded with RNase-free DNase I. The probes were purified by
phenol/chloroform treatment and were gel-filtrated. Tissue total RNA
(20 µg) from DR and DS rats on 0.3% and 8% NaCl diets (n=7,
respectively) was hybridized with the three probes (5x105
cpm each) for 12 hours at 45°C. Nonannealed nucleic acids were then
digested with RNase A (40 µg/µL) and RNase T1 (125 U/mL) at 30°C
for 1 hour. Proteinase K and sodium dodecyl sulfate (SDS) were
added to the mixture at 37°C for 30 minutes. RNase-resistant
hybrids were purified by phenol/chloroform treatment, were
ethanol-precipitated, and were resuspended in a gel-loading buffer
containing 80% formamide. RNase-protected fragments were fractionated
on a urea-degenerated 5% polyacrylamide gel and exposed to
Kodak X-Omat AR5 film with an intensifying screen at -80°C or to an
imaging plate. The plate was analyzed by a Phosphor Imager BAS
2000 (Fuji Film).
In Situ Hybridization
In situ hybridization with nonradioactive digoxygenin
(DIG)–labeled cRNA probe was performed as described
previously.25 Briefly, DR and DS rats on 0.3% and 8%
NaCl diets (n=2 for each group) were perfused first with normal saline
and then with 4% paraformaldehyde/phosphate-buffered
saline. The kidneys were removed, immersed in the same fixative, and
then immersed in 30% sucrose solution. The organs were embedded in
optimal cutting temperature compound, frozen in isopentane/liquid
nitrogen, and stored at -80°C. Cryosections (6 µm thick) were
prepared by cryostat and were thaw-mounted onto Vectabond–coated glass
slides (Vector Laboratories Inc). The sections were postfixed in 4%
paraformaldehyde, treated with proteinase K (1 µg/mL)
at 37°C for 20 minutes, acidified with 0.25% acetic acid, and
dehydrated in graded ethanol. DIG-labeled antisense cRNA probe was
synthesized using a DIG RNA labeling kit (Boehringer Mannheim)
with the same template and polymerase as those used for the RNase
protection assay. Control sense probe was also constructed in the same
way. After the transcription, template DNA was degraded with RNase-free
DNase I. The sections were hybridized with DIG-labeled probes (500
ng/mL) dissolved in a hybridization buffer composed of 50% formamide,
2x SSC (1x SSC=150 mmol/L NaCl, 15 mmol/L sodium citrate,
pH 7.0), 1 µg/µL tRNA, 1 µg/µL sonicated salmon sperm DNA, 1
µg/µL bovine serum albumin, 10% dextran sulfate, and 1.2
mol/L dithiothreitol in a humidified chamber at 42°C for 16 hours.
Slides were then washed in 4x, 2x, 1x, and 0.5x SSC at room
temperature for 10 minutes each and 0.5x SSC at 37°C for 30 minutes.
DIG-labeled hybrids were detected by an enzyme-linked immunoassay kit
(Boehringer Mannheim). After immersion in 1.5% blocking
solution, the slides were exposed to anti-DIG alkaline phosphatase
conjugate diluted at 1:1000 for 30 minutes. The hybrids were visualized
as blue precipitates by the subsequent alkaline phosphatase–catalyzed
color reaction in a solution containing 5-bromo-4-chloro-3-indolyl
phosphate and nitroblue tetrazolium.
Northern Blot Analysis
Northern blotting for ANP and BNP was performed as described
previously.24 Briefly, tissue total RNA (20 µg) from the
four experimental groups (n=7, respectively) was size-fractionated by
formaldehyde-degenerated 1.2% agarose gel electrophoresis. RNAs were
then blotted on a Magnagraph nylon membrane filter and cross-linked by
baking at 80°C for 2 hours. After prehybridization in a solution
containing 50% formamide, 6x SSC, 5x Denhardt's solution (1x
Denhardt's=0.02% each of bovine serum albumin,
polyvinylpyrrolidone, and Ficoll), 100 µg/mL sonicated salmon sperm
DNA, and 1% SDS at 37°C for 2 hours, the filter was hybridized with
random-primed, 32P-labeled cDNA probe for rat ANP or BNP
(5x105 cpm/mL) at 42°C for 16 hours. The hybridized
filters were washed twice in 2x SSC and 0.1% SDS at room temperature
for 5 minutes and twice in 0.1x SSC and 0.1% SDS at 60°C for 1 hour
and then exposed to Kodak X-Omat AR5 film with an intensifying screen
at -80°C and to an imaging plate. The plate was analyzed by
a Phosphor Imager BAS 2000.
Blood Pressure Measurement
Blood pressure was measured directly in some rats from each
group (n=4-5). The left carotid artery was cannulated with a PE-50 tube
with rats under ether anesthesia. The catheter was filled
with heparinized saline (100 IU/mL). Approximately 24 hours after the
operation, arterial pressure was measured with rats in a
conscious and unrestrained condition via a pressure transducer (model
TP-200T, Nihon-Kohden) connected to a thermal array recorder
(WS-641G, Nihon-Kohden). Blood pressure was monitored over 30 minutes
and expressed as mean blood pressure, using at least five measurements
at the stable level.
Plasma ANP Concentration
Plasma ANP concentration was determined by a radioimmunoassay
kit (Peninsula Laboratories Inc). After blood pressure measurement, 1
mL of blood was withdrawn from the catheter (n=4-5 for each group).
Blood samples were collected into prechilled polypropylene tubes
containing aprotinin (500 kallikrein inhibiting units [KIU]/mL) and
EDTA (1 mg/mL). After centrifugation, plasma was
applied to pretreated Sep-Pak C18 cartridges, and eluates were
evaporated and reconstituted in radioimmunoassay buffer. The sample, or
standard, was mixed with rabbit antisera against -ANP and incubated
overnight at 4°C. Then 125I-labeled rat ANP was added and
incubated for another 24 hours. Antibody-bound tracer was precipitated
with goat anti-rabbit 
-globulin and normal rabbit serum for 90
minutes. After centrifugation, the radioactivity of the
pellets was counted in a gamma counter.
Statistical Analysis
Data are expressed as mean±SEM. Statistical analysis
was performed by two-way ANOVA and subsequent Tukey's
simultaneous multiple comparison. A value of
P<.05 was considered to be statistically significant.
|
Results |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Blood Pressure and Body Weight
As shown in Fig 1
, mean arterial
pressure did not differ between DR and DS rats fed a 0.3% NaCl diet.
Salt loading (8%) did not affect blood pressure in DR rats but
significantly increased blood pressure in DS rats (P<.01).
Body weight did not differ among the four groups
(Table).
|
|
Effect of Salt Loading on Gene Expression of NPR-A, NPR-B, and
NPR-C in the Kidney
We performed an RNase protection assay to examine the effect of
salt loading on the gene expression of NPR subtypes in the kidney. Fig 2A shows representative autoradiographs.
The bands for NPR-A (323 bp), NPR-B (242 bp), and NPR-C (173 bp) were
obtained at the appropriate positions. The radioactivity of the
protected band showed a linear relation with the amount of RNA used in
the assay. The message levels of NPR-A or NPR-B did not differ between
DR and DS rats and were not altered by salt loading. On the other hand,
NPR-C gene expression was markedly modulated by salt loading. Whereas
there was no strain difference in NPR-C transcript level when the rats
were fed a 0.3% NaCl diet, salt loading caused a reduction of NPR-C
expression in both DR and DS rats. The reduction of NPR-C mRNA was
greater in DS than DR rats. The radioactivity of the protected bands
for NPR-C was measured, and the results were analyzed
statistically (n=7 for each group) (Fig 2B). NPR-C mRNA levels did not
differ between DR and DS rats when they were fed a 0.3% NaCl diet.
Salt loading caused a significant reduction of NPR-C expression in both
DR (-28%, P<.01) and DS (-53%, P<.01) rats,
with the NPR-C mRNA level significantly lower in salt-loaded DS rats
than in salt-loaded DR rats (P<.01).
|
Salt-Induced Change in NPR-C Gene Expression in Extrarenal
Tissues
We further examined the effect of salt loading on NPR-C expression
in the lung, atrium, aorta, adrenal gland, and brain (Fig 3). The dramatic changes seen in the kidney were not
detected in other tissues. However, there was a tissue-specific pattern
of modulation by salt loading. In the lung and aorta, salt loading
induced a slight increase in NPR-C message, although the change was
small compared with that in the kidney. On the other hand, salt loading
did not affect transcript level in the atrium, adrenal gland, and
brain. The responses to salt loading did not differ between DR and DS
rats.
|
In Situ Localization of NPR-C mRNA in the Kidney
We carried out in situ hybridization in the kidney to determine
which cell types were responsible for the salt-induced alteration in
NPR-C gene expression. Fig 4 shows
representative photomicrographs. Positive signals were
detected as purple/black precipitates. The NPR-C antisense probe
hybridized with the glomerulus and was preferentially located in the
podocytes. The location of the expression was the same in DR and DS
rats and was not affected by salt loading. Control experiments with
sense NPR-C probe resulted in negative staining or only low
background.
|
Plasma ANP Concentration
As shown in the Table
, plasma ANP levels did not differ between DR
and DS rats when they were placed on a 0.3% NaCl diet. During high
salt, the concentration of plasma ANP in DS rats was significantly
higher (P<.05) than in DR rats.
Northern Blot Analysis
Messenger RNA for atrial ANP and ventricular BNP was
examined in the four experimental groups. Corresponding to plasma ANP
levels, salt loading did not increase ANP and BNP transcript levels in
DR rats but augmented the expression in DS rats (Fig 5).
|
|
Discussion |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
The present study demonstrates that chronic salt loading attenuates renal NPR-C gene expression, which was greater in DS than DR rats. This finding suggests that NPR-C may play an important role in the regulation of sodium balance and the pathophysiology of salt-sensitive hypertension.
The effect of salt loading on renal NPR-C level is still controversial. Several groups have demonstrated, by radiolabeled ligand binding experiments, that chronic high salt diet decreases the number of NPR-C without changing the number of guanylyl cyclase–coupled receptors in the kidney.26 27 28 However, it has also been reported that salt loading does not change the number of ANP binding sites in the kidney.29 Moreover, Martin et al30 pointed out a pitfall in that the reported salt-induced reduction of NPR-C26 was due to the prior receptor occupation by endogenous ANP under the condition of elevated plasma ANP concentration. Indeed, they demonstrated that after elimination of bound ANP by acid wash treatment, the number of NPR-C between the kidneys of salt-loaded and control rats did not differ. In the present study, we sought to avoid any spurious change caused by technical problems in the ligand binding assay by analyzing changes in mRNA. As indicated, we have demonstrated a reduction in NPR-C mRNA in DR (control) rats. This finding is compatible with our previous in vitro study in which we showed that in cultured bovine vascular endothelial cells, NaCl treatment caused a reduction in NPR-C at the pretranslational level without affecting NPR-A.17 Furthermore, we also showed that NPR-C mRNA expression was lower in the gills of eels adapted to sea water than in those adapted to fresh water.31 Thus, the regulation of NPR-C gene expression may have an important role in maintaining the internal milieu against extrinsic environmental sodium changes.
This is the first study to demonstrate enhanced reduction of NPR-C mRNA by chronic salt loading in the kidneys of DS rats compared with those in DR rats. This finding can be interpreted in several ways as an explanation for salt-sensitive hypertension. First, several lines of evidence suggest that NPR-C has a major role in the removal of the ligands from the circulation.11 According to this "clearance" theory, one of the most probable explanations is that the augmented NPR-C reduction in DS rats may contribute to a compensatory mechanism. NPR-C reduction in the kidney lowers the elimination of the ligands from the renal circulation and subsequently increases the ANP availability at the target sites in the kidney. As a result, the biological actions of ANP via NPR-A are augmented without affecting systemic ANP levels. Thus, natriuresis is appropriately enhanced in response to sodium overload without causing any extrarenal effects in DR rats. In salt-loaded DS rats, the suppression of NPR-C may be augmented to compensate for the impaired renal sodium excretion caused by other primary factor or factors of salt-sensitive hypertension. However, in our previous study, the mere masking of NPR-C by C-ANF did not reproduce the cGMP hyperreactivity that was observed in a condition with reduced NPR-C expression,17 which does not support the clearance theory.
Several recent studies suggest another possibility; the augmented reduction of NPR-C in DS rats in the present study may be a primary factor in the mechanism of salt-sensitive hypertension. Two groups have recently succeeded in the generation of gene-targeted mice of the natriuretic peptide system. John et al15 reported that gene targeting of proANP caused salt-sensitive hypertension. This finding suggests that the ANP system has an important regulatory role in salt-sensitive hypertension. However, Lopez et al16 reported that NPR-A knockout mice exhibited salt-resistant hypertension. This finding was unexpected because a linkage analysis indicated NPR-A as a candidate gene causing salt-sensitive hypertension in DS rats.32 These facts imply that the actions of ANP cannot be explained by the mechanism via NPR-A alone and that NPR-C, the other receptor for ANP, might be involved in the pathogenesis of salt-sensitive hypertension independent of NPR-A. Indeed, the following evidence supports this possibility. ANP modulates blood pressure and fluid balance also by interacting with other vasoactive hormones and neurotransmitters.12 Recent studies indicate that some of this cross talk is mediated through NPR-C without the interference of NPR-A: For example, ANP inhibits endothelin production,33 inhibits catecholamine release,34 and enhances nitric oxide production35 through NPR-C. NPR-C has also been suggested to mediate the interaction with the renin-angiotensin system.36 37 On the basis of these findings, the reduction in NPR-C in DS rats may alter the equilibrium between vasoconstrictive/antinatriuretic and vasorelaxant/natriuretic systems, resulting in hypertension and elevated plasma ANP. Moreover, some groups12 13 14 have proposed that NPR-C affects cAMP, phosphatidylinositol, or other unknown signaling pathways. Therefore, it is possible that NPR-C contributes to the etiology of salt-sensitive hypertension directly through some novel unknown mechanism.
Our result from in situ hybridization provides additional supportive evidence for the possible involvement of NPR-C in salt-sensitive hypertension of DS rats. On the basis of renal cross-transplantation studies between DS and DR rats, it is generally accepted that the abnormality responsible for the salt sensitivity in DS rats is localized in the kidney.18 Tobian et al38 reported an altered pressure-natriuresis relationship in the kidney of DS rats caused by the impaired renal sodium excretory capability. Analysis of the pattern of the pressure-natriuresis relationship revealed that salt-sensitive hypertension in DS rats can be ascribed to the reduced ultrafiltration coefficient (Kf) per glomerulus, similar to the case of hypertension in the early phase of glomerulonephritis.39 40 41 The reduced single glomerulus Kf can be caused by the reduced surface area per glomerulus or the reduced hydraulic permeability of the glomerular filtration barrier.42 This can be explained by the malfunction of podocytes, since podocytes, together with basement membrane, surround the glomerular capillaries and provide the size and charge barrier for glomerular filtration. In addition, podocytes are thought to modulate the permeability of the glomerular filtration membrane, or the filtration surface area, through the abundant contractile elements in their cytoplasm.43 In the present study, in situ hybridization demonstrated that NPR-C mRNA was localized predominantly in the glomeruli and especially in the podocytes. This finding is consistent with the previously reported RNase protection assay using microdissected nephron fragments, electron microscopy, immunohistochemical staining, and in situ hybridization.25 44 45 46 47 Since the location of the positive signals within the kidney was similar among the four rat groups, podocytes should be responsible for the salt-induced reduction of NPR-C expression. Therefore, the marked reduction of NPR-C in the podocytes in DS rats may contribute to the abnormal function of podocytes resulting in salt-sensitive hypertension. In contrast to our data, it has been reported that NPR-C is detectable in cultured mesangial cells but not in cultured epithelial cells.26 The discrepancy may be due to the subtype switching of natriuretic peptide receptors seen between in vivo and in vitro conditions.48 49 50
In the present study, we found that NPR-C gene expression was regulated by salt loading in a tissue-specific manner; the reduction in NPR-C was seen only in the kidney. Previously, hepatic NPR-C was reported to be reduced by salt loading only in DS rats, although the liver is not a major target of ANP compared with the kidney.51 On the other hand, salt loading did not influence NPR-C gene expression in several tissues, including the atrium, where ANP is synthesized and NPR-C expression is abundant. Furthermore, NPR-C mRNA was slightly increased in the lung and aorta. The mechanism for this tissue-specific modulation of NPR-C is unclear. There is a discrepancy between whole aorta and cultured vascular endothelial cells.17 This discrepancy may be explained by subtype switching between in vivo aorta and cultured cells, as mentioned above.48 49 50 Vascular smooth muscle cells in whole aorta might cause the upregulation observed in the present study.
We have to take into account the possibility that NPR-C reduction is a secondary event caused by several other primary mechanisms. First, the possibility of ligand-mediated receptor downregulation should be considered. Since we observed the changes in mRNA level, the reduction is not an artifact caused by increased endogenous ANP, as mentioned above. The salt-induced NPR-C reduction is not related to ligand-mediated receptor downregulation in DR rats because mRNA levels of ANP and BNP, as well as plasma ANP concentration, were not increased by salt loading in this group. On the other hand, in DS rats, both plasma ANP and mRNA levels of ANP and BNP were increased. However, ligand-mediated receptor downregulation may not be the case even in DS rats because NPR-C reduction was observed only in the kidney. If the increased ligand level was responsible for the reduction of NPR-C, the reduction should be a general effect and should be seen in every organ. Rather, in DS rats, the ligand production might be stimulated in compensation for the NPR-C decrease in the kidney.
Second, NPR-C expression may be affected incidentally in response to other primary defects. NPR-C gene expression is known to be suppressed by angiotensin II and ß2-adrenergic stimulant,52 53 although either insufficient suppression of angiotensin II or overactivity of ß2-adrenergic receptor has not been reported in salt-loaded DS rats. Of course, our statement here does not mean that a direct effect of NPR-C is definitely proved in the present study. Further investigation, such as the establishment of NPR-C knockout mice and the elucidation of the intracellular signaling pathway of NPR-C, will be necessary to provide more detailed insight into the possible involvement of NPR-C.
In conclusion, our data demonstrate that chronic salt loading attenuated NPR-C gene expression without changing NPR-A or NPR-B levels in the kidney. We also show that renal NPR-C expression is reduced to a greater extent in DS than DR rats. These findings suggest that NPR-C may play a pivotal role in the pathogenesis of salt-sensitive hypertension.
|
Selected Abbreviations and Acronyms |
|---|
|
|
Acknowledgments |
|---|
We are grateful to Dr Mervyn J. Merrilees for reviewing the English in our manuscript.
Received August 27, 1996; first decision October 1, 1996; accepted January 9, 1997.
|
References |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
1. de Bold AJ. Atrial natriuretic factor: a hormone produced by the heart. Science. 1985;230:767-770.
2.
Inagami T. Atrial natriuretic
factor. J Biol Chem. 1989;264:3043-3046.
3.
Brenner BM, Ballermann BJ, Gunning ME, Zeidel
ML. Diverse biological actions of atrial natriuretic
peptide. Physiol Rev. 1990;70:665-699.
4. Rosenzweig A, Seidman CE. Atrial natriuretic factor and related peptide hormones. Annu Rev Biochem. 1991;60:229-255.[Medline] [Order article via Infotrieve]
5.
Koller KJ, Goeddel DV. Molecular biology of the
natriuretic peptides and their receptors.
Circulation. 1992;86:1081-1088.
6. Chinkers M, Garbers DL. Signal transduction by guanylyl cyclases. Annu Rev Biochem. 1991;60:553-575.[Medline] [Order article via Infotrieve]
7. Maack T. Receptors of atrial natriuretic factor. Annu Rev Physiol. 1992;54:11-27.[Medline] [Order article via Infotrieve]
8. Chinkers M, Garbers DL, Chang MS, Lowe DG, Chin HM, Goeddel DV, Schulz S. A membrane form of guanylate cyclase is an atrial natriuretic peptide receptor. Nature. 1989;338:78-83.[Medline] [Order article via Infotrieve]
9. Chang MS, Lowe DG, Lewis M, Hellmiss R, Chen E, Goeddel DV. Differential activation by atrial and brain natriuretic peptides of two different receptor guanylate cyclases. Nature. 1989;341:68-72.[Medline] [Order article via Infotrieve]
10.
Fuller F, Porter JG, Arfsten AE, Miller J, Schilling
JW, Scarborough RM, Lewicki JA, Schenk DB. Atrial
natriuretic peptide clearance receptor: complete sequence
and functional expression of cDNA clones. J Biol
Chem. 1988;263:9395-9401.
11.
Maack T, Suzuki M, Almeida FA, Nussenzveig D,
Scarborough RM, McEnroe GA, Lewicki JA.
Physiological role of silent receptors of atrial
natriuretic factor. Science. 1987;238:675-678.
12.
Levin ER. Natriuretic peptide
C-receptor: more than a clearance receptor. Am J
Physiol. 1993;264:E483-E489.
13.
Anand-Srivastava MB, Sairam MR, Cantin M.
Ring-deleted analogs of atrial natriuretic factor inhibit
adenylate cyclase/cAMP system: possible coupling of
clearance atrial natriuretic factor receptors to
adenylate cyclase/cAMP signal transduction system.
J Biol Chem. 1990;265:8566-8572.
14.
Dai LJ, Quamme GA. Atrial
natriuretic peptide initiates Ca2+ transients
in isolated renal cortical thick ascending limb cells.
Am J Physiol. 1993;265:F592-F597.
15.
John SW, Krege JH, Oliver PM, Hagaman JR, Hodgin JB,
Pang SC, Flynn TG, Smithies O. Genetic decreases in atrial
natriuretic peptide and salt-sensitive
hypertension. Science. 1995;267:679-681.
16. Lopez MJ, Wong SK, Kishimoto I, Dubois S, Mach V, Friesen J, Garbers DL, Beuve A. Salt-resistant hypertension in mice lacking the guanylyl cyclase-A receptor for atrial natriuretic peptide. Nature. 1995;378:65-68.[Medline] [Order article via Infotrieve]
17.
Katafuchi T, Mizuno T, Hagiwara H, Itakura M, Ito T,
Hirose S. Modulation by NaCl of atrial natriuretic
peptide receptor levels and cyclic GMP responsiveness to atrial
natriuretic peptide of cultured vascular
endothelial cells. J Biol Chem. 1992;267:7624-7629.
18.
Dahl LK, Heine M, Thompson K. Genetic influence
of the kidneys on blood pressure: evidence from chronic renal
homografts in rats with opposite predispositions to
hypertension. Circ Res. 1974;34:94-101.
19. Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987;162:156-159.[Medline] [Order article via Infotrieve]
20. Maki M, Takayanagi R, Misono KS, Pandey KN, Tibbetts C, Inagami T. Structure of rat atrial natriuretic factor precursor deduced from cDNA sequence. Nature. 1984;309:722-724.[Medline] [Order article via Infotrieve]
21. Kojima M, Minamino N, Kangawa K, Matsuo H. Cloning and sequence analysis of cDNA encoding a precursor for rat brain natriuretic peptide. Biochem Biophys Res Commun. 1989;159:1420-1426.[Medline] [Order article via Infotrieve]
22. Schulz S, Singh S, Bellet RA, Singh G, Tubb DJ, Chin H, Garbers DL. The primary structure of a plasma membrane guanylate cyclase demonstrates diversity within this new receptor family. Cell. 1989;58:1155-1162.[Medline] [Order article via Infotrieve]
23.
Engel AM, Schoenfeld JR, Lowe DG. A single
residue determines the distinct pharmacology of rat and human
natriuretic peptide receptor-C. J Biol
Chem. 1994;269:17005-17008.
24. Katafuchi T, Takashima A, Kashiwagi M, Hagiwara H, Takei Y, Hirose S. Cloning and expression of eel natriuretic-peptide receptor B and comparison with its mammalian counterparts. Eur J Biochem. 1994;222:835-842.[Medline] [Order article via Infotrieve]
25.
Yamamoto T, Feng L, Mizuno T, Hirose S, Kawasaki K,
Yaoita E, Kihara I, Wilson CB. Expression of mRNA for
natriuretic peptide receptor subtypes in bovine
kidney. Am J Physiol. 1994;267:F318-F324.
26. Ballermann BJ, Hoover RL, Karnovski MJ, Brenner BM. Physiologic regulation of atrial natriuretic peptide receptors in rat renal glomeruli. J Clin Invest. 1985;76:2049-2056.
27.
Gauquelin G, Garcia R, Carrier F, Cantin M, Gutkowska
J, Thibault G, Schiffrin EL. Glomerular ANF receptor
regulation during changes in sodium and water
metabolism. Am J Physiol. 1988;254:F51-F55.
28. Michel H, Meyer-Lehnert H, Backer A, Stelkens H, Kramer HJ. Regulation of atrial natriuretic peptide receptors in glomeruli during chronic salt loading. Kidney Int. 1990;38:73-79.[Medline] [Order article via Infotrieve]
29.
Kollenda MC, Vollmar AM, McEnroe GA, Gerbes AL.
Dehydration increases the density of C receptors for ANP on rat
glomerular membranes. Am J Physiol. 1990;258:R1084-R1088.
30.
Martin ER, Lewicki JA, Scarborough RM, Ballermann
BJ. Expression and regulation of ANP receptor subtypes in rat
renal glomeruli and papillae. Am J Physiol. 1989;257:F649-F657.
31. Takashima A, Katafuchi T, Shibasaki M, Kashiwagi M, Hagiwara H, Takei Y, Hirose S. Cloning, properties, site-directed mutagenesis analysis of the subunit structure, tissue distribution and regulation of expression of the type-C eel natriuretic peptide receptor. Eur J Biochem. 1995;227:673-680.[Medline] [Order article via Infotrieve]
32. Deng Y, Rapp JP. Cosegregation of blood pressure with angiotensin converting enzyme and atrial natriuretic peptide receptor genes using Dahl salt-sensitive rats. Nat Genet. 1992;1:267-272.[Medline] [Order article via Infotrieve]
33.
Hu RM, Levin ER, Pedram A, Frank HJL. Atrial
natriuretic peptide inhibits the production and
secretion of endothelin from cultured endothelial
cells: mediation through the C receptor. J Biol
Chem. 1992;267:17384-17389.
34.
Drewett JG, Ziegler RJ, Trachte GJ.
Neuromodulatory effects of atrial natriuretic factor are
independent of guanylate cyclase in adrenergic neuronal
pheochromocytoma cells. J Pharmacol Exp Ther. 1991;255:497-503.
35. McLay JS, Chatterjee PK, Jardine AG, Hawksworth GM. Atrial natriuretic factor modulates nitric oxide production: an ANF-C receptor-mediated effect. J Hypertens. 1995;13:625-630.[Medline] [Order article via Infotrieve]
36.
Davidson NC, Barr CS, Struthers AD. C-type
natriuretic peptide: an endogenous
inhibitor of vascular angiotensin-converting
enzyme activity. Circulation. 1996;93:1155-1159.
37. Krulewitz AH, Fanburg BL. Stimulation of bovine endothelial cell angiotensin I-converting enzyme activity by cyclic AMP-related agents. J Cell Physiol. 1986;129:147-150.[Medline] [Order article via Infotrieve]
38. Tobian L, Lange J, Azar S. Reduction of natriuretic capacity and renin release in isolated, blood-perfused kidneys of Dahl hypertensive-prone rats. Circ Res. 1978;43(suppl I):I-92-I-98.
39. Sterzel RB, Luft FC, Gao Y, Schnermann J, Briggs JP, Ganten D, Waldherr R, Schnabel E, Kriz W. Renal disease and the development of hypertension in salt-sensitive Dahl rats. Kidney Int. 1988;33:1119-1129.[Medline] [Order article via Infotrieve]
40. Azar S, Weller D, Bruo L, Livingston S, Iwai J. Single nephron dynamics in the normotensive Dahl rat on high salt intake. Circ Res. 1977;25:504A. Abstract.
41. Kimura G, Brenner BM. The renal basis for salt sensitivities in hypertension. In: Laragh JH, Brenner BM, eds. Hypertension: Pathophysiology, Diagnosis and Treatment. 2nd ed. New York, NY: Raven Press Publishers; 1995:1569-1587.
42. Kimura G, Frem GJ, Brenner BM. Renal mechanisms of salt sensitivity in hypertension. Curr Opin Nephrol Hypertens. 1994;3:4-12.
43. Drenckhahn D, Franke RP. Ultrastructural organization of contractile and cytoskeletal proteins in glomerular podocytes of chicken, rat, and man. Lab Invest. 1988;59:673-682.[Medline] [Order article via Infotrieve]
44. Bianchi C, Gutkowska J, Thibault G, Garcia R, Genest J, Cantin M. Distinct localization of atrial natriuretic factor and angiotensin II binding sites in the glomerulus. Am J Physiol. 1986;251:F594-F602.
45. Rambotti MG, Saccardi C, Spreca A. Evidence for particulate guanylate cyclase in rat kidney after stimulation by atrial natriuretic factor: an ultracytochemical study. Histochem J. 1990;22:469-474.[Medline] [Order article via Infotrieve]
46. Kawaguchi S, Uchida K, Ito T, Kozuka M, Shimonaka M, Mizuno T, Hirose S. Immunohistochemical localization of atrial natriuretic peptide receptor in bovine kidney and lung. J Histochem Cytochem. 1989;37:1739-1742.[Abstract]
47.
Wilcox JN, Augustine A, Goeddel DV, Lowe DG.
Differential regional expression of three natriuretic
peptide receptor genes within primate tissues. Mol Cell
Biol. 1991;11:3454-3462.
48.
Uchida K, Mizuno T, Shimonaka M, Sugiura N, Hagiwara H,
Hirose S. Subtype switching of ANP receptors during in vitro
culture of vascular cells. Am J Physiol. 1989;256:H311-H314.
49.
Suga S, Nakao K, Kishimoto I, Hosoda K, Mukoyama M,
Arai H, Shirakami G, Ogawa Y, Komatsu Y, Nakagawa O, Hama N, Imura
H. Phenotype-related alteration in expression of
natriuretic peptide receptors in aortic smooth muscle
cells. Circ Res. 1992;71:34-39.
50. Abe T, Nishiyama K, Snajdar R, He X, Misono KS. Aortic smooth muscle contains guanylate-cyclase-coupled 130-kDa atrial natriuretic factor receptor as predominant receptor form spontaneous switching to 60-kDa C-receptor upon cell culturing. Eur J Biochem. 1993;217:295-304.[Medline] [Order article via Infotrieve]
51. McCartney S, Cramb G. Effects of a high-salt diet on hepatic atrial natriuretic peptide receptor expression in Dahl salt-resistant and salt-sensitive rats. J Hypertens. 1993;11:253-262.[Medline] [Order article via Infotrieve]
52.
Chabrier PE, Robert P, Lonchampt MO, Plas P, Braquet
P. Regulation of atrial natriuretic factor receptors
by angiotensin II in rat vascular smooth muscle
cells. J Biol Chem. 1988;263:13199-13202.
53.
Kishimoto I, Yoshimasa T, Suga S, Ogawa Y, Komatsu Y,
Nakagawa O, Itoh H, Nakao K. Natriuretic peptide
clearance receptor is transcriptionally down-regulated by
ß2-adrenergic stimulation in vascular smooth muscle
cells. J Biol Chem. 1994;269:28300-28308.
This article has been cited by other articles:
 |
|
 |
|
  M. Nagase, S. Shibata, S. Yoshida, T. Nagase, T. Gotoda, and T. Fujita Podocyte Injury Underlies the Glomerulopathy of Dahl Salt-Hypertensive Rats and Is Reversed by Aldosterone Blocker Hypertension, June 1, 2006; 47(6): 1084 - 1093. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. E. Woodard, X. Li, and J. A. Rosado Water deprivation enhances the inhibitory effect of natriuretic peptides on cAMP synthesis in rat renal glomeruli Am J Physiol Renal Physiol, September 1, 2004; 287(3): F418 - F426. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Z. Ni, F. Oveisi, and N. D. Vaziri Nitric Oxide Synthase Isotype Expression in Salt-Sensitive and Salt-Resistant Dahl Rats Hypertension, October 1, 1999; 34(4): 552 - 557. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J.-Z. Sun, S.-J. Chen, E. Majid-Hasan, S. Oparil, and Y.-F. Chen Dietary salt supplementation selectively downregulates NPR-C receptor expression in kidney independently of ANP Am J Physiol Renal Physiol, February 1, 2002; 282(2): F220 - F227. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||