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J Biol Chem, Vol. 275, Issue 20, 15357-15362, May 19, 2000
From the In renal extracts, some renin is present as
"high molecular weight renin," a heterodimeric complex of renin
with the 46-kDa renin-binding protein (RnBP), also known as
N-acyl-D-glucosamine 2-epimerase. Because RnBP
specifically inhibits renin activity, the protein was proposed to play
an important role in the regulation of the renin-angiotensin system
(RAS). Using gene targeting, we have generated mice lacking RnBP and
tested this hypothesis in vivo. In particular, we analyzed
biosynthesis, secretion, and activity of renin and other components of
the RAS in mice lacking RnBP. Despite extensive investigations, we were
unable to detect any major effects of RnBP deficiency on the plasma and
renal RAS or on blood pressure regulation. Contrary to previous
hypotheses, we conclude that RnBP does not play a significant role in
the regulation of renin activity in plasma or kidney. However, RnBP knockout mice excrete an abnormal pattern of carbohydrates in the
urine, indicating a role of the protein in renal carbohydrate metabolism.
The aspartyl protease renin catalyzes the cleavage of
angiotensinogen (AOGEN)1 to
the decapeptide angiotensin (ANG) I, which is further processed by
angiotensin converting enzyme to the octapeptide ANG II. ANG II is the
final effector of the renin-angiotensin system (RAS) and a central
regulator of blood pressure, salt, and water homeostasis, as well as
cardiac and vascular growth. Because conversion of AOGEN to ANG I by
renin is the rate-limiting step in initiating the endocrine cascade of
RAS, much attention has focused on elucidating the mechanisms that may
control renin expression and secretion (1, 2).
Although renin is expressed in a variety of tissues, the kidney is the
sole source of circulating active renin and thus plays a crucial role
in short term regulation of RAS activity. Within the kidney,
specialized myoepithelial cells in the afferent arteriole of the
juxtaglomerular apparatus express and secrete active renin in response
to various stimuli, including a decrease in blood pressure or in ANG II
concentration (1). Previously, a 46-kDa protein was identified in the
kidney that was proposed to play a role in the regulation of renin
activity in this tissue. This protein was called the renin-binding
protein (RnBP). RnBP was identified initially because in renal
homogenates it forms a tight 1:1 complex with renin, designated "high
molecular mass renin" (3). Subsequent studies demonstrated that RnBP
specifically binds renin (Kd 0.2 nM) but
not other aspartyl proteases such as cathepsin D or pepsin. Binding
requires a leucine zipper motif present in RnBP (4). Because RnBP
binding to renin strongly inhibits cleavage of AOGEN to ANG I, RnBP was
suggested to act as renin inhibitor in vivo. Experimental
evidence to support this hypothesis came from studies in mouse
pituitary AtT-20 cells, where expression of RnBP inhibits the secretion
of active renin in a dose-dependent manner (5). In
addition, a polymorphism in intron 6 of the RnBP gene (T61C) was shown
to be associated with a 40% increase in the renin/prorenin ratio in
Caucasian men (6).
Although several hypotheses about a function of RnBP in renin
regulation have been advanced, some experimental evidence argued against such a role. Although infusion of recombinant RnBP into rats
leads to a long lasting decrease in plasma renin activity, neither RnBP
nor high molecular mass renin are normally found in plasma (7).
Furthermore, within cells renin is localized in the intracellular
secretory pathway, whereas RnBP lacks a signal peptide and apparently
resides in the cytoplasm. Finally, RnBP was shown to act as an
N-acyl-D-glucosamine 2-epimerase,
interconverting GlcNAc and N-acetylmannosamine (ManNAc), a
precursor in N-acetylneuraminic acid (NANA) biosynthesis,
suggesting a function in carbohydrate metabolism rather than in the RAS
(8).
To clarify this controversy, we have investigated a potential role of
RnBP in RAS in detail. In particular, we have used gene targeting to
generate mice lacking RnBP and to test the consequences of RnBP
deficiency for renin activity in vivo. The gene targeting approach has been used previously to gain important insights into the
RAS in mice (9). Our studies demonstrate that RnBP-deficient mice are
healthy and normotensive and that lack of RnBP does not affect
expression or activity of renin under normal physiological conditions.
Materials and General Methods--
Rabbit anti-mouse
submaxillary renin antibody reacting with mouse renin-1 and renin-2 was
kindly provided by Joel Menard (INSERM U367, Paris, France).
Experiments involving recombinant DNA technologies were performed
according to standard protocols. Characterization of tissues by
two-dimensional gel electrophoresis or immunoelectron microscopy have
been described (10, 11).
In Situ Hybridization--
Cryosections (10 µm) from mouse and
rat kidneys were fixed in 4% paraformaldehyde (in phosphate-buffered
saline, pH 7.4) for 10 min, deproteinated in 0.2 M HCl for
15 min, and acetylated in 100 mM triethanolamine, pH
8.0/0.25% acetic anhydride. To generate RNA probes, a 0.2-kb
SacI-AccI fragment of the mouse renin-2 cDNA and a
1.3-kb EcoRI-BamHI fragment of the rat RnBP
cDNA were transcribed in vitro using digoxigenin-UTP
(Roche Molecular Biochemicals). Hybridization to kidney sections was
carried out for 16 h at 55-65 °C in a buffer containing 20 mM Tris-HCl, pH 7.6, 1× Denhardt's solution, 100 mM dithiothreitol, 5 mg/ml yeast tRNA, 1 mg/ml
poly(A)+ RNA, 333 mM NaCl, 1 mM
EDTA, 10% dextran sulfate, and 50% formamide. Bound probes were
visualized using alkaline phosphatase-coupled anti-digoxigenin IgG and
nitroblue tetrazolium/5-bromo-4-chloro-3-indolylphosphate (Roche
Molecular Biochemicals). The sections were counterstained with 1%
neutral red.
Targeting of the RnBP Gene Locus--
A 1-kb
XhoI-NcoI and a 7-kb NcoI-HindIII
fragment of the murine RnBP gene were used as short and long homology
regions of the targeting construct and fused to the 5' and 3' ends of
the pol2neobpA expression cassette (neo),
respectively. This results in replacement of approximately 1-kb of
genomic sequences including parts of exons 5 and 6 by neo.
Electroporation of the linearized replacement vector into the embryonic
stem cell line ICp4 (kindly provided by C. Graves, UT Southwestern
Medical Center) and derivation of germ line chimeras by blastocyst
injections were performed according to standard procedures. Mice
genetically deficient for RnBP and wild type controls were kept on a
mixed genetic background of C57Bl/6J X 129SvJ. Experimental procedures
involving animals were conducted in accordance with institutional guidelines.
N-Acyl-D-glucosamine 2-Epimerase Assay--
Mouse
kidneys were homogenized in 9 volumes of 20 mM sodium
phosphate buffer, pH 7.0, containing 1 mM EDTA, 10 µM leupeptin, 1 mM
diisopropylphosphofluoridate, and 4 mM Analysis of the Renin-Angiotensin System--
Blood samples (100 µl) were collected from the retroorbital plexus of unanesthetized
mice into prechilled tubes containing 10 µl of 100 mM
EDTA, pH 7.4. To minimize stress-induced renin secretion, the blood
sampling was completed within 20 s. The plasma was separated by
centrifugation at 3,000 × g for 5 min at 4 °C. Total renin and active renin concentrations were measured by enzyme kinetic assay (with or without prior trypsinization) and by detection of ANG I conversion by radioimmunoassay. For total renin activity, 10 µl of plasma were activated for 15 min on ice with 10 µl of tosyl-phenylalanine chloromethyl keton-treated trypsin (Worthington, Lakewood, NJ) at a final concentration of 2.5 mg/ml. Trypsinization was
stopped by addition of 20 µl of (20 mg/ml) soy bean trypsin inhibitor
(Serva, Heidelberg, Germany). ANG I was generated by incubation of 10 µl of plasma (or trypsinate) for 1 h at 37 °C with 25 µl of
renin-free rat plasma and 25 µl of incubation buffer (200 mM Tris, 50 mM EDTA, 5 mM
phenylmethylsulfonyl fluoride, pH 8.5). The samples were diluted 1:100,
and ANG I concentrations were measured by radioimmunoassay as published
(12). All assays were carried out in triplicate. The concentration of
prorenin was calculated by subtracting the active from the total renin concentration. For determination of kidney renin concentrations, kidney
homogenates were prepared as described for the epimerase assay, and 10 µl of a 1:5000 dilution were assayed. To stimulate renin secretion in
mice, the angiotensin converting enzyme inhibitor cilazapril was
administered in drinking water at 50 mg/liter for 5 days, corresponding
to an estimated dose of 10 mg/kg body weight/day.
Blood Pressure Measurements--
Mice were anesthetized with a
mixture of ketamine/xylazine. A polyurethane catheter (1.2 French) was
placed into the right femoral artery and advanced into the abdominal
aorta below the branching of the renal vessels. The catheter was then
tunneled subcutaneously and exteriorized at the back of the neck. On
the next day, blood pressure was measured in conscious, unrestrained animals as published (13). Mean arterial pressure values were calculated for each animal by averaging all values recorded during a
5-10-min time period.
HPAEC Analysis of Urine Samples--
Urine samples were
heat-inactivated for 3 min and precleared by centrifugation at
10,000 × g for 5 min. 100 µl of 1:100 dilution of
the samples were loaded onto a Dionex Carbopac PA-1 column and
subjected to anion exchange chromatography on a sodium hydroxide/sodium acetate gradient essentially as described for the epimerase assay.
Using RNase protection assay, RnBP has previously been shown to be
co-expressed with renin in rat kidney (7). However, data about the
expression pattern in the mouse or about the exact renal cell type
synthesizing RnBP were lacking. To define the expression pattern of the
murine protein, we initially applied Northern blot analysis to screen
mouse tissues for the presence of RnBP mRNA (Fig.
1). In the mouse, RnBP is almost
exclusively expressed in the kidney with low expression in liver and
lung. Abundant expression in the kidney was also observed for renin (Fig. 1). Next, we tested whether the same renal cell types express RnBP and renin using in situ hybridization. As expected,
renin expression was observed in juxtaglomerular cells (Fig.
2A). Surprisingly, RnBP
mRNA was detected exclusively in glomerular cells, most likely mesangial cells (Fig. 2, B and C), with no
apparent co-expression of both proteins in any renal cell type. This
finding argued against a direct role of RnBP in regulation of renin
expression in juxtaglomerular cells.
To exclude a more indirect effect of RnBP on renin metabolism, we used
homologous recombination in embryonic stem cells to introduce a
targeting construct into the murine RnBP gene (Fig. 3). The neo selection cassette
replaced exons 5 and 6 of the RnBP gene, which encode a leucine zipper
motif required for interaction with renin (14, 15). Mice homozygous for
the disrupted RnBP allele were obtained by injection of embryonic stem
cells into blastocysts and by breeding of mice carrying the gene
disruption in their germ line (Fig.
4A). In mice homozygous for
the disrupted allele (
Normal Blood Pressure and Plasma Renin Activity in Mice Lacking
the Renin-binding Protein, a Cellular Renin Inhibitor*
§,,,,
,
Max-Delbrück-Center for Molecular
Medicine, §§ Department of Molecular Biology and
Biochemistry, Free University, and ** Franz-Volhardt-Clinic,
Humboldt University, D-13125 Berlin, Germany, the Departments of
¶ Medical Biochemistry and Cell Biology, University of
Aarhus, 8000 Aarhus C, Denmark, and ¶¶ Akita
Research Institute for Food and Brewing, Akita 010-1623, Japan
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-mercaptoethanol. Homogenates were centrifuged at 10,000 × g for 10 min
at 4 °C. 20 µl of supernatant were incubated for 20 h at
37 °C with 80 µl of epimerase assay buffer (100 mM
Tris-HCl, 10 mM MgCl2, 5 mM ATP, 10 mg/ml N-acetylmannosamine, pH 7.5). Then the samples were
heat-inactivated and cleared at 10,000 × g for 5 min.
100 µl of a 1:500 dilution of the supernatant were loaded on a
Carbopac PA-1 column (Dionex) and subjected to high performance anion
exchange chromatography (HPAEC) on a sodium hydroxide/sodium acetate
gradient at a flow rate of 1.25 ml/min, with subsequent pulsed
amperometric detection.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (52K):
[in a new window]
Fig. 1.
Expression pattern of RnBP and renin in mouse
tissues. 2 µg of poly(A)+ RNA of the indicated mouse
tissues were subjected to Northern blot analysis using hybridization
probes for mouse RnBP, mouse renin, and human -actin. Both RnBP and
renin are highly expressed in the kidney. The asterisk
denotes an alternative -actin mRNA species found in heart and
skeletal (sk.) muscle.
View larger version (117K):
[in a new window]
Fig. 2.
Localization of renin and RnBP expression in
the kidney. Cryosections from mouse (A) or rat kidneys
(B and C) were analyzed by in situ
hybridization with antisense probes directed against mouse renin-2
(A) or rat RnBP mRNAs (B and C).
Expression of renin is detectable in the juxtaglomerular apparatus
(arrow; A), whereas RnBP is confined to mesangial
cells in the glomeruli (arrow; B and
C). No signal is seen with a sense probe for RnBP
(D) or renin mRNA (not shown). Magnification was ×400
for A, B, and D and ×1000 for
C.
/), no RnBP mRNA was detectable in the
kidney, demonstrating successful inactivation of the RnBP gene locus
(Fig. 4B). RnBP/ mice were viable and fertile.
View larger version (9K):
[in a new window]
Fig. 3.
Gene targeting of the murine RnBP gene
locus. The targeting vector was constructed by replacing a
NcoI (N) fragment encoding exons 5 and 6 (filled boxes) of the murine RnBP gene with the
pol2neobpA cassette (NEO). Homologous
recombination of the vector with the wild type (WT) RnBP
allele is detected by hybridization of NcoI-digested genomic
DNA with a probe from the RnBP gene region. A 2.5-kb NcoI
fragment is diagnostic for the wild type, and a 3.0-kb NcoI
fragment is diagnostic for the disrupted allele.
View larger version (42K):
[in a new window]
Fig. 4.
Southern and Northern blot analysis of
mice. A, 20 µg of genomic DNA of mice of the
indicated genotypes were digested with NcoI and subjected to
Southern blot analysis. Wild type (WT) and disrupted RnBP
alleles (KO) are indicated. B, 20 µg of total
RNA from wild type (lanes 3 and 4) or RnBP /
kidneys (lanes 1 and 2) were analyzed by Northern
blot analysis using a mouse RnBP cDNA fragment. A signal
corresponding to the RnBP mRNA was detected in wild type but not in
knockout tissues. As a control, parallel RNA samples were probed for
glyceraldehyde-3-phosphate dehydrogenase mRNA
(GAPDH).
Next, we investigated whether inactivation of the RnBP gene resulted in
complete absence of RnBP activity in the kidney. In hog kidney
extracts, RnBP activity can be assayed by means of its inhibitory
effect on renin activity in vitro (16). In contrast, the
comparatively low amount of RnBP in mouse kidney extracts precludes
reliable detection of such protein activity (not shown). Therefore, we
assayed mouse renal extracts for
N-acyl-D-glucosamine 2-epimerase activity, the
second known biochemical function of RnBP. Conversion of the substrate
ManNAc to GlcNAc was readily observed when the substrate was incubated
with wild type kidney extracts. In contrast, no conversion was seen
when knockout kidney samples were used demonstrating lack of RnBP
activity (Fig. 5).
|
In plasma, renin is present in several glycoforms that result from a
complex pathway of renin synthesis, maturation, and secretion in
juxtaglomerular cells. This variability in glycosylation pattern has
important physiological consequences because it affects secretion and
plasma half-life of the protein. Thus, renin containing complex oligosaccharides is constitutively secreted by juxtaglomerular cells.
This protein exhibits slow hepatic clearance providing a basal source
of long-lived renin activity. On the other hand, renin molecules with
high mannose oligosaccharides are synthesized and stored within
secretory granules and released upon cellular stimulation. These renin
isoforms have a short plasma half-life and constitute the regulated
source of renin to the circulating RAS (17). Given the importance of
the renin biosynthetic pathways for the RAS, we tested whether RnBP
deficiency may affect glycosylation, intracellular localization, or
secretion of renin in the kidney. Plasma proteins from both wild type
and RnBP knockout mice were separated by two-dimensional gel
electrophoresis, transferred to nitrocellulose filters, and probed with
an anti-mouse renin antibody. As seen in Fig.
6, renin was present in several
glycovariants in mouse plasma. The pI for the mouse protein in this
assay was between 6 and 7, which is slightly more basic than renin from other species (e.g. pI 4.8-5.7 in humans). No difference in
the renin protein pattern was observed when comparing wild type with RnBP/ samples (Fig. 6). In addition, we analyzed the subcellular localization of renin in myoepithelial cells of the juxtaglomerular apparatus using immunoelectron microscopy. Intracellular staining for
renin localized to secretory granules in both wild type and knockout
tissue sections without discernible difference between both genotypes
(Fig. 7).
|
|
Finally, we compared the circulating RAS in wild type and
RnBP-deficient animals to detect possible consequences of RnBP
deficiency. No differences were observed when determining the
concentrations of active renin, prorenin, and AOGEN in plasma or active
renin in kidney (Table I). The mean
arterial blood pressure tended to be slightly lower in knockout mice,
but this trend was statistically not significant (Table I). Inhibition
of angiotensin converting enzyme activity is known to stimulate renin
secretion in vivo. Therefore, we administered the
angiotensin converting enzyme inhibitor cilazapril to mice for 5 days
and measured the concentrations of prorenin and active renin
thereafter. Although the levels of prorenin and active renin in plasma
increased 2- and 30-fold, respectively, no obvious differences were
observed when comparing wild type with RnBP knockout animals. The data
presented in Table I were obtained in strains of mice on a mixed
genetic background of C57Bl/6J and 129SvJ. It is well established that
C57Bl/6J mice carry one renin gene (ren-1), whereas 129SvJ
mice have two (ren-1 and ren-2) (18). To
ascertain that variability in the renin genotypes did not influence the
results obtained, we genotyped wild type and RnBP/ mice in these
studies for ren-1 and ren-2 genes using
polymerase chain reaction. Both groups of mice included similar numbers
of animals carrying one or two renin genes (not shown). Furthermore, we
also performed RAS analysis comparing only wild type and knockout mice
with one or both renin genes. No statistically significant differences
between the genotypes were observed in any of these experiments (not
shown).
|
So far, our studies have focused on the role of RnBP in the regulation
of renin. However, another biochemical activity of the protein has been
uncovered recently (8). RnBP acts as a N-acyl-D-glucosamine 2-epimerase and generates
ManNAc, a precursor in the biosynthesis of NANA. The well characterized
enzyme acting in this enzymatic pathway is the UDP-GlcNAc 2-epimerase,
which catalyzes the formation of ManNAc from UDP-GlcNAc. The importance of this enzyme in the regulation of NANA metabolism is underscored in
patients that express a constitutively active enzyme. As a consequence
of the defective regulation of the sialic acid metabolism, individuals
exhibit developmental delay, coarse facies, and massive urinary
excretion of NANA (sialuria) (21). In contrast to the UDP-GlcNAc
2-epimerase, the RnBP/N-acyl-D-glucosamine
2-epimerase is able to interconvert GlcNAc and ManNAc in an
ATP-dependent fashion. The significance of this reaction in
sialic acid metabolism is presently unknown. Using RnBP/ mice, we
now can address this question in vivo. In initial
experiments, we have analyzed the profile of metabolites excreted in
the urine of RnBP knockout mice and have identified distinct
differences as compared with wild type animals. Although the amount of
NANA is not changed significantly, knockout urine samples exhibit a
distinct peak that is absent in control samples (Fig.
8, peak X). The elution profile and additional studies (not shown) suggest that this peak may
represent an uncharged oligosaccharide. The exact nature of this
metabolite and its relationship to RnBP/GlcNAc 2-epimerase remains to
be shown.
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In the present study, we have carried out an extensive analysis of the RAS in mice lacking RnBP, a proposed cellular renin inhibitor. Despite previous experimental evidence implicating RnBP in the regulation of renin activity, we failed to identify any major effects of RnBP deficiency on the circulating RAS.
The kidney is the prime tissue to provide renin to the circulating RAS. Expression and secretion of renin in the kidney is tightly regulated and essential to control blood pressure and fluid homeostasis (1, 2). Thus, disturbances in renin activity may contribute to the development of hypertension. Given the central role of renin in the (patho-)physiology of RAS, much attention has been focused on identifying factors that may control renin expression and activity in the kidney. One such candidate was the RnBP. Experimental data to support this hypothesis were its co-expression with renin in tissues and established cell lines and its ability to specifically bind and inhibit renin (3, 4). We now have re-evaluated some of these earlier observations. Although both RnBP and renin are highly expressed in the mouse kidneys as shown by Northern blotting (Fig. 1), they are not produced in the same renal cell type. Whereas renin is found in juxtaglomerular cells, RnBP is synthesized most likely in mesangial cells of the glomerulus (Fig. 2). Because RnBP is not a secreted protein, it is therefore unlikely to encounter renin in renal tissues or in plasma. Thus, the observation of renin-RnBP complexes (high molecular mass renin) in kidney extracts may be an experimental artifact of homogenization of the tissue.
In vitro, RnBP specifically interacts with renin through a leucine zipper, a peptide motif involved in protein-protein interaction (4). A similar cluster of hydrophobic amino acids is found in renin (residues 232-253 of the human protein) and may constitute the RnBP-binding site. This tight and specific interaction between RnBP and renin is remarkable and suggests that it is more than coincidental. Although our data seem to exclude a specific interaction of RnBP and renin in the kidney or in the circulating RAS of the mouse, they do not rule out an interaction of both proteins in other systems. In particular, some studies have localized renin molecules in cellular compartments other than the secretory pathway, indicating an intracellular function for the protease. For example, an alternative renin mRNA giving rise to a cytosolic protein lacking the signal peptide has been detected in the adrenal and in the brain (19, 20). Potentially, RnBP may be expressed in low amounts in these tissues and involved in the regulation of intracellular renin activity.
In conclusion, we have tested a functional contribution of RnBP to the
RAS in vivo. Such a role for RnBP has previously been postulated based on a number of in vitro and cell culture
studies. We conclude that RnBP deficiency does not affect renal or
plasma renin activity in the mouse under normal physiological
conditions, excluding a role for this protein in regulation of the
circulating RAS. As such, our studies have resolved a long standing
debate on a potential mechanisms of renin regulation by RnBP. In the future, the RnBP-deficient mouse model will be important to uncover the
role played by RnBP in sialic acid metabolism.
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ACKNOWLEDGEMENTS |
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We are indebted to C. Räder, H. Schulz, D. Bischof, and S. Grüger for expert technical assistance and D. Ganten and M. Bader for critical reading of the manuscript.
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FOOTNOTES |
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* This work was supported by grants from the Deutsche Forschungsgemeinschaft, Klinisch-Pharmakologischer Verbund, Berlin-Brandenburg, and the Danish Medical Research Council.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Supported by a fellowship from the Helmholtz Society. To whom correspondence should be addressed: Max-Delbrück-Center for Molecular Medicine, Robert-Rössle-Str. 10, D-13125 Berlin, Germany. Tel.: 49-30-9406-2589; Fax: 49-30-9406-2110; E-mail: cschmitz@mdc-berlin.de.
Heisenberg fellow of the Deutsche Forschungsgemeinschaft.
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ABBREVIATIONS |
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The abbreviations used are: AOGEN, angiotensinogen; ANG, angiotensin; RAS, renin-angiotensin system; RnBP, renin-binding protein; ManNAc, N-acetylmannosamine; NANA, N-acetylneuraminic acid; kb, kilobase(s); HPAEC, high performance anion exchange chromatography.
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REFERENCES |
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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