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Nonproteolytic Activation of Prorenin Contributes to
Development of Cardiac Fibrosis in Genetic Hypertension
Atsuhiro Ichihara, Yuki Kaneshiro, Tomoko Takemitsu, Mariyo Sakoda, Fumiaki Suzuki,
Tsutomu Nakagawa, Akira Nishiyama, Tadashi Inagami, Matsuhiko Hayashi
Abstract—In contrast to proteolytic activation of inactive prorenin by cleavage of the N-terminal 43 residue peptide, we
found that prorenin is activated without proteolysis by binding of the prorenin receptor to the pentameric “handle region”
I11PLLKK15P. We hypothesized that such activation occurs in hypertensive rats and causes cardiac renin–angiotensin
system (RAS) activation and end-organ damage. To test this hypothesis, we devised methods of specifically inhibiting
nonproteolytic activation by decapeptide spanning the pentameric handle region peptide as a decoy. In stroke-prone
spontaneously hypertensive rats (SHRsp) fed a high-salt diet, arterial pressure started to rise significantly with a marked
increase in the cardiac prorenin receptor mRNA level at 8 weeks of age, and cardiac fibrosis had developed by 12 weeks
of age. By immunohistochemistry using antibodies to the active site of the renin molecule, we demonstrated increased
proteolytic or nonproteolytic activation of prorenin in the heart but not in plasma of SHRsp. Continuous subcutaneous
administration of the handle region peptide completely inhibited the increased staining by antibodies to the active site
of the renin molecule, indicating the increased nonproteolytic but not proteolytic activation of prorenin in the heart of
SHRsp. Administration of the handle region peptide also inactivated tissue RAS without affecting circulating RAS or
arterial pressure and significantly attenuated the development and progression of cardiac fibrosis. These results clearly
demonstrate the significant role of nonproteolytically activated tissue prorenin in tissue RAS activation leading to
cardiac fibrosis and significant inhibition of the cardiac damage produced by chronic infusion of the handle region
peptide. (Hypertension. 2006;47:894-900.)
Key Words: angiotensin 䡲antibodies 䡲renin
Progressive end-organ damage of the heart is a hallmark of
hypertensive diseases, which results in an irreversible
morbid outcome leading to death. Direct mechanical stress by
high perfusion pressure and an activated renin-angiotensin
system (RAS) are considered to play decisive roles in the
development and progression of hypertensive cardiac dam-
age. However, because in many types of essential hyperten-
sion plasma RAS is not elevated and circulating RAS is even
subnormal, we cannot explain the morbidity by elevation of
circulating RAS alone. Alternatively, we postulated elevated
tissue renin activity in critical organs, such as the heart, as a
possible mechanism. We, and others, have found that a prorenin
binding protein exists on the plasma membrane surface in
humans
1–5
and now in rat tissues.
6
The present study focused
on the prorenin receptor described by Nguyen et al,
4
because
its binding to prorenin activates the proenzyme via a confor-
mational change without proteolytically cleaving the 43
amino acid prosegment off the main body of active (mature)
renin.
6
Because the prorenin receptor localizes to sensitive
organs like the heart,
4
we hypothesized that the prorenin
receptor will sequester prorenin and activate it on the cell
surfaces of critical organs susceptible to end-organ damage.
The activated prorenin will generate angiotensin I and II locally,
thereby exerting local actions leading to tissue damage. Because
the exact in vivo mechanism of this process has not been
demonstrated, we devised an intervention technique to inhibit
prorenin binding to the receptor in vivo. A short pentapeptide
sequence in the prosegment of prorenin was identified as the
binding region to the receptor, and the peptide was infused
into hypertensive rats to compete for prorenin binding to the
receptor or binding antibody, thus preventing the nonproteo-
lytic activation of prorenin by the prorenin receptor.
6
It is
noteworthy that such prorenin activation does not occur in
blood by a nonproteolytic mechanism, because the prorenin
receptor is not present in plasma.
4
We demonstrate herein that the elevated mRNA of prore-
nin receptor, nonproteolytically activated prorenin, elevated
tissue angiotensin I and II concentrations, and marked fibrosis
occur in the hypertensive heart. Furthermore, the short peptide
competitive inhibitor almost completely blocked the nonpro-
Received November 24, 2005; first decision December 19, 2005; revision accepted February 13, 2006.
From the Department of Internal Medicine (A.I., Y.K., T.T., M.S., M.H.), Keio University School of Medicine, Tokyo, Japan; Faculty of Applied
Biological Sciences (F.S., T.N.), Gifu University, Gifu, Japan; Department of Pharmacology (A.N.), Kagawa University School of Medicine, Kagawa,
Japan; and Department of Biochemistry (T.I.), Vanderbilt University School of Medicine, Nashville, Tenn.
Correspondence to Atsuhiro Ichihara, Department of Internal Medicine, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo,
160-8582, Japan. E-mail atzichi@sc.itc.keio.ac.jp
© 2006 American Heart Association, Inc.
Hypertension is available at http://www.hypertensionaha.org DOI: 10.1161/01.HYP.0000215838.48170.0b
894
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teolytic activation of prorenin, resulting in the normalization
of angiotensin I and II in the hearts of hypertensive animals
to the levels of normotensive animals without lowering the
plasma concentrations of angiotensin I and II. These results
may be taken as evidence of the important roles of nonpro-
teolytically activated prorenin in tissues showing hyperten-
sive cardiac damage.
Methods
Animals
We maintained male stroke-prone spontaneously hypertensive rats
(SHRsp) and normotensive control Wystar Kyoto rats (WKY;
Charles River Labs, Yokohama, Japan) in a temperature-controlled
room, 23°C, and on a 12:12-hour light-dark cycle. Rats had free
access to 1% NaCl water and a normal-salt– diet rat chow (0.4%
NaCl; CE-2, Nihon Clea). The Keio University Animal Care and Use
Committee approved all of the experimental protocols including the
implantation of both minipumps and radiotelemetry transmitters in
rats at 4 weeks of age. At 4 weeks of age, we subcutaneously
implanted osmotic minipumps (model 2004 for 28-day use, Alzet)
containing saline or a decapeptide, NH2-RILLKKMPSV-COOH, as
the “handle region” of rat prorenin (HRP, 0.1 mg/kg) under sodium
pentobarbital anesthesia (50 mg/kg IP) and divided the rats (100- to
150-g body weight) into 4 groups: SHRsp, SHRsp⫹HRP, WKY, and
WKY⫹HRP. At 8 weeks of age, we replaced the minipump with
another pump filled with the same solution, and 4 weeks later (12
weeks of age) decapitated 6 to 8 rats at 12 weeks of age to collect
their blood and hearts. In our preliminary study, an osmotic
minipump with an NH2-SFGR-COOH (0.1 mg/kg, n⫽3) or NH2-
MTRISAE-COOH (0.1 mg/kg, n⫽3) was also implanted into the
SHRsp group. However, these peptides did not inhibit the develop-
ment of glomerulosclerosis or increase in angiotensin II levels in the
kidneys of SHRsp.
Telemetry Probe Implantation
At 4 weeks of age, we implanted a telemetry transmitter probe
(model TA11PA-C40, Data Sciences International) into rats under
sodium pentobarbital anesthesia (50 mg/kg IP), and the flexible tip of
the probe was positioned immediately below the renal arteries. The
transmitter was then surgically sutured into the abdominal wall, and
the incision was closed. The rats were then returned to their home
cages and allowed to recover for 6 days before starting measure-
ments. We monitored conscious mean arterial pressure, heart rate,
and activity in unrestricted and untethered animals with the
Dataquest IV system (Data Sciences International), which consisted
of the implanted radiofrequency transmitter and a receiver placed
under each cage. The output was relayed from the receiver through
a consolidation matrix to a personal computer. Individual 10-s mean
arterial pressure, heart rate, and activity waveforms were sampled
every 5 minutes throughout the course of the study, and daily
averages and SDs were then calculated.
Morphological and
Immunohistochemical Evaluation
Part of the heart removed from each animal was fixed in 10%
formalin in phosphate buffer (pH 7.4), and paraffin-embedded
sections of the heart were stained by the Masson trichrome method.
For immunohistochemical staining, deparaffinized sections were
pretreated with proteinase K and after boiling the sections in citrate
buffer with microwaving to unmask antigenic sites, and endogenous
biotin was blocked with a Biotin Blocking System (X0590; DAKO
Corp). Next, the sections were immersed in 3% H2O2in methanol to
inhibit endogenous peroxidase and then precoated with 1% nonfat
milk in PBS to block nonspecific binding. For immunohistochemical
staining of total and nonproteolytically activated prorenin, the
antibody to the prorenin prosegment
6
or antibody to the active site of
the renin molecule (1:1000; References 7–9) was applied to the
sections as the primary antibody. The sections were incubated with
a biotin-conjugated antirabbit IgG as the secondary antibody, and the
antibody reactions were visualized with a Vectastain ABC Standard
kit (Vector Laboratories) and an AEC Standard kit (DAKO) accord-
ing to the manufacturers’ instructions. For quantitative evaluation of
total prorenin and nonproteolytically activated prorenin, we counted
the number of cells in which the signal intensity of the reaction
products was visible. The final overall score was calculated as the mean
of the values for 100 ventricular cross-sections per group of rats.
Measurements of Renin and Angiotensin Peptides
Immediately after decapitation, a 3-mL blood specimen was col-
lected into a tube containing 30
L of EDTA (500 mmol/L), 15
L
of enalaprilat (1 mmol/L), and 30
L of o-phenanthroline (24.8
mg/mL) and pepstatin (0.2 mmol/L), and plasma samples were
obtained by centrifugation. Plasma levels of components of the
circulating RAS were determined as described previously.
10
For the
measurement of total cardiac renin, a part of the removed cardiac
ventricle was weighed, placed in 5 mL of buffer containing 2.6 mmol/L
EDTA, 1.6 mmol/L dimercaprol, 3.4 mmol/L 8-hydroxyquinoline
sulfate, 0.2 mmol/L PMSF, and 5 mmol/L ammonium acetate; homog-
enized with a chilled glass homogenizer; and centrifuged. The
homogenate was frozen and thawed 4 times, spun at 5000 rpm for 30
minutes at 4°C, and the supernatant was removed. Then, 500
Lof
plasma obtained from nephrectomized male rats were added to an
equal volume of the supernatant as a substrate for the enzymatic
reaction. The renin activity was determined as described previously.
11
Angiotensin I and II levels in the heart were determined as reported
previously.
12
Real-Time Quantitative RT-PCR Analysis
We extracted total RNA from part of the heart removed from each
animal with an Rneasy Mini kit (Qiagen) and performed a real-time
quantitative RT-PCR with the TaqMan One-Step RT-PCR Master
Mix Reagents kit, an ABI Prism 7700 HT Detection System (Applied
Biosystems), and probes and primers for the rat genes encoding renin,
angiotensinogen, angiotensin-converting enzyme (ACE), collagen I, and
GAPDH, as described previously.
6,13
We used the commercially avail-
able probes and primers for the rat genes encoding collagen III (Applied
Biosystems) and designed the probe and primers for the rat prorenin
receptor (forward, 5⬘-CATTCGACACATCCCTGGTG-3⬘; reverse,
5⬘-AAGGTTGTAGGGACTTTGGGTG-3⬘; and probe, 5⬘-FAM-
AAGTCAAGGACCATCCTTGAGACGAAACAA-TAMRA-3⬘), based
on its cDNA sequence reported in the GenBank database (Accession
No. AB188298 in DNA Databank of Japan), which showed a high
sequence homology with the human renin/prorenin receptor mRNA
described by Nguyen et al.
4
Statistical Analyses
Within-group statistical comparisons were made by 1-way ANOVA for
repeated measures followed by the Newman-Keuls post hoc test.
Differences between 2 groups were evaluated by 2-way ANOVA for
repeated measures combined with the Newman-Keuls post hoc test.
P⬍0.05 was considered significant. Data are reported as mean⫾SEM.
Results
Arterial Pressure and Prorenin Receptor
mRNA Expression
We investigated changes in arterial pressure by implanting
minipumps containing an HRP or saline for 8 weeks in SHRsp
and WKY, in the treatment and control groups, respectively
(Figure 1). Mean arterial pressure in the SHRsp group started to
increase significantly at 8 weeks of age and further increased for
the following 4 weeks as compared with arterial pressure in the
WKY rats, whereas HRP did not affect mean arterial pressure in
either the SHRsp or the WKY group. At 8 weeks of age, when
no organ damage was present in either the SHRsp or the WKY
rats, cardiac prorenin receptor mRNA levels were significantly
Ichihara et al Hypertensive Heart by Activated Prorenin 895
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increased in the SHRsp group as compared with the WKY
group. However, HRP did not affect cardiac prorenin receptor
mRNA levels in either the SHRsp or the WKY group, as
shown in Figure 1. At 12 weeks of age, when significant
organ damage was seen in the SHRsp group, cardiac prorenin
receptor mRNA levels in this group were decreased, having
fallen to levels similar to those in the WKY group.
Heart Damage
We also investigated cardiac morphology, heart weight, and
fibrosis in the SHRsp, SHRsp⫹HRP, WKY, and WKY⫹HRP
groups at 12 weeks of age after 8 weeks of treatment (Figure
2). The areas of fibrosis stained blue by Masson trichrome
were increased in perivascular areas (Figure 2a) and the
myocardium (Figure 2b) in the SHRsp group as compared
with the WKY and WKY⫹HRP groups, whereas in the
SHRsp⫹HRP group, fibrosis remained only slightly higher
than in the control. The ventricular size (Figure 2c) and heart
weight (Figure 2d) were also greater in the 12-week-old
SHRsp group than in the WKY and WKY⫹HRP groups.
HRP mitigated the increases in ventricular size and heart
weight in the SHRsp group.
Circulating RAS
At 12 weeks of age, after 8 weeks of treatment with HRP or
saline, plasma renin activity was significantly higher in the
SHRsp group (3.6⫾1.0 ng/mL per hour) than in the WKY
group (1.7⫾0.6 ng/mL per hour) and HRP had no effect in
either group (Figure 3a). At this point, the plasma prorenin
was significantly higher in the SHRsp group (6.7⫾0.4 ng/mL
Figure 1. Mean arterial pressure determined by
telemetry in conscious unrestrained animals during
the 8-week study period (left) and prorenin recep-
tor mRNA levels at 8 weeks of age (right) in the
SHRsp (n⫽7), SHRsp⫹HRP (n⫽7), WKY (n⫽4),
and WKY⫹HRP (n⫽4) groups. *P⬍0.05 vs 5 weeks
of age. †P⬍0.05 for SHRsp vs WKY.
Figure 2. Inhibition of the development of left ventricular fibrosis by the HRP of prorenin in SHRsp at 12 weeks of age. (a) Representa-
tive short-axis images of intramuscular arteries with perivascular fibrosis stained with Masson trichrome. Scale bars: 50
m. (b) Repre-
sentative short-axis images of myocardium with interstitial fibrosis stained with Masson trichrome. Scale bars: 50
m. (c) Representa-
tive short-axis sections of cardiac ventricle stained with Masson trichrome. (d) Ratio of heart weight/body weight in the SHRsp (n⫽8),
SHRsp⫹HRP (n⫽8), WKY (n⫽6), and WKY⫹HRP (n⫽6) groups at 12 weeks of age. *P⬍0.05 vs WKY and WKY⫹HRP. †P⬍0.05 for
SHRsp⫹HRP vs SHRsp.
896 Hypertension May 2006
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per hour) than in the WKY group (3.9⫾0.8 ng/mL per hour),
and HRP had no effect in either group (Figure 3b). At 12
weeks of age, plasma angiotensin I and II levels were also
higher in the SHRsp group (249⫾74 and 169⫾10 fmol/L,
respectively) than in the WKY group (132⫾26 and 38⫾8
fmol/L, respectively), and HRP had no influence on these
levels in either group (Figure 3c and 3d). Thus, the ratio of
angiotensin II to angiotensin I in plasma appeared to be lower
in the WKY group than in the SHRsp group. The plasma
ACE activity may be lower in the WKY group than in the
SHRsp group.
Cardiac RAS and Collagen I and III
mRNA Levels
At 12 weeks of age, cardiac renin mRNA levels were similar
in the SHRsp, SHRsp⫹HRP, WKY, and WKY⫹HRP
groups, but the cardiac total renin content in the 12-week– old
SHRsp group was significantly higher than in the 12-week–
old WKY group, and HRP had no effect on total cardiac renin
content (Figure 4a and 4b). Cardiac angiotensin I and II
contents were significantly higher in the SHRsp group than
the similarly low levels in the SHRsp⫹HRP, WKY, and
WKY⫹HRP groups (Figure 4c and 4d). At 12 weeks of age, the
cardiac angiotensin I contents in the SHRsp, SHRsp⫹HRP,
WKY, and WKY⫹HRP groups averaged 91⫾3, 43⫾6, 42⫾8,
and 44⫾6 fmol/g, respectively, and their cardiac angiotensin II
contents averaged 92⫾3, 35⫾4, 29⫾6, and 31⫾5 fmol/g,
respectively. Thus, HRP completely inhibited the increases in
the cardiac angiotensin I and II contents in the SHRsp group. At
12 weeks of age, the cardiac mRNA levels for angiotensino-
gen and ACE did not differ significantly among SHRsp,
SHRsp⫹HRP, WKY, and WKY⫹HRP (Figure 4e and 4f).
Cardiac collagen I and III mRNA levels were significantly
higher in the SHRsp group than in the WKY group, and HRP
completely inhibited the increases in cardiac collagen I and III
mRNA levels in the SHRsp group, while not affecting the
cardiac collagen I and III mRNA levels in the WKY group
(Figure 4g and 4h).
Total and Nonproteolytically Activated Prorenin
in the Heart
To estimate cardiac levels of total and nonproteolytically
activated prorenin, we performed an immunohistochemical
analysis of cardiac ventricles collected from rats at 12 weeks
of age. There were significantly greater numbers of prorenin-
positive cells stained with antibody to the prorenin proseg-
ment in the perivascular area of the SHRsp group than in that
of the WKY group. The increased prorenin immunoreactivity
was unaffected by HRP (Figure 5a and 5b). The nonproteo-
lytically activated prorenin-positive cells stained with anti-
body to the active site of the renin molecule were also
increased in the perivascular area in the SHRsp group, but
their numbers were significantly decreased by HRP. The
cardiac level of nonproteolytically activated prorenin in the
SHRsp⫹HRP group was similar to those in the WKY and
WKY⫹HRP groups (Figure 5a and 5c). These results suggest
that the SHRsp heart contains an increased level of nonpro-
teolytically activated prorenin.
Discussion
At 8 weeks of age, when no organ damage was present in the
hearts of either SHRsp or WKY rats, significant increases in
arterial pressure and cardiac prorenin receptor mRNA levels
were observed in the SHRsp group as compared with the
WKY group. At 12 weeks of age, significant cardiac fibrosis
with activated tissue RAS and high levels of activated
Figure 3. Changes in plasma renin activity, prorenin levels, and angiotensin peptides levels in the SHRsp group (n⫽8), SHRsp⫹HRP
(n⫽8), WKY (n⫽6), and WKY⫹HRP (n⫽6) group. (a) Plasma renin activity. (b) Plasma prorenin concentration. (c) Plasma angiotensin I
concentration. (d) Plasma angiotensin II concentration. *P⬍0.05 vs WKY and WKY⫹HRP.
Ichihara et al Hypertensive Heart by Activated Prorenin 897
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prorenin was demonstrated in the SHRsp heart. Because
prorenin receptor binding to prorenin activates the proenzyme
via a conformational change without proteolytically cleaving
the prosegment off the main body of active renin, increased
expression of the prorenin receptor may contribute to cardiac
end-organ damage and tissue RAS activation. In the present
study, chronic subcutaneous administration of HRP com-
pletely inhibited both nonproteolytic activation of prorenin
and activation of tissue RAS without affecting circulat-
ing RAS or arterial pressure. In addition, HRP significantly
attenuated the development and progression of hypertensive
end-organ damage, despite the activated circulating RAS and
sustained high arterial pressure, a possible source of mechan-
ical end-organ stress. These findings suggest a crucial role of
nonproteolytic activation of prorenin in tissue RAS activation
in hypertensives and that nonproteolytic activation of prore-
nin should be considered as an important target of strategies
to prevent hypertensive heart damage. Because cardiac pro-
renin receptor mRNA levels in the SHRsp group decreased to
levels similar to those in the WKY group at 12 weeks of age
when significant organ damage developed in the SHRsp
group only, the prorenin receptor may contribute to the
development but not progression of cardiac damage in the
SHRsp group.
Immunohistochemical studies of tissues with antibody to the
prorenin prosegment showed a higher level of prorenin in the
perivascular area of the heart in the hypertensive SHRsp than in
the normotensive WKY group. This in vivo observation indi-
cates the increased tissue prorenin levels in hypertensive animals
and corroborates previous data showing that exposure to high
pressure inhibits conversion of prorenin to renin in juxtaglomer-
ular cells and subsequently increases intracellular prorenin lev-
els.
14
We previously presented in vitro evidence that binding of
a prorenin-binding protein, such as a prorenin receptor, to the
handle region of the prorenin prosegment activated prorenin
without proteolytic cleavage of prorenin and obtained in vitro
and in vivo evidence that HRP, used as a decoy, out-competes
for handle region binding and thereby inhibits the nonproteolytic
activation of prorenin.
6
In the present study, chronic administra-
tion of the decoy peptide HRP did not alter the number of total
prorenin-positive cells but significantly decreased the active
renin immunoreactivity to a level similar to that in WKY rats. If
the active renin immunoreactivity represents proteolytically
activated renin, the HRP decoy peptide should be unable to
decrease the active renin immunoreactivity, because proteolyti-
cally activated renin does not contain the prorenin prosegment,
including the handle region. However, HRP significantly de-
creased the active renin immunoreactivity, suggesting that the
active renin immunoreactivity represents nonproteolytically ac-
tivated prorenin, which contains the prorenin prosegment and
handle region. These results suggest that prorenin levels were
elevated in the damaged hearts of hypertensive animals and that
a greater amount of prorenin was nonproteolytically activated,
probably via elevated expression of the prorenin receptor.
The tissue levels of angiotensin I and II were also higher in the
SHRsp heart than in the WKY heart, and peptide levels were
completely normalized by HRP treatment, presumably by pre-
venting the binding of prorenin to the prorenin receptor. There
were no changes in other RAS components, suggesting nonpro-
teolytic activation of prorenin to play a key role in tissue RAS
activation in hypertensive animals. In addition to the increased
total renin content in the SHRsp heart, we observed a significant
Figure 4. Changes in components of the RAS, collagen I mRNA, and collagen III mRNA in the heart of the SHRsp (n⫽8), SHRsp⫹HRP
(n⫽8), WKY (n⫽6), and WKY⫹HRP (n⫽6) groups. (a) Cardiac renin mRNA level. (b) Cardiac total renin content. (c) Cardiac angiotensin I
level. (d) Cardiac angiotensin II level. (e) Cardiac angiotensinogen mRNA level. (f) Heart ACE (ACE) mRNA level. (g) Cardiac collagen I
mRNA level. (e) Cardiac collagen III mRNA level. *P⬍0.05 vs WKY and WKY⫹HRP.
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increase in prorenin receptor mRNA levels in the hearts of
8-week– old SHRsp. Therefore, it is likely that increases in both
the prorenin receptor and tissue prorenin are the determining
factors in enhancing nonproteolytic activation.
HRP interferes with prorenin binding to a prorenin receptor as
a decoy peptide and thereby inhibits RAS activation by the
nonproteolytic activation of prorenin. Chronic infusion of the
decoy peptide HRP markedly lowered tissue angiotensin I and II
levels of SHRsp to those of WKY, whereas HRP did not lower
arterial pressure or plasma angiotensins. These results indicate
that inhibition of nonproteolytic activation occurs only in tissues,
that is, not in plasma. The difference can be explained by the
prorenin receptor being present exclusively in tissue, whereas
none is detectable in plasma.
4
Our preliminary study showed
that, as well as in the heart, HRP did not affect the total renin
content in the kidneys of SHRsp, although the renin secreted
from the kidneys did influence the circulating RAS. Thus, HRP
significantly decreased cardiac angiotensin I and II by inhibiting
the nonproteolytic activation of prorenin in the heart but did not
affect plasma angiotensin I or II levels. Because HRP had no
effect on proteolytically activated circulating RAS or increased
arterial pressure in the SHRsp group, increased plasma renin
activity but not the plasma prorenin concentration may account
for the activated circulating RAS and increased arterial pressure
in SHRsp.
There appears to be a difference in cardiac total renin
expression between the 2 strains, SHRsp and WKY. However,
no matter what the details of the underlying mechanisms may be,
it is clear that the total renin content of the heart was higher in the
SHRsp group than in the WKY group despite a similar level of
renin mRNA in the 2 groups. Although Peters et al
15
suggested
that increased tissue prorenin may be because of internalization
by cardiac tissue from plasma, we found higher plasma prorenin
concentrations in the SHRsp group than in the WKY group and
predominant immunostaining of prorenin in the perivascular
area of the heart in the SHRsp group. Further studies will be
needed to clarify the mechanism regulating the proteolytically
activated circulating RAS and the increased cardiac total renin
content in the SHRsp group.
Despite a similar level of cardiac prorenin receptors in the
SHRsp and WKY rats at 12 weeks of age, the number of cardiac
prorenin-positive cells was higher in the SHRsp than in the
Figure 5. Total and nonproteolytically activated prorenin in the hearts of the SHRsp (n⫽8), SHRsp⫹HRP (n⫽8), WKY (n⫽6), and
WKY⫹HRP (n⫽6) groups. (a) Immunohistochemistry of total and nonproteolytically activated prorenin in the heart with antibody to the
prorenin prosegment and antibody to the active site of the renin molecule, respectively. Scale bars: 50
m (prorenin prosegment and
active center of renin at left) and 200
m (active center of renin at right). (b) Quantitive analysis of number of prorenin-positive cells per
100 ventricular cross-sections (VCS). (c) Quantitive analysis of number of nonproteolytically activated prorenin-positive cells per 100
VCS. *P⬍0.05 vs WKY and WKY⫹HRP.
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WKY rats. In addition, the inhibition of prorenin binding to the
prorenin receptor by HRP did not alter the number of cardiac
prorenin-positive cells or the perivascular localization of prore-
nin in the heart of SHRsp. These results suggest that the majority
of cardiac prorenin may exist intracellularly and is not bound to
the prorenin receptor.
In conclusion, the present studies using SHRsp show that the
novel nonproteolytic prorenin activation mechanism has a spe-
cific role in hypertensive end-organ damage in tissues where
tissue prorenin is activated via its binding proteins, such as the
prorenin receptor,
4
rather than via traditional activation by
proteolytic cleavage of the 43 amino acid prosegment.
Perspectives
Lowering of blood pressure, if possible, may be another essential
strategy for the prevention of organ damage in hypertensives, as
suggested by several clinical studies.
16–18
However, achieving
the target blood pressure recommended in guidelines is difficult
for hypertensive patients; some are unable to sufficiently control
their blood pressure and ultimately develop end-organ damage,
despite the best efforts of their physicians.
19
In the present study,
HRP significantly attenuated hypertensive end-organ damage
without reducing high arterial pressure. Thus, we propose that
tissue prorenin activation by the nonproteolytic mechanism can
be an important target of strategies for preventing hypertensive
end-organ damage.
Acknowledgments
This work was supported in part by grants from the Ministry of
Education, Science, and Culture of Japan (14571073, 1503340,
16613002, and 16790474); a grant from the Takeda Science Foun-
dation (Osaka, Japan; to A.I.); Health and Labor Sciences research
grants, Research on Measures for Intractable Diseases; and research
grant HL58205 from the National Institutes of Health.
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900 Hypertension May 2006
by guest on February 19, 2018http://hyper.ahajournals.org/Downloaded from
Tsutomu Nakagawa, Akira Nishiyama, Tadashi Inagami and Matsuhiko Hayashi
Atsuhiro Ichihara, Yuki Kaneshiro, Tomoko Takemitsu, Mariyo Sakoda, Fumiaki Suzuki,
Genetic Hypertension
Nonproteolytic Activation of Prorenin Contributes to Development of Cardiac Fibrosis in
Print ISSN: 0194-911X. Online ISSN: 1524-4563
Copyright © 2006 American Heart Association, Inc. All rights reserved.
is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231Hypertension doi: 10.1161/01.HYP.0000215838.48170.0b
2006;47:894-900; originally published online April 3, 2006;Hypertension.
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