Impaired post-infarction cardiac remodeling in chronic kidney disease is due to excessive renin release.
ABSTRACT The complex pathophysiological interactions between heart and kidney diseases are collectively known as cardiorenal syndrome. The renin-angiotensin system (RAS) may have a pivotal role in the development of cardiorenal syndrome. The aim of this study was to elucidate the RAS activity responsible for adverse post-infarction remodeling and prognosis in mice with renal failure. To establish the type IV cardiorenal syndrome model, 5/6 nephrectomy (NTX) was performed in a surgical procedure, followed by the induction of myocardial ischemia (MI) by a coronary artery ligation 4 weeks later. NTX and MI resulted in deteriorated left ventricular remodeling and RAS activation, which was improved by an aliskiren that appeared to be independent of renal function and blood pressure (BP). Moreover, MI induced in renin and angiotensinogen double-transgenic (Tg) mice showed comparable effects to MI plus NTX mice, including advanced ventricular remodeling and enhancement of RAS, oxidative stress, and monocytes chemoattractant protein (MCP)-1. Aliskiren suppressed these changes in the MI-induced Tg mice. In in vitro study, Nox2 expression was elevated by the stimulation of plasma from NTX mice in isolated neonatal cardiomyocytes. However, Nox2 upregulation was negated when we administered plasma from aliskiren-treated-NTX mice or isolated cardiomyocytes from AT1-deficient mice. Primary mononuclear cells also showed an upregulation in the expression of Nox2 and MCP-1 by stimulation with plasma from NTX mice. Our data suggest that renal disorder results in ventricular dysfunction and deteriorates remodeling after MI through excessive RAS activation. Moreover, renin inhibition improved the changes caused by cardiorenal syndrome.Laboratory Investigation advance online publication, 17 September 2012; doi:10.1038/labinvest.2012.136.
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ABSTRACT: Cardiorenal syndrome (CRS) is characterized as a syndrome involving both the cardiovascular system and kidneys. Due to its complexity and high mortality, it has becoming a significant burden and a universal clinical challenge to society worldwide. The mechanisms underlying CRS are potentially multifactorial, including hemodynamic alterations, neurohormonal activation, inflammation, oxidative stress, iron disorders, anaemia, and mineral metabolic derangements. Despite the understanding and awareness of CRS gaining attention, appropriate approaches to manage CRS remain deficient. Loop diuretic and thiazides, inhibition of the renin-angiotensin system, vitamin D receptor activation and dopamine and natriuretic peptides could potentially be helpful to improve the prognosis of CRS. Ultrafiltration might be an alternative therapeutic strategy for the loss of liquid. However, adenosine receptor antagonists appear to not be superior to furosemide in CRS treatment. Additional novel therapeutic approaches should be further explored.Life sciences 10/2013; · 2.56 Impact Factor
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ABSTRACT: Introduction: Nuclear factor-kappa B (NF-kappaB), which is activated by the inhibitor of NF-kappaB kinase (IKK), is involved in the progression of inflammatory disease. However, the effect of IKK inhibition on the progression of myocarditis is unknown. Objective: We examined the effect of IKK inhibition on the progression of myocarditis. Methods: Lewis rats were immunized with porcine cardiac myosin to induce experimental autoimmune myocarditis (EAM). We administered the IKK inhibitor (IMD-0354, 15mg/kg/day) or vehicle to EAM rats daily. Hearts were harvested 21 days after immunization. Results: Although the untreated EAM group showed increased heart weight to body weight ratio, severe myocardial damage, these changes were attenuated in the IKK inhibitor treated group. Moreover, IKK inhibitor administration significantly reduced NF-kappaB activation and mRNA expression of interferon-gamma (IFN-gamma), interleukin-2 (IL-2) and monocyte chemoattractant protein-1 (MCP-1) in myocardium compared with vehicle administration. In vitro study showed that the IKK inhibitor treatment inhibited T cell proliferation and Th1 cytokines production induced by myosin stimulation. Conclusion: The IKK inhibitor ameliorated EAM by suppressing inflammatory reactions via suppression of T-cell activation.AJP Heart and Circulatory Physiology 10/2013; · 4.01 Impact Factor
Impaired post-infarction cardiac remodeling in chronic
kidney disease is due to excessive renin release
Masahito Ogawa1,2,3, Jun-ichi Suzuki2, Kiyoshi Takayama4, Takaaki Senbonmatsu5, Yasunobu Hirata2,6,
Ryozo Nagai6and Mitsuaki Isobe1
The complex pathophysiological interactions between heart and kidney diseases are collectively known as cardiorenal
syndrome. The renin–angiotensin system (RAS) may have a pivotal role in the development of cardiorenal syndrome.
The aim of this study was to elucidate the RAS activity responsible for adverse post-infarction remodeling and prognosis
in mice with renal failure. To establish the type IV cardiorenal syndrome model, 5/6 nephrectomy (NTX) was performed in
a surgical procedure, followed by the induction of myocardial ischemia (MI) by a coronary artery ligation 4 weeks later.
NTX and MI resulted in deteriorated left ventricular remodeling and RAS activation, which was improved by an aliskiren
that appeared to be independent of renal function and blood pressure (BP). Moreover, MI induced in renin and
angiotensinogen double-transgenic (Tg) mice showed comparable effects to MI plus NTX mice, including advanced
ventricular remodeling and enhancement of RAS, oxidative stress, and monocytes chemoattractant protein (MCP)-1.
Aliskiren suppressed these changes in the MI-induced Tg mice. In in vitro study, Nox2 expression was elevated by the
stimulation of plasma from NTX mice in isolated neonatal cardiomyocytes. However, Nox2 upregulation was negated
when we administered plasma from aliskiren-treated-NTX mice or isolated cardiomyocytes from AT1-deficient mice.
Primary mononuclear cells also showed an upregulation in the expression of Nox2 and MCP-1 by stimulation with plasma
from NTX mice. Our data suggest that renal disorder results in ventricular dysfunction and deteriorates remodeling after
MI through excessive RAS activation. Moreover, renin inhibition improved the changes caused by cardiorenal syndrome.
Laboratory Investigation (2012) 92, 1766–1776; doi:10.1038/labinvest.2012.136; published online 17 September 2012
KEYWORDS: infarction; inflammation; remodeling; renin angiotensin system
It is well known that there is a complex relationship between
cardiovascular and renal diseases. In fact, end-stage renal
disease or decreased estimated glomerular filtration rate
correlates with an increased risk of cardiovascular diseases.1
This clinical condition is known as cardiorenal syndrome. In
addition, the presence of chronic kidney disease (CKD) is a
relatively frequent complication in patients with advanced
heart failure and left ventricular (LV) dysfunction. Its pre-
sence is associated with a worse prognosis following cardio-
vascular diseases.2Although preventing this condition includes
identification and amelioration of the precipitating factors
and connections,3the pathophysiological mechanisms of the
syndrome remain to be elucidated. It is also known that the
presence of CKD increases severity, worsens the response to
treatment, and is associated with poor cardiac and renal
outcomes in cardiorenal syndrome.4,5
The renin–angiotensin system (RAS) has a critical role in
the development of cardiovascular and renal disease in a
clinical setting. It has been reported that the role of RAS in
congestive heart failure (CHF) and CKD is complex owing to
involvement of multiple peptides and receptors. Increased
RAS activity results in the development of CHF by stimulation
of cardiac hypertrophy, apoptosis, and LV dilatation. Although
RAS mediates the development of cardiorenal syndrome, clin-
ical studies indicate that angiotensin receptor blockers do not
reduce cardiovascular events in patients with nephropathy.6
Aliskiren, a new class of the first representative non-peptide
direct renin inhibitor,7is broadly used as an antihypertensive
1Department of Cardiovascular Medicine, Tokyo Medical and Dental University, Tokyo, Japan;2Departments of Advanced Clinical Science and Therapeutics, Tokyo,
Japan;3Research Fellow of the Japan Society for the Promotion of Science, Tokyo, Japan;4NB Health Laboratory, Tokyo, Japan;5Department of Pharmacology, Saitama
Medical University, Tokyo, Japan and6Cardiovascular Medicine, University of Tokyo, Tokyo, Japan
Correspondence: Dr J-i Suzuki, MD, PhD, Departments of Advanced Clinical Science and Therapeutics, University of Tokyo, Tokyo Medical and Dental University, 7-3-1
Hongo, Bunkyo, Tokyo 113-8655, Japan.
Received 13 April 2012; revised 9 July 2012; accepted 1 August 2012
1766Laboratory Investigation | Volume 92 December 2012 | www.laboratoryinvestigation.org
Laboratory Investigation (2012) 92, 1766–1776
& 2012 USCAP, Inc All rights reserved 0023-6837/12 $32.00
drug, as its effects directly suppress the plasma concentration
of Ang peptides.8,9Recent papers reveal the benefit of
aliskiren following myocardial ischemia (MI) independent of
pressor effects in rodent and human studies.10,11RAS activity
during sleep time, which is independent of systemic pressure,
has important role in the progression of cardiac remodeling.
The half-life of aliskiren is very long (24–48h) compared
with an ACE inhibitor (about 6h). Aliskiren treatment would
inhibit the RAS activity independent of systemic pressure
because of its longevity. A clinical trial (ALTITUDE) in type 2
diabetes using cardiorenal endpoints is ongoing,12,13but the
detailed pathophysiological roles of aliskiren have not yet
been elucidated in the experimental models of cardiorenal
Thus, the aim of this study was to elucidate the RAS ac-
tivity responsible for adverse myocardial remodeling with
renal failure and to clarify the effect of aliskiren on this
condition. In this study, we demonstrated that renal disorder
results in ventricular dysfunction and deteriorates remodel-
ing after MI through excessive RAS activation in this car-
diorenal model. Aliskiren treatment suppressed cardiac
dysfunction, LV remodeling, and inflammatory or oxidative
factors compared with the vehicle-treated group. These
results may be a new methodological approach against
prognosis in patients with renal failure suffering acute-MI.
MATERIALS AND METHODS
Male C57BL/6 mice were obtained from Crea, Japan.
Transgenic (Tg) mice were generated using heterozygotes
carrying either the 15-kb human renin gene with the 3-kb
native promoter or the 14-kb human angiotensinogen gene
with the 1.3-kb promoter. Double-Tg mice were created by
cross-mating between human renin-Tg and human angio-
tensinogen-Tg mice. Both mice were backcrossed to the
C57Bl/6 background. Human renin in renin-Tg mice is found
at B34-fold higher concentration than that obtained from
normal human plasma. Plasma Ang II concentration and
plasma renin activity (PRA) are B3.5-fold and 6–9-fold
higher in double-Tg mice than in wild-type (WT) mice.14
Human renin-Tg mice and human angiotensinogen-Tg mice
were provided by RIKEN BRC (Tsukuba, Japan). AT1?/?
mice were also used as described previously.15
All mice were 6–8 weeks of age when the experiment started.
A murine model of 5/6 nephrectomy (NTX) was generated
by a two-step surgical procedure as described previously.16
Two to three arterial branches of the left kidney were ligated
leaving an intact kidney segment. The right kidney was
removed, and the mice received 0.9% NaCl in their drinking
water. Four weeks after NTX, MI was induced by ligation of
the left anterior descending (LAD) coronary artery, and this
was continued for 28 days17(Supplementary Figure 1).
Double-Tg and WT-mice were induced MI without NTX.
We killed the mice on day 7 (n¼8–10 per each group) and
day 28 (n¼17–22 per each group). In total, 4–6 mice in each
assay, and 7–11 mice in pathological analysis were randomly
picked from each group (Supplementary Figure 1).
These experiments conform to the Guide for the Care and
Use of Laboratory Animals in the Tokyo Medical and Dental
University, University of Tokyo.
Animals were assigned randomly into treatment groups and
were administered a subcutaneous injection of aliskiren
(25mg/kg; Novartis Pharma), hydralazine (in drinking water,
30mg/kg), apocynin (in drinking water, 150mg/kg; Sigma-
Aldrich), or a vehicle (PBS; Supplementary Figure 1). These
drugs were administered daily for 8 weeks after NTX. The
aliskiren and apocynin dosage were decided based on pre-
viously papers.8,18The dose of hydralazine was determined to
show the equal effect of decreasing BP comparable with the
Heart rate and BP (systolic pressure) were measured in
conscious mice by using a tail-cuff system (BP-98A, Softron,
Tokyo, Japan). Transthoracic echocardiography was per-
formed with ultrasound equipment (Nemio, Toshiba, Tokyo,
Japan) using a 14-MHz annular array transducer. Hearts were
imaged in the two-dimensional mode in short-axis views at
the level of papillary muscle. EF were calculated as EF¼SV/
Harvested hearts were quickly dipped into 4% paraf-
ormaldehyde. The hearts were embedded in paraffin. Sample
sections (1mm) of the tissue were stained by Mallory and
viewed using a light microscope with a computer-assisted
analyzer (Image Pro Express software).19To demonstrate
remodeling analysis, infarct size, infarct thickness, infarct
length, LV circumference, and septal thickness were measured
with the computer-assisted analyzer. Collagen production
was identified by the Sirius Red, and the area of collagen was
determined in the LV border area. To avoid bias, the
histologist was single blinded to the treatment groups of
Collagen concentrations of whole hearts were measured using
the hydroxyproline assay 1 week after MI operation. The
homogenated samples in acetic buffer were hydrolyzed.
Chloramine T buffer were added to sample and incubated for
20min. Erhlich’s solution was then added to each sample,
and incubated for 20min. Absorbance 550nm was read on a
Post-infarction cardiac remodeling in CKD
M Ogawa et al
www.laboratoryinvestigation.org | Laboratory Investigation | Volume 92 December 20121767
Real-Time (RT) PCR
mRNA of whole hearts were collected using the TRIzol
method 1 week after MI or 4 weeks after NTX. RT-PCR
was performed to determine the mRNA expression of ANP
(assay ID: Mm01255747_g1), monocytes chemoattractant
protein (MCP)-1 (Mm00441242_m1),
(Mm0130243_g1), AT1a (Mm00558224_s1), NADPH oxidase
(Nox)2 (Mm1287742_m1), and Nox4 (Mm00479246_m1).
The mRNA expression of the target gene was normalized to
18s ribosomal RNA (4308329). Quantitative data were calcu-
lated to normalize the control group as baseline using the
comparative CT (DDCT) method.21
Immunohistochemistry (IHC) was performed using a pri-
mary antibody against renin (no. 593), followed by the
Nichirei Simplestain kit
changes were scored on a 0 to 5 basis (no detected to large
quantity detected) by single-blinded histologists.
Blood was collected in heparinized microtubes and subjected
to centrifugation. Plasma creatinine levels were measured
using the enzymatic method by SRL.
Plasma was collected from mice and diluted in an Ang I
generation buffer containing 0.1M phosphate buffer, 5mM
EDTA, and 1mM PMSF. The mixtures were incubated
for 30min. Ang I expression was measured using the Ang
I EIA kit (Peninsula Laboratories). Quantitative data
were obtained to determine endogenous Ang I in each group
by the following equation: quantitative data¼Ang I value–
endogenous Ang I value.
Cell Preparation and Culture
The hearts were isolated from neonatal mice (1–2-day-old),
and minced with PBS. The hearts were then denatured by
collagenase (Roche diagnosis). Isolated cells were plated for
40min. Fibroblasts adhered to the plate, while cardiomyo-
cytes did not. Cardiomyocytes were cultured in MEM med-
ium containing 5% FBS on 3.5-mm plane plastic dishes.
Fibroblasts were cultured in DMEM (containing 10% FBS)
until confluent. Mononuclear cells were collected from the
spleens using cell strainers and were plated in RPMI1640
medium.19Isolated cardiomyocytes or mononuclear cells
were serum staved, and stimulated by murine plasma (5% in
the medium) isolated from NTX, aliskiren-treated NTX on
week 4, or non-NTX mice as a control. Aliskiren or vehicle
(PBS) was administrated to the medium for 30min before
All data are expressed as mean±s.e.m. A survival analysis
was performed using the Kaplan–Meier method with the
log-rank test. Data were compared and differences between
the two groups were analyzed by the t-test. Differences in
data between multiple groups were subjected to one-way or
two-way ANOVA and Bonferroni post-test. A statistical test
was performed using the Prism. Differences with values of
Po0.05 were considered significant.
Aliskiren Suppressed NTX-Induced Hypertension
NTX resulted in hypertension compared with the non-NTX
mice 2 weeks after the operation. Although aliskiren reduced
systolic BP, hydralazine administration resulted in a decline
in BP comparable to aliskiren administration (Figure 1a).
Figure 1 Aliskiren suppressed 5/6 nephrectomy (NTX)-induced
hypertension. (a) Representative data of blood pressure in NTX mice.
Mice were administrated vehicle (dilute blue line; n¼10), aliskiren (red
line; n¼8), or hydralazine (blue line; n¼10). *Po0.05 vs aliskiren,
**Po0.05 vs hydralazine. (b) Representative data of serum creatinine
levels in NTX. Plasma creatinine levels are shown 4 weeks after NTX
in NTX and non-NTX groups (Po0.05). *Po0.05 vs each group.
(c) Representative data of survival rate in NTX. Vehicle: n¼26; aliskiren:
n¼24; hydralazine: n¼13, *Po0.05.
Post-infarction cardiac remodeling in CKD
M Ogawa et al
1768Laboratory Investigation | Volume 92 December 2012 | www.laboratoryinvestigation.org
Plasma creatinine levels were elevated 4 weeks after NTX
compared with the non-NTX group. Neither aliskiren nor
hydralazine administration changed the plasma creatinine
levels (Figure 1b).
Although many control mice died within the first 4 weeks
after NTX (73.91%, n¼24), aliskiren administration sig-
nificantly improved the survival rates of mice with NTX
(percent survival¼92.31%, n¼26; Po0.05 vs vehicle).
However, mice treated with hydralazine showed survival rates
comparable to those of vehicle-treated mice (76.92%, n¼13;
NTX Augmented Renin Expression and Activity
To investigate whether NTX activated renin expression and
activity, we performed IHC and plasma biochemistry analy-
sis. Four weeks post NTX, the remaining kidneys showed
increased renin expression (4.5±0.3, Po0.05) at the juxta-
glomerular area compared with the non-NTX kidneys
(1.0±0.4; Figures 2a and b). Although NTX elevated PRA
compared with the control mice, aliskiren treatment negated
this effect (Figure 2c). Hydralazine treatment did not alter the
PRA value. Although cardiac AT1 transcription was elevated
by nephrectomy compared with the control hearts (Figure 2d),
aliskiren treatment did not change the level.
Aliskiren Suppressed MI plus NTX-induced Myocardial
To clarify the relationship between renal disorder and cardiac
dysfunction, we induced MI by LAD ligation in mice that had
undergone NTX. Cardiac function was analyzed by echo-
cardiogram and performed 1 and 4 weeks post LAD ligation
(Table 1). MI-induced hearts showed myocardial infarction
in large anterior region of LV. NTX had no effect on the
(86.0%±2.7%) compared with those that had not under-
gone NTX (86.0%±1.0%). MI hearts with NTX showed
increased impairment of LVEF (at 1 week: 51.6%±4.7%,
4 weeks: 36.4%±2.2%; n¼8) compared with MI hearts
without NTX (at 1 week: 70.7%±5.9%, 4 weeks: 53.8%±
2.6%; n¼10). However, aliskiren significantly improved
LVEF of MI hearts with NTX (1 week: 67.5%±3.0%,
4 weeks: 65.4%±2.4%; n¼11) compared with the vehicle.
Hydralazine did not improve LVEF of MI hearts with NTX
(1 week: 52.1%±3.0%, 4 weeks: 36.4%±2.2%; n¼7;
Figures 3a and b). Aliskiren treatment also significantly
suppressed ratio of lung/body weight compared with the
vehicle-treated group (Table 2).
LAD ligation resulted in significant pathological remodel-
ing of the LV anterior wall 4 weeks after ischemia induction
(Table 3). Infarction length and septal wall thickness were not
Figure 2 NTX (5/6 nephrectomy) augmented renin expression and activity. (a) Representative immunohistochemistry (IHC) of kidneys from NTX mice.
Dot-line circle indicates glomerular area. Bar shows 50mm. (b) Quantitative data of the IHC. *Po0.05. (c) Representative plasma renin activity in NTX
mice. *Po0.05 (d) Representative Real-TimePCR of hearts from NTX mice. *Po0.05.
Post-infarction cardiac remodeling in CKD
M Ogawa et al
www.laboratoryinvestigation.org | Laboratory Investigation | Volume 92 December 20121769
significantly different between the vehicle- and aliskiren-
treated group. Although vehicle-treated LAD ligation
resulted in LV thinning, aliskiren administration sig-
nificantly reduced this pathological change. Enlarged LV
circumference was also reduced by aliskiren. Collagen
expression was significantly enhanced along the border
by MI with NTX (19.3%±2.4%; n¼8), but this was re-
duced by aliskiren administration (6.8%±2.0%; n¼11;
Po0.05; Figure 3c and Table 3). mRNA and collagen were
collected from the cardiac ventricle on week 1 post MI.
Although MI hearts elevated collagen-type I mRNA levels
and collagen concentration compared with the hearts of
control mice, MI with NTX hearts showed enhanced col-
lagen levels compared with MI hearts without NTX (Figures
3d and e). Aliskiren suppressed the levels of collagen in MI
hearts with NTX.
NTX Enhanced ANP, Oxidative Stress, and MCP-1 in
RAS is known to have a key role in myocardial hypertrophy
and fibrosis via increased oxidative stress and the expression
of inflammatory chemokines. Thus, we examined the levels
of ANP, Nox2, Nox4, and MCP-1 mRNA in MI or non-MI
hearts treated by NTX. mRNA was collected from the cardiac
ventricle 1 week after MI. ANP levels were elevated in the MI
hearts compared with the non-MI hearts. Moreover, hearts
with MI treated by NTX showed enhanced ANP mRNA levels
compared with the MI hearts without NTX. The mRNA
levels of Nox2 and Nox4 were elevated in MI hearts com-
pared with non-MI hearts. Nox2 and Nox4 are isoforms of
Nox, a major enzyme that produces reactive oxygen species
(ROS). In addition, MI-induced hearts treated with NTX
showed further elevation of Nox2, Nox4, and MCP-1
mRNA levels as compared with the MI hearts that were not
treated with NTX (Figures 4a–d). Aliskiren treatment
significantly suppressed these levels compared with the
NTX plus MI group.
It is known that Nox enzymes can be activated by RAS. To
reveal the role of Nox enzymes in MI-induced myocardial
remodeling in renal dysfunction, we administrated apocynin
as a Nox inhibitor to the NTX plus MI mice. Although
apocynin did not alter BP, attenuation of Nox improved
LVEF and myocardial fibrosis in the 28 days after LAD
ligation mice as compared with the vehicle-treated group
(Figures 4e and f).
Double-Tg Mice Deteriorated After MI
Although we demonstrated that NTX deteriorated LV dys-
function through RAS activation, we did not clarify the
relationship between upregulated circulating RAS and the
progression of LV remodeling. To clarify the pathophysiolo-
gical mechanism, we used human renin and angiotensinogen
Tg mice. Systolic BP was consistently elevated in the double-
Tg mice compared with the WT mice (Supplementary
Figure 2A).Thehigh BP resulted in mildcardiac
Table 1 Echocardiography and heart rate
NTX-MI with Vehicle
NTX-MI with Aliskiren
NTX-MI with Hydralazine
585±29.54 592.40±0.10 576.50±72.5 607.63±13.27 638.5±27.76 587.29±18.81 619.50±42.41 653.86±25.93 747.40±4.76 669.14±4.74 512.75±9.56
Abbreviations: EF, ejection fraction; FS, fractional shortening; LVDd, left ventricular end-diastolic diameter; LVSd, left ventricular end-systolic diameter; MI, myocardial ischemia; NTX, 5/6 nephrectomy.
*Po0.05 vs NTX-MI with vehicle.
Post-infarction cardiac remodeling in CKD
M Ogawa et al
1770Laboratory Investigation | Volume 92 December 2012 | www.laboratoryinvestigation.org