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
19. Ogawa M, Suzuki J, Hishikari K, et al. Clarithromycin attenuates
acute and chronic rejection via matrix metalloproteinase suppression
in murine cardiac transplantation. J Am Coll Cardiol 2008;51:
Reddy GK, Enwemeka CS. A simplified method for the analysis
of hydroxyproline in biological tissues. Clin Biochem 1996;29:
Ogawa M, Suzuki J, Kosuge H, et al. The mechanism of anti-
inflammatory effects of prostaglandin E2 receptor 4 activation in
murine cardiac transplantation. Transplantation 2009;87:1645–1653.
Looi YH, Grieve DJ, Siva A, et al. Involvement of Nox2 NADPH
oxidase in adverse cardiac remodeling after myocardial infarction.
23. Cucoranu I, Clempus R, Dikalova A, et al. NAD(P)H oxidase 4 mediates
transforming growth factor-beta1-induced differentiation of cardiac
fibroblasts into myofibroblasts. Circ Res 2005;97:900–907.
Ago T, Kuroda J, Pain J, et al. Upregulation of Nox4 by hypertrophic
stimuli promotes apoptosis and mitochondrial dysfunction in cardiac
myocytes. Circ Res 2010;106:1253–1264.
HayashidaniS, Tsutsui H,Shiomi
chemoattractant protein-1 gene therapy attenuates left ventricular
remodeling and failure after experimental myocardial infarction.
Kaikita K, Hayasaki T, Okuma T, et al. Targeted deletion of CC
chemokine receptor 2 attenuates left ventricular remodeling after
experimental myocardial infarction. Am J Pathol 2004;165:439–447.
25.T, et al. Anti-monocyte
Post-infarction cardiac remodeling in CKD
M Ogawa et al
1776 Laboratory Investigation | Volume 92 December 2012 | www.laboratoryinvestigation.org