Intracellular Angiotensin II Production in Diabetic Rats Is Correlated With Cardiomyocyte Apoptosis, Oxidative Stress, and Cardiac Fibrosis

Abstract

Many of the effects of angiotensin (Ang) II are mediated through specific plasma membrane receptors. However, Ang II also elicits biological effects from the interior of the cell (intracrine), some of which are not inhibited by Ang receptor blockers (ARBs). Recent in vitro studies have identified high glucose as a potent stimulus for the intracellular synthesis of Ang II, the production of which is mainly chymase dependent. In the present study, we determined whether hyperglycemia activates the cardiac intracellular renin-Ang system (RAS) in vivo and whether ARBs, ACE, or renin inhibitors block synthesis and effects of intracellular Ang II (iAng II).
Diabetes was induced in adult male rats by streptozotocin. Diabetic rats were treated with insulin, candesartan (ARB), benazepril (ACE inhibitor), or aliskiren (renin inhibitor).
One week of diabetes significantly increased iAng II levels in cardiac myocytes, which were not normalized by candesartan, suggesting that Ang II was synthesized intracellularly, not internalized through AT(1) receptor. Increased intracellular levels of Ang II, angiotensinogen, and renin were observed by confocal microscopy. iAng II synthesis was blocked by aliskiren but not by benazepril. Diabetes-induced superoxide production and cardiac fibrosis were partially inhibited by candesartan and benazepril, whereas aliskiren produced complete inhibition. Myocyte apoptosis was partially inhibited by all three agents.
Diabetes activates the cardiac intracellular RAS, which increases oxidative stress and cardiac fibrosis. Renin inhibition has a more pronounced effect than ARBs and ACE inhibitors on these diabetes complications and may be clinically more efficacious.

Full text

Intracellular Angiotensin II Production in Diabetic Rats
Is Correlated With Cardiomyocyte Apoptosis, Oxidative
Stress, and Cardiac Fibrosis
Vivek P. Singh,
1,2
Bao Le,
2
Renu Khode,
3
Kenneth M. Baker,
1,2
and Rajesh Kumar
1,2
OBJECTIVE—Many of the effects of angiotensin (Ang) II are
mediated through specific plasma membrane receptors. How-
ever, Ang II also elicits biological effects from the interior of the
cell (intracrine), some of which are not inhibited by Ang receptor
blockers (ARBs). Recent in vitro studies have identified high
glucose as a potent stimulus for the intracellular synthesis of Ang
II, the production of which is mainly chymase dependent. In the
present study, we determined whether hyperglycemia activates
the cardiac intracellular renin-Ang system (RAS) in vivo and
whether ARBs, ACE, or renin inhibitors block synthesis and
effects of intracellular Ang II (iAng II).
RESEARCH DESIGN AND METHODS—Diabetes was in-
duced in adult male rats by streptozotocin. Diabetic rats were
treated with insulin, candesartan (ARB), benazepril (ACE inhib-
itor), or aliskiren (renin inhibitor).
RESULTS—One week of diabetes significantly increased iAng II
levels in cardiac myocytes, which were not normalized by
candesartan, suggesting that Ang II was synthesized intracellu-
larly, not internalized through AT
1
receptor. Increased intracel-
lular levels of Ang II, angiotensinogen, and renin were observed
by confocal microscopy. iAng II synthesis was blocked by
aliskiren but not by benazepril. Diabetes-induced superoxide
production and cardiac fibrosis were partially inhibited by can-
desartan and benazepril, whereas aliskiren produced complete
inhibition. Myocyte apoptosis was partially inhibited by all three
agents.
CONCLUSIONS—Diabetes activates the cardiac intracellular
RAS, which increases oxidative stress and cardiac fibrosis. Renin
inhibition has a more pronounced effect than ARBs and ACE
inhibitors on these diabetes complications and may be clinically
more efficacious. Diabetes 57:3297–3306, 2008
Involvement of the renin-angiotensin (Ang) system
(RAS) in human pathophysiology has expanded to
include several diseases beyond a traditional role in
saltwater homeostasis (1). In diabetes, there is sig-
nificant overactivity of the RAS, which is reversed by
treatment with RAS inhibitors, thus decreasing diabetes
complications (2). Activation of the RAS in diabetes in-
cludes activation of new components, such as the pro(re-
nin) receptor (3), and Ang II–independent effects,
mediated through interaction of pro(renin), with the pro-
(renin) receptor (4). Although circulating renin and Ang II
levels are reduced in diabetes, prorenin levels are en-
hanced severalfold (5,6). Prorenin may have dual effects,
providing for generation of Ang I at tissue sites through
receptor-mediated nonproteolytic activation and directly
through activation of receptor-mediated signaling path-
ways (4,7,8). Ang II–independent RAS actions suggest that
efficacy of RAS inhibitors, Ang receptor blockers (ARBs),
and ACE inhibitors would have limitations in hyperglyce-
mic conditions. Recent meta-analyses of clinical trials
have suggested that currently used RAS blockers may not
provide additional benefits in diabetic compared with
nondiabetic patients (9,10).
We recently reported a novel aspect of the RAS, the
intracellular RAS, having identified an intracellular or
intracrine system (11,12). In cardiac myocytes and fibro-
blasts, we demonstrated the presence of RAS components
and synthesis of Ang II intracellularly (13,14). Hyperglyce-
mia selectively upregulates the intracellular system in
cardiac myocytes, vascular smooth muscle cells (VSMCs),
and renal mesangial cells, where Ang II synthesis is largely
catalyzed by chymase, not ACE (14 –18). We and others
have previously reported that intracellular Ang II (iAng II)
elicits biological effects, some of which are not blocked by
ARBs (19 –22). These observations further support the
speculation that currently available RAS inhibitors may
not provide the anticipated cardiovascular benefits in
diabetic conditions (23). In this study, we have examined
the activation of the cardiac intracellular RAS in a rat
model of diabetes. We also determined the role of iAng II
in diabetes-induced oxidative stress, cardiac myocyte apop-
tosis, and cardiac fibrosis and the efficacy of different RAS
blockers under hyperglycemic conditions.
RESEARCH DESIGN AND METHODS
All animal use was approved by the Institutional Animal Care and Use
Committee of the Texas A&M Health Science Center. The AT
1
receptor
blocker candesartan was obtained from AstraZeneca (Wilmington, DE); the
renin inhibitor aliskiren was from Novartis (Cambridge, MA); the ACE
inhibitor benazepril was from Sigma; and insulin (Humulin N) was from Eli
Lilly (Indianapolis, IN).
Induction of diabetes and treatment of animals. Diabetes was induced by
a single injection of streptozotocin (STZ, 65 mg/kg body wt i.p.) dissolved in
0.1 mol/l sodium citrate– buffered saline (pH 4.5), in adult male Sprague
Dawley rats (250 –300 g). Control animals received buffered saline alone.
Diabetes was confirmed by sustained blood glucose levels ⬎15 mmol/l, as
determined 48 h after STZ injection and on alternate days thereafter. Diabetic
rats, in groups of nine animals, were treated with insulin (2–5 units s.c., twice
daily), candesartan (1 mg/kg i.p.), aliskiren (30 mg/kg orally), or benazepril (10
mg/kg orally) daily for 7 days beginning 48 h after STZ injection. Twenty-four
hours after the last treatment, animals were weighed and anesthetized using
From the
1
Division of Molecular Cardiology, Department of Medicine, Texas
A&M Health Science Center, College of Medicine, Scott & White, Central
Texas Veterans Health Care System, Temple, Texas; and the
2
Department of
Medicine, Scott & White, Temple, Texas; and
3
Department of Pathology,
Scott & White, Temple, Texas.
Corresponding author: Rajesh Kumar, kumar@medicine.tamhsc.edu.
Received 17 June 2008 and accepted 8 September 2008.
Published ahead of print at http://diabetes.diabetesjournals.org on 1 October
2008. DOI: 10.2337/db08-0805.
© 2008 by the American Diabetes Association. Readers may use this article as
long as the work is properly cited, the use is educational and not for profit,
and the work is not altered. See http://creativecommons.org/licenses/by
-nc-nd/3.0/ for details.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked “advertisement” in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
ORIGINAL ARTICLE
DIABETES, VOL. 57, DECEMBER 2008 3297

ketamine/xylazine (50/5 mg/kg), and hearts were isolated and weighed before
perfusion, the latter using the Langendorff methodology.
Isolation of cardiac myocytes and measurement of iAng II. Hearts were
isolated and perfused with Krebs-Henseleit bicarbonate buffer, followed by
digestion with 0.1% (wt/vol) collagenase II. Myocytes were separated from
nonmyocytes by differential centrifugation at 25g. The purity of the myocyte
preparations using this procedure was ⬎90%, as analyzed by fluorescence-
activated cell sorting, using anti-sarcomeric myosin (MF-20) and anti-sarco-
meric actin antibody. The pellet containing myocytes was processed for Ang
II extraction, as described previously (14). Briefly, cells were lysed in ice-cold
1 mol/l acetic acid containing a protease inhibitor cocktail (Sigma) by brief
sonication. The lysate was sedimented at 20,000gfor 10 min, and the
supernatant was dried in a vacufuge, followed by reconstitution in 1% acetic
acid. The samples were applied to a conditioned DSC-18 column (Supelco),
washed, and eluted with methanol. The eluted samples were dried and reconsti-
tuted in PBS for enzyme-linked immunosorbent assay (ELISA). For isolation of
Ang II from plasma, an equal volume of 2% acetic acid was added to plasma,
followed by filtration through Amicon Ultra-15 filters. The filtrate was applied to
DSC-18 columns, and Ang II was eluted as described for the cell lysates. Using the
above procedure, we have obtained ⬎90% recovery of exogenously added Ang II.
Ang II was measured by quantitative, competitive ELISA, using a specific
anti–Ang II antibody (Peninsula Labs), which was previously validated by
high-performance liquid chromatography– chip/mass spectrometric analysis (14).
ELISA was performed on protein-A and anti–Ang II antibody– coated 96-well
dishes. Competitive binding of synthetic biotinylated Ang II, in the presence of the
extracted peptide, was detected with streptavidin– horseradish peroxidase con-
jugate. A standard curve, generated from binding of a constant amount of
biotinylated Ang II with increasing concentrations of nonbiotinylated synthetic
Ang II, was used to calculate the concentration of the peptide in the sample. The
concentration of Ang II in the cell lysates is expressed as femtomoles per
milligram protein and in plasma as femtomoles per milliliter.
Immunohistochemistry of RAS components. Hearts were frozen in OCT
compound (Tissue-Tek; Sakura Finetek) at ⫺80°C for immunofluorescence
staining of Ang II, renin, and anti-angiotensinogen (AGT). Frozen tissue was
cut into 5-␮m sections, which were air-dried, fixed with 4% formaldehyde, and
permeabilized using 0.2% Triton X-100. Nonspecific binding was blocked by 5%
BSAfor1hatroom temperature. The sections were incubated with anti–Ang
II antibody (1:100; Peninsula Labs), anti-renin antibody (1:100; gift from Dr.
Tadashi Inagami [Vanderbilt University, Nashville, TN]), or AGT antibody
(1:500; Swant). The sections were costained for anti-sarcomeric actin and
laminin, where indicated. After washings, the sections were incubated with
respective secondary antibodies. Specificity of the staining was determined by
preadsorption of primary antibodies with the antigen or by using secondary
antibody alone. Images were acquired with a confocal fluorescence micro-
scope (Olympus Fluoview 300). Fluorescence intensities in tissue sections
were determined by digital microscopy software (Slide Book 4.2) after
subtracting background fluorescence.
Cardiac myocyte apoptosis. Apoptotic cardiac myocytes were detected in
paraffin-embedded heart sections using the terminal deoxynucleotide trans-
ferase-mediated dUTP nick-end labeling (TUNEL) assay and cleaved
caspase-3 staining. TUNEL assay was performed using an assay kit (Millipore,
Temecula, CA) per the manufacturer’s instructions. Cytoplasm and nuclei
from the myocytes were counterstained using anti-sarcomeric actin antibody
and DAPI, respectively. For cleaved caspase-3 staining, deparaffinized sec-
tions were subjected to antigen retrieval in 0.01 mol/l citrate buffer (pH 6.0) by
microwaving. After blocking with 5% BSA, the sections were incubated with
rabbit monoclonal anti– cleaved caspase-3 antibody (1:200; Cell Signaling
Technology, Danvers, MA) overnight at 4°C, followed by fluorescein isothio-
cyanate– conjugated goat anti-rabbit IgG (1:200; Molecular Probes). The
number of positively stained nuclei was counted from 20 fields per heart
(⬃25,000 cells) and three hearts per treatment group.
Reactive oxygen species detection in the heart. Superoxide production in
the hearts was detected by dihydroethidium (DHE) staining (Sigma-Aldrich).
Frozen heart sections (20 ␮m thick) were incubated with 10 ␮mol/l DHE at
37°C for 45 min in a humidified chamber protected from light. Fluorescent
images obtained with an Olympus FV300 confocal microscope were analyzed
with Slide Book 4.2. The mean DHE fluorescence intensity of myocyte nuclei
was calculated by dividing the combined fluorescence value of the pixels by
the total number of pixels in 15 randomly selected fields observed with
identical laser and photomultiplier settings.
Cardiac fibrosis. Cardiac interstitial fibrosis was determined by Masson’s
trichrome staining on 5-␮m paraffin-embedded sections. The extent and
degree of fibrosis was subjectively graded on a scale of 0 –4. Grade 0 signified
no apparent collagen fiber proliferation except for small islets of fibrous tissue
around the capillaries, as well as an intercellular single layer of collagenous
tissue, as in normal myocardium. Focal and minimal fibrosis was graded as 1,
mild patchy fibrosis as grade 2, moderate diffuse fibrosis as grade 3, and the
most prominent fibrosis, covering a major area of the specimen, was classified
as 4. A minimum of three sections per heart with five fields per section and
three animals per experimental group were analyzed, and results are pre-
sented as an average grade.
Statistical analysis. Values are expressed as means ⫾SE. ANOVA with
Tukey’s post hoc test was used for statistical analysis. P⬍0.05 was
considered statistically significant.
RESULTS
Hyperglycemia increases intracellular levels of Ang
II in cardiac myocytes from diabetic heart. Diabetes
was induced in adult male rats by STZ injection. One group
of diabetic animals was treated with insulin to confirm that
the observed effects in the experimental groups were
secondary to hyperglycemia. One week of diabetes signif-
icantly reduced body and heart weights, which were
normalized by insulin treatment but not by any of the RAS
inhibitors (Table 1). However, no significant effect on the
heart-to-body weight ratio (Table 1) or plasma Ang II
levels (Fig. 1B) was observed, which is consistent with
previous reports (24,25). Ang II levels in cardiac myocytes,
which were isolated after perfusion of the hearts and
enzymatic dispersion, represented Ang II present intracel-
lularly. To determine the source of iAng II, i.e., intracellu-
lar synthesis or AT
1
-mediated internalization, one group of
diabetic animals was treated with the AT
1
antagonist
candesartan to prevent receptor-mediated uptake. As
shown in Fig. 1A, cardiac myocytes from diabetic rat
hearts demonstrated a 9.4-fold elevation in the levels of
iAng II (183 ⫾13 fmol/mg protein) compared with cells
from control animals (19 ⫾4 fmol/mg protein). Normal-
ization of blood glucose levels by insulin in rats adminis-
tered STZ completely blocked the rise in iAng II levels,
indicating that the latter was a specific effect of hyper-
glycemia. Treatment of diabetic rats with candesartan
partially reduced iAng II levels, suggesting that the major
source of iAng II was intracellular synthesis, which is
consistent with our previous report in neonatal rat ven-
tricular myocytes (NRVMs) (14).
Intracellular synthesis of Ang II is not blocked by an
ACE inhibitor. We and others had previously reported
that several cell types (NRVMs, VSMCs, and renal mesan-
TABLE 1
Effect of diabetes on body and heart weight
Control
Diabetic
No
treatment Insulin Aliskiren Candesartan Benazepril
Body weight (g) 337 ⫾10 263 ⫾8.3* 307 ⫾4.7 248 ⫾8.5* 285 ⫾5.9* 267 ⫾6.9*
Heart weight (mg) 1,168 ⫾54 933 ⫾33* 1,074 ⫾27 867 ⫾37* 988 ⫾15* 901 ⫾16*
Heart weight/body weight (mg/g) 3.46 ⫾0.1 3.55 ⫾0.1 3.49 ⫾0.1 3.50 ⫾0.1 3.47 ⫾0.1 3.37 ⫾0.1
Data are means ⫾SE (n⫽9) after 1 week of induction of diabetes. *P⬍0.05 vs. control.
INTRACELLULAR Ang II IN DIABETES
3298 DIABETES, VOL. 57, DECEMBER 2008

gial cells) use alternative pathways to synthesize Ang II in
high-glucose culture conditions (14 –18). To determine the
mechanism of hyperglycemia-induced cardiac iAng II syn-
thesis in vivo, diabetic rats were treated with either a renin
inhibitor (aliskiren) or an ACE inhibitor (benazepril). As
shown in Fig. 1A, aliskiren completely normalized (33 ⫾7
fmol/mg protein) iAng II levels in diabetic rat cardiac
myocytes, whereas benazepril did not have any effect
(143 ⫾8 fmol/mg protein). These results indicated that the
observed increase in iAng II was not catalyzed by ACE,
which is consistent with intracellular synthesis of Ang II.
None of the RAS blocking drugs significantly altered
plasma Ang II levels, which were measured 24 h after the
last dose. The latter observation corroborated with the
reported 2- to 6-h period for reactive changes in plasma
levels of Ang II, which returned to baseline between 14
and 30 h after drug intake (26 –28). The lack of increase in
plasma Ang II levels in any of the treatment groups further
indicated that the increase in cardiac myocyte Ang II levels
was due to local synthesis.
Immunohistochemical localization of Ang II in rat
heart. To further confirm elevation of Ang II in cardiac
myocytes and intracellular localization, frozen heart sec-
tions were immunostained with anti–Ang II antibody and
visualized using confocal microscopy. Sections were coun-
terstained for laminin to mark cell boundaries (peripheral
green/yellow staining in merged images), anti-sarcomeric
actin to identify cardiac myocytes (red), and DAPI to
identify nuclei (blue). Significantly increased levels of Ang
II staining, which colocalized with anti-sarcomeric actin,
were observed in diabetic rat hearts (Fig. 1C–F). Quanti-
fication of fluorescence intensity revealed about a fivefold
FIG. 1. Ang II levels in cardiac myocytes and plasma. Ang II was measured by a competitive ELISA in cardiac myocytes (A) and plasma (B)of
control rats (Cont); diabetic rats (STZ); and diabetic rats treated with insulin (Ins), aliskiren (Alsk), candesartan (Cand), or benazepril (Bnz).
Values are expressed as means ⴞSE, nⴝ6. C–F: Intracellular localization of Ang II (yellow dots, indicated by white arrow), as determined by
confocal immunofluorescence microscopy, in heart sections from control rats (C), diabetic rats (D), diabetic rats treated with aliskiren (E), and
diabetic rats treated with candesartan (F). Myocyte profiles were identified by costaining with anti-sarcomeric actin (red) and laminin (yellow,
peripheral staining). The blue color indicates nuclear staining by DAPI. Magnification ⴛ1,200. G: Quantitative representation of Ang II
fluorescence intensity in heart sections (from five images per heart and three hearts per group). Values are expressed as means ⴞSE, nⴝ15.
*P<0.05 vs. control, †P<0.05 vs. diabetic rats without any treatment. (Please see http://dx.doi.org/10.2337/db08-0805 for a high-quality digital
representation of this figure.)
V.P. SINGH AND ASSOCIATES
DIABETES, VOL. 57, DECEMBER 2008 3299

increase in iAng II levels (Fig. 1G), consistent with the Ang
II measurement by ELISA (Fig. 1A). Immunohistochemis-
try also confirmed that candesartan partially reduced iAng
II levels, benazepril did not have any effect, and aliskiren
completely prevented the increase in iAng II in diabetic
hearts (Fig. 1G).
Intracellular localization of AGT and renin in cardiac
myocytes. Intracellular synthesis of Ang II would require
the presence of the precursor molecule AGT and process-
ing enzyme, renin, intracellularly. To demonstrate this
possibility, immunohistochemistry was performed on
heart sections using anti-AGT and anti-renin antibodies,
along with counterstaining for anti-sarcomeric actin, as
described previously (24). Elevated levels of AGT and
renin were apparent in cardiac myocytes of diabetic hearts
(green/yellow staining) compared with control, confirming
activation of the intracellular RAS in hyperglycemic con-
ditions (Fig. 2).
iAng II is correlated with hyperglycemia-induced
oxidative stress. Hyperglycemia is known to induce
myocardial oxidative stress, which may be related to
glucose metabolism or activation of cytokines and other
hormones. To determine whether there was a role of iAng
II, superoxide production was detected by DHE staining in
frozen heart sections of diabetic rats treated with different
RAS inhibitors. As shown in Fig. 3, diabetic hearts showed
enhanced superoxide production, which was prevented
in insulin-treated animals. Treatment of diabetic rats
with candesartan or benazepril significantly, but not
completely, reduced oxidative stress, whereas aliskiren
blocked completely. Our previous studies had indicated
that ARBs and ACE inhibitors were ineffective in block-
ing the intracellular RAS, unlike a renin inhibitor that
blocks both the intracellular and extracellular systems
(14,19,22). The observed partial efficacy of candesartan
and benazepril strongly suggested that iAng II contrib-
uted to hyperglycemia-induced oxidative stress in the
myocardium.
iAng II is correlated with hyperglycemia-induced
cardiac myocyte apoptosis. Cardiac myocyte apoptosis
was determined by TUNEL assay and activated caspase-3
immunostaining. Figure 4 shows an increased number of
apoptotic cells in the heart sections. Quantification of
apoptotic cells (Fig. 4Cand F) showed a five- to eightfold
increase in diabetic hearts compared with control by both
TUNEL assay and caspase-3 staining. Normalization of
blood glucose by insulin or blockade of the RAS with the
three different inhibitors significantly reduced the num-
ber of apoptotic cells but did not prevent apoptosis
completely. There was a significant difference between
aliskiren- and benazepril-treated animals, with aliskiren
being more protective.
iAng II is correlated with hyperglycemia-induced
cardiac fibrosis. Cardiac fibrosis is an important patho-
genic factor in diabetes-induced diastolic dysfunction.
Paraffin-embedded heart sections were stained with Mas-
son’s Trichrome, and the degree of blue staining was
evaluated on a scale of 0 – 4, as described in RESEARCH DESIGN
FIG. 2. Representative confocal immunofluorescence images of AGT and renin staining in hearts from control and diabetic rats. Pictures shown
are merged images of staining for anti-sarcomeric actin (red), laminin (peripheral red staining in top), AGT (top) or renin (bottom) (green and
yellow staining), and nuclei (blue). Magnification ⴛ900. (Please see http://dx.doi.org/10.2337/db08-0805 for a high-quality digital representation
of this figure.)
INTRACELLULAR Ang II IN DIABETES
3300 DIABETES, VOL. 57, DECEMBER 2008

AND METHODS. Even after only 1 week of diabetes, the overall
staining for fibrosis was enhanced in hearts from diabetic
rats (grade 1.5) compared with control animals (grade 0)
(Fig. 5). Insulin treatment completely prevented the in-
crease in fibrosis (grade 0.04). Candesartan and benazepril
reduced the degree of fibrosis (grade 0.43 and 0.88, respec-
FIG. 3. Measurement of oxidative stress in heart
sections by DHE staining. These are representative
images of DHE-stained heart sections from control
rats (A); diabetic rats (B); and diabetic rats treated
with insulin (C), aliskiren (D), candesartan (E), or
benazepril (F). Magnification ⴛ60. G: DHE fluores-
cence intensity was calculated from five images per
heart and three hearts per group. Values are ex-
pressed as means ⴞSE (nⴝ15). *P<0.05 vs.
control, †P<0.05 vs. diabetic rats without any
treatment. (Please see http://dx.doi.org/10.2337/
db08-0805 for a high-quality digital representation
of this figure.)
A B
DE
STZ
0
0.1
0.2
0.3
0.4
Cont Ins Alsk Cand Bnz
Activated Caspase- 3
Positive Cells (%)
F
*
†
*
†
*
†
*†
*
*
0
0.1
0.2
0.3
0.4
Cont Ins Alsk Cand Bnz
TUNEL Positive Nuclei
(%)
†
*
†
*†
*
†
*
STZ
C
FIG. 4. Detection of apoptosis in cardiac myocytes by TUNEL assay and cleaved caspase-3 staining. Aand B: TUNEL assay on heart sections from control (A)
and diabetic (B) rats. Dand E: Staining for cleaved caspase-3 in the hearts of control (D) and diabetic (E) rats. Cand F: Quantification of TUNEL
ⴙ
(C) and
cleaved caspase-3
ⴙ
(F) cells (⬃25,000 cells were counted in each case). Values are expressed as means ⴞSE. *P<0.05 vs. control, †P<0.05 vs. diabetic rats
without any treatment. (Please see http://dx.doi.org/10.2337/db08-0805 for a high-quality digital representation of this figure.)
V.P. SINGH AND ASSOCIATES
DIABETES, VOL. 57, DECEMBER 2008 3301

tively), whereas aliskiren had a more pronounced reduc-
tion of fibrosis (grade 0.25) in diabetic rat hearts (Fig. 5).
DISCUSSION
In this study, we observe a dramatic activation of the
intracellular RAS, a novel aspect of the tissue RAS, in
diabetic rat hearts. We also demonstrate that iAng II is
correlated with the development of pathological condi-
tions associated with diabetes. Significantly, we observed
that blockade of the RAS by a renin inhibitor in diabetic
rats provided greater protection from oxidative stress and
cardiac fibrosis compared with inhibition with an AT
1
antagonist or ACE inhibitor.
We first described a physiologically relevant intracellu-
lar RAS in NRVMs (14), defined as the presence of the
precursor protein and enzymes and synthesis of Ang II
inside the cell and which was coupled to a biological
action (11). The regulation and separation of the intracel-
lular from the extracellular RAS becomes very obvious in
hyperglycemic conditions. High glucose promoted accu-
mulation of AGT, renin, and Ang II intracellularly, result-
ing in a dramatic rise in iAng II concentrations without
affecting extracellular Ang II levels (14). Similar to NRVMs,
the intracellular accumulation of RAS components and
iAng II synthesis have also been described in VSMCs and
renal mesangial cells in high-glucose culture conditions
(15,17,29). To extend the in vitro observations, we deter-
mined whether a similar activation of the intracellular RAS
occurs in adult diabetic animals. We observed a significant
increase in intracellular levels of AGT and renin and iAng
II synthesis in diabetic rat hearts, as determined by ELISA
and confocal immunocytochemistry. In a previous human
study, a threefold increase in Ang II staining was described
in hearts from diabetic compared with nondiabetic pa-
tients, which was enhanced an additional 2.5-fold in dia-
betic hypertensive patients (30). However, in that study, it
was not clear whether the increased Ang II staining
represented activation of an intracellular RAS or was due
to internalization of extracellularly synthesized Ang II. We
observed an increase in iAng II levels even in the presence
of AT
1
blockade with candesartan, which strongly sup-
ported activation of the intracellular RAS. The latter
conclusion was further strengthened by an observed in-
crease in intracellular staining for renin and AGT.
The observation of elevated Ang II levels after removal
of the high-glucose stimulus, suggested that iAng II was
highly stable. The reported half-life of Ang II in the heart
is 15 min in vivo and 30 min ex vivo (31). In this regard, it
is important to make a distinction between Ang II that was
internalized versus that which was synthesized intracellu-
larly, because the intracellular location is likely to be
different, which could substantially affect the half-life. The
half-life reported in the literature was for Ang II that was
internalized through AT
1
receptor (31), a major part of
which was likely targeted to lysosomes for degradation
(32). We measured Ang II that was synthesized intracellu-
larly, which is most likely to occur in organelles or at sites
that are not associated with protein degradation.
An interesting and therapeutically significant aspect of
the intracellular RAS is that high glucose–induced iAng II
synthesis in cardiac myocytes is catalyzed by chymase, not
ACE. Chymase levels are significantly elevated in NRVMs
after exposure to high glucose, whereas ACE levels remain
unchanged (14). Similarly, no change in gene expression of
ACE was observed in diabetic rat hearts (33). In the
above-referenced human study (30), diabetic patients who
had elevated iAng II levels were on ACE-inhibitor therapy.
In the latter case, a rise in Ang II levels could be attributed
to an “ACE-escape” phenomenon, which is believed to
occur after prolonged treatment with ACE inhibitors (34).
In the present study, the lack of reduction in iAng II levels
after only 1 week of ACE-inhibitor treatment strongly
suggests an ACE-independent mechanism of iAng II syn-
thesis, corroborating in vitro observations. Upregulation
of vascular chymase in diabetic patients and chymase-
mediated Ang II generation in human and rat VSMCs and
human mesangial cells has been previously reported
(15,17,18,35). Involvement of chymase further strengthens
the concept of an intracellular RAS in diabetes.
Renin inhibition by aliskiren completely prevented hy-
perglycemia-induced iAng II synthesis. The source of renin
in the heart has been an issue of debate (36). Circulating
prorenin levels are elevated severalfold in diabetes, which
B
AC
DEF
FIG. 5. Detection of cardiac fibrosis by Masson’s Trichrome staining in heart sections from control rats (A); diabetic rats (B); and diabetic rats
treated with insulin (C), aliskiren (D), candesartan (E), or benazepril (F). Representative images of five sections per heart and three hearts per
group were observed with a ⴛ40 objective. (Please see http://dx.doi.org/10.2337/db08-0805 for a high-quality digital representation of this figure.)
INTRACELLULAR Ang II IN DIABETES
3302 DIABETES, VOL. 57, DECEMBER 2008

might also contribute to cardiac levels of pro(renin) (37).
Cardiac myocytes have been shown to internalize and
activate prorenin, which could contribute to iAng II syn-
thesis (38,39). In addition, several reports have described
expression of renin by cardiac myocytes, fibroblasts, and
cardiac mast cells (13,40 – 42). Adult rat cardiac myocytes
have been shown to express an intracellular form of
prorenin, which lacked a portion of the preprofragment,
eliminating the need for proteolytic activation (43). We
have previously described a significant increase in intra-
cellular renin levels in NRVMs exposed to high-glucose
conditions (14). In the current study, we observed en-
hanced staining for renin in the heart of diabetic rats.
Thus, inhibition of cardiac iAng II synthesis by aliskiren
was consistent with these observations.
Although aliskiren is the most potent inhibitor of human
renin (half-maximal inhibitory concentration [IC
50
] 0.6
nmol/l), it inhibits rat renin at higher concentrations (IC
50
80 nmol/l) (44). We chose an aliskiren dose of 30 mg 䡠kg
⫺1
䡠
day
⫺1
based on significant blood pressure–lowering ef-
fects of this dose in spontaneously hypertensive rats (45).
Pharmacokinetic studies of aliskiren in Sprague-Dawley
rats (species used in this study) demonstrated that an oral
dose of 30 mg/kg resulted in an area under the curve of
3.06 ⫾1.8 ␮mol 䡠l
⫺1
䡠h
⫺1
, indicating sufficient drug in the
bloodstream (45). Several additional characteristics of
aliskiren could explain the observed effects in diabetic
rats. These include cellular uptake, accumulation on mul-
tiple dosing, longer half-life, and rapid binding to renin
with slow dissociation (46). We observed that neonatal rat
cardiac myocytes internalized aliskiren in a concentration-
dependent manner, levels of which were measured at 1,
24, and 48 h after addition to the culture medium. Maxi-
mum intracellular levels of aliskiren were observed at
24 h, which were not reduced even after 48 h (data not
shown). Aliskiren levels were measured by liquid chroma-
tography tandem mass spectrometry, as previously de-
scribed (47). Similarly, rats that were treated with
aliskiren at a dose of 10 mg 䡠kg
⫺1
䡠day
⫺1
for 2 weeks,
showed a kidney-to-plasma ratio of aliskiren in the range
of 45 to 64 (47). The latter indicated extensive partitioning
of aliskiren to the kidneys, which localized to glomeruli
and the walls of small cortical arteries. Persistent renal
protective effects of aliskiren after discontinuation of
treatment also suggest slow clearance and accumulation
in tissues (48). The above findings suggest similar parti-
tioning of aliskiren in the heart, which might have resulted
in sufficiently high intracellular levels of aliskiren to inhibit
rat renin in our studies. Furthermore, tissue accumulation
of aliskiren may result in effects on target organs, even at
doses that do not affect blood pressure. This was evident
from a recent study wherein 2.5 mg/kg aliskiren did not
produce a statistically significant sustained reduction in
blood-pressure but reduced atherosclerotic lesion size
significantly in hypercholesterolemic Ldlr
⫺/⫺
mice (49).
Higher doses of aliskiren (up to 50 mg/kg) produced
↑AGEs
↑Glucose
↑GAD -3P
↑TCA cycle
Hyperglycemia
Hexosamine
Biosynthetic Pathway
O-glycosylation of
transcription factors
AGT
Renin
Chymase
AGT
Ang IAng II
Oxidative Stress
AT1 AGTAng IAng II
Renin
Chymase
ACE Renin
?
↑DAG ↑PKC
Apoptosis
Fibrosis
?
FIG. 6. Schematic representation of the relationship between hyperglycemia, iAng II, and pathological effects. In hyperglycemia, there is an
increase in glucose oxidation through the tricarboxylic acid cycle in mitochondria, which results in enhanced generation of reactive oxygen
species. Overproduction of superoxide inhibits glyceraldehyde-3-phosphate dehydrogenase activity, resulting in an accumulation of upstream
metabolites of the glycolytic pathway. Increased levels of glyceraldehyde-3-phosphate (GAD-3P) cause activation of PKC isoforms through
diacylglycerol (DAG) production and synthesis of advanced glycation end products (AGEs). There is increased shuttling of glucose through the
hexosamine biosynthesis pathway, resulting in the modification of transcription factors through o-glycosylation. All of these products of
hyperglycemia, i.e., oxidative stress, AGEs, PKC, and o-glycosylation of transcription factors, activate expression of RAS components. Cardiac
myocytes synthesize and retain Ang II intracellularly in hyperglycemia, whereas cardiac fibroblasts increase both intra- and extracellular Ang II.
iAng II could directly increase oxidative stress and cellular apoptosis through unidentified mechanisms and/or could enhance expression of RAS
components through a positive feedback mechanism, resulting in enhanced extracellular Ang II levels as well, particularly via cardiac fibroblasts.
Extracellular Ang II in turn causes oxidative stress, cardiac myocyte apoptosis, and cardiac fibrosis through the AT
1
receptor. Interrupting this
cycle by blocking Ang II synthesis provides protection from hyperglycemia-induced pathological events. ACE inhibitors or ARBs would block only
the extracellular synthesis or actions, respectively, of Ang II; whereas a renin inhibitor would block both intra- and extracellular Ang II synthesis,
the latter providing an explanation for the more pronounced effects of aliskiren observed in this study.
V.P. SINGH AND ASSOCIATES
DIABETES, VOL. 57, DECEMBER 2008 3303

similar results. In the double human renin-AGT transgenic
rat model, a dose (of aliskiren) of 0.03 mg/kg, which did
not decrease blood pressure, reduced albuminuria and
cardiac hypertrophy (50).
iAng II has been shown to produce multiple biological
actions, including cardiac hypertrophy (11,19). Many of
the reported iAng II effects are not prevented by ARBs,
either due to limited cell permeability of these drugs or to
an AT
1
-independent mechanism of iAng II–mediated ef-
fects. We previously demonstrated that iAng II–induced
NRVM cell growth and cardiac hypertrophy was not
inhibited by ARBs (19). Proliferation of Chinese hamster
ovary cells, which are deficient in AT
1
receptor, demon-
strated that some of the effects of iAng II do not require
AT
1
receptor (22). In addition, enhanced transforming
growth factor-␤/smad signaling was reported in kidneys
from diabetic AT
1
-knockout mice (51). These findings,
together with the observation that iAng II synthesis is
chymase dependent, suggest that ARBs and ACE inhibi-
tors do not block the intracellular RAS, which is activated
in diabetes. A renin inhibitor prevents both intracellular
and extracellular Ang II synthesis (14) and thus may prove
more beneficial in diabetic conditions. Consistent with the
latter hypothesis, we observed that aliskiren was more
effective in preventing oxidative stress and cardiac fibrosis
compared with candesartan or benazepril. Effects of
aliskiren were unlikely to be mediated through Ang II–
independent mechanisms because aliskiren does not in-
hibit renin binding to the (pro)renin receptor and
extracellular signal–related kinase activation (8,47). The
effect of these drugs on cardiac myocyte apoptosis dem-
onstrated that aliskiren was significantly more beneficial
than benazepril.
Reversal of cardiac effects in diabetic animals by insulin
treatment indicates that the observed effects were due to
hyperglycemia. Although STZ-induced diabetes is repre-
sentative of type 1 diabetes, we also predict activation of
the intracellular RAS in type 2 diabetes. This is based on
our unpublished observations of no effect of insulin treat-
ment on high glucose–induced iAng II synthesis in NRVMs
and patients with type 2 diabetes, who showed enhanced
iAng II staining in the heart (30).
As depicted in Fig. 6, activation of the RAS appears to be
a major event in hyperglycemia as a result of increased
oxidative stress, increased protein kinase C (PKC) levels,
and/or increased activity of the hexosamine biosynthesis
pathway. iAng II could directly produce oxidative stress
and cellular apoptosis through unidentified mechanisms
and/or could enhance expression of RAS components
through a positive feedback mechanism (52), resulting in
enhanced extracellular Ang II levels as well, particularly
via cardiac fibroblasts (13). Extracellular Ang II in turn
causes oxidative stress, cardiac myocyte apoptosis, and
cardiac fibrosis through the AT
1
receptor. Interrupting this
cycle by blocking Ang II synthesis protects from hypergly-
cemia-induced pathological events. ACE inhibitors or
ARBs would block only the extracellular synthesis or
actions, respectively, of Ang II; whereas a renin inhibitor
would block both intra- and extracellular Ang II synthesis,
the latter providing an explanation for the more pro-
nounced effects of aliskiren observed in this study. In
diabetic heart, the source and target of Ang II could be
represented by multiple cell types. In addition to NRVMs,
we previously demonstrated that cardiac fibroblasts re-
spond to high glucose with enhanced activity of the RAS
and extracellular matrix production (13). Cardiac fibro-
blasts increase both intracellular and extracellular Ang II,
in contrast to cardiac myocytes, which demonstrate only
increased iAng II in high-glucose conditions. Additionally,
Ang II synthesis by cardiac fibroblasts, extracellular as
well as intracellular, is catalyzed by ACE (13). Thus, ACE
inhibitors would block Ang II synthesis by cardiac fibro-
blasts, and ARBs would block autocrine/paracrine effects
of extracellular Ang II. This explains the partial effects of
these agents in diabetic rats. However, these agents would
not block high glucose–stimulated iAng II synthesis or
intracellular actions in cardiac myocytes. In addition to a
direct effect on cardiac myocytes, iAng II synthesized in
cardiac myocytes would likely have indirect functional
effects on other cells by stimulating synthesis and release
of growth factors and cytokines from myocytes (53). Thus,
the intracellular RAS possibly explains progression from
microalbuminuria to proteinuria in diabetic patients on
ACE inhibitor therapy, the mechanism of resistance to
antihypertensive therapy in type 2 diabetes and higher
cardiovascular morbidity and mortality in hypertensive
patients with diabetes (10,54 –56). Although ARBs and
ACE inhibitors would provide some protection to the
cardiovascular system through positive hemodynamic ef-
fects, partial inhibition of the local RAS, or other non-
RAS–related mechanisms, such as an effect on peroxisome
proliferator–activated receptor-␥and the kallikrein-kinin
system (57–59); additional blockade of the intracellular
RAS using agents such as a renin inhibitor might prove
more beneficial in diabetes. Long-term studies that include
cardiac functional analysis will be necessary to validate
the above hypothesis.
ACKNOWLEDGMENTS
We thank the Texas A&M Health Science Center Micros-
copy Imaging Center for providing confocal microscopy
services. R.K. has received an American Heart Association,
Texas Affiliate, Beginning Grant-In-Aid.
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