Content uploaded by Philipp Lutze
Author content
All content in this area was uploaded by Philipp Lutze on May 31, 2016
Content may be subject to copyright.
ORIGINAL ARTICLE
Anti-necrotic and cardioprotective effects of a cytosolic renin
isoform under ischemia-related conditions
Heike Wanka
1
& Doreen Staar
1
& Philipp Lutze
1
& Barbara Peters
1
&
Johanna Hildebrandt
1
& Tim Beck
1
& Inga Bäumgen
1
& Alexander Albers
1
&
Thomas Krieg
2,3
& Katrin Zimmermann
3
& Jaroslaw Sczodrok
1
& Simon Schäfer
1
&
Sigrid Hoffmann
4
& Jörg Peters
1
Received: 25 March 2015 /Revised: 25 June 2015 /Accepted: 1 July 2015 /Published online: 11 August 2015
#
Springer-Verlag Berlin Heidelberg 2015
Abstract
In the heart, secretory renin promotes hypertrophy, apoptosis,
necrosis, fibrosis, and cardiac failure through angiotensin gen-
eration from angiotensinogen. Thus, inhibitors of the renin-
angiotensin system are among the most potent drugs in the
treatment of cardiac failure. Renin transcripts have been iden-
tified encoding a renin isoform with unknown targets and
unknown functions that are localized to the cytosol and mito-
chondria. We hypothesize that this isoform, in contrast to se-
cretory renin, exerts cardioprotective effects in an angiotensin-
independent manner. Cells overexpressing cytosolic renin
were generated by transfection or obtained from CX(exon2-
9)renin transgenic rats. Overexpression of cytosolic renin re-
duced the rate of necrosis in H9c2 cardiomyoblasts and in
primary cardiomyocytes after glucose depletion. These effects
were not mediated by angiotensin generation since an inhibi-
tor of renin activity did not influence the in vitro effects.
siRNA-mediated knockdown of endogenous cytosolic renin
increased the rate of necrosis and aggravated the pro-necrotic
effects of glucose depletion. Isolated perfused hearts obtained
from transgenic rats overexpressing cytosolic renin exhibited
a 50 % reduction of infarct size after ischemia-reperfusion
injury. Cytosolic renin is essential for survival, both under
basal conditions and during glucose starvation. The protective
effects are angiotensin-independent and contrary to the known
actions of secretory renin.
Key messages
& A cytosolic isoform of renin with unknown functions is
expressed in the heart.
& Cytosolic renin diminishes ischemia induced damage to
the heart.
& The protective effects of cytosolic renin contradict the
known function of secretory renin.
& The effects of cytosolic renin are not mediated via angio-
tensin generation.
& Renin-binding protein is a potential target for cytosolic
renin.
Keywords Cytosolic renin
.
Ischemia
.
Cardioprotection
.
Glucose depletion
.
Necrosis
Abbreviations
ANG Angiotensin
E(1–9) Exon(1–9)renin, secretory
E(1A–9) Exon(1A–9)renin, cytosolic
E(2–9) Exon(2–9)renin, cytosolic
I/R Ischemia/reperfusion
TG+ Transgenic rats overexpressing cytosolic renin
TG- Non-transgenic rats
cAMP Cyclic adenosine monophosphate
Electronic supplementary material The online version of this article
(doi:10.1007/s00109-015-1321-z) contains supplementary material,
which is available to authorized users.
* Heike Wanka
wanka@uni-greifswald.de
1
Department of Physiology, University Medicine of Greifswald,
Greifswalder Str. 11C, D-17495 Karlsburg, Germany
2
Department of Medicine, University of Cambridge, Addenbrooke’s
Hospital, Cambridge CB2 0QQ, UK
3
Internal Medicine, University Medicine Greifswald,
D-17489 Greifswald, Germany
4
Medical Research Center, Medical Faculty Mannheim, University of
Heidelberg, D-68135 Mannheim, Germany
J Mol Med (2016) 94:61–69
DOI 10.1007/s00109-015-1321-z
YWHAZ Tyrosine 3 monooxygenase/tryptophan 5
monooxygenase activation protein, zeta
polypeptide
RLU Relative light units
RnBP Renin-binding protein
RAS Renin-angiotensin system
Introduction
Renin is a secretory glycoprotein that, after secretion into the
extracellular space, generates angiotensin (ANG) I from its
only known substrate, angiotensinogen [1]. ANG I is further
cleaved to ANG II, the effector peptide of the renin-
angiotensin system (RAS) that increases oxidative stress and
exerts pro-inflammatory effects [2, 3]. In the heart, ANG II
promotes hypertrophy, apoptosis, necrosis, fibrosis, myocar-
dial remodeling, and hence, cardiac failure [4, 5]. Correspond-
ingly, inhibitors of the RAS are among the most potent drugs
in the treatment of hypertension and cardiac failure, markedly
increasing the life span of patients [6].
In addition to the classical transcript [exon(1–9)], alterna-
tive renin transcripts have been identified in several species
incl uding humans [exon(2-9 ), exon(1A-9), renin-c] [7–9].
The transcript for secretory renin is mainly expressed in the
kidney. The alternative transcripts encode for a non-secretory
cytosolic form of renin and are expressed in extrarenal tissues
[10]. The targeting of proteins to the secretory pathway re-
quires the co-translational transport to the endoplasmic retic-
ulum (ER). In rats, the renin gene consists of nine exons with
the corresponding ER signal sequence, the so-called pre-frag-
ment, being encoded by exon 1. Because the alternative tran-
scripts lack exon 1, the translated renin protein remains in the
cytosol or is taken up by the mitochondria [8]. In humans, the
alternative renin transcript directly starts with exon 2 [9]. In
rats, exon 2 is preceded by a short non-coding sequence of
about 80 base pairs derived from intron A forming the
exon(1A–9)renin transcript. The exon(1A–9)renin and the ex-
on(2–9)renin transcripts are translated into a truncated
prorenin starting at the first in-frame UTG in exon 2. This
protein lacks the pre-fragment and the first 15 amino acids
of the conventional prorenin but still exerts renin activity
[7–9].
The biological functions of cytosolic renin are currently
unknown. In the rat heart, the expression of cytosolic renin
but not of secretory renin was increased after myocardial in-
farction, and first vague hints suggested that cells overexpress-
ing cytosolic renin may be more stable against necrotic cell
death [10, 11]. In this context, it is known that the heart pos-
sesses the ability to adapt to stress by actively enhancing its
resistance to injury. This molecular plasticity is illustrated by
the phenomenon of ischemic preconditioning where the toler-
ance of the heart to an ischemic event is increased by
influencing different signaling pathways [12, 13]. Similar ef-
fects described as part of a genetic reprogramming to fetal
gene profile are also detectable in failing hearts [14, 15]. We
hypothesized that cytosolic renin may play a role in the re-
sponse to energy depletion and that it may reduce the severity
of cardiac damage under ischemic conditions. In this study, we
show that cytosolic renin decreases the rate of necrosis, par-
ticularly under conditions of glucose depletion and attenuates
the infarct size after coronary ligation in isolated beating
hearts.
Methods
Overexpression, downregulation and inhibition of renin
Hearts from CX-exon(2-9)renin transgenic rats (TG+) over-
expressing cytosolic renin under the control of the CX-CMV
promoter and hearts from non-transgenic control rats (TG-)
were used to prepare primary cardiomyocytes [16]. H9c2
cardiomyoblasts (ATCC, CRL-1446) were transfected with
the pIRES1neo vector containing exon(2-9)renin DNA as pre-
viously described [11]. Downregulation of renin in H9c2 cells
was accomplished with the RNA interference method using
80 nmol/L siGENOME SMART pool siRNA to renin (Ther-
mo Scientific Dharmacon) and polyethyleneimine solution
according to the manufacturer’s instructions. Renin transcript
abundance was quantified using qRT-PCR and Western blot
analyses. The renin inhibitor CH732 (10
−9
to 10
−6
mol/L) was
used to block any possible generation of ANG I by exogenous
renin [1.8 μg ANG I/(ml×h)]. After incubation of H9c2 cells
with 10
−6
mol/L CH732 for 24 h, trypsinated cells were
sonificated and separated into a soluble and a membrane frac-
tion by centrifugation at 20,000×g. The pellet and the soluble
fractions potentially containing CH732 were then used to in-
hibit renin enzyme activity in terms of ANG I generation [17]
as determined with renin standard preparations (see
Supplement Material 1). For functional analysis, CH732 was
added to H9c2 cells for 24 h in a concentration of 10
−6
mol/L
followed by the detection of the necrosis rate.
Analysis of metabolic parameters and necrosis in cardiac
cells
Cardiomyoblasts (H9c2, ATCC) and primary cardiomyocytes
were cultured at 37 °C in a humidified atmosphere with 5 %
CO
2
. Plated cells were subjected to control conditions or glu-
cose deprivation for 24 or time dependent for 24, 72, and
120 h, respectively. Consequences of glucose deprivation on
necrosis rate were analyzed by the Cytotoxicity Detection Kit
(LDH) (Roche Applied Science, Germany) as previously de-
scribed [11]. ATP content, glucose consumption and lactate
62 J Mol Med (2016) 94:61–69
accumulation into the medium were detected as described in
the expanded methods online supplement.
Analysis of renin expression and renin/prorenin activities
After treatment of cardiac cells, renin expression was deter-
mined by quantitative real–time PCR (Rotor-Gene SYBR
Green PCR Kit) and Western blot analyses. For quantification
of specific renin transcript isoforms, the following primers
were used: renin reverse primer: position 722-743 of the renin
cDNA and the following forward primers for specific tran-
scripts: exon(1A–9)renin: position 3996-4019 of intron A of
the renin gene, exon(1–9)renin: position 211-233 of the renin
cDNA, exon(2–9)renin: position 421–442 of the renin cDNA.
The threshold cycle number (CT) in combination with the
2
delta CT
method was normalized against YWHAZ expression
and compared to the control.
To detect renin protein, we used a primary rabbit anti-renin
antibody (Bioss Inc, Woburn, MA, USA) in combination with
a horseradish peroxidase (HRP)-conjugated secondary anti-
rabbit antibody (CellSignaling, Merck-Millipore, Billerica,
MA, USA). The proteins were visualized by the enhanced
chemiluminescence method (BioRad Laboratories, Munich,
Germany) using an image capture system (Chemidoc XRS,
BioRad Laboratories, Munich, Germany).
Renin activity was determined as follows. Extracts from
H9c2 cell lines were prepared by ultrasonification. Mem-
branes were pelleted by centrifugation at 20,000× g and
discarded. (Pro)renin activities of the supernatants were deter-
mined by measuring the capability of the extracts to generate
ANG I from excess renin substrate followed by an ANG I
radioimmunoassay as previously described [11, 16]. To fur-
ther test the specificity of the results, aliquots of extracts were
incubated with and without the addition of the rat renin inhib-
itor CH732 [17]. The inhibitor completely abolished the gen-
eration of ANGI in all H9c2 cell lines.
Langendorff experiment
TG+ and TG− rats were deeply anesthetized with thiopental
solution containing heparin to remove their hearts for
Langendorff experiments. Cardiac ischemia was induced by
placing a ligature around the left anterior descending artery for
30 min followed by 120 min reperfusion. A solution of mi-
crospheres was injected retrograde though the aortic root to
demarcate the risk zone. Transverse slices of the hearts were
stained with triphenyltetrazolium chloride (TTC) to assess the
non-infarcted myocardium of the risk zone [18].
Additionally, detailed material and methods are provided in
the online supplement.
Results
Upregulation of cytosolic renin
During periods of ischemia, cells are deprived of extra-
cellular glucose. Here, we demonstrate that in H 9c2
cardiomyoblasts, glucose depletion for 24 h upregulated
the expression of cytosolic exon(1A–9)renin but not of
secretory exon(1– 9)r enin at the transcript and protein
levels (Fig. 1a–c). Although renin expression clearly
increased at mRNA (Fig. 1a) and protein levels
(Fig. 1b, c), the renin activity even decreased to unde-
tectable levels (not shown), likely due to an inhibitory
effect of cytosolic renin-binding protein (RnBP).
Fig. 1 Cytosolic renin expression is increased in cardiac H9c2 cells
under glucose deprivation. Cells were cultured in medium containing
25 mM glucose (white columns) or exposed to glucose deprivation for
24 h (gray columns). a Effect of 24 h glucose deprivation on transcript
levels of cytosolic exon(1A-9)renin and secretory exon(1–9)renin as
detected by RT PCR. Expression of renin transcripts was normalized to
YWHAZ as housekeeping gene. b Western blot demonstrates the effect
of glucose deprivation on renin protein expression. Bands corresponding
to cytosolic prorenin and renin are estimated at 30, 36, and 41 kDa,
respectively (arrows). c Renin protein normalized to total protein
content. Values repres ent means±s.e.m., n =8 experiments each;
*p<0.05, **p<0.01
J Mol Med (2016) 94:61–69 63
Cytosolic renin protects cardiac cells from necrosis
To determine the functional relevance of cytosolic renin,
we evaluated whether or not cytosolic renin can protect
cardiac H9c2 cells from necrosis during glucose deple-
tion. To this end, we generated a number of cell lines
overexpressing cytosolic renin at various degrees rang-
ing from 3- to 2000-fold (confirmed by qRT–PCR as
well as by We stern blot, not shown). We then chose
such cells overexpressing renin by about 5- to 10-fold,
which compares well with the amount of overexpression
after glucose depletion (Fig. 1). Renin mRNA was in-
creased by about 8-fold (Fig. 2a). Nevertheless, renin as
determined by its capacity to generate ANGI from ex-
cess substrate was not increased (Fig. 1c). This may be
attributed to the inhibitory effect of RnBP present in
those cells. However, after trypsin activation, the over-
expression was also obvious at the protein/enzymatic
level (Fig. 1b).
In the presence of glucose, cells overexpressing cytosolic
renin by about 5- to 10-fold (Fig. 2a–c) exhibited low necrosis
rates similar to H9c2 and pIRES vector-transfected cells.
Twenty-four hours of glucose depletion markedly increased
necrosis rates in control cells. In contrast, in cells overexpress-
ing cytosolic renin, the increase of necrosis rate in response to
glucose depletion was abolished (Fig. 2d).
This anti-necrotic effect was further validated in primary
cardiomyocytes derived from transgenic rats overexpressing
cytosolic renin in the heart [16]. Primary cardiomyocytes of
TG+ rats were also protected from necrotic cell death under
conditions of glucose deprivation, however with a delay com-
pared with the H9c2 cell lines (Fig. 3). Anti-necrotic effects
were detectable after 72 and 120 h of glucose deprivation.
Vice versa, knockdown of cytosolic renin expression by
siRNA should diminish survival of cardiomyoblasts. In our
experiments, we used a 48 h knockdown because the efficien-
cy of knockdown decreased time dependently (Fig. 4a). After
knockdown, necrosis rates of H9c2 cells increased already
under basal conditions and the increase after glucose depletion
was still prominent (Fig. 4b).
Cytosolic renin acts ANG-independently
To clarify whether the anti-necrotic effects of cytosolic renin
were ANG-mediated, we applied the rat-specific renin inhib-
itor CH732. CH732 inhibited renin activity dose dependently
and almost completely at a conc entr ation of 10
−6
mol/L
(Fig. 5a). Furthermore, CH732 inhibitory activity was ob-
served in both the soluble and the membrane fraction s of
CH732-pretreated H9c2 cells indicating the uptake of
CH732 into the cells (Fig. 5b). Although CH732 clearly
inhibited ANG generation and was taken up by the cells
(Fig. 5a, b), CH732 did not block the protective anti-necrotic
effects of cytosolic renin overexpression (Fig. 5c).
Cytosolic renin changes metabolic state
Under basal conditions, cells overexpressing cytosolic renin
differed from vector-transfected pIRES cells by a modified
metabolic state as evidenced by a reduced ATP content (788
±59×10
3
vs. 991±18×10
3
RLU; n=9; p<0.01), a higher glu-
cose consumption (10.80±0.43 vs. 6.08±0.66 mM/10
6
cells/
24 h; n=8; p<0.001), and an increased accumulation of lactate
in the medium (9.28±1.74 vs. 5.14±0.49 mM/10
6
cells/24 h;
n=8; p<0.05). Furthermore, glucose deprivation for 24 h de-
creased ATP content in vector-transfected control cells but not
Fig. 2 Cytosolic renin protects H9c2 cells from necrotic cell death. a
Transcript levels of cytosolic exon(2–9)reninnormalizedtoYWHAZas
detected by RT PCR. b, c Enzymatic inactive (prorenin) and active renin
of H9c2 cell lines. In extracts from exon(2–9)renin overexpressing H9c2
cells, the prorenin level (a, dark column) was significantly increased
compared to H9c2 and pIRES control cells (each n=6). There were no
differences in renin activity (b). d Effect of cytosolic renin overexpression
on LDH release. H9c2 cells (white column), pIRES control cells (gray
columns) and H9c2 cells overexpressing cytosolic exon(2-9)renin (dark
columns) were maintained in medium containing 25 mM glucose (+) or
exposed to glucose depletion (-) for 24 h (each n=9). The values shown
are mean±s.e.m. Statistical analysis was performed by one way ANOVA
with *p<0.05 and ***p<0.001 as indicated and
###
p<0.001 effect
between exon(2-9) and pIRES control cells grown under glucose
depletion
64 J Mol Med (2016) 94:61–69
in cells overexpressing cytosolic renin (controls: from 991±
18 to 855±21×10
3
vs. E(2–9) cells: from 788±59 to 841±
55×10
3
RLU; n=9; p<0.01).
Protective effects of cytosolic renin in isolated perfused
hearts
We finally applied an ex vivo model of myocardial infarction
to further investigate the protective effects of cytosolic renin
under conditions of ischemia reperfusion (I/R) induced by
coronary ligation. For these experiments, we used two inde-
pendently generated transgenic rat lines and two non-
transgenic control lines. In Sprague–Dawley and Wistar rat
hearts, the infarct size relative to the area at risk was about
40 %. In both strains of TG+ rats, infarct size was reduced to
about 20 % of the risk area, representing a 50 % reduction of
the infarct size compared to controls (Fig. 6).
Discussion
There is no doubt that renin-angiotensin systems reside locally
within the heart [19, 20]. Even the existence of intracellular
acting RAS has been postulated, but this is presently subject of
controversial discussions [21]. However, it is difficult to un-
derstand how secretory proteins such as renin and
angiotensinogen can be located in the cytosol, in mitochon-
dria, or in nuclei since these proteins are inevitably packed
into secretory vesicles for externalization or internalized via
vesicle-mediated uptake . Meanwhile, for renin but not for
angiotensinogen, alternative transcripts have been discovered
coding for a renin isoform that is translated at free ribosomes
[7–9]. This renin isoform is located in the cytosol and can be
imported into mitochondria [8]. Here, we report that this cy-
tosolic renin protects cardiac cells from necrosis and reduces
infarct sizes in isolated perfused hearts. We also demonstrate
that the anti-necrotic effect of cytosolic renin is not mediated
by cleaving ANG I from angiotensinogen and suggest the
renin-binding protein (RnBP) as a possible target for cytosolic
renin. The fact that renin expression increased by glucose
starvation (mRNA and protein levels) whereas renin activity
remained unchanged supports the hypothesis that in the cyto-
sol renin activity is blocked, and RnBP is a likely candidate
[22] since RnBP forms heterodimers with renin particularly
under energy depletion [23].
During ischemia, the expression of cytosolic renin is up-
regulated in vivo [10]. Here , we demonstrate that glucose
starvation as one aspect of ischemia also upregulates the en-
dogenous expression of cytosolic renin but not of secretory
renin, indicating that especially the cytosolic renin isoform is
part of an adaptive response to starvation. Applying different
models of overexpression and downregulation of cytosolic
renin, we come to the conclusion that endogenous cytosolic
Fig. 3 Anti-necrotic effect of cytosolic renin in primary cardiomyocytes.
Cardiac cells overexpressing cytosolic renin obtained from TG 294 and
TG 307 (n=7 each) (gray columns) and cardiac cells from control TG−
rats ( n=7) (white columns) were maintained in medium containing
25 mM glucose or exposed to glucose depletion (0 mM) for 24 h (a),
72 h (b), or 120 h (c), respectively. Cardiomyocytes of TG+ rats were
protected from necrotic cell death caused by glucose deprivation after 72
and 120 h. Data are presented as means±s.e.m.; *p<0.05 vs. TG−;
**p<0.01 vs. TG−;
#
p<0.05 vs. basal conditions;
##
p<0.01 vs. basal
conditions
J Mol Med (2016) 94:61–69 65
renin protects cardiac cells under ischemia and ischemia-
related conditions. For the present studies, we used CX-ex-
on(2–9)renin transgenic rats overexpressing cytosolic renin
about 5 -fold in the h eart and accordingly exon(2–9)renin
transfected H9c2 cardiomyoblasts showing a 5- to 10-fold
overexpression of cytosolic renin. We previously demonstrat-
ed already that overexpression of cytosolic renin in cells as
well as in rats resulted in an increase of renin particularly in
the cytosolic or mitochondrial fractions [11, 16]. The degree
of overexpression of cytosolic renin corresponds well with
that seen after myocardial infarction in vivo [10, 24]. To ex-
clude or at least minimize genetic background effects, we
chose two independently generated transgenic rat strains, line
294 and 307, as well as two different control strains (Wistar,
Sprague–Dawley rats from different providers) for the exper-
iments. The rationale was that all rats without overexpression
of cytosolic renin should be susceptible to ischemia indepen-
dently of further genetic differences, whereas all rats with
overexpression of cytosolic renin were protected, again inde-
pendently of further genetic differences. In transgenic rats, the
Fig. 4 Renin knockdown increased necrotic cell death in H9c2 control
cells. a Time dependent efficiency of siRNA-mediated downregulation of
exon(1A-9)renin transcript validated by qRT-PCR. Renin expression was
normalized to YWHAZ as housekeeping gene and related to untreated
scramble controls. b Effect of 48 h downregulation of endogeneous
cytosolic renin on LDH release of control and glucose-deprived H9c2
cells. Values represent means±s.e.m. of n=8 experiments. Statistical
analysis was performed by one way (a) and two-way ANOVA (b)with
*p< 0.05; ***p <0.001 as indicated, and
§§
p<0.01 effect of siRNA-
mediated downregulation vs. untreated cells grown under the same
conditions
Fig. 5 Inhibition of renin activity by CH732 did not prevent anti-necrotic
effects of cytosolic renin. a Dose-response curve of CH732 on the
efficacy to block ANG I generation as determined by the
radioimmunoassay. Complete inhibition of renin activity was reached at
a CH732 concentration of 10
-6
mol/L (n=6). b Inhibitory potential of
H9c2 cell fractions pretreated with 10
-6
mol/L CH732 for 24 h. The
data confirm that the renin inhibitor wa s taken up by the cells and
appeared in the cytosolic fraction where it effectively reduced
exogenous renin activity (n=6). c Anti-necrotic, protective effects of
cytosolic renin under glucose deprivation were not impaired by CH732
pretreatment. Experiments wereperformedwithpIREScells(gray
columns) and E(2-9)renin overexpressing cells (dark columns) for 24 h
without and with a concentration of 10
-6
mol/L (n=9). Values represent
means±s.e.m. Statistical analysis was performed by one way (a, b)and
two-way ANOVA (c)with*p>0.05; **p<0.01; ***p< 0.001 as indicated
and ###p<0.001 effect of cytosolic renin vs. pIRES control cells grown
under the same conditions
66 J Mol Med (2016) 94:61–69
levels of circulating renin and prorenin were not different from
controls. Rats overexpressing cytosolic renin showed no signs
of inflammation, fibrosis, hypertrophy, or cardiac failure, ex-
cluding harmful effects of cytosolic renin in vivo [16].
In the presence of glucose, H9c2 cells overexpressing cy-
tosolic renin exhibited low necrosis rates similar to vector-
transfected control cells, confirming the lack of toxicity of
cytosolic renin under basal conditions in vitro. During glucose
starvation, overexpression of cytosolic renin prevented the
rise in necrosis rates. O n the other hand, t he siRNA-
mediated knockdown of renin expression in H9c2 cells was
already harmful under basal conditions by increasing the ne-
crosis rate. These data confirm the specificity of our findings.
Compared to H9c2 cells, primary cardiomyocytes showed a
markedly higher basal necrosis rate which may be due to the
isolation procedure. However, they seem to be more resistant
to glucose depletion in general because starvation did not
enhance necrosis rate. Overexpression of cytosolic renin in
cardiomyocytes obtained from transgenic rats resulted in a
marked reduction of necrosis rates compared to non-
transgenic primary cardiomyocytes, demonstrating the
cardioprotective potential of cytosolic renin. The data support
the hypothesis that endogenous cytosolic renin substantially
enhances the survival of cardiac cells both under basal and
especially under glucose-deprived conditions in vitro. The
cardioprotective effects of cytosolic renin may be particularly
beneficial during transient ischemic periods in patients with
coronary heart disease.
To clarify the underlying protective mechanisms, especial-
ly to check for potential intracrine ANG effects, we used the
rat renin inhibitor CH732 [17]. Although the inhibitor clearly
inhibited ANG I generation in vitro and was taken up into the
cells, CH732 did not block the protective effects of cytosolic
renin in glucose-deprived cells. These data demonstrate that
the cardioprotective effects of cytosolic renin are ANG-inde-
pendent. In contrast, the glucose depletion-induced rise in
necrosis rate was higher in CH732-pretreated pIRES cells than
in untreated pIRES control cells. In this context, the existence
of an intracellular RAS with intra-cytoplasmatic ANG II ac-
tions has long been postulated (for review, see [21]). There is
evidence for the presence of renin, angiotensin-converting en-
zyme, ANGs, and ANG receptors in the cytosol, nuclei, and
mitochondria. A complete mitochondrial RAS has been re-
ported in various cell types including mouse cardiac myocytes
[25]. Intracellular application of renin decreased junctional
conductance between cardiac cells, which was potentiated
by concomitantly dialyzed angiotensinogen. Also, ANG II
injected into the cytoplasm increased cytosolic calcium, which
was blocked by concomitant injection of an AT1 receptor
blocker [26, 27]. Furthermore, in isolated nuclei, ANG II stim-
ulated the de novo synthesis of RNA via an ANG receptor-
dependent mechanism [28]. Overexpression of
angiotensinogen increased the mitogenic index, which was
blocked by the AT1 receptor blocker losartan and co-
expression of ANG II together with the AT1 receptor en-
hanced cell proliferation and increased the activity of cAMP
response element-binding protein (CREB) [29, 30]. It is im-
portant to note that in these studies, ANG II was given intra-
cellular by microinjections or by dialysis or produced from
engineered constructs encoding a non-secretory
angiotensinogen variant or an ANG II fusion protein. It still
remains unclear how ANG can be generated within the cyto-
plasm and how it can reach the mitochondria or the nucleus,
assuming that angiotensinogen is a secretory protein and
should not be localized in the cytosol. Our data neither support
nor exclude the idea of intracellular actions of ANGs; however ,
the fact that the renin inhibitor CH732 did not abolish the
protective effects of cytosolic renin demonstrates that these
effects are not mediated by ANGs. The effect of CH732 on
control cells, however, supports the view that additionally
intracellular ANG may have so far unknown effects.
In diabetic rats, a hyperglycemia-associated upregulation
of the RAS contributes to an increase of intracellular produced
ANG II that correlates with cardiomyocyte apoptosis, en-
hanced oxidative stress, and cardiac fibrosis [31]. The group
also demonstrated that the renin inhibitor aliskiren prevented
intracellular ANG II synthesis and reduced harmful effects
more efficiently than other RAS blockers in cardiac fibroblast
under glucose load [32]. In their study, an increase of renin
protein levels was observed in response to glucose load
in vitro. Whether or not this was the result of increased
Fig. 6 Hearts of cytosolic renin transgenic rats are protected against
ischemia-reperfusion injury ex vivo. Effect of 30 min ischemia
followed by 120 min reperfusion on infarct size in isolated perfused
Langendorff hearts of two control rat lines (SD; W) and two
independently generated transgenic rat lines overexpressing cytosolic
renin in the heart (line 294; 307). Infarct size was reduced in transgenic
rats (line 294: n=8 and line 307: n=9) compared to control rats (each n=
10). Data are presented as means±s.e.m.; *p<0.05
J Mol Med (2016) 94:61–69 67
expression of secretory or cytosolic renin still remains to be
investigated. Since the increased renin protein levels were
accompanied by increased intracellula r angiotensin levels
and harmful effects as shown by Singh et al., it is likely that
under glucose load in this experiment, the expression of
secretory renin was increased rather than of cytosolic renin.
The intracellular interaction partners for cytosolic renin
still need to be identified. One possible target is the cyto-
solic RnBP that has already been shown to interact with
renin in vitro particularly under conditions with depletion
of high-energy nucleotides [23, 33]. Such conditions may
prevail during ischemia. RnBP ac ts as an epimerase,
interconverting N-acetyl-D-glucosamine and N-acetyl-D-
mannosamine. This enzymatic activity of RnBP is inhibited
by renin [34]. By regulating the levels of N-acetyl-D-glu-
cosamine, RnBP may affect the glycosylation and
sialylation of many proteins. As a first hint, downregulation
of cytosolic renin increased the level of free sialic acids
released into the medium and a mild siRNA-mediated
downregulation of RnBP had similar protective effects as
overexpression of cytosolic renin (data not shown). Thus,
cytosolic renin may be an endogenous inhibitor of N–ace-
tyl–D–glucosamine epimerase activity. However, other
mechanisms of action also need to be considered.
An important principle of cardioprotection against ische-
mia is the adequate provision of ATP [35]. Opie first described
the so-called glucose hypothesis according to which an en-
hanced uptake and metabolism of glucose delays cellular
damage [36]. In agreement with this hypothesis, our cells
overexpressing cytosolic renin exhibited an enhanced basal
exogenous glucose uptake and increased lactate accumulation
in the medium compared to H9c2 and pIRES control cells
indicating a metabolic alteration. This alteration is not associ-
ated with an anaerobic exploitation of glucose because the
ratio of lactate release to glucose uptake did not differ between
the cell lines. Instead, this effect may be achieved by a raised
generation or storage of glycogen since glucose was the sole
fuel in medium of the cell culture experiments. This assump-
tion is supported by our results that following glucose starva-
tion cells overexpressing cytosolic renin exhibited an en-
hanced preservation of the ATP content. In conclusion, these
data suggest that cells overexpressing cytosolic renin may be
better adapted to fight fuel deficiencies than control cells pro-
viding them with stronger protection against acute ischemia-
mediated cell death.
We finally applied an ex vivo model of myocardial infarc-
tion to investigate the effects of cytosolic renin under condi-
tions of I/R. For these experiments, we used two independent-
ly generated transgenic rat lines and two different non-
transgenic control lines in an attempt to minimize artifacts
due to transgene-induced insertional mutagenesis or due to
differences in the genomic background. The remarkable re-
duction in infarct size in hearts from transgenic rats compared
to that in hearts from control rats demonstrates that cytosolic
renin protects the heart also from I/R injury. Additional studies
are necessary to determine the mechanisms and signal path-
ways by which cytosolic renin elicit cardioprotection. Several
in vivo studies have shown that aliskiren, a clinically used oral
renin inhibitor, is able to abrogate the detrimental cardiac ef-
fects of I/R in rats independent of blood pressure lowering [37,
38]. Such studies underline the harmful effects of angiotensin
generation and hence the potential benefit of renin inhibitors.
Our data do not contradict these observations since we dem-
onstrate that inhibition of renin activity does not play a role for
the functions of cytosolic renin including those presented in
this study.
In conclusion, cytosolic renin has the potential of becoming
a new therapeutic target for the treatment of cardiac diseases
as it decreases necrotic cell death and reduces infarct size after
I/R. It is quite likely that this new cytosolic renin system is not
cardi o-specific but may also protect other cell types from
death under ischemic conditions.
Acknowledgements The work was supported by a grant from the Ger-
man Research Foundation to J. Peters (PE 366/11-1). B. Sturm and D.
Albrecht performed technical assistance.
Conflict of interest The authors declare that they have no competing
interests.
References
1. Hackenthal E, Paul M, Ganten D, Taugner R (1990) Morphology,
physiology, and molecular biology of renin secretion. Physiol Rev
70:1067–1116
2. Paul M, Poyan Mehr A, Kreutz R (2006) Physiology of local renin-
angiotensin systems. Physiol Rev 86:747–803
3. Unger T (2002) The role of the renin-angiotensin system in the
development of cardiovascular disease. Am J Cardiol 89:3A–9A,
discussion 10A
4. Aoki H, Richmond M, Izumo S, Sadoshima J (2000) Specific
role of the extracellular signal-regulated kinase pathway in an-
giotensin II-induced cardiac hypertrophy in vitro. Biochem J
347(Pt 1):275–284
5. Mehta PK, Griendling KK (2007) Angiotensin II cell signaling:
physiological and pathological effects in the cardiovascular system.
Am J Physiol Cell Physiol 292:C82–97
6. Werner C, Baumhakel M, Teo KK, Schmieder R, Mann J, Unger T,
Yusuf S, Bohm M (2008) RAS blockade with ARB and ACE in-
hibitors: current perspective on rationale and patient selection. Clin
Res Cardiol 97:418–431
7. Lee-Kirsch MA, Gaudet F, Cardoso MC, Lindpaintner K (1999)
Distinct renin isoforms generated by tissue-specific transcription
initiation and alternative splicing. Circ Res 84:240–246
8. Clausmeyer S, Sturzebecher R, Peters J (1999) An alternative tran-
script of the rat renin gene can result in a truncated prorenin that is
transported into adrenal mitochondria. Circ Res 84:337–344
9. Sinn PL, Sigmund CD (2000) Identification of three human renin
mRNA isoforms from alternative tissue-specific transcriptional ini-
tiation. Physiol Genomics 3:25–31
68 J Mol Med (2016) 94:61–69
10. Clausmeyer S, Reinecke A, Farrenkopf R, Unger T, Peters J (2000)
Tissue-specific expression of a rat renin transcript lacking the cod-
ing sequence for the prefragment and its stimulation by myocardial
infarction. Endocrinology 141:2963–2970
11. Wanka H, Kessler N, Ellmer J, Endlich N, Peters BS, Clausmeyer S,
Peters J (2009) Cytosolic renin is targeted to mitochondria and
induces apoptosis in H9c2 rat cardiomyoblasts. J Cell Mol Med
13:2926–2937
12. Heusch G (2013) Cardioprotection: chances and challenges of its
translation to the clinic. Lancet 381:166–175
13. Hausenloy DJ (2013) Cardioprotection techniques: precondition-
ing, postconditioning and remote conditioning (basic science).
Curr Pharm Des 19:4544–4563
14. De Mello WC (2006) Cardiac intracrine renin angiotensin system.
Part of genetic reprogramming? Regul Pept 133:10–12
15. Kim SH, Kim HS, Lee MM (2002) Re-expression of fetal troponin
isoforms in the postinfarction failing heart of the rat. Circ J 66:959–
964
16. Peters J, Wanka H, Peters B, Hoffmann S (2008) A renin transcript
lacking exon 1 encodes for a non-secretory intracellular renin that
increases aldosterone production in transgenic rats. J Cell Mol Med
12:1229–1237
17. Peters J, Kranzlin B, Schaeffer S, Zimmer J, Resch S, Bachmann S,
Gretz N, Hackenthal E (1996) Presence of renin within
intramitochondrial dense bodies of the rat adrenal cortex. Am J
Physiol 271:E439–450
18. Maas O, Donat U, Frenzel M, Rutz T, Kroemer HK, Felix SB,
Krieg T (2008) Vardenafil protects isolated rat hearts at reperfusion
dependent on GC and PKG. Br J Pharmacol 154:25–31
19. Peters J, Clausmeyer S (2002) Intracellular sorting of renin: cell
type specific differences and their consequences. J Mol Cell
Cardiol 34:1561–1568
20. Peters J (2008) Secretory and cytosolic (pro)renin in kidney, heart,
and adrenal gland. J Mol Med (Berl) 86:711–714
21. Peters J (2013) Cytosolic (pro)renin and the matter of intracellular
renin actions. Front Biosci (Schol Ed) 5:198–205
22. Takahashi S, Inoue H, Fukui K, Miyake Y (1994) Structure and
function of renin binding protein. Kidney Int 46:1525–1527
23. Takahashi S, Hori K, Ogasawara H, Hiwatashi K, Sugiyama T
(2006) Effects of nucleotides on the interaction of renin with
GlcNAc 2-epimerase (renin binding protein, RnBP). J Biochem
140:725–730
24. Passier RC, Smits JF, Verluyten MJ, Daemen MJ (1996) Expression
and localization of renin and angiotensinogen in rat heart after
myocardial infarction. Am J Physiol 271:H1040–1048
25. Abadir PM, Foster DB, Crow M, Cooke CA, Rucker JJ, Jain A,
Smith BJ, Burks TN, Cohn RD, Fedarko NS et al (2011)
Identification and characterization of a functional mitochondrial
angiotensin system. Proc Natl Acad Sci U S A 108:14849–14854
26. De Mello WC (1998) Intracellular angiotensin II regulates the in-
ward calcium current in cardiac myocytes. Hypertension 32:976–
982
27. Haller H, Lindschau C, Erdmann B, Quass P, Luft FC (1996)
Effects of intracellular angiotensin II in vascular smooth muscle
cells. Circ Res 79:765–772
28. Tadevosyan A, Maguy A, Villeneuve LR, Babin J, Bonnefoy A,
Allen BG, Nattel S (2010) Nuclear-delimited angiotensin receptor-
mediated signaling regulates cardiomyocyte gene expression. J Biol
Chem 285:22338–22349
29. Cook JL, Zhang Z, Re RN (2001) In vitro evidence for an intracel-
lular site of angiotensin action. Circ Res 89:1138–1146
30. Cook JL, Re R, Alam J, Hart M, Zhang Z (2004) Intracellular
angiotensin II fusion protein alters AT1 receptor fusion protein dis-
tribution and activates CREB. J Mol Cell Cardiol 36:75–90
31. Singh VP, Le B, Khode R, Baker KM, Kumar R (2008) Intracellular
angiotensin II production in diabetic rats is correlated with cardio-
myocyte apoptosis, oxidative stress, and cardiac fibrosis. Diabetes
57:3297–3306
32. Singh VP, Baker KM, Kumar R (2008) Activation of the intracel-
lular renin-angiotensin system in cardiac fibroblasts by high glu-
cose: role in extracellular matrix production. Am J Physiol Heart
Circ Physiol 294:H1675–
1684
33
. Inoue H, Fukui K, Takahashi S, Miyake Y (1990) Molecular clon-
ing and sequence analysis of a cDNA encoding a porcine kidney
renin-binding protein. J Biol Chem 265:6556–6561
34. Takahashi S, Kumagai M, Shindo S, Saito K, Kawamura Y (2000)
Renin inhibits N-acetyl-D-glucosamine 2-epimerase (renin-binding
protein). J Biochem 128:951–956
35. Schulz R, Cohen MV, Behrends M, Downey JM, Heusch G (2001)
Signal transduction of ischemic preconditioning. Cardiovasc Res
52:181–198
36. Opie LH (1990) Myocardial ischemia—metabolic pathways and
implications of increas ed glycolysis. Cardiovasc Drugs Ther
4(Suppl 4):777–790
37. Zhang W, Han Y, Meng G, Bai W, Xie L, Lu H, Shao Y, Wei L, Pan
S, Zhou S et al (2014) Direct renin inhibition with aliskiren protects
against myocardial ischemia/reperfusion injury by activating nitric
oxide synthase signaling in spontaneously hypertensive rats. J Am
Heart Assoc 3, e000606
38. Koid SS, Ziogas J, Campbell DJ (2014) Aliskiren reduces myocar-
dial ischemia-reperfusion injury by a bradykinin B2 receptor- and
angiotensin AT2 receptor-mediated mechanism. Hypertension 63:
768–773
J Mol Med (2016) 94:61–69 69
