Dipeptidyl peptidase 3 modulates the renin–Angiotensin system in mice

Abstract

Dipeptidyl peptidase 3 (DPP3) is a zinc-dependent hydrolase involved in degrading oligopeptides with 4–12 amino acid residues. It has been associated with several pathophysiological processes, including blood pressure regulation, pain signaling, and cancer cell defense against oxidative stress. However, the physiological substrates and the cellular pathways that are potentially targeted by DPP3 to mediate these effects remain unknown. Here, we show that global DPP3 deficiency in mice (DPP3-/-) affects the renin–angiotensin system (RAS). LC-MS–based profiling of circulating angiotensin peptides revealed elevated levels of angiotensin II, III, IV, and 1–5 in DPP3-/- mice, while blood pressure, renin activity, and aldosterone levels remained unchanged. Activity assays using the purified enzyme confirmed that angiotensin peptides are substrates for DPP3. Aberrant angiotensin signaling was associated with substantially higher water intake and increased renal reactive oxygen species (ROS) formation in the kidneys of DPP3-/- mice. The metabolic changes and altered angiotensin levels observed in male DPP3-/- mice were either absent or attenuated in female DPP3-/- mice, indicating sex-specific differences. Taken together, our observations suggest that DPP3 regulates the RAS pathway and water homeostasis by degrading circulating angiotensin peptides.

Full text

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Dipeptidyl peptidase 3 modulates the renin–angiotensin system in mice
Shalinee Jha1§, Ulrike Taschler2§, Oliver Domenig3, Marko Poglitsch3, Benjamin Bourgeois4, Marion
Pollheimer5, Lisa M. Pusch2, Grazia Malovan1, Saša Frank4, Tobias Madl4,6, Karl Gruber2, Robert
Zimmermann2,6 and Peter Macheroux1*
1Institute of Biochemistry, Graz University of Technology, NAWI Graz, Petersgasse 12, 8010 Graz,
Austria
2Institute of Molecular Biosciences, University of Graz, NAWI Graz, Heinrichstrasse 31 and
Humboldtstrasse 50, 8010 Graz, Austria
3Attoquant Diagnostics GmbH, Campus Vienna Biocenter 5, 1030 Vienna, Austria
4Gottfried Schatz Research Center, Division of Molecular Biology and Biochemistry,
Medical University of Graz, Neue Stiftingtalstrasse 6, 8010 Graz, Austria
5Diagnostic & Research Institute of Pathology, Medical University of Graz, Neue Stiftingtalstrasse 6,
8010 Graz, Austria
6BioTechMed Graz, 8010 Graz, Austria
*to whom correspondence should be addressed:
Prof. Dr. Peter Macheroux, Graz University of Technology, Institute of Biochemistry, Petersgasse
12/2, A-8010 Graz, Austria; Tel.: +43-316-873 6450; Fax: +43-316-873 6952;
Email: peter.macheroux@tugraz.at
§These authors have contributed equally to this work.
Running title: Physiological role of DPP3
Keywords: angiotensin II, dipeptidyl peptidase 3 (DPP3), metalloprotease, mouse, oxidative stress,
peptidase, renal physiology, renin angiotensin system, kidney function, sex-specific difference
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Abstract
Dipeptidyl peptidase 3 (DPP3) is a zinc-
dependent hydrolase involved in degrading
oligopeptides with 4–12 amino acid residues. It
has been associated with several
pathophysiological processes, including blood
pressure regulation, pain signaling, and cancer
cell defense against oxidative stress. However,
the physiological substrates and the cellular
pathways that are potentially targeted by DPP3
to mediate these effects remain unknown.
Here, we show that global DPP3 deficiency in
mice (DPP3-/-) affects the renin–angiotensin
system (RAS). LC-MS–based profiling of
circulating angiotensin peptides revealed
elevated levels of angiotensin II, III, IV, and
1–5 in DPP3-/- mice, while blood pressure,
renin activity, and aldosterone levels remained
unchanged. Activity assays using the purified
enzyme confirmed that angiotensin peptides
are substrates for DPP3. Aberrant angiotensin
signaling was associated with substantially
higher water intake and increased renal
reactive oxygen species (ROS) formation in
the kidneys of DPP3-/- mice. The metabolic
changes and altered angiotensin levels
observed in male DPP3-/- mice were either
absent or attenuated in female DPP3-/- mice,
indicating sex-specific differences. Taken
together, our observations suggest that DPP3
regulates the RAS pathway and water
homeostasis by degrading circulating
angiotensin peptides.
Introduction
Dipeptidyl peptidase 3 (DPP3, EC 3.4.14.4) is
a metalloprotease that specifically cleaves
dipeptides at the N-terminus of peptides with 4
to 12 amino acids. It is ubiquitously expressed
in both, prokaryotes and eukaryotes. DPP3 is
part of the central human proteome, i.e. it
belongs to a set of proteins ubiquitously and
abundantly expressed in all human cells (1).
The crystal structures of bacterial, yeast, and
human DPP3 have been reported (2–4). All
these structures are composed of an upper and
a lower domain separated by a wide cleft
which has been shown to be the substrate
binding site (4, 5). The conserved (HEXXGH)
and (EECRAE/D) motifs are part of the upper
domain, and are involved in the coordination
of a catalytically essential zinc ion in the
binding site (3).
A variety of small bioactive peptides, such as
met-enkephalin and angiotensin (I and II), are
substrates of DPP3 although the full range of
substrate peptides remains undefined (1, 5).
Consequently, DPP3 has been implicated in
pain modulation (6, 7) and blood pressure
regulation (8, 9). In addition, DPP3 exhibits a
moonlighting activity in the kelch-like ECH-
associated protein 1 (Keap1)-nuclear factor
erythroid 2-related factor 2 (Nrf2) signaling
pathway, which appears to play a role in stress
responses through transcriptional regulation of
the antioxidant response element (ARE) (10).
Despite the structural and biochemical
evidence indicating an intriguing involvement
of DPP3 in peptide processing and signaling as
well as in the response to oxygen stress, its
physiological role and potential involvement in
disease-related processes is currently
unknown.
Recently, it was reported that adult DPP3
knockout mice exhibited a growth defect,
increased bone loss and significantly elevated
bone marrow cellularity. Deletion of DPP3
also resulted in oxidative stress and alterations
of bone microenvironment favoring osteoclast
over osteoblast lineage. The osteoclasts
showed increased reactive oxygen species
(ROS) production, which made them prone to
apoptosis (11). A previous study further
established that DPP3 administration to
Angiotensin II (Ang II)-induced hypertensive
mice could significantly diminish systolic
blood pressure, cardiac hypertrophy, and
myocardial fibrosis in an extent at par with the
effect of the angiotensin receptor blocker
candesartan. It was also observed that DPP3
effectively reduced urine albumin excretion,
kidney damage, and the renal protein levels of
the pro inflammatory molecule monocyte
chemo-attractant protein-1 and the pro-
coagulant platelet activator inhibitor (9). Taken
together, the enzyme’s ability to degrade
various bioactive peptides, DPP3 may have
complex effects and influence basic
physiological processes, particularly those
affecting cellular metabolism and oxidative
stress.
Ang II, which is the most prominent substrate
reported for DPP3, is the principal effector of
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the renin-angiotensin system (RAS). RAS
plays a pivotal role in the pathophysiological
modulation of renal and cardiovascular
processes (12, 13). Ang II regulates
vasoconstriction and is responsible for
maintaining homeostasis in the heart and
kidney (14). In addition, Ang II is a potent
stimulator of NAD(P)H oxidase, which
augments formation of ROS in various tissues.
Ang II-mediated ROS production has been
associated with cell growth, apoptosis, cell
migration, and expression of inflammatory and
extracellular matrix genes (15). An imbalance
between the production of ROS and the
antioxidant defense to eliminate these toxic
intermediates leads to oxidative stress. There is
a plethora of evidence demonstrating the
importance of oxidative stress in Ang II-
induced metabolic disorders like hypertension,
diabetes mellitus, and chronic kidney disease
(16–19). Although blockade of the RAS is the
most commonly adopted strategy to slow
progression of cardiovascular and associated
renal diseases, a better understanding of the
novel aspects of the RAS is of paramount
importance for the development of innovative
therapies that target pathologies inflicted by
anomalies of this pathway.
In the present study, we attempted to elucidate
the physiological role of DPP3 in the RAS
system through characterization of DPP3
knockout mice (DPP3-/-). Our observations
suggest that DPP3 regulates the RAS pathway
and water homeostasis by degrading
circulating angiotensin peptides. Interestingly,
the lack of DPP3 affected only the phenotype
of male mice, with the effects either being
absent or much weaker in female mice. This
sex-specific difference points at a link between
the endocrine system and the physiological
role of DPP3. The characterization of DPP3 in
this study establishes that it has strong
metabolic implications through the modulation
of the RAS pathway, a property that could be
useful in the management of several
cardiovascular and related metabolic
pathologies.
Results
Generation and gross characterization of
DPP3 knockout mice
To investigate the function of DPP3 in vivo,
we generated mice globally lacking DPP3.
Mice were generated using ES cells from
EUCOMM containing a β-galactosidase
cassette (lacZ) and a promotor-driven selection
cassette (neo) between exon 5-6 of the DPP3
gene. The selection cassettes as well as exon 6
of DPP3 were flanked by loxP sites. Mice
bearing the targeted allele were crossed with
transgenic mice expressing Cre-recombinase
under the control of a cytomegalovirus (CMV)
promotor resulting in deletion of neo and exon
6 (Figure 1A). DPP3 was detected by Western
blotting in most investigated tissues of wild-
type controls (DPP3+/+), but not in tissue
lysates of DPP3-/- mice (Figure 1B). A
comparison of DPP3 activity in various tissues
of DPP3+/+ and DPP3-/- mice using the artificial
substrate Arg-Arg-2-naphthylamide clearly
demonstrated blunted activity in DPP3-/- tissue
lysates (Figure 1C).
In accordance with a previously published
study (11), gross characterization revealed that
male DPP3-/- mice exhibit lower body weight
(Figure 2A) and less fat mass than litter-mates
(Figure 2B). Food and water consumption as
well as energy expenditure and spontaneous
locomotor activity were monitored in
metabolic cages over a period of 150 h.
Cumulative analysis revealed a slight increase
in food intake in male DPP3-/- mice (Figure
2C) due to increased food consumption during
the dark phase (Figure 2F). Cumulative water
intake was significantly elevated (Figure 2D)
due to a 27% and 46% increase in drinking
during the light and dark phase respectively
(Figure 2G). Cumulative (Figure 2E) and daily
(Figure 2H) locomotor activity remained
unchanged. As shown in Figure 3A-F, oxygen
consumption and carbon-dioxide production
were comparable between genotypes, while the
respiratory exchange ratio (RER) was slightly
increased in the knockout mice during the dark
phase. We also calculated energy expenditure
(EE) based on the amount of oxygen consumed
and carbon dioxide produced, using the
formula: EE (kJ per day) = 15.818*VO2 +
5.176*VCO2/1000*24 (20) and was found to
remain unaltered between the genotypes
(DPP3+/+, 42.6 ± 2.3 kJ per day vs. DPP3-/-,
43.7 ± 2.8 kJ per day; total energy
expenditure).
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Mice consume substantially lower amounts of
water after food deprivation. To investigate if
drinking behavior of male DPP3-/- mice was
also altered upon food restriction, we
monitored water consumption during a 13h
fasting period. Under these conditions, water
intake of DPP3-/- mice was ~ 4-fold higher as
compared to DPP3+/+ mice suggesting that
DPP3-deficiency is associated with polydipsia
in male animals (Figure 4).
DPP3 acts as a modulator of the RAS
Our observations indicate that DPP3 is
involved in the regulation of water homeostasis
and recently published data suggested a role of
DPP3 in angiotensin degradation (9, 21).
Accordingly, we generated a serum “RAS-
Fingerprint” consisting of 10 different
angiotensin peptides using ultra-pressure-
liquid chromatography-tandem mass
spectrometry (LC-MS/MS). Serum analysis
revealed that male DPP3-/- mice had higher
concentrations of most angiotensin
metabolites. As shown in Figure 5A, the
concentration of Ang II was twice as high as in
wild-type mice. Interestingly, downstream
peptides, in particular Ang (1-5), Ang III, and
Ang IV accumulated 5.4-, 4.2-, and 5.3-fold,
respectively, suggesting that DPP3 has
multiple natural substrates among angiotensin
metabolites and that DPP3 deletion leads to
perturbation of the entire RAS in males. Serum
aldosterone levels (Figure 5B) and serum renin
activity (Figure 5C) remained unchanged.
Deletion of DPP3 enhances oxidative stress in
male mice
Ang II is known to promote ROS production in
kidney, and therefore we determined the
generation of reactive oxygen intermediates
using 2′,7′-dichlorodihydrofluorescein
diacetate (H2DCFDA). DPP3 deletion led to a
significantly enhanced fluorescence signal
from the ROS reporter dye H2DCFDA in the
kidney lysates prepared from male mice
(Figure 6A). Accumulation of ROS in the
DPP3-/- kidneys also triggered a trend towards
higher malondialdehyde (MDA) levels, a
marker of lipid peroxidation (Figure 6B).
Catalase activity was significantly increased in
kidney homogenates of male DPP3-/- mice,
indicating increased generation of H2O2
(Figure 6C). We also observed that despite this
increase in renal ROS, the kidney morphology
was not significantly altered between the
genotypes (Figure 7). We observed normal
glomeruli with mesanglial normocellularity
and thin basement of the capillary loops.
Moreover, renal tubules and interstitium
appeared normal, lacking signs of
inflammation or fibrosis.
DPP3 deficiency does not affect blood
pressure
The extensive changes in the RAS
accompanied by increased oxidative stress
levels in kidneys of DPP3-/- mice prompted us
to assess potential effects on blood pressure,
which is one of the major physiological output
parameters of the RAS. Toward that end, we
measured blood pressure in 18-22 weeks old
DPP3+/+ and DPP3-/- mice by the tail-cuff
method. There was no significant difference
between DPP3+/+ and DPP3-/- mice, both for the
systolic (DPP3+/+, 145.6 ± 12.1 mm Hg vs.
DPP3-/-, 134.2 ± 22.7 mm Hg) and diastolic
(DPP3+/+, 112.6 ± 12.4 mm Hg vs. DPP3-/-,
99.0 ± 22.5 mm Hg) blood pressure (Figure 8).
Purified DPP3 catalyzes the turnover of
multiple angiotensin peptides
The pleiotropic effect of DPP3 deletion on
angiotensin peptides suggests that the enzyme
does not only accept Ang II as a substrate but
may also degrade other peptides in this
pathway. Thus, we investigated the activity of
DPP3 against different angiotensin peptides.
Kinetic parameters of angiotensin peptides
were obtained by single injection calorimetry
measurements that are based on the exothermal
hydrolysis of peptide bonds by purified
recombinant human DPP3 which shares 93%
sequence identity with the mouse DPP3.
Figure 9 displays the curves for the rate of the
reaction, calculated from integrated raw data
using the Enzyme Kinetics - Single Injection
fitting model. The graphs show the rate of
angiotensin conversion as a function of its
concentration. The insets depict the raw data of
heat change due to the conversion of substrate
peptides. The dependence of the reaction rate
on angiotensin concentration followed typical
Michaelis–Menten kinetics, and the parameters
- enthalpy heat change (ΔH), turnover number
(kcat) and Michaelis-Menten constant (KM) -
were found by the fitting model in the
MicroCal PEAQ-ITC Analysis Software
(Table 1). Among the six angiotensin peptides
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tested, Ang I, Ang II, Ang (1-5), and Ang (1-7)
showed an exothermic reaction, indicating
their turnover by purified hDPP3 (Figure 9A-
C). However, in the case of Ang I, data
extraction was not possible due to the weak
exothermic signal observed, indicating that
Ang I is a poor substrate of DPP3. In contrast,
Ang III and Ang IV displayed both
endothermic and exothermic behavior, a
characteristic observed for slow substrates like
the peptide tynorphin and its derivatives
(Figure 9D-F) (5). These experiments clearly
confirm that DPP3 does not only act on Ang II
but also efficiently hydrolyzes Ang (1-5) and
Ang (1-7).
DPP3-deficiency has only minor impact on
the phenotype of female mice
In contrast to male mice, female DPP3-/- mice
did not show any significant difference in body
weight (Figure 10A) or body mass composition
(Figure 10B). Moreover, food intake (Figure
10C) and water consumption (Figure 10D)
were not different between the genotypes.
Most importantly, serum levels of Ang II
remained unaltered in female DPP3-/- mice as
compared to wild-type, while the levels of Ang
III, Ang IV, and Ang (1-5) were 2.4-, 2-, and
7.5-fold higher, respectively (Figure 10E).
Contrary to male DPP3-/- mice, the amount of
ROS generated was significantly lower in the
kidney lysates of DPP3-/- female mice,
indicating sex-specific differences (Figure
10F).
Discussion
In this study, we demonstrate that deletion of
DPP3 in mice caused widespread
physiological and biochemical changes. In
addition to the previously observed alterations
in body weight and bone morphology (11), the
lack of DPP3 clearly affects the RAS pathway,
which is associated with increased water
consumption. This is characteristic for mouse
models lacking genes associated with the RAS
like ACE or angiotensinogen, as they have
diminished ability to concentrate urine due to
impaired renal development (22, 23).
However, we found no obvious changes in
kidney morphology, indicating that, at least in
the young mice investigated in this study, renal
function is normal.
Male DPP3-/- mice displayed increased levels
of equilibrium angiotensin peptides including
Ang II as well as a specific qualitative shift in
angiotensin metabolite profiles, characterized
by selective and profound increases of Ang 1-
5, Ang III, and Ang IV. These profound
differences in angiotensin profiles indicate that
a general up-regulation of the RAS at the level
of renin can be excluded as an underlying
cause for the observed Ang II increase. Ang II
is an integral part of the RAS and mediates
various physiological responses. High Ang II
levels increases reactive oxygen species and
oxidative stress, and depresses mitochondrial
energy metabolism (15, 16, 18, 19, 24). There
are several reports suggesting that high
circulating Ang II concentration is a stimulus
for thirst in a variety of species, which is
consistent with our findings in male DPP3-/-
mice (14, 25, 26). Since Ang II (16, 18, 24, 27,
28) mediates ROS production, which can
contribute to oxidative stress, we measured
putative stress markers in the kidneys of mice.
Congruously, the level of ROS generation and
catalase activity were elevated in the
knockouts. These results confirm our
hypothesis that increased Ang II creates
oxidative stress in the male DPP3-/- mice.
In addition to regulating stress response via
modulation of Ang II, it is likely that DPP3 is
part of endogenous defense system against
oxidative stress. DPP3 is known to promote
nuclear migration of transcription factor Nrf2
by displacing Keap1 (10, 29). The Keap1-Nrf2
pathway is a key regulator of cellular stress
response caused by ROS. Under basal
conditions, Nrf2 is bound to Keap1. Upon
activation by oxidative stressors, Nrf2
translocates to the nucleus of the cell where it
activates the ARE, thereby regulating the
transcription of genes responsible for
antioxidant and anti-inflammatory defense
(30–33). It has been reported that
overexpression of DPP3 could potently
activate the ARE in neuroblastoma cells (IMR-
32 cells) leading to increased expression levels
of NAD(P)H: quinone oxidoreductase 1
(NQO1), a phase II detoxifying enzyme
regulated by ARE. It was also found that DPP3
overexpression efficiently attenuated the toxic
effects of H2O2 and rotenone, demonstrating
the cytoprotective effect of DPP3 against
oxidative stress (34). Furthermore, it was
demonstrated that the DPP3 plasma levels are
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associated with the survival rate of patients
suffering from sepsis (35), cardiogenic shock
(36), heart failure (37), and acute kidney injury
(38). Our results are also consistent with a
recent study, which reported that lack of DPP3
leads to impaired bone homeostasis and
enhanced oxidative stress in the bone tissue
(11), indicating an ubiquitous antioxidant
activity of DPP3.
Increase in the Ang II levels leads to
vasoconstriction and thereby, severe
hypertension (17, 19, 27). However, both male
and female DPP3-/- mice showed no change in
the blood pressure using tail-cuff method under
normal dietary conditions. As measuring blood
pressure using the tail-cuff method has certain
limitations (e.g. stress response of mice),
further studies will be necessary to investigate
a potential fine tuning of blood pressure
regulation in DPP3-/- mice. The unchanged
blood pressure also points towards the
involvement of the cardioprotective arm of
RAS as a compensatory mechanism (8). In
vitro studies using recombinant human DPP3
additionally identified Ang (1-7) and Ang (1-5)
as substrates of the enzyme. Although the level
of Ang (1-7) was not significantly increased in
the knockout, it is interesting to note that it is
the main metabolite of the alternate RAS and
plays an important role as physiological
antagonist of Ang II, having vasodilatory and
antihypertensive properties (8). Ang (1-7) is
produced by carboxy- or endopeptidases like
neutral endopeptidase (NEP), prolyl
endopeptidase (PEP), angiotensin converting
enzyme 2 (ACE 2), or prolyl carboxypeptidase
(PCP) from Ang I and Ang II and is
metabolized to Ang (2-7) and Ang (3-7) by
aminopeptidases and to Ang (1-5) by ACE. In
contrast to Ang (1-7), Ang (1-5) showed a very
pronounced change between wild-type and
knockout mice exhibiting a more than five-fold
higher concentration in the latter. The specific
increase of Ang (1-5) in DPP3-/- mice points to
a clear preference of DPP3 towards Ang (1-5)
over Ang (1-7). It was shown that Ang (1-5)
stimulated the secretion of atrial natriuretic
peptide (ANP) via Mas receptor, a mode of
action similar to that of Ang (1-7) (39). The
ANP system is a hormonal system that
participates in the regulation of body fluid and
electrolyte balance. It acts antagonistically to
the RAS and causes anti-hypertension and anti-
oxidative stress (40, 41). It is conceivable that
Ang (1-5) also plays a role as a vasodilatory
and antihypertensive peptide.
Female DPP3-/- mice displayed a less
pronounced phenotype in comparison to male
mice, indicating sex-specific differences.
Accumulating evidence suggests that there are
sex differences in the tissue expression and
activity of several RAS components, with the
female sex hormone, estrogen, downregulating
Ang II and upregulating Ang (1-7) pathways
(42). It was shown that in C57BL/6J mice, the
Ang II‐induced increase in blood pressure is
greater in males than in females (42). Also,
healthy men had greater pressor and renal
vasoconstrictor responses to acute Ang II
infusion compared to women (43). Similarly,
chronic Ang II infusion induced hypertension
in male but not female mice (44, 45), possibly
due to a shift in the balance from Ang II
towards Ang (1-7) pathways because of
estrogen-mediated protection (46). In high-fat
fed C57BL/6J mice, females maintained
circulating Ang (1-7) levels and were protected
from hypertension and metabolic
complications induced by Ang II (47). These
studies are in line with our observations from
the serum RAS-fingerprint which show that the
female DPP3+/+ and DPP3-/- mice had 6-, and
9-fold higher Ang (1-7) respectively, than the
male mice. For Ang (1-5), the levels were 2-,
and 3-fold higher than in male DPP3+/+ and
DPP3-/- mice, respectively. This is indicative of
an upregulation of the protective RAS arm in
female mice.
On a similar note, numerous studies have
discussed the association of DPP3 to estrogen.
It was reported that 17ß-estradiol (E2), the
predominant estrogen present in the serum,
influences the expression level of DPP3 in
vivo. The hepatic DPP3 levels were found to
be markedly reduced following ovariectomy in
16-weeks old CBA/H mice and E2
administration abolished this effect and
increased DPP3 protein expression (48). It was
also found that DPP3 is instrumental in
estrogen-mediated protection against oxidative
stress in female CBA/H mice (49). DPP3
accumulated in the nucleus in liver tissue
lysates of healthy female mice exposed to
hyperoxia, at a level comparable to the nuclear
accumulation of Nrf2. Further, the combined
induction of hyperoxia and E2 administration
had a synergistic effect on the nuclear
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accumulation of DPP3. In ovariectomized
females exposed to hyperoxia,
supplementation of E2 enhanced DPP3 levels,
accompanied by an upregulation of other
cytoprotective proteins like sirtuin-1 and heme
oxygenase-1, resulting in attenuated oxidative
stress (49). Lack of DPP3 was found to
augment bone loss caused by estrogen
deprivation in the ovariectomized mouse
model of human postmenopausal osteoporosis
(11). This interplay between DPP3, RAS, and
estrogen demands additional studies, however,
it is very likely that the female DPP3-/- mice are
protected from oxidative stress due to the
presence of estrogen. The male DPP3-/- mice on
the other hand show an aggravated response to
Ang II-induced oxidative stress due to this lack
of antioxidant and cytoprotective function.
In summary, the generation and
characterization of a mouse model with global
deletion of DPP3 has revealed a significant
perturbation of the levels of peptides in the
RAS. The changes in peptide levels were
found to be associated with polydipsia and
augmented levels of ROS. Our findings
identify DPP3 as a pleiotropic and sex-specific
modulator of RAS and emphasize its role in
oxidative stress response.
Experimental procedures
Ethics Statement
All animal experiments were approved by the
Austrian Federal Ministry for Science,
Research, and Economy (protocol number
BMWF-66.007/7-ll/3b/), the ethics committee
of the University of Graz, and conducted in
compliance with the council of Europe
Convention (ETS 123).
Animals and generation of DPP3 knockout
mice
All studies were conducted in age-matched
DPP3-/- and wild-type control male and female
mice on C56BL/6J background. Unless stated
otherwise, the results describe the effects of
DPP3 deletion on male mice. Mice were bred
and maintained at regular housing
temperatures (23 ± 1 °C) and 14-h light/10-h
dark cycle. Animals had ad libitum access to
water and chow diet (4.5% fat, 34% starch,
5.0% sugar, and 22.0% protein; Ssniff
Spezialdiaeten). Breeding and genotyping were
done according to standard procedures. For
generation of DPP3-/- mice, targeted mutant ES
cells were obtained from EUCOMM and
injected into blastocysts of C57BL/6J mice.
Chimeric animals with a high degree of coat
color chimerism were bred with C57BL/6J
mice. The construct containing a β-
galactosidase cassette (lacZ) and a promotor-
driven selection cassette (neo) was inserted
into the DPP3 gene. Additionally, the
construct contained two flippase recognition
target (FRT) sites for flippase recombination
enzyme (FLP)-mediated recombination
flanking lacZ and neo. The selection cassette
and exon 6 (essential for DPP3 function),
flanked by loxP sites, were removed by
breeding with transgenic C57BL/6J mice
expressing cre-recombinase under the control
of a cytomegalovirus (CMV) promotor (CMV-
Cre). Cre-lox recombination resulted in
deletion of neo and exon 6 leaving the lacZ
reporter gene intact. Mice totally lacking
DPP3 were bred by crossing mice
heterozygous for the mutant DPP3 allele
lacking neo and exon 6.
Serum and tissue lysate preparation
Animals were anesthetized with isoflurane and
blood was collected by the retro-orbital
puncture. Immediately following collection,
blood was allowed to clot and serum was
isolated by centrifugation. For tissue
collection, mice were sacrificed by cervical
dislocation and tissues were surgically
removed and washed with cold PBS.
Homogenization was performed on ice in
solution A (0.25M sucrose, 1 mM EDTA, 20
μM dithiothreitol, 0.1% Triton X-100, 20
μg/mL leupeptin, 2 μg/mL antipain, 1 μg/mL
pepstatin, pH 7.0) using an Ultra Turrax (IKA,
Staufen, Germany). 20,000 g infranatant was
used for further experiments. Protein
concentrations in the tissue lysates were
estimated using the Protein Assay Dye Reagent
(Bio-Rad, Munich, Germany) using bovine
serum albumin as standard. Serum and tissue
samples were stored at -80 °C until further
analysis.
SDS PAGE and Western blotting
Tissue lysates were diluted in Laemmli’s
sample buffer, and 20 µg of total protein/lane
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was subjected to sodium dodecyl sulfate-
polyacrylamide gel electrophoresis (SDS-
PAGE) using 10% SDS-polyacrylamide gels.
The resolved proteins were transferred onto
polyvinylidene difluoride (PVDF) membranes
(VWR, Pennsylvania, USA) using a Trans-
Blot SD transfer cell (Bio-Rad, CA, USA).
Following transfer, membranes were washed
with Tris-buffered saline containing 0.01%
Tween-20 (TBST) and then blocked in 5%
non-fat milk for 1 h at room temperature. The
membranes were then incubated overnight
with anti-DPP3 rabbit polyclonal antibody
(1:1,500, Proteintech Europe, Manchester, UK)
in TBST containing 5% non-fat milk at 4 ºC.
After washing three times for 10 min in TBST,
membranes were incubated with peroxidase-
labeled secondary antibody (1:5,000; Cell
Signaling Technology®, Danvers, MA, USA)
for 1 h at room temperature. The immunoblots
were developed using enhanced
chemiluminescent western blotting substrate
solution (Pierce-Thermo Fisher Scientific,
Waltham, MA, USA).
DPP3 activity assay in mouse tissue lysates
DPP3 activities in tissue lysates were
determined by fluorometrically (excitation,
332 nm; emission, 420 nm) measuring the
liberation of 2-naphthylamine at 37 °C in a
mixture containing 25 µl of 200 µM Arg-Arg-
2-naphthylamide as substrate in TBS buffer
(50 mM Tris, 100 mM NaCl, pH 8.2) and
tissue lysate equivalent to 20 µg of total
protein in a reaction mixture of 235 µl (White,
Tissue Culture treated Krystal 2000 96-well
plate from Porvair sciences, Norfolk, UK). The
activity assay was performed by continuous
measurement of fluorescence of 2-
naphthylamide for 30 min (Fluorescent plate
reader from Molecular Devices, Sunnyvale
CA, USA). The reaction was started by the
addition of the substrate. The samples were
measured in triplicates.
Body composition and metabolic
phenotyping
Lean and fat mass of mice were analyzed by
NMR (the minispec, NMR Analyzer, Bruker,
Ettlingen, Germany). To measure spontaneous
physical activity, O2 consumption, CO2
production, food and water intake, mice were
housed in metabolic cages allowing continuous
measurement of these parameters (LabMaster,
TSE Systems GmbH, Bad Homburg,
Germany). For measurements of energy
balance, animals were familiarized with these
cages for at least 72 hours before data
collection.
Analysis of angiotensin peptides in serum
Serum equilibration was performed at 37 °C
followed by stabilization of equilibrium
angiotensin levels and subsequent
quantification by LC-MS/MS analysis (50).
Briefly, stable isotope-labeled internal
standards for each Ang metabolite [Ang I (1-
10), Ang II (1-8), Ang (1–7), Ang (1–5), Ang
III (2–8), Ang IV (3–8), Ang (1-9), Ang (3-7),
Ang (2-7), Ang (2-10)] were added to
stabilized serum samples at a concentration of
200 pg/mL. Following C18-based solid-phase-
extraction, samples were subjected to LC-
MS/MS analysis using a reversed-phase
analytical column (Acquity UPLC® C18,
Waters, USA) operating in line with a XEVO
TQ-S triple quadrupole mass spectrometer
(Waters Xevo TQ/S, Milford, Massachusetts,
USA) in multiple reaction monitoring modes.
The internal standard was used to correct for
analyte recovery across the sample preparation
procedure in each sample. Analyte
concentrations were calculated from integrated
chromatograms considering the corresponding
response factors determined in appropriate
calibration curves in serum matrix.
Determination of ROS generation
The intracellular ROS level was detected by
using 2′,7′-dichlorodihydrofluorescein
diacetate (H2DCFDA) (Sigma Aldrich). When
oxidized by various active oxygen species, it is
irreversibly converted to the fluorescent form,
DCF (51, 52). ROS in kidney tissue was
estimated by diluting tissue lysate equivalent to
100 µg of total protein in ice-cold 40 mM Tris-
HCl buffer (pH 7.4). The samples were divided
into two equal fractions. In one fraction 40 µl
of 10 µM H2DCFDA in methanol was added
for ROS estimation. Another fraction with 40
µl of methanol was used as a control for tissue
auto-fluorescence. All samples were incubated
at 37 °C for 15 min and fluorescence was
determined at 485 nm excitation and 525 nm
emission using a fluorescence plate reader
(Molecular Devices, Sunnyvale, CA, USA). To
quantitate ROS levels, relative
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9
dichlorofluorescein fluorescence was used as a
standard.
Detection of lipid peroxidation activity
The extent of lipid peroxidation in kidney was
assessed using thiobarbituric acid reactive
substances (TBARS) as an index. For the
assay, kidney tissue equivalent to 1 mg of total
protein was incubated with 20% TCA and
0.67% TBA. The reaction mixture was heated
at 100 °C for 30 min and then cooled in an ice-
bath for 10 min. The samples were then
centrifuged at 3,000 rpm for 15 min. The
supernatant was collected to measure
absorbance at 532 nm. The formation of
TBARS was expressed using malondialdehyde
(MDA) equivalent as a standard.
Catalase activity
Catalase activity was measured as described in
(53). Briefly, kidney tissue equivalent to 1 mg
of total protein in 0.01 M PBS was incubated
with 0.2 M H2O2. The reaction was stopped by
adding 5% dichromate solution at 30 sec
intervals. The samples were heated at 60 °C for
10 min where the blue precipitate formed was
decomposed to a green solution. Consumption
of H2O2 was determined by recording
absorbance at 570 nm. A standard curve
containing 0 to 100 μM of H2O2 was prepared
to determine the amount of H2O2 present in
each sample.
Histological analysis of kidney
For the analysis of morphological differences,
kidneys were fixed in 4% neutral buffered
formaldehyde solution for 24 h, embedded in
paraffin, and further processed for periodic
acid–Schiff (PAS) staining (2 µm thick
sections).
Blood pressure measurements
The CODA 8-channel non-invasive tail-cuff
technique (Kent Scientific, EMKA
Technologies, Paris, France) was used for
blood pressure measurements in young adult
male mice from 12-16 weeks of age. This
system uses Volume Pressure recording (VPR)
to detect blood pressure based on volume
changes in the tail (54). VPR cuffs were
checked routinely before the start of the
experiments. Heating pads were preheated to
35 °C before and during the measurements.
Each measurement consisted of 5 acclimation
cycles followed by 15 cycles during which the
systolic and diastolic pressure were measured.
Acclimation cycles were not used in blood
pressure analysis.
Isothermal titration calorimetry (ITC)
Calorimetric activity of six different
angiotensin peptides as purported substrates of
DPP3 were further assayed using a MicroCal
PEAQ-ITC (Malvern Panalytical Ltd,
Worcestershire, United Kingdom). The
peptides used for this assay, Ang I (1-10), Ang
II (1-8), Ang III (2-8), Ang IV (3-8), Ang (1-
5), and Ang (1-7), were commercially
purchased (Bachem, Bubendorf, Switzerland).
For this, the single injection method was used
where 5µl of 2 mM angiotensin peptide was
titrated into 200 µl of 20 µM purified hDPP3.
The purification of recombinant hDPP3 was
done as described in (35). Both the ligand and
the purified protein were prepared in 50 mM
Tris-HCl at pH 8.0 containing 100 mM NaCl.
The experiments consisted of a single injection
of the ligand continuously over a period of 10
seconds, including an initial delay of 180
seconds before the injection and a spacing of
300 seconds after the injection. The
experiments were performed at 25 °C with a
stirring speed of 500 rpm. The thermodynamic
characterization of this binding interaction was
obtained using MicroCal PEAQ-ITC Analysis
Software.
Statistical analysis
All data are expressed as mean ± standard
deviation (SD). Results were assessed using
two-tailed unpaired Student’s t-test (GraphPad
Prism 5, San Diego, USA). A P-value less than
0.05 was considered significant.
Data availability
All data presented and discussed are contained
within the article.
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10
Acknowledgements
This work was supported by a grant from the Austrian Science Foundation (FWF) through grants
W901 (Doctoral Program “Molecular Enzymology”) to KG, PM, and RZ. The authors are also
grateful for the support by the interuniversity program in natural sciences (NAWI Graz). We thank
Emilia Strandback, Karin Koch, and Chaitanya R. Tabib for experimental advice and discussions. We
also thank Margarete Lechleitner for her help in blood pressure measurements.
Conflict of interest
The authors declare that they have no conflicts of interest with the contents of this article.
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Footnotes
The abbreviations used are:
DPP3, dipeptidyl peptidase 3; DPP3+/+, wild-type; DPP3-/-, DPP3-knockout; RAS, renin-angiotensin
system; Ang, angiotensin; TCA, tricarboxylic acid; ROS, reactive oxygen species; LC-MS/MS, liquid
chromatography-tandem mass spectrometry; ITC, isothermal titration calorimetry; PCA, principal
component analysis; OPLS-DA, orthogonal projections to latent structures discriminant analysis;
Sirt3, sirtuin-3; PARP, poly (ADP-ribose) polymerase; ACE, angiotensin converting enzyme; NEP,
neutral endopeptidase; PEP, prolyl endopeptidase; PCP, prolyl carboxypeptidase
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Tables
Table 1: Kinetic parameters obtained from single injection ITC experiments with hDPP3 and
angiotensin peptides. The Michaelis-Menten constant KM and maximum turnover number kcat are
obtained from the fit to a Michaelis–Menten curve for the reaction rate as a function of substrate
concentration. The enthalpy of interaction (ΔH) (also called heat of reaction) is a direct measurement
of the rate at which heat is exchanged with the surroundings. Data represents mean ± SD. All data
points are 3 or more technical replicates from 2 biological replicates.
Kinetic, calorimetric parameters of hDPP3 and angiotensin interaction
ΔH (kcal/mol)
kcat (s-1)
KM (µM)
Ang II
-1.97 ± -0.06
0.25 ± 0.007
8.40 ± 0.40
Ang (1-7)
-1.52 ± -0.10
0.14 ± 0.003
1.95 ± 0.20
Ang (1-5)
-1.84 ± -0.04
0.35 ± 0.002
12.50 ± 2.20
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Figures
Figure 1. Generation and validation of DPP3 knockout mice. (A), Strategy for the generation of
DPP3-knockout mice. B-C, Demonstration of the lack of DPP3 in male DPP3-/- mice by Western blot
(B) and activity assays using Arg-Arg-2-naphthylamide as an artificial substrate (C) (BAT = brown
adipose tissue; SI = small intestine; SM = skeletal muscle; WAT = white adipose tissue) (12-16 weeks
of age; n= 3/group). n.d.= non detectable, **p<0.01, ***p<0.001 versus wild-type mice based on
unpaired two-sided Student’s t-test. Data is representative of 3 technical replicates from 3 biological
replicates and presented as mean ± SD.
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Figure 2. DPP3-/- mice display lower body weight and altered food and water intake. (A) Body
weight and (B) body composition of male mice fed a regular chow diet. C-H, Cumulative (top panel)
and total (bottom panel) food intake (C and F, respectively), water consumption (D and G,
respectively) and locomotive motion (E and H, respectively), were measured in metabolic cages over
the light and dark phases in male DPP3-/- and DPP3+/+ mice (12-16 weeks of age; n=6/group) fed a
regular chow diet over a period of six consecutive days. *p<0.05, **p<0.01 versus wild-type mice
based on unpaired two-sided Student’s t-test. Data are representative for two independent cohorts and
presented as mean ± SD.
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Figure 3. DPP3 knockout mice display unaltered energy expenditure. Cumulative and daily
oxygen consumption (A and D respectively) and carbon-dioxide production (B and E respectively),
RER (C) and EE (F) were measured in metabolic cages over the light and dark phases in male DPP3-/-
and DPP3+/+ mice (12-16 weeks of age; n=6/group) fed a regular chow diet over a period of six
consecutive days. *p<0.05, **p<0.01 versus wild-type mice based on unpaired two-sided Student’s t-
test. Data are representative for two independent cohorts and presented as mean ± SD.
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Figure 4. DPP3-/- mice exhibit significantly elevated water consumption during fasting. (A)
Cumulative and (B) daily water intake during a 13h fasting period was measured in metabolic cages in
male DPP3-/- and DPP3+/+ mice (12-16 weeks of age; n=6/group). *p<0.05, **p<0.01 versus wild-type
mice based on unpaired two-sided Student’s t-test. Data represents mean ± SD.
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Figure 5. DPP3-/- mice exhibit increased circulating angiotensin metabolites. (A) Concentration of
RAS peptides, (B) aldosterone, and (C) renin activity in serum measured by liquid chromatography-
mass spectrometry in male DPP3-/- and DPP3+/+ mice (12-16 weeks of age; n = 8/group). *p<0.05,
**p<0.01, ***p<0.001 versus wild-type mice based on unpaired two-sided Student’s t-test. Data
represents mean + SD.
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Figure 6. DPP3 knockout renders mice susceptible to oxidative stress. Quantification of (A) ROS
production, (B) Lipid peroxidation and (C) catalase activity in kidney homogenates of male DPP3-/-
and DPP3+/+ mice (12-16 weeks of age; n = 5/group). *p<0.05, **p<0.01 versus wild-type mice based
on unpaired two-sided Student’s t-test. Experiments were performed in technical triplicates and
represent mean + SD.
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Figure 7. DPP3 deletion does not affect morphology of mouse kidney. PAS (periodic acid Schiff) -
stained slides of renal histology showing unremarkable glomeruli and tubuli in (A) DPP3-/- and (B)
DPP3+/+ mice. Left 10x, right 20x.
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Figure 8. DPP3 deficiency does not lead to changes in blood pressure. Systolic and diastolic blood
pressure were measured by tail-cuff method in male DPP3+/+ and DPP3-/- mice (18-22 weeks of age; n
= 8/group). Data are presented as mean + SD.
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Figure 9. RAS peptides can be “good” or “bad” substrates of DPP3. RAS peptides turned over by
DPP3, thus acting as “good” substrates (A-C). Raw data showing the heat change of the reaction as a
function of time (top panel) and fitted curve for the rate of reaction (bottom panel) of (A) Ang II, (B)
Ang 1-7, and (C) Ang 1-5. Some RAS peptides demonstrated both endothermic and exothermic
behavior, thus acting as “slow” substrates of DPP3 (D-F). The biphasic peaks were most likely due to
binding to DPP3 and a subsequent slow turnover event. Raw data showing the heat change of the
reaction in the case of slow substrates, (D) tynorphin, (E) Ang III, and (F) Ang IV. Curve fitting was
not possible in the case of slow substrates. The reaction was started by injecting 5µl, 2 mM
angiotensin peptides to the calorimetric cell containing 20 µM hDPP3. Data represents 3 or more
technical replicates from 2 biological replicates.
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Figure 10. DPP3 exerts a sex-specific effect on the knockout mice. (A) Body weight and (B) body
composition of female mice fed a regular chow diet. (C) Daily food intake and (D) water consumption
was measured in metabolic cages over the light and dark phases in DPP3-/- and DPP3+/+ female mice
(12-16 weeks of age; n=6/group) fed a regular chow diet over a period of six consecutive days. (E)
Concentration of RAS peptides in serum measured by liquid chromatography-mass spectrometry in
DPP3-/- and DPP3+/+ female mice (12-16 weeks of age; n=8/group) and (F) Quantification of ROS
production in DPP3-/- and DPP3+/+ female mice (12-16 weeks of age; n = 5/group). *p<0.05, **p<0.01
versus wild-type mice based on unpaired two-sided Student’s t-test. Data represents mean ± SD.
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Gruber, Robert Zimmermann and Peter Macheroux
Marion Pollheimer, Lisa M. Pusch, Grazia Malovan, Sasa Frank, Tobias Madl, Karl
Shalinee Jha, Ulrike Taschler, Oliver Domenig, Marko Poglitsch, Benjamin Bourgeois,
Dipeptidyl peptidase 3 modulates the renin-angiotensin system in mice
published online June 16, 2020J. Biol. Chem.
10.1074/jbc.RA120.014183Access the most updated version of this article at doi:
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