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ORIGINAL PAPER
Interaction between irbesartan, peroxisome
proliferator-activated receptor (PPAR-γ), and adiponectin
in the regulation of blood pressure and renal function
in spontaneously hypertensive rats
S. Afzal &M. A. Sattar &Edward J. Johns &
Mohammed H. Abdulla &Safia Akhtar &
Fayyaz Hashmi &Nor Azizan Abdullah
Received: 15 August 2015 / Accepted: 8 June 2016 /Published online: 12 July 2016
#University of Navarra 2016
Abstract Adiponectin exerts vasodilatory effects.
Irbesartan, an angiotensin receptor blocker, possesses
partial peroxisome proliferator-activated receptor gam-
ma (PPAR-γ) agonist activity and increases circulating
adiponectin. This study explored the effect of irbesartan
alone and in combination with adiponectin on blood
pressure, renal hemodynamic excretory function, and
vasoactive responses to angiotensin II and adrenergic
agonists in spontaneously hypertensive rat (SHR).
Irbesartan was given orally (30 mg/kg/day) for 28 days
and adiponectin intraperitoneally (2.5 μg/kg/day) for
last 7 days. Groups of SHR received either irbesartan
or adiponectin or in combination. A group of Wistar
Kyoto rats (WKY) served as controls. Metabolic data
and plasma samples were taken on days 0, 21, and 28. In
acute studies, the renal vasoconstrictor actions of
angiotensin II (ANGII), noradrenaline (NA), phenyl-
ephrine (PE), and methoxamine (ME) were determined.
SHR control rats had a higher mean blood pressure than
the WKY (132 ± 7 vs. 98 ± 2 mmHg), lower plasma and
urinary adiponectin, creatinine clearance, urine flow rate
and sodium excretion, and oxidative stress markers
compared to WKY (all P< 0.05) which were progres-
sively normalized by the individual drug treatments and
to a greater extent by combined treatment. Responses to
intrarenal administration of NA, PE, ME, and ANGII
were larger in SHR (P<0.05) than WKY by 20–25 %.
Irbesartan enhanced (P< 0.05) responses to NA and PE,
while adiponectin blunted responses to all vasoconstric-
tors (all P< 0.05). Combined treatment in SHR further
decreased the renal vascular responses to ANGII. These
findings suggest that an interactive relationship may
exist between PPAR-γ, alpha adrenoceptors, and
ANGII in the renal vasculature of the SHR.
Keywords Angiotensin II .Adiponectin .Peroxisome
proliferator-activated receptor.Renal hemodynamics .
Spontaneously hypertensive rat (SHR)
Introduction
Angiotensin II is importantly involved in contributing to
the development of hypertension and the associated
pathophysiology of vascular and renal function.
Consequently, the use of angiotensin II type 1 receptor
(AT1) blockers has proved extremely useful as
J Physiol Biochem (2016) 72:593–604
DOI 10.1007/s13105-016-0497-1
Institute The present experiment was conducted in
Cardiovascular and Renal Physiology Lab, School of
Pharmaceutical Sciences, Universiti Sains Malaysia, Penang,
11800, Malaysia
S. Afzal (*):M. A. Sattar :S. Akhtar :F. Hashmi
Cardiovascular and Renal Physiology Lab, School of
Pharmaceutical Sciences, Universiti Sains Malaysia,
Penang 11800, Malaysia
e-mail: samalik77@hotmail.com
E. J. Johns :M. H. Abdulla
Department of Physiology, University College Cork, Cork, Ireland
N. A. Abdullah
Department of Pharmacology, Universiti Malaya, Kuala
Lumpur 50603, Malaysia
antihypertensive agents as well as ameliorating the de-
velopment of cardiovascular and renal lesions [2]. The
AT1 receptor blocker, irbesartan, has been shown to
possess reno-protective actions expressed as improved
glomerular filtration rate (GFR) and fluid excretion
[24,30]. The neural control of the kidney is enhanced
by angiotensin II [27,28] as it facilitates the effective-
ness of sympathetic neurotransmission within the kid-
ney which belies an interaction between α1-
adrenoceptors and angiotensin II at the vasculature and
tubules [1].
PPAR-γis a member of peroxisome proliferator-
activated receptor, subfamily of transcription factors
which is a positive regulator of adiponectin gene ex-
pression [17]. The adiponectin gene is mainly expressed
in adipose tissue, vascular endothelial, and renal glo-
merular cells [25]. PPAR-γincreases and upregulates
adiponectin levels in plasma by stimulating the expres-
sion of proteins involved in adiponectin secretion such
as DsbA-L (present in kidney) [9]. Adiponectin is an
adipokine which mediates its action by stimulating nitric
oxide (NO) release from the vascular endothelium there-
by causing vasodilation [8]. Adiponectin has also been
found to inhibit most chronic changes contributing to
vascular disease in hypertension, and its action can be
modulated by AT1 blockers [33].
There have been reports that PPAR-γactivation by
irbesartan increased adiponectin expression since block-
ade of PPAR-γby its antagonist inhibited irbesartan-
induced adiponectin expression, although the concen-
trations used to induce adiponectin expression are
higher than those required for AT1R blockade [10]and
a consequent increase in the level of plasma adiponectin.
Such an action has not been reported with other antihy-
pertensive agents [34]. Interestingly, the concentrations
of AT1 receptor blockers required to exert this action are
higher than those used to reduce blood pressure sug-
gesting that this action may not be mediated via the AT1
receptor. In this study, irbesartan has been used as a
partial PPAR-γagonist; there is a body of evidence that
among ARBs, there is an order of potency to activate
PPAR-γactivating property (telmisartan > irbesartan >
losartan), ranging from low, median, to high concentra-
tion doses required for these ARBs respectively, as well
as non-activating PPAR-γproperty in case of
eprosartan. By contrast, thiazolidinedione derivative
(pioglitazone) acts as full ligand for PPAR-γ[26].
Reduced plasma adiponectin concentrations have been
found in hypertension and shown to be an associated
risk factor for hypertension [14]. Experimentally, low-
dose chronic infusion of angiotensin II to induce hyper-
tension causes a decrease in circulating levels of
adiponectin [18,23]. Thus, there is a body of evidence
linking the development of hypertension with reduced
production of adiponectin and lack of NO although the
exact interactions and mechanisms are unclear.
The question therefore arises as to whether in a rat
model of hypertension, adiponectin could contribute to
the blood pressure lowering and reno-protective action
of an AT1 receptor blocker. The hypothesis explored
was that in the spontaneously hypertensive rat (SHR),
circulating adiponectin levels would be reduced and that
chronic administration of exogenous adiponectin either
alone or in combination with AT1 receptor blocker,
irbesartan, would lower blood pressure or normalize
renal hemodynamic responsiveness to vasoconstrictor
agents to a greater extent following combined
irbesartan/adiponectin than with either compound alone.
Material and methods
Experimental animals and protocol
Twenty-four male SHRs, and six Wistar Kyoto rats,
body weight (230–255 g) were bred and maintained in
the Animal Care Facility, Universiti Sains Malaysia,
Penang, Malaysia, fed commercial rat chow (Gold
Coin Sdn. Bhd., Malaysia) and tap water ad libitum.
Animal handling and all procedures during this study
were approved from the Animal Ethics Committee,
Universiti Sains Malaysia. The animals were acclima-
tized for 3 days in individual metabolic cages prior to
24-h monitoring of water intake and fluid excretion.
Rats were randomly divided into five groups (n=6),
and systolic blood pressure (SBP), diastolic blood pres-
sure (DBP), mean arterial pressure (MAP), and heart
rate (HR) were measured non-invasively using the tail
cuff method, CODA equipment (Kent Scientific
Corporation, Torrington, CT) on days 0, 21, and 28.
Overnight urine collections were performed using the
metabolic cages before the start of treatment protocol on
days 0, 21, and 28 of the experiment. Water intake and
body weight were also measured on each day of urine
collection. Blood samples (2 ml) were collected from
the tail vein on the same days, and plasma was obtained
following centrifugation at 3500 rpm for 10 min. Plasma
and urine sample were stored at −30 °C for biochemical
594 S. Afzal et al.
analysis. Glomerular filtration rate (GFR) was calculat-
ed as creatinine clearance. Only SHRs exhibiting SBP ≥
150 mmHg were selected for the study. Five groups of
rats were studied: one of Wistar Kyoto rats as control
animals and four of SHR (n= 6 ineach group) treated as
follows:
Wistar Kyoto rats: treated with vehicle; SHR: treat-
ed with vehicle
SHR + Irb: given irbesartan (30 mg/kg) by oral
gavage for 28 days starting from day 1; SHR +
Adp: given adiponectin 2.5 μg/kg/day, intraperito-
neal, from day 21 to day 28
SHR + Irb + Adp: given irbesartan (30 mg/kg) by
oral gavage for 28 days starting from day 1 and
adiponectin 2.5 μg/kg/day, intraperitoneal, from
day 21 to day 28.
Stock solutions were prepared fresh every day.
Irbesartan tablets (Approvel, Sanofi, Aventis,
France) were dissolved in distilled water to yield a
stock solution of 30 mg/ml. Full-length recombi-
nant adiponectin obtained from Chemtron
Biotechnology Sdn, Bhd was dissolved in 200 μl
phosphate-buffered saline [13,19].
Acute hemodynamics
Overnight fasted rats (water ad libitum) were anesthe-
tized with 60 mg/kg i.p. sodium pentobarbitone
®
using PP240 (Protex, Kent, UK). Carotid artery was
cannulated (PE 50, Portex, Kent, UK) and coupled to a
pressure transducer (P23 ID Gould, Statham
Instruments, UK) connected to computerized data ac-
quisition system (Power Lab®, AD Instruments,
Sydney, Australia) for blood pressure measurement.
Similarly, left jugular vein was cannulated (PE 50,
Portex, Kent, UK) for infusion of doses of anesthesia
and vasoactive agents (Perfusor secura FT 50 ml, B.
Braun). A midline incision was carried out for the ex-
posure of the left kidney. On the dorsal surface of the
posterior end of kidney, a laser Doppler flow probe
(OxyFlow, AD Instruments) was placed for measure-
ment of renal cortical blood perfusion (RCBP) continu-
ously throughout the experiment which was directly
linked to the data acquisition system. A cannula
(PE50, Portex) was inserted via the left common iliac
artery, in a way for the close entry of its tip to access the
renal artery for administration of vasoactive agents, such
as noradrenaline (NA), phenylephrine (PE),
methoxamine (ME), and angiotensin II (Ang II) into
the renal artery. The baseline measurement of renal
arterial pressure was monitored through another pres-
sure transducer (model P23 ID Gould; Statham
Instruments) linked to a computerized data acquisition
system (Power Lab; AD Instruments). Meanwhile, 6 ml/
kg/hour saline was continuously infused for keeping the
cannula patent. One hourstabilization period was spared
before baseline systemic hemodynamic values were
acquired, which comprised SBP, DBP, MAP, HR, and
RCBP. The baseline blood pressure values were deter-
mined from the values at the beginning of each re-
sponse. Once baseline measurements were acquired
over 30 min, dose–response curves were generated as
follows:
NA at 25, 50, and 100 ng; PE at 0.25, 0.50,
and 1 µg; ME at 0.5, 1, and 2 µg; and Ang II
at 2.5, 5, and 10 ng.
A washout period of 10 min was allowed
between each agonist. Sodium pentobarbitone
(1 ml, i.v.) was used to sacrifice the animals at
the termination of the experimental study.
Vasoactive agents
NA (Sanofi Winthrop, Surrey, UK), PE (Knoll,
Nottingham, UK), ME (Wellcome, London, UK), and
Ang II (CIBA-GEIGY, Basel, Switzerland) were used in
the renal vasoconstrictor experiment. All drugs were
prepared as stock solutions in normal saline on the day
of the experiment and stored at 4 °C.
Biochemical analysis of stored plasma and urine
samples
Plasma and urinary creatinine concentrations were mea-
sured spectrophotometrically (Jaffe’s reaction), while
sodium and potassium concentrations were measured
using a flame photometer (Jenway Ltd., Felsted, UK).
Plasma levels of adiponectin were measured on the
acute surgery day 29 using an ELISA kit, (Chemtron
Biotechnology Sdn, Bhd) according to the manufac-
turer’s protocol. Creatinine clearance (Cr.Cl), fractional
excretion of sodium (FENa), and absolute urinary
Interaction between irbesartan, PPAR-γ, and adiponectin 595
(Nembutal , CEVA, Libourne, France). A midline inci-
sion was done for trachea exposure and cannulation
sodium excretion (UNaV) and NA/K ratio was calculat-
ed using the standard equations. Urinary sodium-to-
potassium ratio was calculated by dividing the urinary
sodium concentration by potassium concentration.
Statistical analysis
All data are expressed as mean ± SEM. The vasocon-
strictor responses caused by Ang II and adrenergic
agonists were taken as the average values caused by
each dose of the agonists administered in ascending
and descending orders. The statistical analysis of the
dose–response data utilized two-way ANOVA followed
by the Bonferroni post hoc test using the statistical
package Superanova (Abacus Inc., Sunnyvale, CA,
USA). However, the analysis of body weight, fluid
intake, urine flow rate (UFR), plasma ADP, and baseline
hemodynamic parameters measured during the acute
experiment were analyzed using repeated measures
one-way ANOVA followed by the Bonferroni post hoc
test for the differences between treatment period weeks.
Difference of 5 % was considered significant between
the mean values.
Results
Physiological and biochemical indices
Basal body weight was the same in all groupsat the start
of the treatment period (Table 1), and the age-dependent
body weight gains over the course of treatment were not
different between the vehicle-treated WKY and SHR
groups (all P> 0.05). There was no significant differ-
ence in body weight of the SHR + Irb, but not the SHR +
Irb + Adp, SHR + Adp groups versus control SHR on
day 28 only. In contrast, body weight of both groups
decreased significantly (P< 0.05) after 1-week treat-
ment of adiponectin (day 28) (both P< 0.05). The in-
crease in body weight of SHR + Adp and SHR + Irb +
Adp group was significantly less as compared to SHR +
Irb group (both P< 0.05) (Table 1). Water intake and
urine flow rate (Table 1) were significantly lower in the
SHR vehicle compared to the WKY control group on all
3 days of observation (all P<0.05;Table1). However,
the administration of irbesartan resulted in a higher
water intake in the SHR + Irb group on day 28
(P< 0.05), whereas urine flow rate remained was un-
changed relative to the control SHR group. In the SHR +
Adp and SHR + Irb + Adp groups, 1-week adiponectin
treatment significantly caused water intake to be lower
and urine flow rate to be higher on day 28 compared to
the SHR control and SHR + Irb groups (all P<0.05).
Urine flow rate increased significantly in the SHR +
Adp and SHR + Irb + Adp groups versus SHR + Irb
group (P<0.05).
Non-invasive techniques for the monitoring of con-
scious rat blood pressure were used in our study to
measure the systolic blood pressure on days 0, 21, and
28, respectively, as shown in Table 2. Systolic SBP on
all days was significantly higher in SHR group com-
pared to WKY group (all P< 0.05). Treatment with
irbesartan for 21 days and adiponectin for 7 days starting
from day 21 significantly reduced SBP in SHR-treated
groups (all P< 0.05). The combined treatment of
irbesartan and adiponectin resulted in further lowering
of SBP as compared to separate treatment in SHR-
treated groups (P< 0.05) and comparable to WKY con-
trol group (Table 2). Plasma sodium concentration of all
the experimental animals was also measured in the
current study on days 0, 21, and 28 (Table 2). Plasma
sodium concentration was significantly higher in SHR
group compared to WKY group on days 21 and 28 only
(both P< 0.05). No significant difference was observed
in SHR + Irb group as compared to SHR control on
respective days (P> 0.05), whereas in SHR + Adp and
SHR + Irb + Adp groups, adiponectin treatment signif-
icantly decreased plasma sodium concentration on day
28 only (P< 0.05) and comparable to WKY control
group (Table 2).
Baseline systemic hemodynamics
At the end of treatment period, and before start of acute
experiment, SBP, MAP, and HR were higher in all SHR
groups compared to the WKY group (all P<0.05).In
the SHR + Irb and SHR + Adp groups, SBP, MAP, and
HR were significantly (all P< 0.05) lower than those of
the SHR control group (Table 3).Thecombinedtreat-
ment with irbesartan and adiponectin resulted in signif-
icantly lower SBP and MAP in the SHR + Irb + Adp
group compared to the SHR + Irb and SHR + Adp
groups (all P< 0.05). There was no difference in HR
between the SHR + Irb + Adp and SHR + Adp groups
which were significantly lower than those of both the
SHR and SHR + Irb groups (both P<0.05; Table 3).
RCBP in the SHR control group was significantly
(P< 0.05) lower compared to the WKY control group
596 S. Afzal et al.
(P< 0.05) but was significantly higher in the SHR + Irb,
SHR + Adp, and SHR + Irb + Adp groups (allP<0.05).
RCBP was not different in the SHR + Irb + Adp versus
WKY control group but was higher than those of the
SHR + Irb and SHR + Adp groups (both P< 0.05;
Table 3).
Tabl e 1 Effect of exogenous
irbesartan, adiponectin, and a
combination of irbesartan and
adiponectin on body weight,
water intake, urine flow rate
(UFR) and urinary adiponectin in
spontaneously hypertensive rats
Notes: Acute experimental data.
The values are mean ± SEM
(n¼6). Statistical analysis was
done by a repeated measure one-
way ANOVA followed by
Bonferroni post hoc test.
*P< 0.05 versus WKY,
**P< 0.05 versus SHR,
***P< 0.05 versus SHR+Irb
Parameters Groups Days of observation
Day 0 Day 21 Day 29
Body weight (g) WKY 245 ± 5 275 ± 4 289 ± 8
SHR 242 ± 3 267 ± 3 280 ± 6
SHR+Irb 248 ± 4 255 ± 7 271 ± 9
SHR+Adp 244 ± 2 263 ± 3 247 ± 8*
,
**
,
***
SHR+Irb+Adp 249 ± 7 258 ± 5 239 ± 6*
,
**
,
***
Water intake (ml/d) WKY 53.1 ± 1.6 55.1 ± 1.7 58.1 ± 1.6
SHR 31.8 ± 1.4* 33.6 ± 1.9* 37.0 ± 1.8*
SHR+Irb 31.7 ± 1.4* 41.7 ± 1.2* 44.5 ± 1.5*
,
**
SHR+Adp 32.1 ± 1.8* 36.6 ± 1.7* 22.5 ± 1.9*
,
**
SHR+Irb+Adp 31.1 ± 1.6* 39.6 ± 1.6* 21.1 ± 1.5*
,
**
UFR (mL/min/kg) WKY 36.3 ± 1.3 37.7 ± 0.9 39.4 ± 1.0
SHR 26.8 ± 0.5* 25.7 ± 0.7* 28.4 ± 0.5*
SHR+Irb 26.3 ± 0.5* 29.5 ± 1.1* 31.6 ± 0.9*
SHR+Adp 25.9 ± 0.7* 26.6 ± 0.5* 52.5 ± 1.3*
,
**
,
***
SHR+Irb+Adp 27.1 ± 0.8* 37.7 ± 1.1** 52.93 ± 1.4*
,
**
,
***
Urinary adiponectin
excretion rate
(μmol/min/kg)
WKY 1.46 ± 0.03 1.49 ± 0.02 1.54 ± 0.05
SHR 0.71 ± 0.05* 0.67 ± 0.07* 0.77 ± 0.08*
SHR+Irb 0.71 ± 0.05* 0.88 ± 0.09*
,
** 1.11 ± 0.05*
,
**
SHR+Adp 0.69 ± 0.07* 0.70 ± 0.06* 2.23 ± 0.07**
,
***
SHR+Irb+Adp 0.73 ± 0.06* 1.17 ± 0.04*
,
** 2.29 ± 0.05**
,
***
Tabl e 2 Systolic blood pressure and plasma sodium level of Spontaneously hypertensive rats after treatment with exogenous irbesartan,
adiponectin, and a combination of irbesartan and adiponectin
Parameters Groups Days of observation
Day 0 Day 21 Day 28
Systolic blood pressure (mmHg) WKY 112 ± 6 115 ± 4 110 ± 7
SHR 159 ± 4* 162 ± 3* 157 ± 8*
SHR+Irb 164 ± 5* 144 ± 4*€137 ± 2*
,
**
SHR+Adp 162 ± 4* 160 ± 5* 129 ± 3*
,
**
,
***
SHR+Irb+Adp 166 ± 7* 145 ± 6* 115 ± 2**
,
***#
Plasma Sodium
Level (mmol/L)
WKY 122.8 ± 1.37 123.3 ± 1.84 124.4 ± 1.73
SHR 123.85 ± 1.25 130.56 ± 1.971* 131.58 ± 2.310*
SHR+Irb 124.35 ± 1.65 128.37 ± 1.83 128.25± 3.56
SHR+Adp 123.56 ± 1.97 130.67 ± 2.17 121.37 ± 0.97**
,
***
SHR+Irb+Adp 124.52 ± 1.57 129.56 ± 2.37 120.19 ± 1.03**
,
***
Notes: Conscious blood pressure measurement data. The values are mean ± SEM (n¼6). Statistical analysis was done by a repeated measure
one-way ANOVA followed by Bonferroni post hoc test. *P< 0.05 versus WKY, **P<0.05 versusSHR, ***P< 0.05 versus SHR+Irb,
#P<0.05 versusSHR+Adp
Interaction between irbesartan, PPAR-γ, and adiponectin 597
Hormone measurement
Plasma adiponectin was significantly (P< 0.05) higher
in WKY control compared to the SHR control group
taken on day 29 on the acute study day (Fig. 1).
Irbesartan treatment for 4 weeks and adiponectin treat-
ment for 1 week significantly increased the plasma
adiponectin levels in the SHR + Irb and SHR + Adp
groups as compared to the control SHR group (both
P< 0.05) (Fig. 1). Plasma adiponectin level of SHR +
Irb + Adp is significantly higher compared to SHR,
SHR + Irb, and SHR + Adp (Fig. 1).
Renal hemodynamic parameters
Cr.Cl in the SHR, SHR + Irb, SHR + Adp, and SHR +
Irb + Adp groups were significantly lower versus WKY
control over the whole 28-day period of observation
(Fig. 2a) but in the irbesartan and adiponectin groups
increased (all P< 0.05) over the treatment periods.
Absolute urinary sodium (UNaV) excretion (Fig. 2b)
in the SHR, SHR + Irb, SHR + Adp, and SHR + Irb +
Adp groups were significantly lower versus WKY con-
trol, whereas irbesartan treatment for 4 weeks and
adiponectin treatment for 1 week significantly increased
the UNaV in the SHR + Irb and SHR + Adp groups as
compared to the control SHR group only (both
P< 0.05), but in SHR + Irb + Adp group, on day 28, it
was no different from the WKY control group (Fig. 2b).
FENa (Fig. 2c) of all treated and untreated SHR groups
was similar on day 0. Irbesartan treatment increased
FENa on days 21 and 28 (all P< 0.05), while in the
SHR + Irb + Adp group, on day 28, it was no different
from that of the control WKY.
The urinary Na/K (Fig. 2d) of the untreated SHR
group remained significantly lower on days 0, 21, and
28 as compared with the WKY group (all P<0.05).
Moreover, the SHR + Adp groups had a lower Na/K
on day 28 compared to the WKY group (P< 0.05).
However, it was evident that the SHR + Irb, SHR +
Adp, and SHR + Irb + Adp groups exhibited signifi-
cantly (all P< 0.05) higher Na/K ratio on day 28
Tabl e 3 Baseline systemic hemodynamic effects of exogenous
adiponectin, irbesartan, and combination of irbesartan and
adiponectin on systolic blood pressure (SBP), mean arterial blood
pressure (MAP), heart rate (HR), and renal cortical blood perfu-
sion (RCBP) in spontaneously hypertensive rats
Parameters Experimental groups
WKY SHR SHR + Irb SHR + Adp SHR + Irb + Adp
SBP (mmHg) 110.0 ± 7 157.0 ± 8* 137.0 ± 2*
,
** 129.0 ± 3*
,
** 115.0 ± 2**
,
***
,#
MAP (mHg) 94.0 ± 5 132.0 ± 7* 103.0 ± 2*
,
** 106.0 ± 2*
,
** 98.0 ± 2**
,
***
,#
HR (beat/min) 323.0 ± 9 386.0 ± 13* 370.0 ± 4*
,
** 335.0 ± 3*
,
**
,
*** 327.0 ± 4**
,
***
RCBP (bpu) 247.0 ± 11 167.0 ± 9* 219.0 ± 3*
,
** 231.0 ± 2*
,
** 266.0 ± 2**
,
***
,#
Acute experimental data. The values are mean ± SEM (n= 6). Statistical analysis was done by a repeated measure one-way ANOVA
followed by Bonferroni post hoc test
bpu, blood perfusion units
*P< 0.05 versus WKY, **P<0.05versusSHR, ***P< 0.05 versus SHR + Irb, #P<0.05versusSHR +Adp
1
0
2
4
6
8
10
12
14
16
WKY
SHR
SHR+IRB
SHR+ADP
SHR+IRB+ADP
*
##
!$
**
*#
Plasma adiponectin ( g/ml)
Fig. 1 Adiponectin
concentration expressed as μg/ml
values mean ± SEM. Statistical
analysis was done by one-way
ANOVA followed by Bonferroni
post hoc test (n=6). *P<0.05
versus WKY, #P< 0.05 versus
SHR, !P< 0.05 versus SHR + Irb,
$P< 0.05 versus SHR + Adp
598 S. Afzal et al.
compared with the SHR control group. On day 28, there
were no significant differences in Na/K between the
SHR + Irb + Adp group versus WKY control group
(P<0.05).
Renal cortical vasoconstrictor response
Adrenergic agonists
The intrarenal injection of NA (Fig. 3a), PE (Fig. 3b),
and ME (Fig. 3c) resulted in dose-dependent renal cor-
tical vasoconstrictions in all experimental groups of rats.
The overall mean % decrease in RCBP was significantly
larger in the SHR compared to WKY control (all
P< 0.05; Fig. 3a, b, c) for all adrenergic agonists. The
SHR + irbesartan group had a significantly greater de-
crease in RCBP (both <0.05) to NA and PE (Fig. 3a, b
respectively), but not ME (Fig. 3c), compared to control
SHR. Conversely, the renal cortical vasoconstrictions
induced by NA, PE, and ME were significantly attenu-
ated (all P< 0.05) in the SHR + Adp compared to the
control SHR. Similarly, the magnitudes of the NA, PE,
and ME-induced renal vasoconstrictions (Fig. 3a, b,c,
respectively) in the SHR + Irb + Adp group were sig-
nificantly smaller (all P< 0.05) compared to the control
SHR and SHR + Irb groups. Adiponectin, whether
given alone or in combination with irbesartan to the
SHR, significantly reduced the magnitude of the adren-
ergically induced reductions in RCBP compared to
those obtained in the untreated SHR (all P< 0.05;
Fig. 3).
Angiotensin II
The overall Ang II-induced renal vasoconstriction in
control WKY was significantly smaller (P< 0.05) than
that of the untreated SHR. In addition, compared to
control SHR, the magnitude of the Ang II-induced de-
crease in RCBP was significantly blunted (P<0.05) in
0
2
4
6
8
WKY
SHR
SHR+IRB
SHR+ADP
SHR+IRB+ADP
*
*
!
Day 0 Day 21 Day 29
*******
**# ##
!
*
##
Cr.Cl.mL/min/kg
0.0
0.1
0.2
0.3
***
***
***
**
*
###
#
#
!
!
Day 0 Day 21 Day 29
$
UNa v mmol/hr/kg
0.0
0.2
0.4
0.6
***
*
*
*
****
*
#####
!
!
$
Day 0 Day 21 Day 29
FE Na%
0.0
0.5
1.0
1.5
2.0
2.5
***
*
*
*
*****
*
##
Day 0 Day 21 Day 29
#
*
!
$
Na:K ratio
ab
cd
Fig. 2 Cr.Cl (a), UNaV (b), FENa (c), and Na/K (d) of WKY,
SHR, SHR + Irb, SHR + Adp, and SHR + Irb + Adp. Values are
mean ±SEM. Statistical analysis was done by a repeated measure
one-way ANOVA followed by Bonferroni post hoc test in respec-
tive days (n=6). *P<0.05 versus WKY, #P< 0.05 versus SHR,
!P<0.05 versus SHR + Irb, #P< 0.05 versus SHR + Adp. Cr.Cl
creatinine clearance, UNaV absolute urinary sodium excretion,
FENa fractional sodium excretion, Na:K urinary sodium potassi-
um ratio
Interaction between irbesartan, PPAR-γ, and adiponectin 599
the SHR + Irb-treated group and to a greater extent in the
SHR + Adp group. The renal vasoconstriction induced
by Ang II in the group given both irbesartan and
adiponectin was significantly smaller (P< 0.05) as com-
pared to separate treatment (Fig. 3d). The magnitude of
vasoconstriction produced by Ang II in the SHR + Irb +
Adp treated group was significantly blunted compared
to the WKY control group (Fig. 3d).
Discussion
The thrust of this investigation was to determine wheth-
er in a rat model of hypertension, the SHR, the blood
pressure, and renal responsiveness to adrenergic ago-
nists and angiotensin II were in any way dependent on
circulating levels of adiponectin and the integrity of the
angiotensin AT1 receptor. This study revealed several
important findings. Firstly, plasma adiponectin concen-
trations and urinary excretion were depressed in the
SHR but were partially restored to those found in the
normotensive WKY rats following the administration of
the AT1 receptor antagonist, irbesartan. This would
support previous reports that adiponectin, via activation
of PPAR-γ, in some way prevents activation of the AT1
receptor. Importantly, the administration of the
adiponectin was at a rate sufficient to raise plasma levels
and urinary excretion comparable to those found in the
WKY. Secondly, adiponectin administration to the SHR
decreased blood pressure and increased renal hemody-
namics and fluid excretion similar to those achieved
with irbesartan. These responses are consistent with
the hypothesis that both compounds block the actions
of angiotensin II at different points along the signaling
pathway. Thirdly, the renal vasoconstrictor responses to
the adrenergic agonists and angiotensin II were en-
hanced in the SHR relative to the WKY, while in the
presence of adiponectin or combined adiponectin and
irbesartan, the responses were attenuated to a similar
degree. This would indicate that within the kidney
25
50
100
-90
-80
-70
-60
-50
-40
-30
-20
-10
0
*
WKY
SHR
SHR+IRB
SHR+ADP
*
*
*
SHR+IRB+ADP
#
#
!
!
Noradrenaline (ng)
% Drop in RCBP
0.25
0.50
1.00
-110
-100
-90
-80
-70
-60
-50
-40
-30
-20
-10
0
*
WKY
SHR
SHR+IRB
SHR+ADP
SHR+IRB+ADP
*
*
*
#
#!
!
Phenylephrine (µg)
% Drop in RCBP
a
cd
b
0.5
1.0
2.0
-100
-90
-80
-70
-60
-50
-40
-30
-20
-10
0
*
WKY
SHR
SHR+IRB
SHR+ADP
SHR+IRB+ADP
*
*
*
Methoxamine (µg)
!
!
#
#
% Drop in RCBP
25
50
100
-100
-90
-80
-70
-60
-50
-40
-30
-20
-10
0
*
WKY
SHR
SHR+IRB
SHR+ADP
*
*
*
#
#
#$
Angiotensin II(ng)
SHR+IRB+ADP
% Drop in RCBP
Fig. 3 Dose–response curve of the renal vasoconstrictor re-
sponses (% drop) to graded doses of NA (a), PE (b), ME (c),
and Ang II (d)inWKY,SHR,SHR+Irb,SHR+Adp,SHR+Irb
+ Adp. Statistical analysis was done by two-way ANOVA
followed by Bonferroni post hoc test (n= 6). Significance is be-
tween the overall mean of responses due to three doses of agonist
in each group. *P<0.05 versus WKY, #P<0.05 versus SHR,
!P<0.05 versusSHR+Irb, #P< 0.05 versus SHR + Adp
600 S. Afzal et al.
vasculature, adiponectin acted to depress the signaling
following activation of adrenoceptors even when AT1
receptors were blocked.
SHRs mimic a model for human essential hyperten-
sion because of main cardiovascular and renal charac-
teristics [22]. Body weight of both control WKY and
SHR increased steadily and to a similar degree over
28 days of the experimental period. In the SHR + Irb
group, there was a small but statistically insignificant
increase in body weight. Irbesartan may regulate adipo-
cyte particle size due to its PPAR-γactivating proper-
ties, but the dose rate and treatment period in the current
study were not sufficient to produce a significant de-
crease in body weight. By contrast, Zorad et al. [35]
reported marked decreases in body weight following
candesartan administration in normotensive WKY
which was associated with increasing PPAR-γexpres-
sion and adiponectin plasma concentration. This differ-
ence between the observation of Zorad et al. (2006) and
those herein may be attributed to the dose rate and
treatment duration of ARBs. However, a significant
decrease in body weight was observed in SHR + Adp
and in SHR + Irb + Adp groups after 1 week treatment
of adiponectin. In the current study and in previous
reports, the administration of ADP decreases body
weight probably due to ADP stimulating fatty acid
oxidation and glucose utilization by activating AMP-
activated protein kinase [21], which eventually leads to
a decrease in adipose tissue mass. Regarding water
intake, irbesartan treatment caused an age-dependent
increase in fluid intake, which is in agreement with that
reported in SHRs after angiotensin II AT1 receptor
blockade [4]. By contrast, adiponectin treatment in-
creased urine flow rate which could be possibly due to
an inhibitory effect of adiponectin on antidiuretic hor-
mone release. Irbesartan treatment caused a significant
increase in plasma adiponectin concentration most like-
ly as a result of its PPAR-γagonistic activity. This
observation would be consistent with previous reports
suggesting that irbesartan can induce adiponectin pro-
duction independent of its AT1 receptor antagonistic
action [12,16]. Moreover, treatment with irbesartan as
a combination therapy with adiponectin further en-
hanced plasma and urinary adiponectin levels.
Nonetheless, the adiponectin concentrations achieved
were likely in the physiological/pathophysiological
range.
In current study, we observed that the untreated
SHR group had significantly higher SBP (P<0.05)
compared to WKY groups throughout the 28 days of
experimental period (Table 2). The higher blood pres-
sure in the SHR compared to the WKY was de-
creased as a consequence of the 1-week adiponectin
administration. The underlying reason is unclear, but
one possibility is an increase in the production of NO
which can be stimulated by adiponectin [10]. The
combined irbesartan/adiponectin treatment reduced
blood pressure in the SHRs to an even greater degree
suggesting a synergistic action of two compounds
due possibly to the upregulation of PPAR-γreceptors
as indicated above. Irbesartan at the dose used in the
present study dose (30 mg/kg/day) is toward the
maximal dose for blockade of the rennin angiotensin
system, while its PPAR-γactivity will also partici-
pate in blood pressure reduction observed in the SHR
as also reported previously [32]. Treating the SHRs
with adiponectin significantly reduced the SBP,
MAP, and heart rate at the end of treatment period
(Table 3) and compared with control SHR which may
be related to the inhibitory effects of adiponectin on
sympathetic activity. Adiponectin has been reported
to be present in cerebrospinal fluid [20], and admin-
istration of adiponectin via central regulation reduces
blood pressure, sympathetic nerve activity, and HR
[29]. Although there was no reduction in HR in the
SHR + Irb-treated group, the adiponectin/irbesartan
combination therapy decreased HR to the same ex-
tent as observed SHR + Adp group which would
indicate adiponectin being solely responsible.
In the present study, 3 weeks of irbesartan adminis-
tration improved creatinine clearance in the SHRs which
paralleled previous reports of Rich et al. [3]. Moreover,
the 1-week treatment with adiponectin also resulted in a
higher creatinine clearance compared to control SHR
group. Interestingly, the increase in creatinine clearance
in the 1-week irbesartan/adiponectin combination ther-
apy group was no different from that observed in the
SHR + Adp group.This would suggest that in relation to
the filtration mechanisms in kidney, adiponectin has a
minor role.
Kidney regulates the long-term BP through
changed renal sodium management to save the
sodium-retaining properties [11]. It was observed
that UNaV was lower in the SHR group from days
0to28ascomparedtotheWKYwhichappearedto
be an effect independent of the higher blood pres-
sure in the SHRs [5]. This possibly indicated a long-
term fluid retention which would contribute to an
Interaction between irbesartan, PPAR-γ, and adiponectin 601
expansion of extracellular fluid volume and an in-
crease in blood pressure. This would be supported
by the decreased FENa which is indicative of greater
renal tubular Na reabsorption in SHRs. Treatment
with either irbesartan for 28 days or adiponectin for
7 days caused FENa and UNaV to increase com-
pared to untreated SHRs and to an even greater
extent in the combined therapy SHR + Irb + Adp
group. These observations consistent with both an-
giotensin II and adiponectin were acting to depress
tubular fluid reabsorptive processes. Angiotensin II
is generated, and its receptors are present along the
proximal tubule, and their activation stimulates so-
dium reabsorption, and blockade of these receptors
will increase sodium excretion. In terms of
adiponectin, much less is known and the exact loca-
tion of receptors along the nephron and the mecha-
nism of its action are unclear. Urinary Na/K serves
as an indirect marker of kidney function of aldoste-
rone acting on the renal cortical collecting tubules
[3] to stimulate sodium reabsorption. The SHRs
exhibited a significantly lower Na/K indicative of
increased aldosterone activity possibly driven by
angiotensin II. This finding is consistent with the
report that SHRs have increased plasma angiotensin
II levels despite normal plasma rennin activity [6]
which could stimulate aldosterone production and
cause relative sodium retention. ANG II may also
enhance the production of superoxide anions [7]
which also have sodium-retaining properties which
could also contribute to the development of hyper-
tension. In support of this view, treatment with
irbesartan for 28 days and adiponectin for 1 week
increased the Na/K ratio which is consistent with
adiponectin decreasing aldosterone and angiotensin
II activity in the SHRs.
Renal cortical blood perfusion was lower in the
SHRs compared to the WKY indicating an increased
renal vasoconstriction along the vasculature. This
finding correlates with the pattern of creatinine
clearance measured in the SHR and WKY in the
chronic studies. Irbesartan treatment resulted in a
higher RCBP in SHR which is similar to that report-
ed following losartan administration in conscious
SHR and was an effect independent of NO [15]. In
the acute study investigating vascular responsive-
ness, the partial blockade of the angiotensin II re-
sponses in SHR could be due to the systemic action
of irbesartan, whereas angiotensin II was delivered
locally in to the kidney and was at a relatively high
dose. Adiponectin treatment decreased the magni-
tude of renal vascular responses to both alpha ad-
renergic agonists and Ang II which suggests a cross
talk relationship between renal vascular adiponectin
receptors (Adipo R1 and AdipoR2), AT1, and α1-
adrenoceptors in attenuating the vasoconstrictor re-
sponses in the renal vasculature, which may be
related to the inhibitory effects of adiponectin on
sympathetic activity. During higher levels of sympa-
thetic nervous activity, α1-adrenoceptors are down-
regulated and there appears to be cross talk relation-
ship between RAAS and sympathetic nervous sys-
tem (SNS) at different levels [12]. Interestingly,
vascular AT1-receptors and α1-adrenoceptors posi-
tively interact with each other and produce an en-
hanced response to their respective substrate [31].
From our experimental results, it may be possible to
speculate that an interactive/cross talk relationship
exists between PPAR-γand adrenergic neurotrans-
mission at the renal vasculature of the SHRs.
Conclusion
The study highlighted the following:
1. A degree of synergism exists between adiponectin
and ARBs (PAR-γagonist).
2. Adiponectin reduces blood pressure and improves
renal hemodynamics in SHRs.
3. The renal vasculature of SHRs exhibits an
interactive/cross talk relationship between
irbesartan (PPAR-γagonist), alpha adrenoceptors,
and ANG II.
4. Adiponectin alters/attenuated renal vascular reactiv-
ity, possibly through eNOs stimulation, which may
underly a reno-protective role of adiponectin main-
taining normal renal physiology.
Acknowledgments The authors fully acknowledge the
Universiti Sains Malaysia, Research grant no. 1001/PFARMASI/
815078, provided by Universiti Sains Malaysia for this work.
Sheryar Afzal is a recipient of USM Fellowship from the
Institute of Post-Graduate Studies (IPS), Universiti Sains Malaysia
(USM), Penang, Malaysia, and this support is gratefully
acknowledged.
602 S. Afzal et al.
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