Pioglitazone Prevents Hypertension and Reduces Oxidative

Stress in Diet-Induced Obesity

Anca D. Dobrian, Suzanne D. Schriver, Ali A. Khraibi, Russell L. Prewitt

AbstractThe objective of this study was to determine the effect of pioglitazone on blood pressure (BP) and oxidative
balance in obese, hypertensive, Sprague-Dawley rats and to identify some of the molecular mechanisms involved. After
12 weeks of a moderately high-fat diet, rats diverged into obesity-prone (OP) and obesity-resistant (OR) groups (n6
per group). At the end of the diet, peroxisome proliferator activated receptor- (PPAR) mRNA expression and activity
in the renal cortex and medulla of OP rats were significantly lower compared with that in OR rats. Pioglitazone treatment
increased PPAR expression and activity in OP rats, suggesting a possible direct ligand-related effect of pioglitazone.
As opposed to the untreated OP group, which showed moderate hypertension (systolic BP1595.3 mm Hg) after 12
weeks, pioglitazone-treated rats were normotensive (systolic BP123.92.7 mm Hg). Insulin production was reduced
by 2-fold in the OP group treated with pioglitazone. Urinary isoprostanes and renal lipid peroxides were also reduced
in OP rats treated with pioglitazone compared with untreated counterparts. Also, expression of p47phox and gp91phox,
Downloaded from http://hyper.ahajournals.org/ by guest on May 12, 2017

both increased in OP versus OR rats, was reduced in the former by pioglitazone treatment. In addition, pioglitazone
treatment increased nitrate/nitrite excretion and expression of renal endothelial and neuronal nitric oxide synthase.
Collectively, the results show that pioglitazone treatment prevented hypertension and renal oxidative stress both by
reducing free-radical production and by increasing nitric oxide production/availability. (Hypertension. 2004;43:48-56.)
Key Words: kidney oxidative stress free radicals nitric oxide vitamins

sion of PPAR in these segments might have important

O besity is a widespread and increasingly prevalent con-
dition associated with a large number of comorbid
diseases, one of the most important of which is obesity-
induced hypertension. A pleiotropic class of molecules in-
volved in regulation of gene expression in a variety of
metabolic and cardiovascular conditions is the peroxisome
proliferatoractivated receptors (PPARs). PPARs are ligand-
activated transcription factors that form heterodimers with the
9-cis retinoic acid receptor RXR.1 PPAR is one of the three
PPAR isoforms and is one of the major regulators of
adipogenesis.2 In addition, PPAR exerts pleiotropic effects
on blood pressure, lipid metabolism, and insulin action.
Recent genetic analysis showed that 2 dominant-negative
mutations in PPAR were associated with severe hyperten-
sion in humans.3,4 Consistent with these findings, thiazolidi-
nendiones, the insulin-sensitizer drugs (pioglitazone, rosigli-
tazone) that are also high-affinity PPAR ligands,5 have been
shown to lower blood pressure (BP) in a variety of hyperten-
sive animal models6 8 as well as in diabetic and nondiabetic,9
hypertensive humans. However, the mechanism underlying
the antihypertensive effects of PPAR agonists is not known.
PPAR and RXR have been found constitutively expressed in
the inner medullary collecting ducts, thick ascending limb,
glomerulus, and renal medullary microvascular endothelial
cells in rats,10,11 rabbits, and humans.12,13 Constitutive expres-
regulatory effects on renal sodium and water reabsorption.
Free-radical production is associated with both obesity and
hypertension in humans and animal models.14,15 One of the
mechanisms by which free radicals can sustain hypertension
is a reduction of nitric oxide (NO) availability at the level of
the thick ascending limb16 or macula densa,17 therefore
leading to increased sodium reabsorption. NO can readily
interact with O2 anion, rendering the former unable to
perform its physiologic vasodilatory effect.18 A recent report
indicates that a major source of O2 in the rat kidney is a
phagocytic-like NAD(P)H oxidase.19 All of the major
NAD(P)H oxidase subunits have been identified in the rat
kidney, and p47phox was shown to be increased in the
prehypertensive spontaneously hypertensive rat, suggesting a
possible causative role in this form of genetic hypertension.20
Also, several recent reports showed the ability of PPAR
agonists to modulate expression of different NAD(P)H oxi-
dase subunits. Inoue et al21 showed that PPAR activators
increase (Cu,Zn)superoxide dismutase expression while de-
creasing p22phox message in human endothelial cells. Also,
activation of PPAR by NO in macrophages was shown to
downregulate p47phox and to attenuate the respiratory
burst.22 In addition, troglitazone was shown to reduce reactive
oxygen species generation by leukocytes and improve post-

Received May 13, 2003; first decision May 29, 2003; revision accepted October 16, 2003.
From the Department of Physiological Sciences, Eastern Virginia Medical School, Norfolk, Va.
Correspondence to Anca Dobrian, PhD, Assistant Professor, Department of Physiological Sciences, Eastern Virginia Medical School, 700W Olney Rd,
Norfolk, VA 23507. E-mail dobriaad@evms.edu
2003 American Heart Association, Inc.
Hypertension is available at http://www.hypertensionaha.org DOI: 10.1161/01.HYP.0000103629.01745.59

48
 Dobrian et al Pioglitazone Prevents Hypertension in Obesity 49

ischemic flow-mediated vasodilation in obese humans.23

However, there are no data on a potential PPAR-related
effect on free-radical or NO production in the kidney in
obese, hypertensive animal models or humans.
In this study, we aimed to assess the contribution of
oxidative stress to BP regulation in a rat model of diet-
induced obesity and hypertension and to identify some of the
mechanisms underlying the hypotensive effects of pioglita-
zone treatment.

Methods
Animals and Treatments
All procedures involving animals were approved by the Institutional
Animal Care and Use Committee of Eastern Virginia Medical
School. Male Sprague-Dawley rats (300 to 350 g) were fed a
moderately high-fat diet (MHF; 32% kcal as fat; Research Diets) or
a purified, low-fat diet (LF; 10.6% kcal as fat) (controls) for 12
weeks. After 1 week, randomly selected rats were placed on either
the MHF or LF diet supplemented with either pioglitazone (0.1%
wt/wt, or 10 mg kg1 d1; Actos, Hanna Pharmaceuticals) or
Downloaded from http://hyper.ahajournals.org/ by guest on May 12, 2017

vitamin E (0.5% wt/wt as -tocopherol acetate, or 200 mg kg1

d1; Sigma). Rats that were fed the MHF diet with or without the
indicated treatments diverged into distinct groups based on body
weight gains. Assignment of rats into obesity-prone (OP, n6) and
obesity-resistant (OR, n6) groups was performed as previously
described.24,25 In brief, construction of body-weight gain histograms
was performed at the end of the study for MHF and LF diet groups.
OR rats were defined as those with weight gains equal to or less than
the heaviest controls, and the OP rats, as those with greater weight
gains.

BP Measurements
The onset and development of hypertension were assessed by the
tail-cuff method with a sphygmomanometer (Narco Biosystems
Electro-Sphygmomanometer), as previously described.24,26
Immunohistochemistry
Formalin-fixed, paraffin-embedded sections were processed for an-
tigen retrieval and incubated with a primary polyclonal antibody for
1:1 Cys,His,Lys-4-hydroxy-2-nonenal (HNE) adducts (Calbiochem;
dilution 1:750). The slides were stained with reagents from a
commercially available kit (Immuno Pure Metal Enhanced DAB
substrate kit, Pierce).
RNA Isolation and RT-PCR
Kidneys were homogenized in ice-cold guanidinium isothiocyanate and
processed for total RNA extraction according to Chomczynski.27 RNA
(1 g) was reverse-transcribed (RT) and amplified by polymerase chain
reaction (PCR) as previously described.26 The following pairs of
forward and reverse primers were used: PPAR1,2, 5-TCCGTG
ATGGAAGACCACTC-3 and 5-CCCTTGCATCCTTCACAAGC-
3; and -actin, 5-CGTAAAGACCTCTATGCCAA-3 and 5-
CTCCAGTGCCAAGGTC TGAA-3. The neuronal (nNOS) and endo-
thelial (eNOS) NO synthase primers were described previously.26,28 The
bands corresponding to PCR products were measured by densitometry
on an EagleEye system (Stratagene).
Western Blotting
The tissue was homogenized and fractionated by centrifugations at
800g (to pellet debris), at 10 000g (to pellet intracellular organelles),
and at 150 000g, after which the membrane pellet was reconstituted
in Tris-sucrose buffer, pH 7.4. Sample protein was assessed with the
bicinchoninic acid method with a kit (Sigma). Equal amounts of
protein (20 g per lane) were separated on 10% sodium dodecyl
sulfatepolyacrylamide gel electrophoresis gels and subsequently
electroblotted. The following primary antibodies were used: anti-
p47phox (Santa Cruz, 1:1000 dilution), anti-gp91phox (Transduction
Figure 1. Systolic BP in OP, OR, and control (C) rats treated
with pioglitazone (Pio; A) or vitamin (Vit) E (B) for 12 weeks com-
pared with respective untreated groups (n6 per group). Data
represent meanSEM. *Significant vs OR and control; #signifi-
cant compared with treated OP group.
Laboratories, 1:2000 dilution), and anti-eNOS and anti-nNOS
(Transduction Laboratories, 1:1500 dilution). Bands were visualized
by enhanced chemiluminescence (ECL, Amersham Life Science).
Other Assays
Urinary free isoprostanes and thiobarbituric acidreactive substances
(TBARS) were measured as previously described.26 Plasma insulin
and urine nitrite/nitrate (NOx) were measured with kits from Linco
Research Inc and Cayman Chemical, respectively. Sodium excretion
was assessed by flame photometry.
Statistical Analysis
Comparisons between group means were performed by 2-way
ANOVA and Tukey post hoc analysis test with InStat software
(GraphPad Software). The null hypothesis was rejected when
P0.05.
Results
Effect of Pioglitazone and Vitamin E Treatments
on BP in OP, OR, and Control Rats
In accordance with previous data,24 the OP rats showed a
significant elevation in systolic BP after week 7 on the diet,
which remained higher (159.36.5 mm Hg) compared with that
in OR (136.24.5 mm Hg) and control (128.63.2 mm Hg)
groups until the end of the experiment (Figure 1A). When
pioglitazone was added to the diets, OP rats maintained their BP
 50 Hypertension January 2004
Body Weight, Plasma Insulin, and Urinary Sodium, Water, and NOx Excretion in OP, OR, and Control (C) Rats With and Without
Pioglitazone or Vitamin E Treatment
Group Body Weight, g, at Week 12 Insulin, ng/mL
OP 692.18.1* 15.40.9*
OR 570.322.6 8.21.5
C 569.719.3 5.71.4
OPpioglitazone 757.935.2* 4.20.7
ORpioglitazone 617.520.8 3.10.4
Cpioglitazone 647.315.8 3.30.4
OPvitamin E 675.212.1* 13.82.7*
ORvitamin E 544.022.3 6.71.4
Cvitamin E 550.211.7 6.31.1
Six rats per group were analyzed for each parameter. Terminal blood was collected after 12 weeks of diet; urine was collected for 24 hours, on ice, in metabolic
cages. Results are meanSEM.
*Significant compared with corresponding OR and C.
Significant compared with pioglitazone-treated group. The null hypothesis was rejected when P0.05.
Downloaded from http://hyper.ahajournals.org/ by guest on May 12, 2017
within the normal range until the end of the experiment
(131.34.5mm Hg). Pioglitazone also had a moderate hypoten-
sive effect in OR (1252.2 mm Hg) and control
(119.55.2mm Hg) rats (Figure 1A).
To directly assess the effect of oxidative stress on the
development of hypertension, 2 separate groups of rats were
placed on either the MHF or LF diet supplemented with
-tocopherol acetate. Unlike pioglitazone, vitamin E failed to
completely prevent the elevation in BP in OP rats; however,
it significantly reduced it compared with the untreated OP
group after 7 weeks (Figure 1B). At the end of the experi-
ment, OP rats treated with vitamin E had an average systolic
BP of 140.24.5mm Hg. By the end of experiment, no
significant differences were detected between the OR and
control groups with and without vitamin E (Figure 1B).
Body Weight, Circulating Insulin, and Sodium
Excretion in OP, OR, and Control Rats With and
Without Pioglitazone Treatment
The divergence into 2 different populations, OP and OR,
according to body weights occurred similarly in both treated
and untreated groups (Table). However, the pioglitazone-
treated rats gained significantly more weight compared with
untreated as well as vitamin Etreated groups by the end of
the diet (Table).
In accordance with previous data,29 in OP rats after 12
weeks on the diet, plasma insulin was increased 2-fold
compared with that in OR rats (Table). As expected, piogli-
tazone prevented the elevation in plasma insulin in OP rats
(Table). The groups treated with vitamin E had no change in
circulating insulin levels compared with nontreated counter-
parts (Table). Measurements of urine volume and sodium
indicated an increased sodium and water excretion in
pioglitazone-treated OP rats compared with controls by the
end of the diet (the Table), whereas vitamin E treatment did
not change sodium and water excretion (Table).
PPAR Expression in the Kidney in OP, OR, and
Control Rats With and Without Pioglitazone or
Vitamin E Treatment
We examined PPAR mRNA and protein expression in renal
tissue in rats with or without pioglitazone or vitamin E
Urine Volume/24 h, mL Urine Na, mmol/24 h Urine NOx, mol/24 h
28.52.3 15.61.53# 2.10.08*
30.42.2 16.11.13 5.40.29
19.82.2 16.20.46 4.40.23
51.93.6* 23.71.17* 4.10.06*
36.72.2 16.01.45 5.80.15
22.02.2 18.70.67 4.70.10
27.32.6 16.91.34 2.70.09*
29.32.0 16.70.58 5.50.10
22.02.5 14.81.25 4.50.19
treatment. Semiquantitative RT-PCR for PPAR1,2 showed a
30% higher expression in the cortex and a 3-fold increased
expression in the medulla of OR rats compared with OP rats
(Figure 2C). Also, PPAR expression in the control group
was comparable to the levels detected in OP rats (Figure 2C).
In OR and control groups, the level of PPAR expression was
higher in the renal medulla than in the cortex (Figure 2).
Pioglitazone increased PPAR mRNA in the cortex and
medulla of the OP rats but had no effect in the OR group
(Figures 2A through 2D). However, vitamin E treatment of
OP, OR, and control rats did not significantly alter mRNA
expression in either the cortex or medulla compared with
corresponding untreated groups (Figures 2A through 2D).
Effect of Pioglitazone on Oxidative Stress and
NAD(P)H Oxidase Subunit Expression in OP, OR,
and Control Rats
We tested whether PPAR activation had an effect on 2
different parameters of oxidative stress as well as on NO
production. Results indicated that both urine free isopros-
tanes, a recognized index for systemic free-radical produc-
tion, and kidney TBARS, an index of lipid peroxidation, were
moderately though significantly inhibited by pioglitazone
treatment after 12 weeks of diet feeding in OP rats (Figure 3).
To compare the effect of pioglitazone with that of a known
antioxidant, we measured the effect of vitamin E treatment on
oxidative stress in OP, OR, and control groups. Vitamin E
had a comparable effect in reducing urine free isoprostane
formation (Figure 3A) and a more potent effect in reducing
lipid peroxidation in renal tissue (Figure 3B). In addition,
HNE immunostaining of renal tissue showed strong staining
of the proximal and distal tubules in the cortex of OP
compared with OR rats (Figure 4). In OP rats treated with
pioglitazone, there was a noticeable reduction in HNE stain-
ing in the renal cortex (Figure 4). Vitamin E had a compa-
rable effect in the ability to reduce HNE staining in OP rats
(not shown).
NAD(P)H oxidase has been shown to be one of the major
sources of free radicals in the kidney.19 To test whether piogli-
tazone exerted a genomic effect on different NAD(P)H oxidase
components, we measured protein expression for p47phox,
 Dobrian et al
Downloaded from http://hyper.ahajournals.org/ by guest on May 12, 2017
p67phox, gp91phox, and p22phox by Western blotting in OP,
OR, and control groups of rats with or without pioglitazone
treatment. Both p47phox and gp91phox showed increased ex-
pression in OP compared with OR rats, and pioglitazone
treatment decreased p47phox and gp91phox expression in the
former (Figure 5), suggesting a possible relation between the
level of expression for PPAR and the 2 NAD(P)H oxidase
subunits. Neither p22phox nor p67phox significantly differed in
OP versus OR rats or changed with pioglitazone treatment (not
shown). To verify the specificity of the pioglitazone effect, we
also measured p47phox and gp91phox protein expression in the
Pioglitazone Prevents Hypertension in Obesity 51
Figure 2. Semiquantitative RT-PCR for
PPAR in kidney cortex and medulla of
OP, OR, and control (C) rats without
treatment (top gel) or treated with piogli-
tazone or vitamin E for 12 weeks (bottom
gel). Graphs illustrate densitometric data
from n6 rats per group. PPAR product
has 350 bp (top band on agarose gels),
and -actin has 332 bp (lower band).
Data represent meanSEM. *Significant
vs OP, and control; #significant vs
pioglitazone-treated counterpart; signifi-
cant vs vitamin Etreated counterpart.
vitamin Etreated groups. Vitamin E treatment did not signifi-
cantly change p47phox or gp91phox protein expression in OP,
OR, or control groups (Figure 5).
NOx Production and NOS Expression in OP
Rats With or Without Pioglitazone or
Vitamin E Treatment
In accordance with previous reports,26 NOx excretion was
lower in OP versus OR and control rats, suggesting reduced
NO production and/or availability in the former (Table).
Pioglitazone treatment increased the excreted NO metabolites
 52 Hypertension January 2004
Downloaded from http://hyper.ahajournals.org/ by guest on May 12, 2017
Figure 3. Oxidative stress in OP, OR, and control rats untreated
or treated with either pioglitazone or vitamin E for 12 weeks.
*Significant compared with OR and control; #significant com-
pared with the respective pioglitazone- and vitamin Etreated
groups. Results are meanSEM for 6 rats/group.
in OP rats, with no significant effect in OR and control rats,
whereas vitamin E did not significantly alter NOx excretion in
any of the 3 groups (Table). To further test whether changes
in NOx production were related to a change in expression of
the constitutively expressed NOS isoforms in the kidney,
eNOS and nNOS expression was measured in OP, OR, and
control rats with or without pioglitazone or vitamin E
treatment. We previously reported26 that OP rats have in-
creased eNOS expression in the renal cortex and medulla.
When treated with pioglitazone, OP rats displayed a further
increase in both eNOS and nNOS mRNA in both kidney
cortex and medulla (Figure 6). Also, pioglitazone treatment
increased eNOS and especially nNOS expression in both the
cortex and medulla of OR and control rats. In contrast,
vitamin E treatment did not significantly change either eNOS
or nNOS mRNA expression in the cortex and medulla of OP,
OR, and control groups (Figure 6). The change in mRNA
expression was mirrored by a similar increase in protein
expression for both NOS isoforms (Figure 7). The most
prominent changes were detected for the eNOS isoform in
both the cortex and medulla (Figure 7).
Discussion
In this study, we showed that pioglitazone treatment pre-
vented the development of hypertension and renal oxidative
stress in a rat model of diet-induced obesity. Also, our results
indicate that compared with vitamin E, a well-known antiox-
idant, pioglitazone is more efficient in reducing BP and
increasing sodium excretion in obese versus lean rats, while
being equally efficient in reducing urinary isoprostanes,
kidney TBARS, and HNE adducts. One possible explanation
for these different effects is the ability of pioglitazone, as
opposed to vitamin E, to increase renal NOS expression and
NOx excretion in treated OP rats. In addition, pioglitazone,
but not vitamin E treatment, significantly reduced expression
of p47phox and gp91phox in OP rats, suggesting that both a
reduction in oxidative stress and an improvement in NO
production/bioavailability are important in efficiently reduc-
ing BP in this model.
Thiazolidinediones are known to exert pleiotropic actions
both in vivo and in vitro, some of which occur through their
ability to activate PPAR.5 To the best of our knowledge, this
Figure 4. Immunohistochemical staining for
HNE in the renal cortex of OR and OP rats
with and without pioglitazone treatment. In
sections from OP but not OR rats, strong
staining was detected in cortical tubules and
the thick ascending loop of Henle. Pioglita-
zone treatment reduced HNE staining in the
renal cortex of OP rats. Bottom right pho-
tomicrograph represents control without pri-
mary antibody. Bar10 m.
 Dobrian et al
Downloaded from http://hyper.ahajournals.org/ by guest on May 12, 2017
is the first study to address the in vivo regulation of PPAR
expression in the kidney in an obese, hypertensive model in
response to a dietary challenge. Our results indicate that a
high-fat diet upregulated PPAR mRNA expression in lean
(OR) but not in obese (OP) rats. A similar finding was
reported in a genetic mouse model of obesity, in which lean
but not obese mice exposed to a high-fat diet displayed a
4-fold increase in adipose tissue PPAR expression.30 Nev-
ertheless, because PPAR expression was measured only as
an end point in the experiment when the rats were already
obese, further studies are necessary to delineate whether the
reduced PPAR expression in response to a high-fat diet in
OP rats is a late effect induced by the obese state per se or
whether it occurs earlier, before the onset of obesity. Inter-
estingly, although the high-fat diet failed to upregulate
PPAR expression in OP rats, pioglitazone treatment ele-
Pioglitazone Prevents Hypertension in Obesity 53
Figure 5. Western blotting for gp91phox
and p47phox in total kidney homoge-
nates from OP, OR, and control (C)
groups without treatment or treated with
pioglitazone or vitamin E for 12 weeks.
Top graphs represent densitometric anal-
ysis of n6 rats per experimental group.
Results are meanSEM. Bottom shows
representative immunoblots. The same
amount of protein (20 g) was loaded in
each lane. *Significant vs OR and con-
trol; #significant vs pioglitazone-treated
group; significant vs vitamin Etreated
group.
vated PPAR expression in OP rats. A possible explanation is
that pioglitazone indirectly affects PPAR expression by its
ability to act as an insulin sensitizer. Circulating insulin levels
were 3.5-fold higher in the nontreated versus pioglitazone-
treated OP group. One major intracellular signaling pathway
for insulin involves activation of the phosphatidylinositol
3-kinase (PI3K) cascade.31 A recent report demonstrates that
PI3K overexpression decreases PPAR activity in 3T3-L1
adipocytes.32 It is possible that increased circulating insulin,
through activation of intracellular PI3K, is responsible for the
reduced PPAR expression measured in OP rats, and piogli-
tazone treatment indirectly increases PPAR expression by
reducing circulating insulin and improving insulin sensitivity.
However, at least in part, pioglitazone seems to act indepen-
dently of insulin, because the control normoinsulinemic
group treated with pioglitazone had increased PPAR acti-
 54 Hypertension January 2004
Downloaded from http://hyper.ahajournals.org/ by guest on May 12, 2017

Figure 6. eNOS and nNOS mRNA expression in OP, OR, and control (C) rats with or without pioglitazone or vitamin E treatment.
Graphs represent meanSEM of densitometric data from n6 rats per group. Bottom shows representative agarose gels. *Significant
vs OR and control; #significant vs corresponding pioglitazone-treated group; significant vs corresponding vitamin Etreated group.

vation compared with the nontreated control. Also, oxidative
stress does not seem to be responsible for the reduced PPAR
expression and activity in OP rats, because vitamin E signif-
icantly lowered oxidative stress in the treated OP group but
failed to increase the expression or activity of renal PPAR in
the latter.
Interestingly, recent studies suggest a direct role for free
radicals and NO availability in the renal cortex and medulla
in sodium reabsorption and development of hypertension.16,33
The inability of vitamin E, as opposed to pioglitazone
treatment, to increase sodium excretion or totally prevent the
development of hypertension in OP rats could be explained
 Dobrian et al
Downloaded from http://hyper.ahajournals.org/ by guest on May 12, 2017
by its inability to upregulate NOS expression or increase NOx
excretion. Our results suggest that vitamin E treatment, despite
reducing oxidative stress, could not significantly improve NO
bioavailability. In contrast, pioglitazone treatment both reduced
oxidative stress and significantly increased NOx excretion in OP
rats. The result could be explained by the ability of pioglitazone
to also enhance NO availability by a mechanism independent of
free-radical production. Our results indicate that pioglitazone
treatment upregulates the expression of eNOS and nNOS at both
the RNA and protein levels in the renal cortex and medulla of
OP rats. The effect might or might not be directly mediated by
PPAR activation.
Finally, the mechanisms involved in the reduction of
oxidative stress by pioglitazone are largely unknown. A
recent article has demonstrated the ability of both pioglita-
zone and rosiglitazone to reduce nitrotyrosine formation in a
mouse model of rheumatoid arthritis.34 Yet another mecha-
Pioglitazone Prevents Hypertension in Obesity 55
Figure 7. Effect of pioglitazone (Pio)
treatment on eNOS and nNOS protein
expression in OP rats. Densitometry and
representative Western immunoblots of
nNOS (top) and eNOS (bottom) in renal
cortex and medulla in OP rats with or
without pioglitazone treatment. Results
represent meanSEM of 6 rats per
group.
nism suggested by our data seems to be via downregulation of
gp91phox and p47phox protein expression. The results
showed upregulation of these 2 NAD(P)H oxidase subunits in
OP versus OR and control groups without treatment. The
pioglitazone-treated OP group had reduced expression of
both proteins in the kidney compared with their nontreated
counterparts. It is difficult to conclude whether the effect of
pioglitazone on NAD(P)H oxidase subunits was mediated by
PPAR activation. One recent article indicated that activation
of PPAR in macrophages results in downregulation of
p47phox and attenuation of the respiratory burst.22 Also,
p47phox was identified in a variety of renal epithelial,
mesangial, and vascular cells and was shown to be increased
in prehypertensive spontaneously hypertensive rats.20 There-
fore, the latter could be a key target involved in regulation of
superoxide production by PPAR agonists in both renal and
vascular cells.
 56 Hypertension January 2004
Perspectives
This study shows for the first time the in vivo regulation of
PPAR expression and activity in the kidney by a high-fat
diet and pioglitazone treatment. Also, to the best of our
knowledge, this is the first report on the ability of pioglita-
zone, possibly via a PPAR ligand dependent mechanism, to
prevent renal oxidative stress and to improve NO production
in diet-induced obese rats. This effect is possibly achieved by
upregulation of renal eNOS and nNOS, by as-yet-unknown
mechanisms, as well as by reduced superoxide production
because of downregulation of NAD(P)H oxidase subunits.
Owing to the ability of pioglitazone to prevent hypertension
and increase sodium excretion, further studies will be impor-
tant to determine the major cell types involved and the exact
mechanisms by which PPAR could regulate sodium and
water handling by the kidney.
Acknowledgments
This study was supported by National Institutes of Health grants
Downloaded from http://hyper.ahajournals.org/ by guest on May 12, 2017
HL68726 and HD38240 and a postdoctoral fellowship from the
Mid-Atlantic Affiliate of the American Heart Association to Dr Anca
Dobrian.
References
1. Mangelsdorf DJ, Thummel C, Beato M, Herrlich P, Schutz G, Umesono
K, Blumberg B, Kastner P, Mark M, Chambon P. The nuclear receptor
superfamily: the second decade. Cell. 1995;83:835 839.
2. Spiegelman BM. PPAR-: adipogenic regulator and thiazolidinedione
receptor. Diabetes. 1998;47:507514.
3. Barroso I, Gurnell M, Crowley VE, Agostini M, Schwabe JW, Soos MA,
Maslen GL, Williams TD, Lewis H, Schafer AJ, Chatterjee VK,
ORahilly S. Dominant negative mutations in human PPAR associated
with severe insulin resistance, diabetes mellitus and hypertension. Nature.
1999;402:880 883.
4. Savage DB, Tan GD, Acerini CL, Jebb SA, Agostini M, Gurnell M,
Williams RL, Umpleby AM, Thomas EL, Bell JD, Dixon AK, Dunne F,
Boiani R, Cinti S, Vidal-Puig A, Karpe F, Chatterjee VK, ORahilly S.
Human metabolic syndrome resulting from dominant-negative mutations
in the nuclear receptor peroxisome proliferator-activated receptor-.
Diabetes. 2003;52:910 917.
5. Forman BM, Chen J, Evans RM. The peroxisome proliferator-activated
receptors: ligands and activators. Ann N Y Acad Sci. 1996;804:266 275.
6. Walker AB, Chattington PD, Buckingham RE, Williams G. The thiazo-
lidinedione rosiglitazone (BRL-49653) lowers blood pressure and
protects against impairment of endothelial function in Zucker fatty rats.
Diabetes. 1999;48:1448 1453.
7. Uchida A, Nakata T, Hatta T, Kiyama M, Kawa T, Morimoto S, Miki S,
Moriguchi J, Nakamura K, Fujita H, Itoh H, Sasaki S, Takeda K,
Nakagawa M. Reduction of insulin resistance attenuates the development
of hypertension in sucrose-fed SHR. Life Sci. 1997;61:455 464.
8. Yoshimoto T, Naruse M, Nishikawa M, Naruse K, Tanabe A, Seki T,
Imaki T, Demura R, Aikawa E, Demura H. Antihypertensive and vasculo-
and renoprotective effects of pioglitazone in genetically obese diabetic
rats. Am J Physiol. 1997;272:E989 E996.
9. Fullert S, Schneider F, Haak E, Rau H, Badenhoop K, Lubben G, Usadel
KH, Konrad T. Effects of pioglitazone in nondiabetic patients with
arterial hypertension: a double-blind, placebo-controlled study. J Clin
Endocrinol Metab. 2002;87:55035506.
10. Nicholas SB, Kawano Y, Wakino S, Collins AR, Hsueh WA. Expression
and function of peroxisome proliferator-activated receptor- in mesangial
cells. Hypertension. 2001;37:722727.
11. Yang T, Michele DE, Park J, Smart AM, Lin Z, Brosius FC 3rd,
Schnermann JB, Briggs JP. Expression of peroxisomal proliferator-acti-
vated receptors and retinoid X receptors in the kidney. Am J Physiol.
1999;277:F966 F973.
12. Guan Y, Zhang Y, Davis L, Breyer MD. Expression of peroxisome
proliferator-activated receptors in urinary tract of rabbits and humans.
Am J Physiol. 1997;273:F1013F1022.
13. Guan Y, Zhang Y, Schneider A, Davis L, Breyer RM, Breyer MD.
Peroxisome proliferator-activated receptor- activity is associated with
renal microvasculature. Am J Physiol Renal Physiol. 2001;281:
F1036 F1046.
14. Van Gaal LF, Vertommen J, De Leeuw IH. The in vitro oxidizability of
lipoprotein particles in obese and non-obese subjects. Atherosclerosis.
1998;137(suppl):S39 S44.
15. Perticone F, Ceravolo R, Candigliota M, Ventura G, Iacopino S, Sinopoli
F, Mattioli PL. Obesity and body fat distribution induce endothelial
dysfunction by oxidative stress: protective effect of vitamin C. Diabetes.
2001;50:159 165.
16. Ortiz PA, Garvin JL. Interaction of O2 and NO in the thick ascending
limb. Hypertension. 2002;39:591596.
17. Schnackenberg CG, Welch WJ, Wilcox CS. TP receptor-mediated vaso-
constriction in microperfused afferent arterioles: roles of O2 and NO.
Am J Physiol Renal Physiol. 2000;279:F302F308.
18. Pryor WA, Squadrito GL. The chemistry of peroxynitrite: a product from
the reaction of nitric oxide with superoxide [see comments]. Am J
Physiol. 1995;268:L699 L722.
19. Li N, Yi FX, Spurrier JL, Bobrowitz CA, Zou AP. Production of
superoxide through NADH oxidase in thick ascending limb of Henles
loop in rat kidney. Am J Physiol Renal Physiol. 2002;282:F1111F1119.
20. Chabrashvili T, Tojo A, Onozato ML, Kitiyakara C, Quinn MT, Fujita T,
Welch WJ, Wilcox CS. Expression and cellular localization of classic
NADPH oxidase subunits in the spontaneously hypertensive rat kidney.
Hypertension. 2002;39:269 274.
21. Inoue I, Goto S, Matsunaga T, Nakajima T, Awata T, Hokari S, Komoda
T, Katayama S. The ligands/activators for peroxisome proliferator-acti-
vated receptor- (PPAR) and PPAR increase Cu2,Zn2-superoxide
dismutase and decrease p22phox message expressions in primary endo-
thelial cells. Metabolism. 2001;50:311.
22. Von Knethen A, Brune B. Activation of peroxisome proliferator-activated
receptor- by nitric oxide in monocytes/macrophages down-regulates
p47phox and attenuates the respiratory burst. J Immunol. 2002;169:
2619 2626.
23. Garg R, Kumbkarni Y, Aljada A, Mohanty P, Ghanim H, Hamouda W,
Dandona P. Troglitazone reduces reactive oxygen species generation by
leukocytes and lipid peroxidation and improves flow-mediated vasodila-
tation in obese subjects. Hypertension. 2000;36:430 435.
24. Dobrian AD, Davies MJ, Prewitt RL, Lauterio TJ. Development of
hypertension in a rat model of diet-induced obesity. Hypertension. 2000;
35:1009 1015.
25. Lauterio TJ, Barkan A, DeAngelo M, DeMott-Friberg R, Ramirez R.
Plasma growth hormone secretion is impaired in obesity-prone rats before
onset of diet-induced obesity. Am J Physiol. 1998;275:E6 E11.
26. Dobrian AD, Davies MJ, Schriver SD, Lauterio TJ, Prewitt RL. Oxidative
stress in a rat model of obesity-induced hypertension. Hypertension.
2001;37:554 560.
27. Chomczynski P. A reagent for the single-step simultaneous isolation of
RNA, DNA and proteins from cell and tissue samples. Biotechniques.
1993;15:532534, 536 537.
28. Dobrian AD, Schriver SD, Prewitt RL. Role of angiotensin II and free
radicals in blood pressure regulation in a rat model of renal hypertension.
Hypertension. 2001;38:361366.
29. Lauterio TJ, Davies MJ, DeAngelo M, Peyser M, Lee J. Neuropeptide Y
expression and endogenous leptin concentrations in a dietary model of
obesity. Obes Res. 1999;7:498 505.
30. Vidal-Puig A, Jimenez-Linan M, Lowell BB, Hamann A, Hu E,
Spiegelman B, Flier JS, Moller DE. Regulation of PPAR gene
expression by nutrition and obesity in rodents. J Clin Invest. 1996;97:
25532561.
31. Cantley LC. The phosphoinositide 3-kinase pathway. Science. 2002;296:
16551657.
32. Watanabe M, Inukai K, Katagiri H, Awata T, Oka Y, Katayama S.
Regulation of PPAR transcriptional activity in 3T3-L1 adipocytes.
Biochem Biophys Res Commun. 2003;300:429 436.
33. Makino A, Skelton MM, Zou AP, Roman RJ, Cowley AW Jr. Increased
renal medullary oxidative stress produces hypertension. Hypertension.
2002;39:667 672.
34. Shiojiri T, Wada K, Nakajima A, Katayama K, Shibuya A, Kudo C,
Kadowaki T, Mayumi T, Yura Y, Kamisaki Y. PPAR ligands inhibit
nitrotyrosine formation and inflammatory mediator expressions in
adjuvant-induced rheumatoid arthritis mice. Eur J Pharmacol. 2002;448:
231238.
 Pioglitazone Prevents Hypertension and Reduces Oxidative Stress in Diet-Induced Obesity
Anca D. Dobrian, Suzanne D. Schriver, Ali A. Khraibi and Russell L. Prewitt

Hypertension. 2004;43:48-56; originally published online November 24, 2003;

doi: 10.1161/01.HYP.0000103629.01745.59
Downloaded from http://hyper.ahajournals.org/ by guest on May 12, 2017

Hypertension is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
Copyright 2003 American Heart Association, Inc. All rights reserved.
Print ISSN: 0194-911X. Online ISSN: 1524-4563

The online version of this article, along with updated information and services, is located on the
World Wide Web at:
http://hyper.ahajournals.org/content/43/1/48

Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally published
in Hypertension can be obtained via RightsLink, a service of the Copyright Clearance Center, not the Editorial
Office. Once the online version of the published article for which permission is being requested is located,
click Request Permissions in the middle column of the Web page under Services. Further information about
this process is available in the Permissions and Rights Question and Answer document.

Reprints: Information about reprints can be found online at:

http://www.lww.com/reprints

Subscriptions: Information about subscribing to Hypertension is online at:

http://hyper.ahajournals.org//subscriptions/