Download Dietary Iron Restriction Prevents Hypertensive

Dietary Iron Restriction Prevents Hypertensive
Cardiovascular Remodeling in Dahl Salt-Sensitive Rats
Yoshiro Naito, Shinichi Hirotani, Hisashi Sawada, Hirokuni Akahori,
Takeshi Tsujino, Tohru Masuyama
See Editorial Commentary, pp 381–382
Downloaded from http://hyper.ahajournals.org/ by guest on June 12, 2017
Abstract—Iron accumulation is associated with the pathogenesis of several cardiovascular diseases. However, the
preventive effects of iron restriction (IR) against cardiovascular disease remain obscure. We investigated the effects of
dietary IR on cardiovascular pathophysiology and the involved mechanism in Dahl salt-sensitive rats. Dahl salt-sensitive
rats were provided either a normal or high-salt (HS) diet. Another subset of Dahl salt-sensitive rats were fed an HS with
iron-restricted (HS⫹IR) diet for 11 weeks. Dahl salt-sensitive rats given an HS diet developed hypertension, heart
failure, and decreased a survival rate after 11 weeks on the diet. In contrast, IR attenuated the development of
hypertension and heart failure, thereby improving survival rate. Dietary IR suppressed cardiovascular hypertrophy,
fibrosis, and inflammation in HS rats. The phosphorylation of Akt, AMP-activated protein kinase, and endothelial nitric
oxide synthase was decreased in the aorta of HS rats, whereas they were ameliorated by the IR diet. Aortic expression
of the cellular iron import protein transferrin receptor 1, and the iron storage protein ferritin H-subunit, was upregulated
in HS rats. IR also attenuated proteinuria and increased oxidative stress in the HS group. NG-nitro-L-arginine methyl
ester abolished the beneficial effects of IR and decreased survival rate in HS⫹IR rats. Dietary IR had protective effects
on salt-induced hypertension, cardiovascular remodeling, and proteinuria through the inhibition of oxidative stress, and
maintenance of Akt, AMP-activated protein kinase, and endothelial nitric oxide synthase in the aorta. IR could be an
effective strategy for prevention of HS-induced organ damage in salt-sensitive hypertensive patients. (Hypertension.
2011;57:497-504.) ● Online Data Supplement
Key Words: anemia 䡲 dahl salt-sensitive rats 䡲 heart failure 䡲 hypertension 䡲 iron restriction
䡲 nitric oxide synthesis 䡲 oxidative stress
I
ron plays an important role in maintaining physiological
homeostasis in the body (ie, enzymatic reactions and
oxygen transport). However, excess iron can lead to free
radical damage by the Fenton reaction, resulting in tissue
damage. During the past decade, iron has been associated
with the pathogenesis of some cardiovascular diseases. For
instance, iron deposition is related to development of atherosclerosis, and an experimental study showed that an irondeficient diet reduces atherosclerotic lesions in apolipoprotein-E– deficient mice. 1 In addition, a multi-center,
randomized, controlled, single-blinded clinical trial showed
that cancer risk and cancer-specific and all-cause mortality
were lower in the iron reduction (IR) group than in the control
group in patients with peripheral arterial disease.2 These
results suggest that excess total body iron stores are associated with the occurrence of cancer and longevity in patients
with peripheral arterial disease.
Caloric restriction has been shown to extend longevity by
retarding the aging process.3 Calorie restriction also reduces
blood pressure and dyslipidemia.4 In cardiovascular diseases,
several experiments with animal studies have shown that
calorie restriction has protective effects against cardiac hypertrophy, salt-induced cardiac remodeling,5 and ischemiareperfusion injury.6 However, the effects of only IR on
cardiovascular disease, particularly salt-induced organ damage, remain largely unknown. In the present study, we
investigated the effects of dietary IR on salt-induced cardiovascular pathophysiology and its involved mechanism in
Dahl salt-sensitive rats. Our observations indicate that dietary
IR has salutary effects on salt-induced hypertension, cardiovascular remodeling, and proteinuria by reducing oxidative
stress and by improving impairment of vascular Akt, AMPactivated protein kinase (AMPK), and endothelial nitric oxide
synthase (eNOS) signaling.
Received July 16, 2010; first decision August 10, 2010; revision accepted December 22, 2010.
From the Cardiovascular Division, Department of Internal Medicine (Y.N., S.H., H.S., H.A., T.M.), Hyogo College of Medicine, Nishinomiya, Japan;
Department of Pharmacy (T.T.), Hyogo University of Health Sciences, Kobe, Japan.
Correspondence to Yoshiro Naito, Cardiovascular Division, Department of Internal Medicine, Hyogo College of Medicine, 1-1 Mukogawa-cho,
Nishinomiya 663-8501, Japan. E-mail [email protected]
© 2011 American Heart Association, Inc.
Hypertension is available at http://hyper.ahajournals.org
DOI: 10.1161/HYPERTENSIONAHA.110.159681
497
498
Hypertension
A
March 2011
B
BW
g
400
Downloaded from http://hyper.ahajournals.org/ by guest on June 12, 2017
Systtolic Blood Prressure
Hemoglobin
*
200
*
100
0
C
*†
300
6
10
18 age
(weeks)
14
*
200
*
*
*†
*†
100
0
6
10
18 age
(weeks)
14
*†
10
*
*
*†
5
D
SBP
mmHg
300
15
0
6
10
14
18
age
(weeks)
Survival Rate
%
100
Survival Ratte
Body Weightt
500
Hb
g /d L
20
80
Figure 1. Effect of dietary iron restriction on physiological parameters and prognosis in Dahl saltsensitive rats. Time course of (A) body weight, (B)
hemoglobin, (C) SBP, and (D) survival rate in the
control (white circle, n⫽8), HS (black circle, n⫽10),
and HS⫹IR (gray circle, n⫽8) groups. *P⬍0.05 vs
the control group at the corresponding time point;
†P⬍0.05 vs the HS group at the corresponding
time point.
60
*†
40
20
0
6
10
14
Methods
Animals
Five-week-old male Dahl salt-sensitive rats (Japan SLC) were fed a
control diet (0.3% NaCl) for 1 week. Afterward, rats were randomly
assigned to 3 groups and were given a normal salt diet (control; 0.3%
NaCl; n⫽12), a high-salt (HS) diet (8% NaCl; n⫽12), or an HS with
iron-restricted diet (HS⫹IR; n⫽12) for 12 weeks. Regular rat chow
was supplemented with approximately 0.003% of FeC6H5O7䡠H2O.
Rats of the HS⫹IR group were given a diet not supplemented with
FeC6H5O7䡠H2O as previously described.7 In a separate study, 10week-old HS⫹IR group rats, which had been fed that diet from 6
weeks of age, were divided into 2 groups and were given a HS⫹IR
diet with plain drinking water (n⫽6) or NG-nitro-L-arginine methyl
ester (L-NAME; Sigma-Aldrich), a specific nitric oxide (NO) synthase inhibitor (0.25 mg/mL in drinking water; n⫽6). Rats were
maintained on a 12-hour light/dark cycle and had free access to food
and water. All our experimental procedures were approved by the
Animal Research Committee of Hyogo College of Medicine. An
expanded Methods section is available in the online data supplement
at http://hyper.ahajournals.org.
Gene Expression Analysis
Total RNA was extracted from the tissue using TRIzol reagent
(Invitrogen).8 Real-time polymerase chain reactions were performed
using the ABI PRISM 7900 with TaqMan Universal PCR Master
Mix and TaqMan Gene Expression Assays (Applied Biosystems).7
Glyceraldehyde-3-phosphatedehydrogenase was used as an internal
control.
Western Blot Analysis
The total protein homogenate from the aorta was separated by
sodium dodecyl sulfate polyacrylamide gel electrophoresis and
transferred onto polyvinylidene fluoride membranes. The expression
levels of proteins were detected by an enhanced chemiluminescence
kit (Thermo Scientific). Here, the antibodies used were against rabbit
antiphospho-Akt (Ser473), Akt, phospho-AMPK (Thr172), pan
␣AMPK, phospho-eNOS (Ser1177), phospho-extracellular signal–
regulated kinase (ERK) (Thr202/Tyr204), ERK (Cell Signaling
Technology; dilution 1:1000), rabbit anti-eNOS (Santa Cruz; dilution 1:1000), mouse antitransferrin receptor 1 (TfR1; Zymed Laboratories; dilution 1:1000), goat antiferritin H-subunit (Santa Cruz;
dilution 1:200), and mouse anti-␤-actin (Abcam; dilution 1:1000).
18 age
(weeks)
Histological Analysis
Aorta and kidney tissues were quickly embedded in Tissue-Tek
OCT compound (Sakura Finetechnical Co.) and were snap frozen
in liquid nitrogen. Aortic sections were stained with Masson’s
trichrome (MT) and immunohistochemically stained with a primary mouse anti-CD68 antibody (AbD Serotec; dilution 1:1000)
and a primary mouse anti-TfR1 antibody (Zymed Laboratories;
dilution 1:400). Nonimmune immunoglobulin G of the same
species was used as a negative control. Superoxide detection was
performed on transverse cross-sections 8-␮m thick, incubated
with dihydroethidium (DHE; 10 ␮mol/L, 37° C for 30 minutes;
Molecular Probes).
Statistical Analysis
Values are reported as the means⫾SEM. Statistical analysis was
performed using one-way ANOVA. ANOVA (Kruskal-Wallis test,
followed by Mann–Whitney U test) was used for statistical comparisons. Survival rate was assessed by the Kaplan–Meier survival
curves. We considered differences to be significant when the
probability value was ⬍0.05.
Results
Effects of Iron Restriction on
Physiological Parameters
Body weight decreased significantly in the HS group after
14 weeks of age compared with the other groups, while the
HS⫹IR group did not lose body weight until 18 weeks of
age, and the degree of loss was smaller than in the HS
group at the same time (Figure 1A); this suggests that IR
in the presence of HS diet prevented the onset of cachexia
with the development of heart failure. Dietary IR-induced
anemia was measured by hemoglobin content (g/dL) in all
groups studied, whereas blood hemoglobin was comparable between control and HS groups until 10 weeks of age
and began to decrease in the HS group thereafter. Finally,
blood hemoglobin was lower in the HS⫹IR group than in
the HS group at 18 weeks of age (Figure 1B). Conversely, HS
diet resulted in a progressive increase in systolic blood pressure
(SBP) after diet, while SBP did not begin to increase in the
Iron Restriction and Salt-Sensitive Hypertension
Relative Expression
Collagen III
MT
CD68
Control
HS
E
3
2
*
†
1
0
HS
+
IR
Control HS
HS+IR
C
1.5
*
1.0
†
0.5
0
Control HS
HS
+
IR
499
CD68
3
*
2
*†
1
0
HS
+
IR
Control HS
H
G
F
D
TGF-β
Relative Expression
B
A
Relative Expression
Naito et al
p-Akt
p-AMPK
p-eNOS
Akt
AMPK
eNOS
p-ERK
1
*
*
0
Control HS
HS
+
IR
†
0.6
*
0
HS
Control HS +
IR
†
1
*
0
Control HS
HS
+
IR
p-E
ERK/ERK
2
2
2
1.2
p-e N
NOS/eNOS
Downloaded from http://hyper.ahajournals.org/ by guest on June 12, 2017
p--Akt/Akt
†
p-A M
MPK/AMPK
ERK
3
*
*
1
0
Control HS
HS
+
IR
Figure 2. Effect of dietary iron restriction on aortic histology, gene expression, and phosphorylation of aortic signal pathway in Dahl
salt-sensitive rats. A, Representative images of MT and CD68 staining of the aorta sections. Scale bars: 50 ␮m. Aortic gene expression
of (B) Collagen III, (C) TGF-␤, and (D) CD68 in the control (white bar, n⫽6), HS (black bar, n⫽6), and HS⫹IR (gray bar, n⫽6) groups.
Gene expression of Collagen III, TGF-␤, and CD68 were normalized with glyceraldehyde-3-phosphatedehydrogenasegene expression,
and relative levels of gene expression are shown in the graph. Expression of phosphorylated (top) and total (bottom) state of (E) Akt, (F)
AMPK, (G) eNOS, and (H) ERK in the aortas of the control (white bar, n⫽6), HS (black bar, n⫽6), and HS⫹IR (gray bar, n⫽6) groups.
Top: Representative Western blot analysis. Bottom: Densitometric analysis. Expression of phosphorylated Akt, AMPK, eNOS, and ERK
was standardized on the basis of total Akt, AMPK, eNOS, and ERK expression, and relative levels of expression are plotted in the
graphs. *P⬍0.05 vs the control group; †P⬍0.05 vs the HS group.
HS⫹IR group until 14 weeks of age and the increase was
smaller than in the HS group (Figure 1C); this indicates that IR
in the presence of HS diet inhibited the increase in SBP.
During the experimental period, some rats died in the HS
group; however, none of rats died in the HS⫹IR group.
Kaplan–Meier analysis showed that survival rate of the
HS⫹IR group was greater than that of the HS group (Figure
1D).
Iron Restriction Reduced Vascular Hypertrophy, Fibrosis,
and Inflammation in High Salt-Induced Hypertension
Since IR rats did not develop hypertension, we evaluated
vascular hypertrophy, fibrosis, and inflammation in these
groups. MT staining showed vascular hypertrophy and increased fibrotic area in the HS group compared with the
control group, while these changes were dramatically less
pronounced in the HS⫹IR group (Figure 2A). Consistently,
aortic mRNA expression of collagen type III and transforming growth factor-␤ was increased in the HS group but was
suppressed in the HS⫹IR group (Figure 2 B,C). There was a
marked increase in CD68 staining and gene expression in the
aorta of the HS group compared with the control group, but
much less compared with the HS⫹IR group. CD68 positive
staining was mainly seen in the adventitia (Figure 2A,D).
These results indicate that dietary IR attenuated high saltinduced vascular remodeling.
To clarify the mechanisms by which IR has beneficial
effects against the development of hypertension, we eval-
uated molecular signal pathways in the aorta of the HS⫹IR
group. The phosphorylation of Akt at Ser473 in the aorta
was decreased in the HS group compared with the control
group, whereas this change was prevented in the HS⫹IR
group (Figure 2E). The phosphorylation of AMPK at
Thr172 and eNOS at Ser1177 in the aorta was also
decreased in the HS group compared with the control
group; however, these changes were not observed in the
HS⫹IR group (Figure 2F,G). In contrast, the phosphorylation of ERK at Thr202/Tyr204 in the aorta increased in
both HS and HS⫹IR groups compared with the control
group (Figure 2H).
TfR1 Expression Was Increased in the Aorta Under
High-Salt Diet
To investigate how iron intake affects HS-induced hypertension, we evaluated intracellular iron transport proteins,
such as TfR1 and ferritin H- and L-subunits in the aorta of
these groups. TfR1 is required for the uptake of
transferrin-bound iron into the cells.9 Interestingly, aortic
TfR1 gene and protein expression was significantly increased in both HS and HS⫹IR groups compared with the
control group, but the extent to which it increased was
higher in the HS group than in the HS⫹IR group (Figure
3A–C). Immunohistochemical analysis showed that TfR1
was largely expressed in the media (Figure 3C). Aortic
ferritin H-subunit gene expression was increased in the HS
groups compared with the control group, while ferritin
Hypertension
Relative Expression
A
March 2011
B
TfR1
*
5
4
*†
3
2
1
0
HS
+
IR
Control HS
Table 1. Physiological and Echocardiac Parameters in all
Groups at 18 Weeks of Age
TfR1
β-actin
Relative Expression
500
Parameter
4
*
*†
2
0
Control
HS
HS
+
IR
C
TfR1
negative
control
2
*
*†
1
0
Control HS
HS+IR
Ferritin L
E
Relative Expression
Relative Expression
HS
Ferritin H
D
HS
+
IR
F
2
1
*†
0
Control
HS
HS
+
IR
Control HS
HS
+
IR
G
Ferritin H
β-actin
ug/dry g
2
100
*
Iron content
Relative Expression
Downloaded from http://hyper.ahajournals.org/ by guest on June 12, 2017
Control
*†
1
50
0
0
Control HS
HS
+
IR
H
Control
HS
HS+IR
Figure 3. Expression of intracellular iron transport proteins in Dahl
salt-sensitive rats. Aortic (A) gene and (B) protein expression of
TfR1 in the control (white bar, n⫽6), HS (black bar, n⫽6), and
HS⫹IR (gray bar, n⫽6) groups. C, Representative images of TfR1
staining of the aorta sections. Scale bars: 50 ␮m. Aortic gene
expression of (D) ferritin H- and (E) ferritin L-subunits, (F) protein
expression of ferritin H-subunit, and (G) iron contents in the control
(white bar, n⫽6), HS (black bar, n⫽6), and HS⫹IR (gray bar, n⫽6)
groups. H, Representative images of DHE staining of the aorta sections. Scale bars: 50 ␮m. Gene expression of TfR1, ferritin H- and
ferritin L-subunits was normalized with glyceraldehyde-3phosphatedehydrogenasegene expression, and relative levels of gene
expression are shown in the graph. Protein expression of TfR1 and
ferritin H-subunit was standardized on the basis of ␤-actin expression,
and relative levels of expression are plotted in the graphs. *P⬍0.05 vs
the control group; †P⬍0.05 vs the HS group.
Control
HS
HS⫹IR
LV weight/tibia length (mg/mm)
22.2⫾0.5
32.9⫾0.9*
28.8⫾0.7*†
Lung weight/tibia length (mg/mm)
40.5⫾1.1
108.1⫾5.3*
42.1⫾0.7†
Diastolic wall thickness of LV
posterior wall (mm)
1.8⫾0.0
2.4⫾0.0*
2.2⫾0.1*†
LV end-diastolic dimension (mm)
8.2⫾0.1
8.9⫾0.1
8.4⫾0.2
LV end-systolic dimension (mm)
5.2⫾0.1
5.7⫾0.4
5.1⫾0.2
LV fractional shortening (%)
34.8⫾1.1
34.6⫾2.9
39.8⫾2.5
Early diastolic filling wave (cm/s)
84.9⫾5.0
117.2⫾10.1*
96.8⫾5.2
Peak filling velocity at atrial
contraction (cm/s)
36.3⫾2.2
49.0⫾8.8
72.6⫾3.0*†
E/A ratio
2.4⫾0.2
2.8⫾0.7
1.3⫾0.1*†
Deceleration time (msec)
39⫾2
31⫾1*
46⫾4†
E/A ratio indicates the ratio of peak early diastolic filling velocity and peak
filling velocity at atrial contraction; Control, Dahl salt-sensitive rats fed normal
salt diet; HS, Dahl salt-sensitive rats fed high-salt diet; HS⫹IR, Dahl
salt-sensitive rats fed high-salt with iron-restricted diet; LV, left ventricle.
*P⬍0.05 vs Control group.
†P⬍0.05 vs HS group. n⫽6 per group.
L-subunit gene expression was not increased in the HS
group. In contrast, both ferritin H- and L-subunits gene
expression was decreased in the HS⫹IR group compared
with the other groups (Figure 3D,E). Aortic ferritin
H-subunit protein expression was consistent with the gene
expression (Figure 3F). Tissue iron content of the aorta
increased in the HS group, while it decreased in the
HS⫹IR group, relative to the control group (Figure 3G).
To investigate further whether dietary IR exerts local
antioxidant effect in the aorta, we examined superoxide
production by staining of the aorta with DHE. The aorta in the
HS group showed a higher fluorescent signal compared with
other groups. The HS⫹IR group showed significantly decreased vascular production of superoxide (Figure 3H).
Effects of Iron Restriction on Cardiac Function
As shown in Table 1, HS diet induced a marked increase in
left-ventricle-(LV)-weight-to-tibia-length ratio compared
with the control group at 18 weeks of age, demonstrating
cardiac hypertrophy. Lung-weight-to-tibia-length ratio was
also increased in the HS group relative to the control group,
indicating pulmonary congestion. However, IR⫹HS diet
inhibited the increase in LV-weight-to-tibia-length ratio and
lung-weight-to-tibia-length ratio, suggesting that IR attenuated development of heart failure.
Echocardiographic analysis showed that LV hypertrophy
was evident at 18 weeks of age in the HS group, whereas it
was attenuated in the HS⫹IR group. LV cavity size and
fractional shortening were comparable among the 3 groups. E
wave was higher and deceleration time shortened in the HS
group compared with the other groups, whereas A wave was
higher and deceleration time was prolonged; this resulted in
decreasing E/A ratio in the HS⫹IR group relative to the other
groups at 18 weeks of age.
Histological analysis revealed that the cross-sectional
area of cardiomyocytes increased in both HS and HS⫹IR
Rela
ative Expresssion
D
HS
E
ANP
60
*
40
*†
20
0
Control
HS
HS
+
IR
5
*†
400
200
0
Control HS
HS+IR
Rela
ative Expresssion
Control
4
F
*
4
3
†
2
1
0
Control
HS
HS
+
IR
Downloaded from http://hyper.ahajournals.org/ by guest on June 12, 2017
groups at 18 weeks of age compared with the control
group, but the increase in cross-sectional area of cardiomyocytes was attenuated more in the HS⫹IR group than in
the HS group (Figure 4A,B). In addition, cardiac interstitial fibrosis increased in the HS and HS⫹IR groups at 18
weeks of age compared with the control group; however, it
was reduced significantly in the HS⫹IR group compared
with the HS group (Figure 4A,C); this suggests that
IR⫹HS diet inhibited increased LV hypertrophy and
interstitial fibrosis. Consistent with these changes, dietary
IR suppressed the increased expression of atrial natriuretic
peptide, collagen type I, and CD68 mRNA in the heart of
the HS group (Figure 4D–F). These results also supported
the finding that dietary IR attenuated the development of
LV hypertrophy and heart failure.
Iron Restriction Attenuated Renal Injury Under
High-Salt Diet
Next, we evaluated proteinuria and urinary 8-Hydroxy-2⬘deoxyguanosine (8-OHdG) concentration in these animals.
Proteinuria and urinary 8-OHdG/creatinin ratio increased in
the HS group compared with the other groups, while they
were suppressed in the HS⫹IR group compared with the HS
group (Figure 5A,B). DHE staining showed that there was
increased superoxide production in the kidney of the HS
group compared with the other groups; whereas in the
HS⫹IR group, there was decreased renal production of
superoxide (Figure 5C). These data indicated that dietary IR
inhibited the development of renal injury and oxidative stress
under HS diet.
Abolished the Beneficial Effects of Iron
Restriction Under High-Salt Diet
To determine whether IR has beneficial effects through
NO-mediated pathway, we next explored the effects of
L-NAME in the HS⫹IR group. After administration of
L-NAME, SBP markedly increased in the HS⫹IR group and
became similar to that of the HS group (Figure 6A). In
addition, L-NAME treatment abrogated the decreased proteinuria in the HS⫹IR group (Figure 6B). Consistent with these
changes, L-NAME led to the decreased survival rate in the
2
†
1
0
HS
+
IR
Collagen I
*
3
Control HS
HS
+
IR
CD68
5
*
4
†
3
2
1
0
Control
HS
501
Figure 4. Effect of dietary iron restriction
on cardiac histology and gene expression
in Dahl salt-sensitive rats. A, Representative images of hematoxylin and eosin and
MT staining of the heart sections. Scale
bars: 10 ␮m for hematoxylin and eosin
and 100 ␮m for MT staining. Quantitative
analysis of (B) cardiac myocyte crosssectional area and (C) myocardial interstitial fibrosis in the control (white bar), HS
(black bar), and HS⫹IR (gray bar) groups.
Cardiac gene expression of (D) atrial natriuretic peptide, (E) Collagen I, and (F) CD68
in the control (white bar, n⫽6), HS (black
bar, n⫽6), and HS⫹IR (gray bar, n⫽6)
groups. Gene expression of atrial natriuretic
peptide, collagen type I, and CD68 was normalized with glyceraldehyde-3phosphatedehydrogenasegene expression,
and relative levels of gene expression are
shown in the graph. *P⬍0.05 vs the control
group; †P⬍0.05 vs the HS group.
%
HS
+
IR
HS⫹IR group (Figure 6C). Taken together, L-NAME abolished the beneficial effects of IR under HS diet. Dietary IR
had protective effects on salt-induced organ damage through
NO-mediated pathway.
Discussion
This study demonstrated that dietary IR has beneficial
effects on cardiovascular remodeling in Dahl salt-sensitive
rats. IR attenuated the development of hypertension, LV
hypertrophy, heart failure, and proteinuria, thereby improving survival rate in Dahl salt-sensitive rats through the
inhibition of oxidative stress; it also maintained Akt,
AMPK, and eNOS signaling in the aorta. L-NAME abolished the beneficial effects of IR.
TP/Cre
A
mg/mg
To
otal protein/crreatinin Ratio
o
MT
*
600
Area
Fibrosis A
Cross-sectional Area
C
HE
C
µm2
800
Rela
ative Expresssion
B
A
Iron Restriction and Salt-Sensitive Hypertension
ng/mg
*
40
30
20
†
10
0
Control
HS
8-OHdG/Cre
B
8-OHdG/creatinin Ratio
Naito et al
HS
+
IR
1.0
*
0.5
†
0
Control
HS
HS
+
IR
C
L-NAME
Control
HS
HS+IR
Figure 5. Effect of dietary iron restriction on proteinuria and oxidative stress in Dahl salt-sensitive rats. A, Urinary total protein/
creatinin ratio, (B) urinary 8-OHdG/creatinin ratio, and (C) representative images of DHE staining of the kidney sections in the
control (white bar, n⫽6), HS (black bar, n⫽6), and HS⫹IR (gray
bar, n⫽6) groups. Scale bars: 50 ␮m. TP/Cre, urinary total protein/creatinin ratio; 8-OHdG/Cre, urinary 8-OHdG/creatinin ratio.
*P⬍0.05 vs the control group; †P⬍0.05 vs the HS group.
Systolic B
Blood Pressu
ure
A
March 2011
B
SBP
mmHg
300
200
†
*
100
0
HS
HS
+
IR
HS
+
IR
+
L-NAME
C
TP/Cre
mg/mg
40
Survival Rate
%
100
30
†
20
*
10
Surv
vival Rate
Hypertension
Total Protein/creatinin R
Ratio
502
80
60
40
L-NAME
20
†
0
HS
HS
+
IR
HS
+
IR
+
L-NAME
0
6
10
14
18
age
(weeks)
Figure 6. L-NAME abolished the protective effects of iron restriction under HS diet in Dahl salt-sensitive rats. Comparison of (A) systolic
blood pressure, (B) urinary total protein/creatinin ratio, and (C) survival rate in the HS (black bar and circle, n⫽6), HS⫹IR (gray bar and
circle, n⫽6), and HS⫹IR with L-NAME treatment (silver bar and square, n⫽6) groups. *P⬍0.05 vs the HS group; †P⬍0.05 vs the HS⫹IR
group.
Downloaded from http://hyper.ahajournals.org/ by guest on June 12, 2017
Iron is a vital element in life. However, it may participate
in diverse pathological processes by catalyzing the formation
of reactive oxygen free radicals. Iron accumulation promotes
increased free radicals and oxidative stress, which eventually
lead to cell and tissue damage. Therefore, it is important to
consider the influence of iron on the pathophysiology of
various diseases. Iron is involved in the pathogenesis of
several cardiovascular diseases. It has been reported that the
iron deposition shown in human atherosclerotic lesions10 and
body iron stores is related to the risk of carotid atherosclerosis.11 Thus, iron accumulation in atheroma may be associated
with the progression of atherosclerosis, and the reduction of
iron accumulation may be effective for the development of
atherosclerosis. In the current study, we elucidated the effects
of dietary IR on cardiovascular remodeling in Dahl saltsensitive rats. HS-loading on salt-sensitive individuals caused
hypertension, LV hypertrophy, heart failure, and renal injury.
Of note, we demonstrated that IR attenuated the development
of these diseases and improved survival rate in Dahl saltsensitive rats.
Calorie restriction was previously reported to be beneficial
for cardiac remodeling in Dahl salt-sensitive rats5; however,
there was no report to investigate the effects of only IR
against HS-induced cardiac remodeling. To our knowledge,
this is the first article to report that only IR was effective for
cardiovascular remodeling in Dahl salt-sensitive rats. IR led
to only a slight decrease in body weight, consistent with a
previous observation,7 whereas body weight decreased more
in the HS group than in the HS⫹IR group. At 18 weeks of
age, Dahl salt-sensitive rats showed cachexia with the development of heart failure, similar to previous reports.12 Taking
these findings into consideration, IR⫹HS diet prevented the
onset of cachexia with the development of heart failure.
Although IR improved survival rate in Dahl salt-sensitive
heart failure rats, dietary IR induced iron deficiency anemia
measured by hemoglobin content, in agreement with previous
observation.7 Interestingly, blood hemoglobin began to decrease in the HS group at 10 weeks of age. Finally, blood
hemoglobin was lower in the HS⫹IR group than in the HS
group at 18 weeks of age. Because previous studies have
reported that plasma volume increased in Dahl salt-sensitive
heart failure rats,13 several factors such as fluid retention and
increased plasma volume seem to influence anemia in the HS
group.
Iron deficiency is a contributing factor in heart failure7,14;
however, IR attenuated development of heart failure in Dahl
salt-sensitive rats. SBP increased in the HS group, while IR
inhibited the increase in SBP, despite an HS diet. As a result,
it appears that IR attenuated the development of LV hypertrophy, decompensated pressure-overload hypertrophy, and
heart failure. To clarify the mechanisms by which IR benefits
the development of hypertension, we elucidated molecular
signaling pathways in the aorta of these animals. In the
current study, the phosphorylation of Akt, AMPK, and eNOS
in the aorta decreased in the HS group, whereas it maintained
the phosphorylation of Akt, AMPK, and eNOS in the aorta of
the HS⫹IR group. On the contrary, the phosphorylation of
ERK in the aorta was increased in both HS and HS⫹IR
groups compared with the control group. Thus, IR appears to
prevent the development of hypertension through Akt,
AMPK, and eNOS signaling but not through ERK signaling.
The phosphorylation of eNOS at Ser 1177 is associated with
increased production of NO.15 Both Akt and AMPK are
reported to phosphorylate directly eNOS at Ser 1177, NO
production, and vasorelaxation.16,17 In addition, cross-talk
between Akt and AMPK is reported to be important for eNOS
phosphorylation at Ser 1177.18 Therefore, Akt and AMPK
may be upstream kinases of vascular eNOS phosphorylation
in the IR-mediated protection of endothelial function. In
contrast, oxidative stress also causes endothelial dysfunction
through regulation of eNOS.19 Thus, we investigated the
urinary 8-OHdG/creatinin ratio and superoxide production of
the aorta with DHE staining among the groups. As expected,
the urinary 8-OHdG/creatinin ratio and vascular superoxide
production increased in the HS group, whereas IR significantly reduced both systemic and vascular oxidative stress
under HS diet. Taken together, these data provide evidence
that the attenuation of oxidative stress also contributes to the
vascular protective effect of IR. Oxidative stress could
regulate the phosphorylation of Akt, AMPK, and eNOS in the
aorta,20 whereas aortic ERK signaling is reported not to be
dependent on the attenuation of oxidative stress in the
Naito et al
Downloaded from http://hyper.ahajournals.org/ by guest on June 12, 2017
hypertensive rats.21 Therefore, we may observe differential
response between the signaling axis of Akt, AMPK, eNOS,
and that of ERK.
To investigate how iron intake affects HS-induced hypertension, we evaluated cellular iron transport proteins, such as
TfR1 and ferritin H- and L-subunits, in the aorta of these
animals. Low-iron conditions normally lead to upregulated
TfR1 and downregulated ferritin expression. Conversely,
high-iron conditions usually downregulate TfR1 and upregulate ferritin expression.9 We observed these changes in the
aorta of the HS⫹IR group as normal, while this was not the
case in the HS group. Interestingly, both TfR1 and ferritin
H-subunit expression were upregulated in the aorta of the HS
group, suggesting that dysregulation of cellular iron transport
proteins occurs in the aorta of the HS group. More iron is
necessary for cells for their growth and metabolism than for
resting cells. Upregulated aortic TfR1 may increase iron
uptake into the cell and participate in vascular remodeling in
the HS group. Since oxidative stress was reported to upregulate TfR1 expression,22 oxidative stress may be related to
aortic TfR1 upregulation in the HS group. Meanwhile, ferritin
is a major intracellular iron-storage protein. Ferritin
H-subunit has ferroxidase activity, which is required for iron
sequestration, while ferritin L-subunit has no ferroxidase
activity.23,24 Only ferritin H-, but not L-subunit, expression
was increased in the aorta of the HS group, which may
sequester excess free iron molecules to minimize generation
of iron-catalyzed reactive oxygen species.
Clinically, treatment with intravenous iron improved exercise capacity and symptoms of heart failure in patients with or
without anemia,25 although excess total body iron stores are
associated with higher cancer risk and mortality.2 In this
study, both HS and HS⫹IR group showed anemia, whereas
the HS⫹IR group showed good prognosis. Iron is essential
for several biological processes, such as enzymatic reactions
and oxygen delivery, while its excess is involved in pathophysiology. Therefore, although iron supplementation may be
beneficial for patients with heart failure, the role of iron in
maintaining cardiac function in hypoxia and anemia has not
been further considered. In addition, studies using intravenous iron treatment are needed to assess long-term safety in
renal and cardiac disease.
In conclusion, dietary IR has protective effects on HSinduced hypertension and cardiovascular remodeling through
the reduction of oxidative stress and maintenance of Akt,
AMPK, and eNOS signaling in the aorta. IR could be an
effective strategy for prevention of HS-induced organ damage in salt-sensitive hypertensive patients.
Perspectives
We have shown that dietary IR has preventive effects on
salt-induced cardiovascular remodeling in Dahl salt-sensitive
rats. In addition, we found that dysregulation of intracellular
iron transport proteins, such as upregulation of TfR1 and
ferritin H-subunit, occurs in the aorta of Dahl salt-sensitive
rats with an HS diet. This is the first article to our knowledge
that reveals the effect of dietary IR on hypertensive cardiovascular remodeling. Notably, we have shown that IR attenuated the development of hypertension, LV hypertrophy,
Iron Restriction and Salt-Sensitive Hypertension
503
heart failure, and renal injury, thereby improving survival rate
in Dahl salt-sensitive rats; this occurred through inhibition of
oxidative stress and by maintaining Akt, AMPK, and eNOS
signaling in the aorta. Based on our findings, understanding
the beneficial effects of dietary IR on salt-induced cardiovascular remodeling may lead to a new therapeutic strategy for
prevention of HS-induced organ damage in salt-sensitive
hypertensive patients.
Acknowledgments
We are grateful to Noriko Kumon and Naoko Sasaki for their
technical support.
Sources of Funding
This study was supported by a Grant-in-Aid for Young Scientists (B)
from the Ministry of Education, Culture, Sports, Science, and
Technology of Japan (Y.N.).
Disclosures
None.
References
1. Lee TS, Shiao MS, Pan CC, Chau LY. Iron-deficient diet reduces atherosclerotic lesions in apoE-deficient mice. Circulation. 1999;99:
1222–1229.
2. Zacharski LR, Chow BK, Howes PS, Shamayeva G, Baron JA, Dalman
RL, Malenka DJ, Ozaki CK, Lavori PW. Decreased cancer risk after iron
reduction in patients with peripheral arterial disease: results from a
randomized trial. J Natl Cancer Inst. 2008;100:996 –1002.
3. Kenyon C. The plasticity of aging: Insights from long-lived mutants. Cell.
2005;120:449–460.
4. Zimmerman J, Kaufmann NA, Fainaru M, Eisenberg S, Oschry Y, Friedlander Y, Stein Y. Effect of weight loss in moderate obesity on plasma
lipoprotein and apolipoprotein levels and on high density lipoprotein
composition. Arteriosclerosis. 1984;4:115–123.
5. Seymour EM, Parikh RV, Singer AA, Bolling SF. Moderate calorie
restriction improves cardiac remodeling and diastolic dysfunction in the
Dahl-SS rat. J Mol Cell Cardiol. 2006;41:661– 668.
6. Shinmura K, Tamaki K, Saito K, Nakano Y, Tobe T, Bolli R. Cardioprotective effects of short-term caloric restriction are mediated by adiponectin via activation of AMP-activated protein kinase. Circulation.
2007;116:2809 –2817.
7. Naito Y, Tsujino T, Matsumoto M, Sakoda T, Ohyanagi M, Masuyama T.
Adaptive response of the heart to long term anemia induced by iron
deficiency. Am J Physiol Heart Circ Physiol. 2009;296:H585–H593.
8. Naito Y, Tsujino T, Fujioka Y, Ohyanagi M, Iwasaki T. Augmented
diurnal variations of the cardiac renin-angiotensin system in hypertensive
rats. Hypertension. 2002;40:827– 833.
9. Daniels TR, Delgado T, Rodriguez JA, Helguera G, Penichet ML. The
transferrin receptor part I: Biology and targeting with cytotoxic antibodies
for the treatment of cancer. Clinical Immunology. 2006;121:144 –158.
10. Pang JH, Jiang MJ, Chen YL, Wang FW, Wang DL, Chu SH, Chau LY.
Increased ferritin gene expression in atherosclerotic lesions. J Clin Invest.
1996;97:2204 –2212.
11. Kiechl S, Willeit J, Egger G, Poewe W, Oberhollenzer F. Body iron stores
and the risk of carotid atherosclerosis: prospective results from the
Bruneck study. Circulation. 1997;96:3300 –3307.
12. Klotz S, Hay I, Zhang G, Maurer M, Wang J, Burkhoff D. Development
of heart failure in chronic hypertensive Dahl rats: focus on heart failure
with preserved ejection fraction. Hypertension. 2006;47:901–911.
13. Simchon S, Manger WM, Brown TW. Dual hemodynamic mechanisms
for salt-induced hypertension in Dahl salt-sensitive rats. Hypertension.
1991;17:1063–1071.
14. Dong F, Zhang X, Culver B, Chew HG Jr., Kelley RO, Ren J. Dietary iron
deficiency induces ventricular dilation, mitochondrial ultrastructural aberrations and cytochrome c release: involvement of nitric oxide synthase
and protein tyrosine nitration. Clin Sci (Lond). 2005;109:277–286.
504
Hypertension
March 2011
15. Fleming I, Busse R. Molecular mechanisms involved in the regulation of
the endothelial nitric oxide synthase. Am J Physiol Regul Integr Comp
Physiol. 2003;284:R1–R12.
16. Dimmeler S, Fleming I, Fisslthaler B, Hermann C, Busse R, Zeiher AM.
Activation of nitric oxide synthase in endothelial cells by Akt-dependent
phosphorylation. Nature. 1999;399:601– 605.
17. Chen ZP, Mitchelhill KI, Michell BJ, Stapleton D, Rodriguez-Crespo I,
Witters LA, Power DA, Ortiz de Montellano PR, Kemp BE. AMP-activated protein kinase phosphorylation of endothelial NO synthase. FEBS
Lett. 1999;443:285–289.
18. Kovacic S, Soltys CL, Barr AJ, Shiojima I, Walsh K, Dyck JR. Akt
activity negatively regulates phosphorylation of AMP-activated protein
kinase in the heart. J Biol Chem. 2003;278:39422–39427.
19. Cai H, Harrison DG. Endothelial dysfunction in cardiovascular diseases:
the role of oxidant stress. Circ Res. 2000;87:840 – 844.
20. Hu Z, Chen J, Wei Q, Xia Y. Bidirectional actions of hydrogen peroxide
on endothelial nitric-oxide synthase phosphorylation and function:
co-commitment and interplay of Akt and AMPK. J Biol Chem. 2008;
283:25256 –25263.
21. Ding L, Chapman A, Boyd R, Wang HD. ERK activation contributes to
regulation of spontaneous contractile tone via superoxide anion in
22.
23.
24.
25.
isolated rat aorta of angiotensin II-induced hypertension. Am J Physiol
Heart Circ Physiol. 2007;292:H2997–H3005.
Tampo Y, Kotamraju S, Chitambar CR, Kalivendi SV, Keszler A, Joseph
J, Kalyanaraman B. Oxidative stress-induced iron signaling is responsible
for peroxide-dependent oxidation of dichlorodihydrofluorescein in endothelial cells: role of transferrin receptor-dependent iron uptake in apoptosis. Circ Res. 2003;92:56 – 63.
Levi S, Luzzago A, Cesareni G Cozze A, Franceschinelli F, Albertini A,
Arosio P. Mechanism of ferritin iron uptake: activity of the H-chain and
deletion mapping of the ferro-oxidase site: a study of iron uptake and
ferro-oxidase activity of human liver, recombinant H-chain ferritins, and
of two H-chain deletion mutants. J Biol Chem. 1988;263:18086 –18092.
Torti FM, Torti SV. Regulation of ferritin genes and protein. Blood.
2002;99:3505–3516.
Anker SD, Comin Colet J, Filippatos G, Willenheimer R, Dickstein K,
Drexler H, Lüscher TF, Bart B, Banasiak W, Niegowska J, Kirwan BA,
Mori C, von Eisenhart Rothe B, Pocock SJ, Poole-Wilson PA, Ponikowski P; FAIR-HF Trial Investigators. Ferric carboxymaltose in patients
with heart failure and iron deficiency. N Engl J Med. 2009;361:
2436 –2448.
Downloaded from http://hyper.ahajournals.org/ by guest on June 12, 2017
Dietary Iron Restriction Prevents Hypertensive Cardiovascular Remodeling in Dahl
Salt-Sensitive Rats
Yoshiro Naito, Shinichi Hirotani, Hisashi Sawada, Hirokuni Akahori, Takeshi Tsujino and
Tohru Masuyama
Downloaded from http://hyper.ahajournals.org/ by guest on June 12, 2017
Hypertension. 2011;57:497-504; originally published online January 24, 2011;
doi: 10.1161/HYPERTENSIONAHA.110.159681
Hypertension is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
Copyright © 2011 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/57/3/497
Data Supplement (unedited) at:
http://hyper.ahajournals.org/content/suppl/2011/01/21/HYPERTENSIONAHA.110.159681.DC1
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/
Online Supplement
Dietary Iron Restriction Prevents Hypertensive Cardiovascular Remodeling in
Dahl Salt-Sensitive Rats
Short title: Iron restriction and salt-sensitive hypertension
Yoshiro Naitoa, Shinichi Hirotania, Hisashi Sawadaa, Hirokuni Akahoria, Takeshi
Tsujinob, and Tohru Masuyamaa
a
Cardiovascular Division, Department of Internal Medicine, Hyogo College of
Medicine, Nishinomiya, Japan, b Department of Pharmacy, Hyogo University of Health
Sciences, Kobe, Japan
Corresponding author: Yoshiro Naito MD, PhD
Cardiovascular Division, Department of Internal Medicine, Hyogo College of Medicine
1-1 Mukogawa-cho, Nishinomiya, 663-8501, Japan
Phone number: +81-798-45-6553, Fax number: +81-798-45-6551
E-mail address: [email protected]
Methods
Assessments
of
Blood
Pressure,
Blood
Cell
Count,
Urinary
8-Hydroxy-2'-deoxyguanosine, and Tissue Iron Content: Systolic blood pressure and
heart rate were measured by a non-invasive computerized tail-cuff system in conscious
rats (MK-2000, Muromachi Kikai, Tokyo, Japan) (1). Peripheral blood cell count was
measured using an automatic cell count analyzer (Pentra 60 LC-5000, Horiba, Kyoto,
Japan). 24-hour urine samples were collected in metabolic cages for measuring urinary
volume, protein, and 8-Hydroxy-2'-deoxyguanosine (8-OHdG) and creatinine levels.
Urinary 8-OHdG levels were assessed by enzyme-linked immune sorbent assay (Japan
Institute for the Control of Aging, Shizuoka, Japan). The iron content of the aorta was
determined by atomic absorption.
Echocardiography: Rats were anesthetized with ketamine HCl (50 mg/kg) and
xylazine HCl (10 mg/kg), and assessed by transthoracic echocardiography (Aplio,
Toshiba Medical Systems Co., Odawara, Japan). Left ventricle (LV) cavity size, wall
thickness, and LV fractional shortening were calculated. We also measured peak early
diastolic filling velocity (E), peak filling velocity at atrial contraction (A), their ratio
(E/A) and deceleration time from pulsed Doppler mitral flow velocity pattern (2).
Gene Expression Analysis: Total RNA was extracted from the tissue using TRIzol
reagent (Invitrogen, Carlsbad, CA, USA) (3). DNase-treated RNA was
reverse-transcribed into cDNA using random primers (Applied Biosystems, Foster City,
CA, USA). Real-time PCR reactions were performed using the ABI PRISM 7900 with
TaqMan Universal PCR Master Mix and TaqMan Gene Expression Assays (Applied
Biosystems) (1). TaqMan Gene Expression Assays were used as primers and probes for
each gene were as follows: atrial natriuretic peptide (assay ID Rn00561661_m1),
collagen type I (assay ID Rn00801649_g1), collagen type III (assay ID
Rn01437683_m1), transforming growth factor-β (assay ID Rn99999016_m1), CD68
(assay ID Rn01495634_g1), transferrin receptor1 (assay ID Rn01474701_m1), ferritin
H-subunit (assay ID Rn00820640_g1), ferritin L-subunit (assay ID Rn00821071_g1),
and Glyceraldehyde-3-phosphatedehydrogenase (GAPDH) (assay ID Rn99999916_s1).
GAPDH was used as an internal control.
Histological Analysis: Heart tissues were fixed with buffered 4% paraformaldehyde,
embedded in paraffin, and cut into 4-µm-thick sections. Hematoxylin-eosin and
Masson’s trichrome staining were performed using serial sections. Photomicrographs
were quantified with the use of NIH Image-J software to measure the cross-sectional
area of cardiomyocytes and to assess the fibrosis area of myocardium. 100 randomly
selected cardiomyocytes in the LV were measured for cross-sectional area (1).
References
1. Naito Y, Tsujino T, Matsumoto M, Sakoda T, Ohyanagi M, Masuyama T. Adaptive
response of the heart to long term anemia induced by iron deficiency. Am J Physiol
Heart Circ Physiol. 2009; 296: H585-H593.
2. Masuyama T, Yamamoto K, Sakata Y, Doi R, Nishikawa N, Kondo H, Ono K,
Kuzuya T, Sugawara M, Hori M. Evolving changes in Doppler mitral flow velocity
pattern in rats with hypertensive hypertrophy. J Am Coll Cardiol. 2000; 36:
2333-2338.
3. Naito Y, Tsujino T, Fujioka Y, Ohyanagi M, Iwasaki T. Augmented diurnal variations
of the cardiac renin-angiotensin system in hypertensive rats. Hypertension. 2002;
40: 827-833.