Effect of salt on hypertension and oxidative stress in a rat model of diet-induced obesity
Anca D. Dobrian,
Suzanne D. Schriver,
Terrie Lynch, and
Russell L. Prewitt
Department of Physiological Sciences, Eastern Virginia Medical School,
Norfolk, Virginia 23507
Submitted 31 October 2002
; accepted in final form 6 June 2003
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ABSTRACT
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High-salt diet is known to induce or aggravate hypertension in animal
models of hypertension and in humans. When Sprague-Dawley rats (n =
60) are fed a moderately high-fat diet (32% kcal fat, 0.8% NaCl) for 10 wk,
about one-half develop obesity [obesity prone (OP)] and mild hypertension,
whereas the other half [obesity resistant (OR)] maintain body weight
equivalent to a low-fat control (C) and are normotensive. The aim of this
study was to test the effect of high-NaCl diets (2 and 4% NaCl) on the
development of hypertension and obesity, oxidative stress, and renal function.
Both 2 and 4% NaCl induced an early increase in systolic blood pressure of OP
but not OR or C rats. High-salt intake induced an increase in the size and
reduction in number of adipocytes, concomitant to a twofold increase in
circulating leptin in OP rats. Aortic superoxide generation indicated a
2.8-fold increase in the OP high-salt vs. normal-salt groups, whereas urine
isoprostanes were not significantly increased. Also, hydroxynonenal protein
adducts in the kidney were highly increased in OP rats on 2 and 4% NaCl,
indicating oxidative stress in the renal tissue. Urine albumin was increased
threefold in the OP on 2% NaCl and fourfold in the same group on 4% NaCl vs.
0.8% NaCl. Kidney histology indicated a higher degree of glomerulosclerosis in
OP rats on high-salt diets. In summary, high-salt diet accelerated the
development but did not increase the severity of hypertension; high salt
increased oxidative stress in the vasculature and kidney and induced kidney
glomerulosclerosis and microalbuminuria. Also, the OP rats on high salt
displayed adipocyte hypertrophy and increased leptin production.
glomerulosclerosis; kidney; leptin; sodium dietary
OBESITY IS A complex, multifactorial disease that is associated
with essential hypertension in
78% of men and
65% of women, as
indicated by the data from the Framingham Heart Study
(25). Another important
contributor to hypertension in humans is the excessive consumption of dietary
salt, and epidemiological studies have demonstrated a significant but weak
relation between salt intake and hypertension
(32,
33). Some, but not all,
interventional studies have shown that salt restriction may lower blood
pressure (BP) (19,
33). Some recent studies
report correlation among hypertension, salt sensitivity, and insulin
resistance in obese humans
(38), whereas others fail to
observe a significant relationship
(8). Animal models of obesity,
hypertension, and insulin resistance display differences with respect to salt
sensitivity. In Zucker rats, there is a clear correlation between salt intake
and the severity of hypertension
(4,
47), whereas in chronic
hyperinsulinemic Sprague-Dawley (SD) rats, hypertension is not salt sensitive,
albeit a shift in pressure-natriuresis relationship was reported
(2). One important contributor
to hypertension in salt-sensitive animal models and humans seems to be the
endothelial dysfunction, in particular the altered vascular reactivity due to
an impairment in nitric oxide (NO) production
(22,
31,
36). High-salt intake is able
to decrease both plasma levels and urinary excretion of nitrates
(3,
16). One possible explanation
is a reduced availability rather than decreased production of NO. The ability
of NO to quickly interact with superoxide anion, forming the potent oxidant
peroxynitrite, is well documented
(43). Increased superoxide
production in both vasculature and kidney was extensively reported in various
forms of hypertension in experimental models and humans
(4042,
46). Moreover, we reported
that obese hypertensive rats on high-fat diet also display increased oxidative
stress and reduced NO bioavailability
(12). Also, salt sensitivity
was associated with increased oxidative stress in rats
(5,
49). Apart from the effects on
BP regulation, elevated salt intake was associated with cardiovascular and
renal changes leading to end-organ damage
(6). Moreover, a recent report
connects salt intake with oxidative stress and nephrosclerosis in
Dahl-sensitive hypertensive rats
(48). Another important factor
involved in BP regulation in obesity is leptin
(24). Leptin was shown to have
both a vasopressor effect at peripheral level and, infused in high doses, a
hypertensive effect acting at central level
(24). However, a recent report
suggests that leptin may not contribute to arterial pressure sensitivity to
salt in hyperleptinemic obese rats
(7). The aim of our study was
to assess the effect of high-fat, high-salt diets on the development of
hypertension and oxidative stress in a rat model of diet-induced obesity.
Moreover, the effect on vascular hypertrophy and kidney sclerosis was
assessed. Additionally, the effect of salt on adiposity and leptin production
was also measured.
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METHODS
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Animals
All procedures involving animals were approved by the Institutional Animal
Care and Use Committee of Eastern Virginia Medical School. Eighteen male SD
rats (300350 g) individually caged were randomly selected to be fed a
moderately high-fat diet (MHF) with 0.8% sodium (32% kcal as fat, Research
Diets, New Brunswick, NJ), whereas six rats (controls) were fed purified
low-fat (LF) diet with 0.8% sodium (10.6% kcal as fat, Research Diets) for 10
wk. An identical number of rats was placed on MHF and LF diets, each
containing either 2 or 4% sodium (high-salt diets). Food and water were
provided ad libitum throughout the experiment. Body weights (BW) and lengths
were measured initially and then weekly together with food intake. Rats fed
the MHF diet on both low and high salt diverged into distinct groups based on
BW gains. Assignment of rats into obesityprone (OP) (n = 8) and
obesity-resistant (OR) (n = 8) groups was performed as described
previously (12).
Systolic BP
The onset and development of hypertension were assessed by using the
tail-cuff method with a Narco Biosystems Electro-Sphygnomanometer (Houston,
TX). BP was measured under conscious conditions at the beginning of the
experiment and at 1, 5, 8, and 10 wk of diet. The average of five pressure
readings was recorded for each measurement.
Assessment of Oxidative Stress
Superoxide anion production was measured in isolated aortic rings using a
method previously described
(12,
21). Briefly, 5-mm aortic
rings were preincubated in Krebs-bicarbonate buffer, at 37°C, for 30 min
and then transferred to a cocktail containing 5 µmol/l lucigenin and
immediately measured, every 2 min, for 15 min total, using a scintillation
counter set in the out-of-coincidence mode. The readings were plotted and the
area under the curve was integrated. Results were normalized per milligram of
DNA measured using the Hoechst 33258 dye as described
(27). The specificity of the
reaction was tested by the ability of 50 U/ml of SOD to quench the
chemiluminescence at the end of the measurement.
Free 8-isoprostane F2
. Isoprostanes
were measured by EIA using a kit from Cayman Chemicals as previously described
(12). Urine collected in
metabolic cages over a 24-h period was supplemented with 0.05% butylated
hydroxytoluene and spiked with 8-[3H]isoprostane. The samples (1
ml) were passed on an affinity column (Cayman Chemicals) and only the free
isoprostanes were eluted using 95% methanol. The eluate was evaporated to
dryness under a stream of N2 and the pellet was resuspended in a
1-ml assay buffer. Each sample was assayed in duplicate at two different
dilutions and corrected for individual recovery of
8-[3H]isoprostane, and the results were averaged. Nitrate/nitrite
was assayed both in plasma and urine (diluted 1:50 in PBS) using a LDH
colorimetric method with a kit from Cayman Chemicals.
Immunohistochemistry for 4-hydroxy-2-nonenal. Kidneys were fixed
in 10% buffered formaline for 3 h and paraffin embedded. The sections were
incubated with a polyclonal antibody recognizing 1:1 Cys, His, Lys-4
hydroxy-2-nonenal "Michael" adducts (Calbiochem, dilution 1:750).
The slides were then reacted with biotinylated secondary goat anti-rabbit
antibody (1:500 dilution; Vector Laboratories, Burlin-game, CA), with the
ABC-Elite avidin reagent (Vector Laboratories), and finally with the Immuno
Pure Metal Enhanced DAB Substrate kit (Pierce, Rockford, IL).
Vascular Hypertrophy and Kidney Sclerosis
Aortic wall area. Thin sections of the paraffin-embedded tissue
were stained for 1 min with toluidine blue and analyzed as described
previously (10).
Kidney histology. Kidneys were fixed in 10% buffered formalin for
4 h and embedded in paraffin. Sections were stained using the periodic
acid-Schiff (PAS) reagent and counterstained with hematoxylin. To evaluate the
degree of segmental sclerosis, three independent investigators examined the
slides in a blind fashion, mixing the slides after covering the protocol
numbers. In each case, 80100 glomeruli were examined for each slide and
individually graded on a scale of 0 to 2+ according to the degree of
glomerular sclerosis. Grade 0 was a normal looking glomerulus;
grade 1+ was characterized by mild expansion of mesangial matrix, no
occlusion in the glomerular capillaries or adhesion to Bowman's capsule; and
grade 2+ included expansion of the mesangial matrix, usually focal
with adhesion to Bowman's capsule and some degree of capillary occlusion. A
score representing the sum of grades was obtained for each rat.
Adipocyte Morphometry
Adipose tissues from the same depot and group were pooled and collagenase
was digested according to Rodbell and Krishna
(39). Adipocytes were washed
several times to remove collagenase and centrifuged to separate adipocytes
from preadipocytes, stromal cells, and vascular membranes. Cell diameter of
1,200 cells was measured with the Image 1 Analysis System (Universal
Image, West Chester, PA). Mean cell diameter was used to estimate mean cell
volume. Cell size (µg lipid/cell) was calculated by multiplying cell volume
(pl) by lipid density (
0.915 g/ml). Cell lipid content was determined
according to the method of Dole
(14). Cell lipid content and
cell size were used to calculate cell number.
Statistics
Data are means ± SE. To determine the significance between different
groups, two-way or three-way ANOVA was performed followed by Tukey's post hoc
test. P < 0.05 was considered statistically significant.
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RESULTS
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Effect of Salt on BW, Adiposity, Adipocyte Morphometry, and
Leptin
After 10 wk of diet, BW in the OP groups on both high- and regular-salt
diets was significantly higher than those in the corresponding OR and control
(C) groups (Table 1). In
addition, no significant difference in BWs was detected between each of the
OP, OR, and C groups on 0.8, 2, and 4% NaCl, respectively
(Table 1). The result is in
accordance with daily food intake data, indicating that high-salt diets did
not result in increased food consumption in OP, OR, or C rats on the
respective diets vs. their counterparts on the low-salt diet
(Fig. 1, A and
B). However, from the beginning of the experiment until
week 8, the OP rats ate significantly more than OR rats on a similar
diet (Fig. 1, A and
B). Also, the average food intake in all experimental
groups reaches a peak after 3 wk on the respective diets, followed by a
decrease by week 5 and a subsequent relatively stable level until the
end of the experiment (Fig. 1, A
and B), indicating that the highest salt intakes occurred
in the first 35 wk on the diet. The increased BWs in the OP groups
compared with OR and C groups were also mirrored by the elevated adiposity.
Both the epididymal and retroperitoneal fat depots were significantly
increased in the OP groups compared with OR and C, but no significant
differences were recorded between the high- and low-salt groups
(Table 1). Furthermore, the
obesity index was higher in the OP groups compared with OR and C and was not
influenced by the salt intake (Table
1). In contradistinction, the adipocyte morphometry and number
were different among the OP, OR, and C groups placed on low- vs. high-salt
diets. For all OP, OR, and C, the 2% NaCl diet induced an increase by
1220% in cell volume and 1215% in cell size with the highest
effect on OP adipocytes (Table
2). Also, a decrease in adipocyte number was measured for OP
(
30%) and OR (
12%) groups on 2 vs. 0.8% NaCl diet, with no
difference for C adipocytes (Table
2). This indicates hypertrophy of adipocytes from the OP rats,
significantly exacerbated by the high-salt intake. In accordance with previous
findings (29), our results
indicate an increase in circulating leptin for OP rats compared with OR and C
after 1014 wk of diet (Table
2). Interestingly, both the 2 and 4% NaCl diets significantly
increased, by
40%, plasma leptin in the OP rats, and only the 4% NaCl
diet induced a significant increase in the leptin levels in OR and C rats,
compared with their counterparts on 0.8% NaCl
(Table 2). The latter result
suggests that obesity and high salt are both important in regulation of leptin
levels. Moreover, the finding that the 4% NaCl, but not 2% NaCl, diet
increased leptin levels in both OR and C lean groups of rats suggests that
even in the absence of obesity, and independently of the amount of fat in the
diet, a high enough content of NaCl (in our particular experiment 4 vs. 2%)
could modulate the leptin levels.

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Fig. 1. Average daily food intake in obesity-prone (OP), obesity-resistant (OR),
and control (C) groups on 2% NaCl (A) and 4% NaCl (B)
compared with 0.8% NaCl groups. Food consumption was measured weekly and
corrected for spillage for each individual rat. Average amount per day was
plotted for the week when systolic blood pressure had also been measured.
*Significant compared with OR in the same salt group (P <
0.05).
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Effect of High-Salt Diet on BP and Plasma Renin Activity in OP, OR,
and C Rats
Systolic BP measured in the conscious rats at the beginning of the diet
indicated an average of 122 ± 3.9 mmHg. Starting with week 5,
the OP rats on both 2 and 4% NaCl displayed a significant increase in BP with
an average of 160.2 ± 5.2 and 156.5 ± 4.4 mmHg, respectively, as
opposed to all the other groups that were either normotensive or borderline
hypertensive (Fig. 2, A and
B). At week 8, the OP rats on 0.8% NaCl diet
were moderately hypertensive with an average BP of 154 ± 3.2 mmHg,
whereas the OP rats on both 2 and 4% NaCl did not show a further significant
increase in their systolic BP compared with week 5
(Fig. 2, A and
B). By the end of the experiment (week 10), all
three OP groups (on 0.8, 2, and 4% salt) had a similar increase in BP that
averaged
158 mmHg. Also, the OR and C groups on high- and normal-salt
diets were normotensive (Fig. 2, A
and B). In the OP rats on 0.8% NaCl, the increase in
systolic BP was paralleled by an approximately twofold increase in plasma
renin activity (PRA), as measured at the end of the experiment
(Fig. 2C). The 2% NaCl
diet induced a
40% reduction in PRA in the OP rats and slightly decreased
PRA in the OR and C rats (Fig.
2C). In addition, the 4% NaCl diet induced a significant
reduction in PRA in OP, OR, and C groups compared with their respective
counterparts on 0.8 and 2% NaCl diets (Fig.
2C). The ability of the OP rat groups to adequately
respond to the different increase in dietary salt at week 10 may
explain the lack of difference in the systolic BP between the three OP groups
at that time point.

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Fig. 2. Systolic blood pressure (BP) in OP, OR, and C on 2% NaCl (A) and
4% NaCl (B) compared with 0.8% NaCl diets. Systolic BP was monitored
from the beginning of the study using the tail-cuff method. OP rats on high
salt have significantly increased BP starting with week 5, whereas OP
rats on regular salt are hypertensive starting with week 8 on the
diet. Plasma renin activity (C) was measured in terminal plasma
samples from OP, OR, and C rats on 0.8% NaCl, 2% NaCl, and 4% NaCl, using a
RIA method. Data represent means ± SE of 6 rats/group. * Significant
compared with OR and C groups; #significant compared with counterpart on 2%
salt; *significant compared with counterpart on 4% salt (P <
0.05).
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Oxidative Stress in Rats Fed Regular- and High-Salt Diets
The systemic oxidative stress measured as the excreted free 8-isoprostane
F2
in 24-h urine samples indicated an
2.5-fold increase in the OP groups on both normal (0.8% NaCl)- and high
(2 and 4% NaCl)-salt diets, compared with the respective OR and C groups
(Fig. 3A), indicating
that high salt does not further increase systemic oxidative stress in the
obese rats. However, the ability of thoracic aortic rings to generate
superoxide anions, measured as lucigenin chemiluminescence, is double in OP
rats on both 2 and 4% NaCl vs. OP rats on regular salt, indicating an increase
in oxidative stress in the large vessels of obese animals
(Fig. 3B). Also, a
significant increase induced in response to high salt was measured in C rats
and the same trend was present in the OR rats
(Fig. 3B). The latter
result indicates that high salt increased superoxide formation independent of
obese state and the amount of dietary fat. In addition, the high-salt intake
and obesity, but not dietary fat, seem to have a synergistic effect on
superoxide generation. Also, the urinary nitrate/nitrite is four- to fivefold
decreased in OP rats on both regular and salt-supplemented diets, compared
with the OR and C counterparts (Fig.
3C). The result indicates that salt intake does not
further decrease nitrite/nitrate excretion, despite its significant effect on
superoxide generation in the vasculature. Therefore, nitrite/nitrite formation
seems to be modulated mainly by the obese state per se and not critically by
the high-fat or high-salt content in the diets. Kidney immunohistochemistry
using a polyclonal antibody for 2-hydroxy-4-nonenal protein adducts indicates
a similar staining pattern in all groups on both regularand high-salt diets;
however, the intensity of the staining is much higher in the OP, OR, and C
rats on 4% vs. regular-salt diets (Fig. 4,
GL). The most intense staining is noticed
in the distal tubules, thick ascending limb, and to a lesser extent in the
cortical proximal tubules, whereas it is virtually absent in the glomeruli. As
shown in Fig. 4,
GL, the staining is more prominent in all
OP, OR, and C rats on high salt (Fig. 4,
GI) vs. regular salt
(Fig. 4,
JL), suggesting an increased local free
radical production in the kidney cortex induced by high-sodium intake. The
control in which the primary antibody was replaced with nonimmune serum shows
no staining (Fig.
4M).

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Fig. 3. Free urinary isoprostanes (A), aortic superoxide generation
(B), and nitrate/nitrite excretion (C) in OP, OR, and C rats
on 0.8% NaCl, 2% NaCl, and 4% NaCl diets. Urinary free isoprostanes were
measured using an EIA method. In both OP groups on high- and regular-Na diet,
the isoprostanes are significantly increased compared with the respective OR
and C groups. Superoxide anion generation by aortic rings was measured by
lucigenin chemiluminescence as described under METHODS. High-Na
intake induces an increase in superoxide production in the OP group but not in
the OR and C. Nitrate/nitrite excretion, measured by a colorimetric LDH
method, indicates an approximately fourfold reduction in OP rats on both
regular- and high-salt diets, compared with OR and C groups. Salt intake does
not further reduce nitrate/nitrite excretion. *Significant compared with OR
and C; #significant vs. 2% NaCl counterpart; *significant vs. 4% NaCl
counterpart (P < 0.05).
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Fig. 4. Periodic acid-Schiff (PAS)-hematoxylin staining of the kidney cortex in
high-salt OP (A), OR (B), and C (C) rats and
normal-salt OP (D), OR (E), and C (F) rats.
Immunohistochemistry using a 2-hydroxy-4 nonenal (HNE) antibody in 4% NaCl OP
(G), OR (H), and C (I) rats and normal (0.8%)-salt
OP (J), OR (K), and C (L) rats; method control
using nonimmune serum instead of primary antibody (M). PAS staining
indicates glomerulosclerosis and increased matrix deposition in the cortex of
OP rats on high-salt (A) and to a lesser extent in OP rats on
regular-salt diet. Also, HNE staining is more intense in the OP, OR, and C
rats on high salt (GI) vs. normal salt
(JL), with the same distribution pattern. Bar = 10
µm.
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Effect of Salt on Vascular Hypertrophy, Kidney Sclerosis, and
Excretory Function
Vascular hypertrophy was measured as aortic cross-sectional wall area.
Results indicated that in all the OP groups (0.8% NaCl, 2% NaCl, and 4% NaCl),
there is a significant increase in wall area compared with the respective OR
and C groups (Fig.
5A). However, high salt did not induce a further increase
in wall area in OP rats, suggesting no additional effect on vascular
hypertrophy (Fig. 5A).
To address the possible morphological changes in the kidney, we used
PAS-hematoxylin staining followed by morphometric analysis. In accordance with
our previous data (11), in OP
rats on regular-salt diet, a mild sclerosis with most of the lesions in a
relatively early stage was noticed, as opposed to OR and C rats that displayed
a normal kidney histology (Fig. 4,
DF). The OP rats on 4% NaCl displayed
numerous and more advanced lesions of the glomeruli as well as significant
matrix deposition throughout the cortex
(Fig. 4A). The
glomerular lesions displayed capillary loop collapse, mesangial matrix
expansion, and sometimes adhesion to Bowman's capsule. In addition,
interstitial fibrosis and glomerular membrane thickening were noticed
(Fig. 4A). The changes
noticed in the 2% NaCl groups were somewhat intermediate between the 4 and
0.8% NaCl counterparts (not shown). In OR and C rats, a normal histological
appearance was observed regardless of the amount of NaCl in the diets.
Morphometric analysis indicated a mean ± SE mesangial score of 16.9
± 1.4 for the OP rats on 4% NaCl compared with 14.2 ± 0.8 and
10.9 ± 1.2 for the OP rats on 2% NaCl and 0.8% NaCl, respectively. The
scores for the OR rats on 4, 2, and 0.8% NaCl were 9.4 ± 1.1, 8.7
± 1.2, and 8.4 ± 1.3, respectively, and the scores for the C
rats on 4, 2, and 0.8% NaCl were 8.8 ± 1.4, 8.2 ± 1.2, and 8.4
± 1.3, respectively. To test the possible changes in the renal
function, protein, creatinine, and albumin excretion were measured. OP rats on
both high-salt diets did not display overt proteinuria or significantly
increased protein excretion compared with OP rats on low salt. Also, the
creatinine values were similar among all groups. However, OP rats on 2% NaCl
had mild albuminuria (5.6 ± 0.42 mg/24 h) compared with OP on regular
salt (2.12 ± 0.47 mg/24 h) (Fig.
5B). In addition, the OP group on 4% NaCl had a
significantly higher albumin excretion (7.8 ± 0.53 mg/24 h) compared
with both 2% NaCl and 0.8% NaCl counterparts
(Fig. 5B). The results
indicate that the changes in renal morphology, paralleled by albuminuria, are
dependent on the salt content in OP rats only, suggesting a synergistic effect
for salt and obesity but not for the dietary fat.

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Fig. 5. Vascular hypertrophy (A) and albumin excretion (B) in OP,
OR, and C rats on normal (0.8%)- and high (2 and 4%)-salt diets. Wall area
(A) was measured on aortic sections stained with toluidine blue, with
the use of a video-based image system with edge-tracking software. Albumin
(B) was measured using an ELISA kit in 24-h urine samples, collected
in metabolic cages at the end of the study. Data represent means ± SE
of 6 rats/group. *Significant compared with OR and C; #significant compared
with 2% NaCl counterpart; *significant compared with 4% NaCl counterpart
(P < 0.05).
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DISCUSSION
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This rat model of diet-induced obesity was shown to develop moderate
hypertension subsequent to the accretion of visceral adiposity, suggesting a
role for metabolic factors associated with obesity in the development of
hypertension (11,
12). We also reported
increased oxidative stress in the vasculature, plasma, and urine of obese
animals at both early (3 wk) and late (>10 wk) stages of the diet
(11,
12). Oxidative stress was
documented in a variety of animal models, such as the spontaneously
hypertensive (42,
44), Dahl-sensitive
(45,
46), or ANG II-infused rat
(28). In addition, an
important role for free radicals in BP regulation was shown in a model of
lead-induced hypertension (9,
50), in 1K1C
[PDB]
renal
hypertension (13), in chronic
renal failure (51), and in a
model of glutathione-depleted rats
(52). Several mechanisms were
proposed for explaining the effect of free radicals production on BP
regulation. It was recently demonstrated that endogenously produced superoxide
anion can decrease NO bioavailability in the thick ascending limb and
therefore increase NaCl reabsorption and induce hypertension
(37). Also, chronically
increased oxidative stress induced in the medulla of uninephrectomized
Sprague-Dawley rats was shown to lower medullary blood flow leading to
hypertension (34).
Hypertension in spontaneously hypertensive rats seems to involve reduced NO
availability in macula densa cells
(54). Some forms of
hypertension in humans, including essential hypertension associated with
obesity, are influenced by an increase in the amount of dietary salt intake
(17,
53). The aim of the present
study was to assess the effect of increased salt intake on the development of
hypertension and associated mechanisms involving oxidative stress and
end-organ damage in obese rats. The data showed that the increase in dietary
sodium up to 2 or 4% induces a more rapid elevation in BP, after only 5 wk of
diet, instead of 8 wk in OP rats on a regular-salt diet. Although we do not
have PRA data at week 5, it is reasonable to assume that the latter
result could be explained by the inability of OP rats to adequately respond to
an increase in the dietary salt early (at week 5) on the diet.
Conversely, the lack of difference in the BP between OP rats on the three
different diets at the end of the experiment is reflected in the ability of OP
rats to reduce their PRA according to the different levels of salt intake.
Also, the similar daily food intake for OP rats on 0.8, 2, and 4% NaCl at any
time point throughout the experiment rules out the possibility of a higher
salt intake in the early (up to week 5) as opposed to late part of
the diet, which could have accounted for the earlier increase in BP in the 2
and 4% NaCl vs. 0.8% NaCl OP group. Another possible explanation for the
earlier increase in BP in OP rats on a high-salt diet may be related to the
pressor effect of leptin, as recently reported
(23). Our own data or data
reported by others (29)
indicate that OP rats are hyperleptinemic by the end of the diet. Data showed
that high-salt diets induced a significant 40% increase in plasma leptin in OP
rats. Interaction between high-salt intake and obese state significantly
increased circulating leptin in the OP groups on high- vs. regular-salt diet.
Also, there was a lower, although significant, increase in plasma leptin in
lean OR and C groups on 4% NaCl, compared with 0.8% NaCl, suggesting that
high-salt intake could contribute to elevated plasma leptin independently of
obesity and high dietary fat. A recent report by Correia et al.
(7) demonstrated that high
amounts of circulating leptin can act centrally to increase BP in rats.
Increased leptin may act centrally as a pressor agent in the initial stages of
the diet, before full onset of obesity, but it is unable to have any effect
later, possibly due to the onset of leptin resistance.
Also, high-salt diet induced a significant increase in the adipocyte size,
especially in the OP rats. Adipocyte hypertrophy may potentiate the insulin
resistance in OP rats, due to the increased fatty acids efflux and increased
circulating triglycerides. It was shown that salt increases circulating levels
of fatty acids (17), and our
data indicating adipocyte hypertrophy suggest a possible increase in
circulating fatty acids.
Oxidative stress was reported previously for this animal model in both
prehypertensive (11) stage and
after the development of moderate hypertension
(12). In the present study, we
tested whether salt has an effect on free radicals formation in the obese
rats. Data indicated that 2 and 4% NaCl diets did not enhance free
isoprostanes excretion in OP, OR, or C rat groups compared with their
counterparts on 0.8% NaCl diet. However, the superoxide production by aortic
rings is significantly increased in OP rats on both high-salt diets vs. 0.8%
NaCl group. Urine isoprostanes are considered a reliable marker to quantify
systemic oxidative stress
(30). However, recent reports
indicate that in rats, under certain conditions such as increased oxygen
tension (26) or
NADPH-stimulated free radical production
(15),
F2
isoprostanes were not increased, although
other oxidative stress parameters were elevated. Although this study does not
provide data to support this hypothesis, it is possible that the increased
vascular superoxide production mainly originates from a vascular NAD(P)H
oxidase (18,
35) and hence isoprostanes
F2
could not accurately reflect the increased
aortic oxidative stress. Nevertheless, increased superoxide production in the
aorta does not seem to affect vascular hypertrophy. The wall area in OP rat
groups on high- and regular-salt diets is increased vs. the OR and C, but no
differences were measured among the three OP groups on 4, 2, and 0.8% NaCl.
The results suggest that vascular remodeling is due to the elevation in BP
rather than directly related to free radicals production in rats on high-salt
diet. The presence of increased hydroxynonenal protein adducts in the kidneys
of OP rats on high-salt vs. normal-salt diets indicates elevated free radicals
production in the renal tissue in the former. One possible source of free
radicals in the kidney may originate from high-leptin production by the local
infiltrates of adipose tissue. Leptin was shown to induce oxidative stress in
the endothelial cells in culture
(1). Therefore, it is possible
that increased local leptin production may contribute to reactive oxygen
species generation. High dietary fat does not appear to have a direct effect,
because both the OR and C groups displayed similar levels of lipid
peroxidation. Conversely, high-salt intake (4% NaCl) induced increased renal
lipid peroxidation in all study groups (OP, OR, and C), suggesting a role for
high salt independent from obesity and dietary fat. However, the higher lipid
peroxidation in the OP group on 4% NaCl vs. 0.8% NaCl and the higher
peroxidation in all OP groups compared with their OR and C counterparts on
similar diets indicate a possible synergistic effect of obesity and salt on
renal lipid peroxidation.
A direct or indirect effect of high-salt intake, possibly via free radicals
production, could be responsible for the kidney glomerulosclerosis in the OP
rats. Salt was shown to induce smooth muscle cells and myoblasts hypertrophy
in vitro (20). Also, oxidative
stress seems to be directly involved in the renal dysfunction in Dahl
salt-sensitive rats (48).
Therefore, it is reasonable to assume that the higher degree of renal damage
in the OP rats on high salt vs. normal salt is likely to be independent of a
pressor effect and rather due to the production of local excess leptin and/or
free radicals. In conclusion, our results indicate that 1) high-salt
diet induces an earlier increase in systolic BP in OP rats (5 wk on 4 and 2%
NaCl vs. 8 wk on 0.8% NaCl), possibly due to the inability of OP rats to
reduce their renin production in response to increased NaCl intake early in
the diet; 2) salt does not affect fat accretion, but it induces
adipocyte hypertrophy and increased leptin production, independently from
dietary fat and in synergy with obese state; 3) high-NaCl intake
induces increased vascular and renal oxidative stress, independently from
dietary fat and synergistically to obesity; and 4) high-salt diet
accelerates kidney sclerosis, which correlates with renal oxidative stress,
but it is, at least in part, independent of a direct pressor effect and does
not affect vascular hypertrophy, which is probably the direct result of high
arterial pressure. In this model, the concurrent effect of metabolic factors
related to obese state and high-salt intake seems to induce kidney sclerosis
and moderate hypertension. The finding could be relevant for human pathology,
indicating that increased salt intake in obese individuals with moderate
hypertension may lead to accelerated end-organ damage.
 |
DISCLOSURES
|
---|
This study was supported by National Institutes of Health Grant HL-54810, a
Grant-in-Aid from the American Heart Association (AHA), and a postdoctoral
fellowship from the MidAtlantic Affiliate of the AHA to Dr. A. D. Dobrian.
 |
FOOTNOTES
|
---|
Address for reprint requests and other correspondence: A. Dobrian, Eastern
Virginia Medical School, Dept. of Physiological Sciences, 700W Olney Rd,
Norfolk, VA 23507 (E-mail:
dobriaad{at}evms.edu).
The costs of publication of this article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section 1734
solely to indicate this fact.
 |
REFERENCES
|
---|
- Bouloumie A,
Marumo T, Lafontan M, and Busse R. Leptin induces oxidative stress in
human endothelial cells. FASEB J
13: 12311238,
1999.[Abstract/Free Full Text]
- Brands MW,
Hildebrandt DA, Mizelle HL, and Hall JE. Hypertension during chronic
hyperinsulinemia in rats is not salt-sensitive.
Hypertension 19:
I83I89, 1992.[ISI][Medline]
- Campese VM,
Tawadrous M, Bigazzi R, Bianchi S, Mann AS, Oparil S, and Raij L. Salt
intake and plasma atrial natriuretic peptide and nitric oxide in hypertension.
Hypertension 28:
335340, 1996.[Abstract/Free Full Text]
- Carlson SH,
Shelton J, White CR, and Wyss JM. Elevated sympathetic activity
contributes to hypertension and salt sensitivity in diabetic obese Zucker
rats. Hypertension 35:
403408, 2000.[Abstract/Free Full Text]
- Cheng ZJ,
Vaskonen T, Tikkanen I, Nurminen K, Ruskoaho H, Vapaatalo H, Muller D, Park
JK, Luft FC, and Mervaala EM. Endothelial dysfunction and salt-sensitive
hypertension in spontaneously diabetic Goto-Kakizaki rats.
Hypertension 37:
433439, 2001.[Abstract/Free Full Text]
- Chobanian AV and Hill M. National Heart, Lung, and Blood Institute Workshop on Sodium
and Blood Pressure: a critical review of current scientific evidence.
Hypertension 35:
858863, 2000.[Free Full Text]
- Correia ML,
Morgan DA, Sivitz WI, Mark AL, and Haynes WG. Leptin acts in the central
nervous system to produce dose-dependent changes in arterial pressure.
Hypertension 37:
936942, 2001.[Abstract/Free Full Text]
- Cubeddu LX,
Hoffmann IS, Jimenez E, Roa CM, Cubeddu RJ, Palermo C, and Baldonedo RM.
Insulin and blood pressure responses to changes in salt intake. J
Hum Hypertens 14, Suppl 1:
S32S35, 2000.[ISI][Medline]
- Ding Y, Gonick
HC, Vaziri ND, Liang K, and Wei L. Lead-induced hypertension. III.
Increased hydroxyl radical production. Am J Hypertens
14: 169173,
2001.[ISI][Medline]
- Dobrian A, Wade
SS, and Prewitt RL. PDGF-A expression correlates with blood pressure and
remodeling in 1K1C
[PDB]
hypertensive rat arteries. Am J Physiol Heart
Circ Physiol 276:
H2159H2167, 1999.[Abstract/Free Full Text]
- Dobrian AD,
Davies MJ, Prewitt RL, and Lauterio TJ. Development of hypertension in a
rat model of diet-induced obesity. Hypertension
35: 10091015,
2000.[Abstract/Free Full Text]
- Dobrian AD,
Davies MJ, Schriver SD, Lauterio TJ, and Prewitt RL. Oxidative stress in a
rat model of obesity-induced hypertension.
Hypertension 37:
554560, 2001.[Abstract/Free Full Text]
- Dobrian AD,
Schriver SD, and Prewitt RL. Role of angiotensin II and free radicals in
blood pressure regulation in a rat model of renal hypertension.
Hypertension 38:
361366, 2001.[Abstract/Free Full Text]
- Dole VP.
The fatty acid pool in adipose tissue. J Biol Chem
236: 31213124,
1961.[ISI]
- Fessel JP,
Porter NA, Moore KP, Sheller JR, and Roberts LJ II. Discovery of lipid
peroxidation products formed in vivo with a substituted tetrahydrofuran ring
(isofurans) that are favored by increased oxygen tension. Proc Natl
Acad Sci USA 99:
1671316718, 2002.[Abstract/Free Full Text]
- Fujiwara N,
Osanai T, Kamada T, Katoh T, Takahashi K, and Okumura K. Study on the
relationship between plasma nitrite and nitrate level and salt sensitivity in
human hypertension: modulation of nitric oxide synthesis by salt intake.
Circulation 101:
856861, 2000.[Abstract/Free Full Text]
- Goodfriend TL,
Ball DL, Weinberger MH, Moore TJ, Weder AB, and Egan BM. Salt loads raise
plasma fatty acids and lower insulin. Hypertension
17: 958964,
1991.[Abstract]
- Gorlach A,
Brandes RP, Nguyen K, Amidi M, Dehghani F, and Busse R. A gp91phox
containing NADPH oxidase selectively expressed in endothelial cells is a major
source of oxygen radical generation in the arterial wall. Circ
Res 87:
2632, 2000.[Abstract/Free Full Text]
- Grobbee DE and
Hofman A. Does sodium restriction lower blood pressure? Br Med
J 293:
2729, 1986.[ISI][Medline]
- Gu JW, Anand V,
Shek EW, Moore MC, Brady AL, Kelly WC, and Adair TH. Sodium induces
hypertrophy of cultured myocardial myoblasts and vascular smooth muscle cells.
Hypertension 31:
10831087, 1998.[Abstract/Free Full Text]
- Gyllenhammar H. Lucigenin chemiluminescence in the assessment
of neutrophil superoxide production. J Immunol Methods
97: 209213,
1987.[ISI][Medline]
- Hayakawa H,
Coffee K, and Raij L. Endothelial dysfunction and cardiorenal injury in
experimental salt-sensitive hypertension: effects of antihypertensive therapy.
Circulation 96:
24072413, 1997.[Abstract/Free Full Text]
- Haynes WG,
Morgan DA, Walsh SA, Sivitz WI, and Mark AL. Cardiovascular consequences
of obesity: role of leptin. Clin Exp Pharmacol Physiol
25: 6569,
1998.[ISI][Medline]
- Haynes WG,
Sivitz WI, Morgan DA, Walsh SA, and Mark AL. Sympathetic and cardiorenal
actions of leptin. Hypertension
30: 619623,
1997.[Abstract/Free Full Text]
- Kannel WB,
Brand N, Skinner JJ Jr, Dawber TR, and McNamara PM. The relation of
adiposity to blood pressure and development of hypertension. The Framingham
study. Ann Intern Med 67:
4859, 1967.[ISI][Medline]
- Klein T,
Neuhaus K, Reutter F, and Nusing RM. Generation of 8-epi-prostaglandin
F(2
) in isolated rat kidney glomeruli by a radical-independent
mechanism. Br J Pharmacol 133:
643650, 2001.[Abstract/Free Full Text]
- Labarca C and
Paigen K. A simple, rapid, and sensitive DNA assay procedure.
Anal Biochem 102:
344352, 1980.[ISI][Medline]
- Laursen JB,
Rajagopalan S, Galis Z, Tarpey M, Freeman BA, and Harrison DG. Role of
superoxide in angiotensin II-induced but not catecholamine-induced
hypertension. Circulation 95:
588593, 1997.[Abstract/Free Full Text]
- Lauterio TJ,
Davies MJ, DeAngelo M, Peyser M, and Lee J. Neuropeptide Y expression and
endogenous leptin concentrations in a dietary model of obesity.
Obes Res 7:
498505, 1999.[Abstract]
- Lawson JA,
Rokach J, and FitzGerald GA. Isoprostanes: formation, analysis and use as
indices of lipid peroxidation in vivo. J Biol Chem
274: 2444124444,
1999.[Free Full Text]
- Luscher TF,
Raij L, and Vanhoutte PM. Endothelium-dependent vascular responses in
normotensive and hypertensive Dahl rats. Hypertension
9: 157163,
1987.[Abstract]
- MacGregor GA. Sodium and potassium intake and blood
pressure. Hypertension 5:
III79III84, 1983.[Medline]
- MacGregor GA,
Markandu ND, Sagnella GA, Singer DR, and Cappuccio FP. Double-blind study
of three sodium intakes and long-term effects of sodium restriction in
essential hypertension. Lancet
2: 12441247,
1989.[ISI][Medline]
- Makino A,
Skelton MM, Zou AP, Roman RJ, and Cowley AW Jr. Increased renal medullary
oxidative stress produces hypertension. Hypertension
39: 667672,
2002.[Abstract/Free Full Text]
- Mohazzab KM,
Kaminski PM, and Wolin MS. NADH oxidoreductase is a major source of
superoxide anion in bovine coronary artery endothelium. Am J
Physiol Heart Circ Physiol 266:
H2568H2572, 1994.[Abstract/Free Full Text]
- Nishida Y, Ding
J, Zhou MS, Chen QH, Murakami H, Wu XZ, and Kosaka H. Role of nitric oxide
in vascular hyperresponsiveness to norepinephrine in hypertensive Dahl rats.
J Hypertens 16:
16111618, 1998.[ISI][Medline]
- Ortiz PA and
Garvin JL. Interaction of O(2)() and NO in the thick ascending
limb. Hypertension 39:
591596, 2002.[Abstract/Free Full Text]
- Rocchini AP. Obesity hypertension, salt sensitivity and
insulin resistance. Nutr Metab Cardiovasc Dis
10: 287294,
2000.[ISI][Medline]
- Rodbell M and
Krishna G. Preparation of isolated fat cells and fat cell
"ghosts"; methods for assaying adenylate cyclase activity and
levels of cyclic AMP. Methods Enzymol
31: 103114,
1974.[Medline]
- Russo C,
Olivieri O, Girelli D, Faccini G, Zenari ML, Lombardi S, and Corrocher R.
Anti-oxidant status and lipid peroxidation in patients with essential
hypertension. J Hypertens 16:
12671271, 1998.[ISI][Medline]
- Sagar S, Kallo
IJ, Kaul N, Ganguly NK, and Sharma BK. Oxygen free radicals in essential
hypertension. Mol Cell Biochem
111: 103108,
1992.[ISI][Medline]
- Schnackenberg CG, Welch WJ, and Wilcox CS. Normalization of
blood pressure and renal vascular resistance in SHR with a membrane-permeable
superoxide dismutase mimetic: role of nitric oxide.
Hypertension 32:
5964, 1998.[Abstract/Free Full Text]
- Squadrito GL and Pryor WA. The formation of peroxynitrite in vivo from nitric oxide and
superoxide. Chem Biol Interact
96: 203206,
1995.[ISI][Medline]
- Suzuki H, Swei
A, Zweifach BW, and Schmid-Schonbein GW. In vivo evidence for
microvascular oxidative stress in spontaneously hypertensive rats.
Hydroethidine microfluorography. Hypertension
25: 10831089,
1995.[Abstract/Free Full Text]
- Swei A, Lacy F,
Delano FA, Parks DA, and Schmid-Schonbein GW. A mechanism of oxygen free
radical production in the Dahl hypertensive rat.
Microcirculation 6:
179187, 1999.[ISI][Medline]
- Swei A, Lacy F,
DeLano FA, and Schmid-Schonbein GW. Oxidative stress in the Dahl
hypertensive rat. Hypertension
30: 16281633,
1997.[Abstract/Free Full Text]
- Tallam LS and
Jandhyala BS. Significance of exaggerated natriuresis after angiotensin
AT1 receptor blockade or angiotensin-converting enzyme inhibition
in obese Zucker rats. Clin Exp Pharmacol Physiol
28: 433440,
2001.[ISI][Medline]
- Trolliet MR,
Rudd MA, and Loscalzo J. Oxidative stress and renal dysfunction in
salt-sensitive hypertension. Kidney Blood Press Res
24: 116123,
2001.[ISI][Medline]
- Tsutsui H, Ide
T, Hayashidani S, Kinugawa S, Suematsu N, Utsumi H, and Takeshita A.
Effects of ACE inhibition on left ventricular failure and oxidative stress in
Dahl salt-sensitive rats. J Cardiovasc Pharmacol
37: 725733,
2001.[ISI][Medline]
- Vaziri ND,
Liang K, and Ding Y. Increased nitric oxide inactivation by reactive
oxygen species in lead-induced hypertension. Kidney
Int 56:
14921498, 1999.[ISI][Medline]
- Vaziri ND,
Oveisi F, and Ding Y. Role of increased oxygen free radical activity in
the pathogenesis of uremic hypertension. Kidney Int
53: 17481754,
1998.[ISI][Medline]
- Vaziri ND, Wang
XQ, Oveisi F, and Rad B. Induction of oxidative stress by glutathione
depletion causes severe hypertension in normal rats.
Hypertension 36:
142146, 2000.[Abstract/Free Full Text]
- Weinberger MH. Salt sensitivity of blood pressure in humans.
Hypertension 27:
481490, 1996.[Abstract/Free Full Text]
- Wilcox CS.
Reactive oxygen species: roles in blood pressure and kidney function.
Curr Hypertens Rep 4:
160166, 2002.[ISI][Medline]