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Antioxidant SMe1EC2 may attenuate the disbalance of sodiumhomeostasis in the organism induced by higher intakeof cholesterol
Lucia Mezesova • Veronika Jendruchova-Javorkova •
Jana Vlkovicova • Zuzana Kyselova •
Jana Navarova • Stefan Bezek • Norbert Vrbjar
Received: 5 October 2011 / Accepted: 2 March 2012 / Published online: 22 March 2012
� Springer Science+Business Media, LLC. 2012
Abstract The study was focused to the influence of
higher intake of cholesterol on properties of the renal
Na,K-ATPase, a key system in maintaining the homeo-
stasis of sodium in the organism. Feeding for 4 weeks with
cholesterol-enriched food for rats afflicted with hereditary
hypertriglyceridemia by itself enhanced the activity of
Na,K-ATPase, probably as a consequence of higher num-
ber of active enzyme molecules as suggested by 32 %
increase of Vmax value. This may be hypothesized as a
reason for the increased retention of sodium. Three-week-
lasting treatment of animals kept on high cholesterol diet
with antioxidant SMe1EC2 in a dose of 10 mg kg-1 day-1
normalized the function of renal Na,K-ATPase to the level
comparable in hypertriglyceridemic rats fed with the
standard diet. Therefore, our results suggest that the anti-
oxidant SMe1EC2 in the applied dose seems to be effective
in the attenuation of cholesterol-induced retention of
sodium. Treatment for 3 weeks with Fenofibrate in a dose
of 100 mg kg-1 day-1 reversed the function of renal
Na,K-ATPase only slightly.
Keywords Sodium pump � Kidney � Antioxidant �Fibrate � Hypertriglyceridemia
Introduction
Hypertriglyceridemia as an independent risk factor for
coronary heart disease is involved in the development of
atherosclerosis and hypertension. It was shown that
reduction of triglycerides is associated with decreased
number of cardiovascular events particularly among
patients with diagnosed heart disease [1, 2]. An important
class of lipid-lowering agents is represented by fibrates
markedly lowering triglyceride level and modestly raising
the HDL-C level. It has been summarized that fibrate
therapy does not reduce mortality but may reduce nonfatal
coronary events in patients at risk for cardiovascular dis-
ease [3]. Besides the disturbance of cardiovascular system,
dyslipidemia also is a contributory factor in the progression
of glomerular injury in nephrotic syndrome as documented
by increased mean serum level of total cholesterol (TC),
LDL-C, triglycerides, and significantly decreased level of
HDL-C [4].
The kidney as a major player in the maintenance of salt
and water homeostasis accounting for the absorption of
approximately 70 % Na? and water-filtered load is highly
enriched in the Na,K-ATPase or the so-called sodium
pump which transports three Na? ions out of the cell and
two K? ions into the cell using the energy derived from
hydrolysis of one molecule of ATP [5]. Previous studies
using Prague hereditary hypertriglyceridemic (HTG) rats,
as an often-used experimental model, documented increase
of blood pressure [6], cardiac fibrosis [7], alterations in
distribution of connexin 43 in aorta [8], and in cardiac
tissue, the latter being connected to higher susceptibility to
arrhythmias [9]. In addition in HTG rats, hyperinsulinemia
was documented [10]. Hyperinsulinemia is accompanied
with increased abundance of Na,K-ATPase in kidneys as
documented previously. This finding was hypothesized as a
L. Mezesova � V. Jendruchova-Javorkova � J. Vlkovicova �N. Vrbjar (&)
Institute for Heart Research, Department of Biochemistry,
Slovak Academy of Sciences, Dubravska cesta 9, P.O. Box 104,
840 05 Bratislava 45, Slovak Republic
e-mail: usrdnorb@savba.sk
Z. Kyselova � J. Navarova � S. Bezek
Institute of Experimental Pharmacology and Toxicology, Slovak
Academy of Sciences, Dubravska cesta 9, 841 04 Bratislava,
Slovak Republic
123
Mol Cell Biochem (2012) 366:41–48
DOI 10.1007/s11010-012-1281-3
possible reason for the increased retention of Na? [11].
Therefore, the aim of our study was to determine if an
abnormal triglyceride metabolism might play a causal role
in alterations of properties of the renal Na,K-ATPase, a key
enzyme involved in the maintenance of sodium ions in the
organism. Using genetically hypertensive rats of the Prague
hereditary HTG strain, we investigated whether the per-
turbation of lipid metabolism induced by high cholesterol
intake and/or their lowering by chronic treatment with
fenofibrate and the antioxidant SMe1EC2 are followed by
reciprocal changes in the properties of the enzyme.
Materials and methods
Animal model
Experiments were performed on adult 4-month-old male
Prague hereditary HTG rats. At the beginning of experi-
ments, the animals were divided into five groups (n = 7 in
each experimental group): First group of HTG rats was fed
with standard diet (H). The second group of HTG rats was
treated by p.o. administration of SMe1EC2 in a dose of
10 mg kg-1 day-1 (HS). The third group of HTG rats was
fed with diet enriched by cholesterol (1 % w/w) and lard
(7.5 % w/w) (HCh). In the fourth group of rats with higher
cholesterol intake at the seventh day of experiment, the p.o.
administration with a dose of 100 mg kg-1 day-1 fenofi-
brate (HChF) was started. The fifth group of rats with high
cholesterol intake was from the same time treated by p.o.
administration of SMe1EC2 in a dose of 10 mg kg-1 day-1
(HChS). The cholesterol was purchased from Sigma-
Aldrich, the fenofibrate was gifted by Zentiva, Slovak
republic, and the SMe1EC2 was synthesized at the Institute
of Experimental Toxicology and Pharmacology. All the rats
were allowed free access to food and drinking water. At the
end of the experiment lasting 28 days, the rats were anes-
thetized by thiopental (Valeant Czech Pharma, Czech
republic) in a dose of 65 mg kg-1.The excised kidneys were
immediately frozen in liquid nitrogen and stored for further
investigations of Na,K-ATPase properties.
All the experiments were in accordance with the
approval of the Veterinary Council of the Slovak Republic
(Decree No. 289, part 139, July 9th 2003), and they con-
formed to Principles of Laboratory Animals Care (NIH
publication 83-25, revised 1985).
Lipid profile
Lipid profile was characterized by serum levels of TC and
triacylglycerols (TAG). These biochemical parameters
were assayed using commercially available diagnostic kits
(RANDOX Laboratories Ltd., UK).
Oxidative status
Malondialdehyde level, as an index of lipid peroxidation,
was measured colorimetrically via reaction with thiobar-
bituric acid as a thiobarbituric acid-reactive substance
(TBARS) [12].
Creatinine clearance
Creatinine was measured in plasma and urine samples
using commercially available diagnostic kit (RANDOX
Laboratories Ltd., UK), and glomerular filtration rate
(GFR) was calculated using the standard formula.
Preparation of plasmalemmal fraction for kinetic
measurements
The plasmalemmal membrane fraction from rat kidney was
isolated according to Jorgensen [13] with slight modifica-
tions. In brief, the renal tissue was homogenized in cold
isolation medium containing (in mmol l-1): 250 sucrose,
25 imidazol, and 1 EDTA (pH 7.4), using a tissue disruptor
(3 9 10 s at a setting of 4, Polytron PT-20). The homog-
enate was centrifuged at 6,0009g for 15 min. The sedi-
ment was re-homogenized and centrifuged again at
6,0009g for 15 min. The collected supernatants from both
centrifugations were re-centrifuged at 48,0009g for
30 min, and the final sediment was re-suspended in the
isolation medium. An aliquot was removed for determi-
nation of proteins by the method of Lowry et al. [14] using
bovine serum albumin as a standard.
Kinetic measurements of Na,K-ATPase
ATP-kinetics of Na,K-ATPase was estimated at the tem-
perature of 37 �C measuring the hydrolysis of ATP by
10 lg plasmalemmal proteins in the presence of increasing
concentrations of substrate ATP (0.16–8.0 mmol l-1). The
total volume of medium was 0.5 ml containing (in
mmol l-1): MgCl2 4, KCl 10, NaCl 100, and imidazole 50
(pH 7.4). After 20 min of pre-incubation in substrate-free
medium, the reaction was started by addition of ATP, and
after 20 min, the reaction was stopped by addition of 0.3 ml
12 % ice-cold solution of trichloroacetic acid. The liberated
inorganic phosphorus was determined according to Taussky
and Shorr [15]. In order to establish the Na,K-ATPase
activity, the ATP hydrolysis that occurred in the presence of
Mg2? only was subtracted. The Na,K-ATPase kinetics for
cofactor Na? was determined by the same method, with
increasing concentration of NaCl (2.0–100.0 mmol l-1).
The amount of ATP was constant (8 mmol l-1). The kinetic
parameters Vmax, Km, and KNa were evaluated from the
obtained data by means of direct nonlinear regression. The
42 Mol Cell Biochem (2012) 366:41–48
123
parameter Vmax represents the maximal velocity, Km, and
KNa values represent the concentrations of ATP or Na?
necessary for inducing half the maximal activation of the
enzyme. All the results were expressed as mean ± S.E.M.
The significance of differences between the individual
groups was determined using the one-way analysis of var-
iance (ANOVA) by Student–Newman–Keuls test. A value
of p \ 0.05 was regarded as significant.
Results
Body weight and kidney weight
The supplementation of high amount of cholesterol in the
diet lasting for 4 weeks did not alter either the body weight
or the kidney weight in HTG rats. Administration of fe-
nofibrate or antioxidant SMe1EC2 also did not affect the
investigated weight parameters. All data are presented in
Table 1.
TC and TAG contents in plasma
Supplementation of high amount of cholesterol in the diet
was followed by surprisingly decreased content of total
serum cholesterol when comparing the HCh group with H
group. However, this decrease was statistically insignifi-
cant. Administration of fenofibrate or antioxidant
SMe1EC2 also did not significantly affect the content of
TC (Table 2).
Concerning the concentration of TAG, we observed a
trend of decrease in the level of TAG in animals with
high cholesterol diet, but this change was statistically
insignificant. The content of TAG in serum was signifi-
cantly decreased in the HChF group when comparing with
the H group and also with the HCh group (Table 2).
The oxidative status
As a measure for possible oxidative damage of renal tissue
the estimation of TBARS was performed in our experi-
mental groups. There were no significant alterations among
the groups (Table 3).
GFR
We used creatinine clearance to estimate the GFR in our
experimental groups of rats. The cholesterol-rich diet
slightly decreased the GFR, but this change was statisti-
cally insignificant. Administration of fenofibrate signifi-
cantly reduced the GFR (by 75 %) as compared with the
HCh group. Administration of antioxidant SMe1EC2 didTable 1 Weight parameters of hereditary HTG rats (H), HTG rats
treated with antioxidant SMe1EC2 in a dose of 10 mg kg-1 day-1
(HS), HTG rats fed with cholesterol (1 % w/w) and lard (7.5 % w/w)
for 4 weeks (HCh), rats fed with high cholesterol for 4 weeks and
treated with antioxidant SMe1EC2 in a dose of 10 mg kg-1 day-1
during the last 3 weeks (HChS), rats fed with high cholesterol for
4 weeks and treated with fenofibrate in a dose of 100 mg kg-1 day-1
during the last 3 weeks (HChF)
Groups of rats Bw (g) Kw (L ? R)
(mg)
Kw (L ? R)/Bw
(mg g-1)
H 293 ± 5 2204 ± 65 7.5 ± 0.2
HS 309 ± 9 2253 ± 25 7.3 ± 0.2
HCh 291 ± 12 2171 ± 77 7.5 ± 0.1
HChS 307 ± 14 2163 ± 36 7.1 ± 0.3
HChF 285 ± 12 2372 ± 93 8.3 ± 0.1
Data represent means ± SEM at the end of experiment, n = 7 in all
groups
Bw body weight, Kw (L ? R) kidney weight (left ? right), Kw(L ? R)/Bw kidney weight/body weight ratio
Table 2 TAG and TC contents in hereditary HTG rats (H), HTG rats
treated with antioxidant SMe1EC2 in a dose of 10 mg kg-1 day-1
(HS), HTG rats fed with cholesterol (1 % w/w) and lard (7.5 % w/w)
for 4 weeks (HCh), rats fed with high cholesterol for 4 weeks and
treated with antioxidant SMe1EC2 in a dose of 10 mg kg-1 day-1
during the last 3 weeks (HChS), rats fed with high cholesterol for
4 weeks and treated with fenofibrate in a dose of 100 mg kg-1 day-1
during the last 3 weeks (HChF)
Groups of rats TAG (nmol l-1) TC (nmol l-1)
H 1.88 ± 0.24 3.12 ± 0.24
HS 1.45 ± 0.25 2.64 ± 0.05
HCh 1.59 ± 0.14 2.93 ± 0.15
HChS 1.08 ± 0.29 2.59 ± 0.15
HChF 0.68 ± 0.14a,b 2.59 ± 0.17
Statistical significance p \ 0.05, comparisons a versus H, b versus
HCh
Table 3 Concentrations of TBARS and proteins in renal tissue in
hereditary HTG rats (H), HTG rats treated with antioxidant SMe1EC2
in a dose of 10 mg kg-1 day-1 (HS), HTG rats fed with cholesterol
(1 % w/w) and lard (7.5 % w/w) for 4 weeks (HCh), rats fed with
high cholesterol for 4 weeks and treated with antioxidant SMe1EC2
in a dose of 10 mg kg-1 day-1 during the last 3 weeks (HChS), and
rats fed with high cholesterol for 4 weeks and treated with fenofibrate
in a dose of 100 mg kg-1 day-1 during the last 3 weeks (HChF)
Groups of rats TBARS
(lmol g-1
tissue)
Proteins
(mg g-1
tissue)
TBARS/
proteins
(nmol mg-1)
H 2.90 ± 0.23 122 ± 2 23.75 ± 2.00
HS 2.99 ± 0.29 120 ± 3 24.94 ± 2.74
HCh 3.09 ± 0.47 135 ± 6 22.90 ± 3.31
HChS 2.20 ± 0.36 123 ± 4 17.85 ± 2.91
HChF 3.37 ± 0.34 137 ± 3 24.59 ± 2.86
Mol Cell Biochem (2012) 366:41–48 43
123
not alter significantly the GFR in HTG animals kept on
either standard or cholesterol-rich diet (Fig. 1).
Kinetics of Na,K-ATPase
Comparison of HTG rats fed with chow enriched in choles-
terol (HCh) with untreated control HTG rats (H) resulted in
variations of kinetic properties of Na,K-ATPase molecule.
When activating the enzyme with increasing concentration of
ATP, we observed a significant increase of the enzyme
activity in the HCh group throughout the whole concentration
range of the substrate. The highest increase 36 % was
observed in the presence of 0.16 mmol l-1 ATP. With
increasing concentration of ATP the effect decreased stepwise
to 32 % observed in the presence of 8 mmol l-1 ATP (Fig. 2).
Evaluation of the above data by the method of nonlinear
regression resulted in statistically significant increase of Vmax
value by 32 %, while the Km value in HCh group remained
unchanged, as compared with HTG rats kept on standard diet
(H) (Fig. 3). Activation of the enzyme with increasing con-
centration of NaCl resulted again in significantly higher
activities in the HCh group. The effect decreased from 42 to
33 % with increasing concentrations of cofactor in the range
2–100 mmol l-1 of NaCl (Fig. 4). Evaluation of kinetic
parameters for activation with Na? revealed an increase of
Vmax value by 32 % in HCh group. The KNa values were
similar in both HCh and H groups (Fig. 5).
The antioxidant SMe1EC2 stimulated the Na,K-ATPase
activity in animals kept on standard diet. When activating
the enzyme with increasing concentration of ATP, we
observed an increase in the activity of the enzyme in HS
compared with the H group. The effect of treatment
increased with increasing concentration of substrate
reaching the maximal stimulation by 16 % in the presence
of 8 mmol l-1 ATP (Fig. 2). Evaluation of the above data
by the method of nonlinear regression resulted in statisti-
cally significant increase of Vmax by 19 % and that of Km
value by 22 % in HS group, compared with the H group
(Fig. 3). When activating the enzyme with increasing
concentration of Na? ions, the stimulatory effect of
SMe1EC2 varied between 20 and 11 % (Fig. 4) resulting
in statistically insignificant alterations in the values of Vmax
and KN (Fig. 5).
On the other hand, in animals fed with cholesterol-rich
diet, the antioxidant SMe1EC2 induced a 30 % decrease of
Na,K-ATPase activity throughout the concentration range of
ATP (0.16–8 mmol l-1 ATP) when comparing the HChS
group with the HCh group (Fig. 2). Consequently, the Vmax
H HS HChHChS
HChF
Cre
atin
ine
clea
ranc
e (m
l·min
-1)
0
1
2
3
4
5
6
a
Fig. 1 Clearance of creatinine in hereditary HTG rats (H), HTG rats
treated with antioxidant SMe1EC2 in a dose of 10 mg kg-1 day-1
(HS), HTG rats fed with cholesterol (1 % w/w) and lard (7.5 % w/w)
for 4 weeks (HCh), rats fed with high cholesterol for 4 weeks, and
treated with antioxidant SMe1EC2 in a dose of 10 mg kg-1 day-1
during the last 3 weeks (HChS), rats fed with high cholesterol for
4 weeks and treated with fenofibrate in a dose of 100 mg kg-1 day-1
during the last 3 weeks (HChF). Statistical significance p \ 0.01;
comparisons a versus HCh
[ATP] (mmol.l -1)
Na,
K-A
TP
ase
acti
vity
(µm
ol P
i . m
g-1
pro
tein
. h
-1)
0
5
10
15
20
25
H
HS
HCh
[ATP] (mmol.l -1)
0.16 0.32 0.48 0.64 0.80
0 2 4 6 8
activ
ity
0
10
20
30
40
HChS
HChF
Fig. 2 Activation of renal Na,K-ATPase by low concentrations of
substrate ATP in hereditary HTG rats (H), HTG rats treated with
antioxidant SMe1EC2 in a dose of 10 mg kg-1 day-1 (HS), HTG rats
fed with cholesterol (1 % w/w) and lard (7.5 % w/w) for 4 weeks
(HCh), rats fed with high cholesterol for 4 weeks and treated with
antioxidant SMe1EC2 in a dose of 10 mg kg-1 day-1 during the last
3 weeks (HChS), and rats fed with high cholesterol for 4 weeks and
treated with fenofibrate in a dose of 100 mg kg-1 day-1 during the
last 3 weeks (HChF). Inset activation of the enzyme in the whole
range of ATP concentration
44 Mol Cell Biochem (2012) 366:41–48
123
was decreased by 30 % in the HChS group with no altera-
tions in the Km value (Fig. 3). When activating the enzyme
with increasing concentration of NaCl, we observed again
lower activities in the HChS group. The effect decreased
from 41 to 34 % inhibition with increasing concentration of
cofactor in the range 2–100 mmol l-1 of NaCl (Fig. 4).
Evaluation of kinetic parameters resulted in unchanged KNa
value, but the Vmax value was lower by 32 % in HChS group
compared with the HCh group (Fig. 5).
Administration of fenofibrate to rats fed with choles-
terol-rich diet induced a diminution of Na,K-ATPase
activity by 10 % in the whole concentration range of ATP
when comparing the HChF group with the HCh group
(Fig. 2). Both kinetic parameters, i.e., the Km and Vmax,
were not changed significantly in HChF group versus the
HCh group (Fig. 3). Similar pattern was observed also for
activation of the enzyme with increasing concentrations of
sodium (Fig. 4) resulting again in no changes in the values
of Km and Vmax when comparing the HChF group with the
HCh group (Fig. 5).
Discussion
The diet enriched in cholesterol (1 % w/w) and lard (7.5 %
w/w) did not induce significant alterations in levels of
plasma cholesterol and TAG suggesting that during the
course of the 4-week experiment, the liver was able to keep
the lipid metabolism on the level like in animals on stan-
dard diet. In addition, the higher intake of cholesterol also
did not induce significant alteration in renal function as
shown by stable value of creatinine clearance. However,
administration of fenofibrate was followed by significant
deterioration of GFR. This finding is consistent with data
from various clinical and experimental studies [16–18].
The second compound used for treatment of experimental
animals in our experiments was the antioxidant SMe1EC2.
For this compound, a remarkable antioxidant efficacy was
observed in rat brain homogenates exposed to iron/ascor-
bate system by means of protection of lipids and creatine
kinase against the oxidative impairment [19]. Administra-
tion of SMe1EC2 protected also the endothelial function. It
significantly decreased endothelemia of diabetic rats and
improved endothelium-dependent relaxation of arteries
with slightly decreased ROS-production [20]. Our data
indicate the possible organ specificity of the antioxidant
SMe1EC2 as suggested by the lack of significant influence
of the above compound on the antioxidant status of the
kidney as shown by similarities of TBARS in HChS and
H HS HChHChS
HChFVm
ax (
µ mo
l Pi.m
g-1 p
rote
in.h
-1)
0
10
20
30
40
50
H HS HChHChS
HChF
Km
(m
mo
l.l-1 A
TP
)
0.0
0.2
0.4
0.6
0.8
1.0
1.2
a
a
d
a
b,c
b bb
Fig. 3 Kinetic parameters of renal Na,K-ATPase during activation
with ATP in hereditary HTG rats (H), HTG rats treated with
antioxidant SMe1EC2 in a dose of 10 mg kg-1 day-1 (HS), HTG rats
fed with cholesterol (1 % w/w) and lard (7.5 % w/w) for 4 weeks
(HCh), rats fed with high cholesterol for 4 weeks and treated with
antioxidant SMe1EC2 in a dose of 10 mg kg-1 day-1 during the last
3 weeks (HChS), and rats fed with high cholesterol for 4 weeks and
treated with fenofibrate in a dose of 100 mg kg-1 day-1 during the
last 3 weeks (HChF). Statistical significance p \ 0.05; comparisons:
a versus H, b versus HS, c versus Hch, and d versus HchS
[NaCl] (mmol.l-1)
Na,
K-A
TP
ase
acti
vity
(µm
ol P
i . m
g-1
pro
tein
. h
-1)
0
5
10
15
20
25
H
HS
HCh
[NaCl] (mmol.l-1)
0 2 4 6 8 10
0 20 40 60 80 100
activ
ity
0
10
20
30
40
HChS
HChF
Fig. 4 Activation of renal Na,K-ATPase by low concentrations of
cofactor Na? in hereditary HTG rats (H), HTG rats treated with
antioxidant SMe1EC2 in a dose of 10 mg kg-1 day-1 (HS), HTG rats
fed with cholesterol (1 % w/w) and lard (7.5 % w/w) (HCh) for 4
weeks, rats fed with high cholesterol for 4 weeks, and treated with
antioxidant SMe1EC2 in a dose of 10 mg kg-1 day-1 during the last
3 weeks (HChS), rats fed with high cholesterol for 4 weeks and
treated with fenofibrate in a dose of 100 mg kg-1 day-1 during the
last 3 weeks (HChF). Inset activation of the enzyme in the whole
range of NaCl concentration
Mol Cell Biochem (2012) 366:41–48 45
123
HCh groups. Maybe the lack of significant effect of
SMe1EC2 on TBARS in HChS group was caused by rel-
atively moderate dosage of applied substance. In another
experiment when higher dosage of 30 mg kg-1 day-1 was
applied, the concentration of TBARS was lower on statis-
tically significant level (data not shown).
Some previous studies demonstrated a deleterious effect
of cholesterol on the activity [21, 22] and also expression
of Na,K-ATPase [23]. Our results concerning the Na,K-
ATPase activity in HTG rats fed for 4 weeks with diet
enriched with cholesterol surprisingly showed an opposite
stimulatory effect. The higher activity of Na,K-ATPase in
HCh rats observed in the presence of all concentrations of
ATP or Na?, respectively, was caused probably by the
higher number of active enzyme molecules as indicated by
the increased Vmax values for both types of enzyme acti-
vation. This increase in the amount of active enzyme
molecules was not accompanied with qualitative alterations
in binding properties of the Na,K-ATPase for ATP and
Na? as indicated by unchanged values of Km and KNa. The
observed accumulation of active Na,K-ATPase molecules
in kidney may represent an adaptation of the enzyme as a
consequence of higher consumption of cholesterol during
the 4 weeks of feeding. Similar adaptation of Na,K-ATPase
to changed physiological conditions was observed also in a
case of short-time-lasting diabetes which was followed by
an increased activity and expression of the enzyme in renal
tissue [24–26].
The increased Na,K-ATPase expression was ascribed to
be involved in development of symptoms of salt and water
retention in patients with proteinuric kidney diseases [27].
It may be hypothesized that the increased presence of
Na,K-ATPase in kidney of HTG rats may be also one of the
causalities inducing increased retention of Na?. In addition
in HTG rats, hyperinsulinemia was documented [10].
Hyperinsulinemia is accompanied with increased abun-
dance of Na,K-ATPase in kidneys as has been documented
previously. This finding was hypothesized as a possible
reason for the increased retention of Na? [11]. For this
increase, the renal Na,K-ATPase was, at least partially,
responsible as shown by the higher enzyme activity in
HTG rats compared with control Wistar rats [28]. There-
fore, the improved extrusion of intracellular sodium out of
the cell, as a consequence of the increased Na,K-ATPase
activity, may represent another impulse for the additional
increase of sodium retention caused by higher cholesterol
consumption during the 4 weeks. This hypothesis is in
agreement with previous observation that hypercholester-
olemia is accompanied with hypernatremia [29]. The
mechanism of the observed increase in Na,K-ATPase
activity due to higher cholesterol intake might be explained
on the basis of observation of Chen et al. [30], who dem-
onstrated that the above enzyme is involved in the control
of the plasma membrane cholesterol distribution. More-
over, the reciprocal regulation of alpha 1 subunit of Na,K-
ATPase by cholesterol was demonstrated as the expression
of alpha 1 subunit was significantly reduced; when the
intracellular cholesterol trafficking was blocked, it was
hypothesized that the decrease in the plasma membrane
cholesterol stimulated the endocytosis and the degradation
of alpha 1 subunit of Na,K-ATPase [31]. Therefore, we
hypothesize that the higher intake in cholesterol may be
followed by higher cholesterol content in plasma mem-
branes, and thereby the endocytosis and the degradation of
Na,K-ATPase might be slowed down resulting in higher
number of active enzyme molecules in renal tissue.
Among the substances used for treatment of hypertri-
glyceridemia one important group is represented by PPARaagonists. Post hoc analyses of several of fibrate studies
have shown that overweight people, with high plasma tri-
glyceride levels and low levels of HDL cholesterol, derive
a disproportionately large reduction in cardiovascular
events when treated with these agents [32]. Experimental
studies revealed that beside other effects, PPARa agonists
attenuated the increase of blood pressure and sodium
retention in DOCA-salt hypertension as was demonstrated
in the case of clofibrate [33, 34]. Combination of other
fibrates (gemfibrozil, WY-14643) with manitol markedly
improved renal preservation [35]. Our data showed a slight
diminution in the number of active Na,K-ATPase mole-
cules as suggested by statistically insignificant decrease of
Vmax values in both types of enzyme activation, when
comparing the HChF with HCh group. The low effective-
ness of fenofibrate may be caused by its lower dose in our
H HS HChHChS
HChFVm
ax (
µ mo
l Pi.m
g-1
pro
tein
.h-1
)
0
10
20
30
40
50
H HS HChHChS
HChF
KN
a (
mm
ol.l
-1 N
aCl)
0
4
8
12
16
20a,b
c
d
Fig. 5 Kinetic parameters of renal Na,K-ATPase during activation
with NaCl in hereditary HTG rats (H), HTG rats treated with
antioxidant SMe1EC2 in a dose of 10 mg kg-1 day-1 (HS), HTG rats
fed with cholesterol (1 % w/w) and lard (7.5 % w/w) (HCh) for 4
weeks, rats fed with high cholesterol for 4 weeks and treated with
antioxidant SMe1EC2 in a dose of 10 mg kg-1 day-1 during the last
3 weeks (HChS), and rats fed with high cholesterol for 4 weeks and
treated with fenofibrate in a dose of 100 mg kg-1 day-1 during the
last 3 weeks (HChF). Statistical significance p \ 0.05; comparisons:
a versus H, b versus HS, c versus HCh, and d versus HChS
46 Mol Cell Biochem (2012) 366:41–48
123
experiment (100 mg kg-1 day-1) compared with 2.5 times
higher dose of clofibrate, revealing significant depression
of Na,K-ATPase activity in proximal tubules [33]. It may
be hypothesized that certain increase of the fenofibrate
dose in future studies might increase its efficiency, thus
resulting in possibly more significant decrease of the
sodium retention in the organism.
Previously, it was reported that HTG rats exhibit some
signs of oxidative damage including increased lipoprotein
oxidability and lipid peroxidation [36]. The increased pro-
duction of reactive oxygen species and decreased availabil-
ity of nitric oxide have been suggested to be responsible for
endothelial dysfunction in HTG rats [37, 38]. Natural anti-
oxidants from plants improved antioxidant status and posi-
tively affected the plasma lipoprotein profile in HTG rats
[39]. Our previous studies revealed a protection of renal
Na,K-ATPase molecule by natural antioxidants extracted
from red wine [40, 41] and also synthetic antioxidants like
stobadine [42, 43] in condition of various patophysiological
overloads like hypertension or diabetes mellitus type 1. In the
present study, administration of antioxidant SMe1EC2 an
analogue of pyridoindole antioxidant stobadine to rats fed
with high cholesterol diet decreased the activity and the
amount of active molecules of Na,K-ATPase as suggested by
comparison of Vmax values in HChS and HCh groups.
Therefore, the antioxidant attenuated the cholesterol-
induced effect on the number of active enzyme molecules in
renal tissue, thus resulting probably in the reduced sodium
retention in rats fed with cholesterol rich diet. The antioxi-
dant SMe1EC2 in the applied dose normalized the function
of renal Na,K-ATPase to the level comparable in HTG rats
fed with standard diet.
In conclusion, higher intake of cholesterol induced an
increase in the number of active Na,K-ATPase molecules
in HTG rats, which may result in the increased retention of
sodium. Treatment with antioxidant SMe1EC2 in the
applied dose normalized the function of renal Na,K-ATP-
ase to the level comparable in HTG rats fed with standard
diet. Fenofibrate in the applied dose reversed the function
of renal Na,K-ATPase only slightly.
Acknowledgments The present study was supported by the Slovak
Grant Agencies VEGA 2/0115/10 and by VEGA 2/0086/08. The
authors thank Mrs. Z. Hradecka for her careful technical assistance.
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