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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