Life Sciences 90 (2012) 4753

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Life Sciences
journal homepage: www.elsevier.com/locate/lifescie

Lack of modulatory effect of simvastatin on indoxyl sulfate-induced activation of

cultured endothelial cells
Chien-Te Lee a,, Yueh-Ting Lee a, Hwee-Yeong Ng a, Terry Ting-Yu Chiou a, Cheng-I Cheng b,
Chien-Chun Kuo a, Chien-Hsing Wu a, Po-Jui Chi a, Wen-Chin Lee a
a
b

Division of Nephrology, Department of Internal Medicine, Kaohsiung Chang-Gung Memorial Hospital and Chang-Gung University College of Medicine, Kaohsiung, Taiwan
Division of Cardiology, Department of Internal Medicine, Kaohsiung Chang-Gung Memorial Hospital and Chang-Gung University College of Medicine, Kaohsiung, Taiwan

a r t i c l e

i n f o

Article history:
Received 26 March 2011
Accepted 8 October 2011
Keywords:
Indoxyl sulfate
Statins
Adhesion molecule
Endothelial cell

a b s t r a c t
Aims: Endothelial dysfunction is a common manifestation of chronic kidney disease (CKD). The proteinbound uremic toxins have emerged as important factors associated with cardiovascular disease and the outcome of CKD. The effect of indoxyl sulfate (IS) on endothelial cells remains unclear.
Main methods: Human umbilical endothelial cells (HUVEC) were incubated using IS at two concentrations:
100 M and 1000 M over two periods of time: 16 and 48 h. HUVEC were also pre-treated with simvastatin
to examine its effect. RT-PCR was used to assess changes in the gene expression of intracellular cell adhesion
molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), Monocyte chemotactic protein-1 (MCP1), E-selectin, and angiotensin receptor type 1 (AT1R). Protein abundance of the investigated molecules was
assessed by immunoblotting.
Key ndings: Treatment with 100 M IS for 16 h induced a 2-fold increase in the expression of ICAM-1, VCAM1, and MCP-1. At a concentration of 1000 M, there was a 23-fold increase. An extended treatment period at
low concentrations was associated with a 23 fold increase and the increase of ICAM-1 and VCAM-1 was
more prominent under high concentration. Results of immunoblotting conrmed an increase in the abundance of ICAM-1, VCAM-1 and MCP-1. No signicant change was noted in E-selectin and AT1R according
to concentration or treatment duration. Pre-treatment with simvastatin did not alter IS-induced changes.
Signicance: IS increased the expression of adhesion molecules of endothelial cells exhibiting a concentration
and duration dependent pattern. Simvastatin did not demonstrate any effect on IS-associated endothelial
activation.
2011 Elsevier Inc. All rights reserved.

Introduction
Chronic kidney disease (CKD) is associated with an increase in
cardiovascular disease (Fort, 2005). Irrespective of underlying renal
disease, patients with renal impairment manifest uremic symptoms
as renal function progressively deteriorates. It has been postulated
that renal failure syndrome is mainly due to a reduction in renal function, and the compounds retained in renal failure, so called uremic
toxins produce a variety of deleterious effects involving many organ
systems (Vanholder et al., 2008a). Protein-bound uremic toxins
have been linked to long term outcomes both in pre- and chronic dialysis patients, indicating their pivotal role in CKD (Bammens et al.,
2006; Barreto et al., 2009).

Corresponding author at: Division of Nephrology, Department of Internal

Medicine, Kaohsiung Chang-Gung Memorial Hospital, Kaohsiung, Taiwan, 123, Ta-Pei
Road, Niao-Sung District, Kaohsiung, 833, Taiwan. Tel.: + 886 7 7317123x8306;
fax: + 886 7 7322402.
E-mail address: chientel@gmail.com (C.-T. Lee).
0024-3205/$ see front matter 2011 Elsevier Inc. All rights reserved.
doi:10.1016/j.lfs.2011.10.014

Endothelial dysfunction is a common feature of cardiovascular disease. With the high prevalence of cardiovascular disease in CKD, it is
believed that renal impairment perpetuates endothelial injury, eventually leading to pathologic vasculature (Rabelink et al., 2010). The
pathogenesis of endothelial dysfunction associated with renal disease
is a multiple process and most of the factors also indicate a risk of
cardiovascular disease. Nevertheless, information regarding whether
uremic toxins can directly cause endothelial injury is limited (Tumur
et al., 2010). Statins are competitive inhibitors of 3-hydroxy3methylglutary coenzyme A (HMG-CoA) reductase. Clinical evidence
strongly supports their administration to lower hyperlipidemia, to improve cardiovascular outcome. Furthermore, with their pleiotrophic
properties, statins are able to modulate inammatory reactions and
have been considered vasculoprotective agents (Wang et al., 2008).
In renal patients, accumulated evidence has supported the effectiveness of statins in reducing proteinuria, thereby providing renoprotection (Douglas et al., 2006).
The current study presents an investigation of the effect of proteinbound uremic toxin: indoxyl sulfate (IS) on endothelial cells. Leukocyte
adhesion molecules expressed in endothelial cells were examined to

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C.-T. Lee et al. / Life Sciences 90 (2012) 4753

evaluate the effect on endothelial activation. We further examined

whether HMG-CoA reductase inhibitor, simvastatin inuences ISinduced changes in endothelial cells.
Materials and methods
Materials

mouse monoclonal antibody (1:80, Santa Cruz, CA, USA), and with
anti-MCP-1 mouse monoclonal antibody (1:150, Santa Cruz, CA, USA),
anti-E-selectin rat monoclonal antibody (1:300, Santa Cruz, CA, USA)
and anti-ATR1 mouse monoclonal antibody (1:200, Santa Cruz, CA,
USA) for determination of MCP-1 E-selectin and ATR1. Finally, the
membrane was incubated with goat anti-mouse IgG (1:5000 for
ICAM-1 and ATR1; 1:3000 for VAM-1 and MCP-1, Jackson, USA) in conjunction with peroxidase-conjugated AfniPure (1:5000). For E-selecin,
goat anti-rat IgG (1:5000, Jackson, USA) was used as secondary antibody. The abundance of these molecules was then quantied by densitometric analysis. Changes in protein abundance were presented as
percentages (%) of control animal values.

Human umbilical vein endothelial cells (HUVEC) were purchased

from Bioresource Collection and Research Center (Hsinchu, Taiwan).
The cells were seeded on 0.1% gelatin-coated culture ask (SigmaAldrich, Louis, MO, U.S.A.) and grown in M199 medium (GIBCO, California, U.S.A). In addition to 10% fetal bovine serum (FBS,GIBCO, California,
U.S.A), 25 U/mL heparin and 30 g/mL endothelial cell growth supplement (ECGS, Millipore Corporation, Billerica, MA, U.S.A.) was also
added to the culture medium. The cultures were maintained at 37 C
in a fully humidied atmosphere of 5% CO2 in air, and the culture medium was changed every 2 days. Cells were then detached using a
trypsin-EDTA solution and subcultured to the next passage. All experiments were performed between cell passage 4 and 7.

Data was presented as means SEMs. Statistical analysis of the

data was performed using SPSS-PC software. Unpaired Student's
t tests were used to compare differences between two groups. A
p value of less than 0.05 was considered statistically signicant for
all tests.

Preparation of reagents

Results

IS and simvastatin were purchased from Sigma (St. Louis, MO.,

USA). To examine the effects of IS, HUVEC were incubated at two concentrations: 100 M and 1000 M respectively for 2 periods of time:
16 h and 48 h. HUVEC was then collected to study gene expression
and analyze proteins. To investigate the effect of simvastatin on ISinduced changes, HUVEC were pre-incubated with 0.5 M and
2.5 M respectively for 24 h and then treated with IS.

Effects on gene expression

Gene expression study

Total RNA was extracted from the HUVEC using a Total RNA Mini
kit following the manufacturer's instructions, and spectrophotometry
at a wavelength of 260 nm was then used to detect total RNA concentrations. RNA was stored at 80 C until use. A total of 1 g RNA of
each sample was reverse-transcribed using a First Strand cDNA Synthesis Kit. Real-time PCR was performed using the Light Cycler instrument with LightCycler TaqMan Master, Universal ProbeLibrary
Probe and primers of the target gene. The results of this study were
normalized with housekeeping gene -actin. The levels of mRNA expression were presented as the ratio of each mRNA to -actin mRNA.
The primer set of studied genes were as follows: ICAM-1(forward:
AACCTCAGCCTCGCTATGG; reverse: ACTTTTGAGGGGGACACAGA),
VCAM-1(forward: AAGGCAGGCTGTAAAAGAATTG; reverse: GTAGACCCTCGCTGGAACAG), MCP-1(forward: TTCTGTGCCTGCTGCTCAT;
reverse: GGGGCATTGATTGCATCT), E-selectin (forward: AAAGGGTAGAATTCTGACAACTGG; reverse: TCCCTCTTGTTTTCCATTTCC), angiotensin receptor type 1 (ATR1, forward: ATTTTGTGAAAGAAGGAGCAAGA,
reverse: TGCTCATTTGGTAGTGAAGTGC).
Protein abundance analysis (Western blotting)
All HUVEC (both the treated and control) were added to a protein
lysis buffer solution containing 20-mM TrisHCl (pH 7.4), 0.1% sodium
dodecyl sulfate (SDS), 5-mM EDTA, 1% Triton X-100, and a protease inhibitor cocktail tablet (Roche, Penzberg, Germany). After determining
the concentration, protein samples were then run on 6% SDSpolyacrylamide gel electrophoresis (PAGE) for ICAM-1, MCP-1, Eselectin and 10% for VCAM-1, ATR1, respectively, to transfer to the
PVDF membranes. -actin was used as the internal control in this
study. For ICAM-1, the membrane was incubated with anti-ICAM-1
mouse monoclonal antibody (1:1000, ABCAM, UK) for 12 h after blocking with 5% skim milk in 0.1% Tris-buffered saline with Tween20 (TBST). For VCAM-1, the membrane was incubated with anti-VCAM-1

Statistic analysis

Treatment with IS on HUVEC with different concentration and period

(Figs. 1 and 2)
The results of RT-PCR are shown in Fig. 1. Cultured HUVEC treated
with IS (100 M) for 16 h induced a signicant increase in the expression
of ICAM-1 (177 9%), VCAM-1 (171 7%), MCP-1 (181 10%) with no
signicant change in E-selectin (107 2%) and AT1R (95 4%). Treatment with higher dose (1000 M) also elicited a marked increase in
ICAM-1 (262 9%), VCAM-1 (224 8%), MCP-1 (261 8%), but no
change in E-selectin (129 3%) and AT1R (124 7%) was noted. An extension of the treatment period to 48 h with IS 100 M was associated
with a signicant increase in ICAM-1 (2867%), VCAM-1 (243 8%),
MCP-1 (193 9%) and expression of E-selectin and AT1R was not inuenced (114 3% and 111 7%). A signicant increase in ICAM-1 (597
9%), VCAM-1 (502 7%), MCP-1 (270 10%) were observed with
1000 M IS with no signicant alteration in E-selectin (128 6%) and
AT1R (93 4%).
Pre-incubation with simvastatin (Figs. 1 and 2)
Pre-incubation with simvastatin at a dosage of 0.5 M followed by
16 h treatment with IS either at concentration of 100 M or 1000 M
increased ICAM-1, VCAM-1 and MCP-1 signicantly (ICAM-1: 183
10%, 296 5%; VCAM-1: 167 8%, 209 6%; MCP-1:199 7%, 244
6%, compared with the control, all p b 0.05). The expression of Eselectin and AT1R was not altered (E-selectin: 117 4%, 109 2%;
AT1R: 122 10%, 89 4%, all p > 0.05). There was no signicant difference between samples with and without simvastatin preincubation. With a higher concentration of simvastatin, 2.5 M, followed by IS of 100 M or 1000 M, there was still signicant increase
in the expression of ICAM-1, VCAM-1 and MCP-1 (ICAM: 212 10%,
281 9%; VCAM: 183 10%, 240 5%; MCP-1: 163 6%, 288 4%;
all p b 0.05). We compared the expression of all molecules, and no signicant difference was observed between the IS alone and the group
having received pre-incubation with simvastatin. The expression of Eselectin and AT1R was not inuenced (E-selectin: 98 6%, 130 4%;
AT1R: 108 6%, 94 8%, all p > 0.05).
After extending the treatment period to 48 h, pre-treatment
with simvastatin 0.5 M followed by 100 M or 1000 M of IS also induced a marked increase in gene expression (ICAM: 244 8%, 622
9%; VCAM: 271 6%, 586 10%; MCP-1: 183 7%, 332 9%; all
p b 0.05). No signicant change was observed in E-selectin and AT1R
(E-selectin: 101 4%, 125 6%; ATR1: 90 3%, 129 9%). Pre-

C.-T. Lee et al. / Life Sciences 90 (2012) 4753

VCAM-1

ICAM-1

300

mRNA expression
(% of control)

mRNA expression
(% of control)

400

300

200

100

200

100

0
IS(uM)

0
0

SIM(uM)

100 1000
0

100

1000

0.5

IS(uM)

SIM(uM)

100 1000

0.5

2.5

2.5

100 1000
0

100

1000

0.5

0.5

100 1000
2.5

2.5

E-selectin

MCP-1
300

200

mRNA expression
(% of control)

mRNA expression
(% of control)

49

200

100

150

100

50

0
IS(uM)

SIM(uM)

100 1000
0

100

1000

0.5

0.5

100 1000
2.5

2.5

IS(uM)

SIM(uM)

100 1000
0

100

1000

0.5

0.5

100 1000
2.5

2.5

AT1R
mRNA expression
(% of control)

150

100

50

0
IS(uM)

SIM(uM)

100 1000
0

100

1000

0.5

0.5

100 1000
2.5

2.5

Fig. 1. Effects of indoxyl sulfate treatment for 16 h on gene expression of ICAM-1, VCAM-1, MCP-1, E-selectin and AT1R with concentration of 100 M and 1000 M. The effects of
pre-incubation with simvastatin 0.5 and 2.5 M are also shown. (* indicates p b 0.05 compared with control).

treatment with simvastatin 2.5 M did not inuence the increase

in the expression of molecules when treated with either 100 M or
1000 M (ICAM-1: 322 7%, 584 10%; VCAM-1: 269 3%, 614
10%; MCP-1: 177 6%, 252 89%; all p b 0.05). No signicant change
was observed in E-selectin and AT1R (E-selectin: 114 5%, 126 3%;
ATR1: 108 5%, 94 3%). Statistical analysis revealed that the increment in ICAM-1, VCAM-1 and MCP-1 were similar between groups
with or without pre-treatment of simvastatin when compared with
the control.
Immunoblotting study (Figs. 37)
Treatment with IS 100 M and 1000 M for 16 h resulted in signicant increase in protein abundance of ICAM-1 (157 4% and
204 5% of control, both p b 0.05), VCAM-1 (158 4% and 212 5,
both p b 0.05) and MCP-1(182 3% and 193 5%, both p b 0.05).
Pre-treatment with simvastatin of either 0.5 M or 2.5 M did not inuence the abundance ICAM-1 (IS 100 M: 168 5% and 218 6%;

IS 1000 M: 235 5% and 251 5%), VCAM-1 (168 4% and 275

6%; 156 4% and 230 5%), MCP-1(158 4% and 177 6%; 188
5% and 201 5%). IS treatment for 48 h at concentration of 100 M
and 1000 M increased ICAM-1 (317 5% and 639 6%, both
p b 0.05), VCAM-1(175 5% and 520 4%, both p b 0.05) and MCP-1
(176 4% and 259 4%, both p b 0.05). Simvastatin 0.5 M did not
inuence IS-induced increase of ICAM-1 (100 M: 382 6%;
1000 M: 594 5%, both p b 0.05), VCAM-1(166 4% and 571 6%,
both p b 0.05), MCP-1(199 6% and 289 5%, both p b 0.05). At a
concentration of 2.5 M, simvastatin did not inuence the increment
of ICAM-1 (100 M: 251 4%; 1000 M: 663 6%, both p b 0.05),
VCAM-1(193 3% and 622 5%, both p b 0.05), MCP-1(193 5%
and 281 6%, both p b 0.05). The abundance of E-selectin and AT1R
did not present a signicant change during treatment with IS either
100 M or 1000 M for 16 h (E-selectin: 101 2% and 137 5%;
AT1R: 102 3% and 119 4%, all p > 0.05) or 48 h (E-selectin: 96
5% and 116 4%; AT1R: 102 3% and 119 4%, all p > 0.05).

50

C.-T. Lee et al. / Life Sciences 90 (2012) 4753

VCAM-1

ICAM-1

750

mRNA expression
(% of control)

mRNA expression
(% of control)

750

500

250

500

250

IS(uM)

SIM(uM)

100 1000
0

100

1000

0.5

0.5

100 1000
2.5

2.5

MCP-1

IS(uM)

SIM(uM)

100

1000

0.5

0.5

100 1000
2.5

2.5

E-selectin

400

200

mRNA expression
(% of control)

mRNA expression
(% of control)

100 1000

300

200

100

150

100

50

0
IS(uM)

SIM(uM)

100 1000
0

100

1000

0.5

0.5

100 1000
2.5

2.5

IS(uM)

SIM(uM)

100 1000
0

100

1000

0.5

0.5

100 1000
2.5

2.5

AT1R
mRNA expression
(% of control)

150

100

50

0
IS(uM)

SIM(uM)

100 1000
0

100

1000

0.5

0.5

100 1000
2.5

2.5

Fig. 2. Effects of indoxyl sulfate treatment for 48 h on gene expression of ICAM-1, VCAM-1, MCP-1, E-selectin and AT1R with concentration of 100 M and 1000 M. The effects of
pre-incubation with simvastatin 0.5 and 2.5 M are also shown. (* indicates p b 0.05 compared with control).

Pretreatment with simvastatin did not induce a signicant change in

E-selectin and AT1R followed by 16 h treatment of IS (100 M and
1000 M) either with 0.5 M (E-selectin: 99 4% and 122 6%;
AT1R: 123 3% and 117 4%, all p > 0.05) or 2.5 M (E-selectin:
116 5% and 108 5%; AT1R: 123 3% and 117 4%, all p > 0.05).
There was no inuence of simvastatin during treatment with
IS (100 M and 1000 M) for 48 h at a concentration of 0.5 M (Eselectin: 120 3% and 111 3%; AT1R: 123 3% and 117 4%,
all p > 0.05) or 2.5 M (E-selectin: 132 6% and 106 4%; AT1R:
123 3% and 117 4%, all p > 0.05).
Discussion
This in vitro study demonstrated that protein-bound uremic toxins
such as IS have a direct deleterious effect on endothelial cells. The expression of surrogate markers of endothelial activation we tested:
ICAM-1 and VCAM-1 increased following IS treatment in a dose and

time-dependent manner. There was a differential effect of IS on different leukocyte adhesion molecules. Another potential target which has
been shown to be involved in cardiovascular and renal disease, the
reninangiotensin system was not inuenced. Furthermore, simvastatin did not exert any benecial effects on IS-induced endothelial
activation.
Cardiovascular disease is the leading cause of death among patients with chronic kidney disease (Foley et al., 2005). Endothelial
dysfunction has emerged as an indicator and marker of cardiovascular risk (Goligorsky, 2006). There are a number of underlying causes
of endothelial dysfunction (Feletou and Vanhoutte, 2006). In patients
with CKD, an increase in inammation along with oxidative stress
is overwhelmingly common, and for this reason, endothelial dysfunction is commonly associated with other traditional risk factors
(Wu-Wong, 2008). Recently, retained molecules accumulating as
renal function deteriorates have been reclassied (Vanholder et al.,
2008b). Among these, protein-bound uremic toxins are compounds

C.-T. Lee et al. / Life Sciences 90 (2012) 4753

16hrs

48hrs

ICAM-1

ICAM-1

actin

actin

IS(uM)

SIM(uM)

100 1000 100 1000 100 1000

0

0.5

0.5

2.5

2.5

IS(uM)

SIM(uM)

100 1000 100 1000 100 1000

0

0.5

0.5

2.5

750

200

*
ICAM-1 protein
(% of control)

ICAM-1 protein
(% of control)

300

51

100

2.5

500

250

Fig. 3. Western blots of indoxyl sulfate treatment on ICAM-1 of endothelial cells with or without simvastatin pre-incubation. (* indicates p b 0.05 compared with control).

that bind to serum protein tightly and are not easily removed via traditional dialysis therapy. Of note, accumulating evidence has shown
that serum levels of toxins, such as IS and p-cresol sulfate, can predict
cardiovascular events and mortality (Meijers et al., 2010; Bammens et
al., 2006; Barreto et al., 2009). In a previous study, we also showed a
relationship between IS and pro-inammatory cytokines (Lee et al.,
2010). The linkage between retained compounds and clinical outcome has motivated researchers to explore the probable pathogenic
role of uremic toxins in CKD.
It has been proposed that IS plays an active role in the progression
of chronic kidney disease (Niwa et al., 1997). More recently, several
studies have demonstrated its deleterious effects on vascular smooth
cells, osteoblastic cells, and cardiac broblasts (Muteliefu et al., 2009;
Nii-Kono et al., 2007; Lekawanvijit et al., 2010). Little information is
available on the direct effect of IS on endothelial cells. Induction of reactive oxygen species and increased expression of adhesion molecules have been found in in vitro studies (Tumur and Niwa, 2009;

Tumur et al., 2010). Ito et al. also demonstrated that IS enhanced

the TNF--activated endothelial cells (Ito et al., 2010). However, no
increase in ICAM-1 and VCAM-1 were noted when cells were treated
by IS alone. It is possible that the expression of these adhesion molecules may differ with or without the presence of monocyte and endothelial interaction. In the present study, treatment with IS elicited
activation of HUVEC, as evidenced by an increase in the expression
of leukocyte adhesion molecules: ICAM-1, VCAM-1 and also the chemokine: MCP-1. The effect was more prominent following either a
higher dose or longer treatment duration in ICAM-1 and VCAM-1.
While the change in MCP-1 tended to be concentration dependent.
The expression of E-selectin was not inuenced by IS treatment. The
reasons for this differential effect are not clear. However, the time
and concentration dependent characteristics observed in VAM-1
and ICVM-1 represent direct toxic effect of IS on endothelial cells
(Tumur et al., 2010). It has been well-recognized that the activation
of reninangiotensin system (RAS) is of crucial importance of

16hrs

48hrs

VCAM-1

VCAM-1

actin
actin
IS(uM)

SIM(uM)

100 1000 100

0

0.5

1000 100 1000

IS(uM)

0.5

SIM(uM)

2.5

2.5

VCAM-1 protein
(% of control)

VCAM-1 protein
(% of control)

100

0.5

1000
0.5

100 1000
2.5

2.5

750

300

200

100 1000 100

500

250

Fig. 4. Western blots of indoxyl sulfate treatment on VCAM-1 of endothelial cells with or without simvastatin pre-incubation. (* indicates p b 0.05 compared with control).

52

C.-T. Lee et al. / Life Sciences 90 (2012) 4753

16hrs

48hrs

MCP-1

MCP-1

actin

actin

IS(uM)

SIM(uM)

100 1000
0

100

1000 100 1000

0.5 0.5

2.5

2.5

IS(uM)

SIM(uM)

0.5

1000 100
0.5

2.5

1000
2.5

300

MCP-1 protein
(% of control)

MCP-1 protein
(% of control)

300

100 1000 100

200

100

200

100

Fig. 5. Western blots of indoxyl sulfate treatment on MCP-1 of endothelial cells with or without simvastatin pre-incubation. (* indicates p b 0.05 compared with control).

cardiovascular and renal diseases and pharmacological inhibition of

RAS is an effective strategy for organ protection (Brewster and
Perazella, 2004; Le and Coffman, 2008). AT1R activation contributes independently to chronic cardiovascular disease by promoting vascular
growth and the proliferation of endothelial dysfunction. Furthermore,
angiotensin II increases the adhesiveness of endothelium through the
expression of adhesion molecules (Skultetyova et al., 2007). Our results
exclude the possibility that the increase in the expression of adhesion
molecules induced by IS was mediated through the activation of RAS.
Although inhibitors of RAS are effective renal modifying agents, AT1R
was not activated by IS. It appears that pharmacological blockers, particularly AT1R antagonists may not reverse IS-induced endothelial injury.
Another major nding is that simvastatin does not inuence the
upregulation of adhesion molecules during IS treatment. This result indicates no benet derived from the pleiotropic effect of statins during
IS-induced endothelial injury. Beyond its lipid-lowering effect, by inhibiting the conversion of HMG-CoA to L-mevalonic acid, statins reduce
the synthesis of isoprenoids participating in many important cellular

16hrs

processes, particularly those associated with cardiovascular disease

(Almuti et al., 2005). A lack of protection against IS-associated endothelial damage suggests that endothelial injury is induced by IS via a pathway that was not affected by simvastatin. Previous studies have
demonstrated the benecial effect of simvastatin on C-reactive protein,
tumor necrosis factor , and the oxidized lipoprotein-induced activation of endothelial cells (Schaefer et al., 2004; Zapolska-Downar et al.,
2004; Liang et al., 2008). The concentration of simvastatin used in our
study was comparable to that of other studies and the duration of simvastatin pre-treatment was also similar. Results of clinical studies using
statins in chronic dialysis population is not effective, as demonstrated in
non-dialysis patients. Two randomized controlled trials failed to support the survival benets of statins in chronic hemodialysis patients
(Fellstrm et al., 2009; Wanner et al., 2005). In these studies, despite
their effectiveness in lowering lipids, there was no signicant improvement in all causes or cardiovascular mortality in this high risk population. Our results provide at least partial evidence showing a lack of
vascular protection in the stains in uremia.

48hrs

E-selectin

E-selectin

actin

actin

IS(uM)

SIM(uM)

100 1000
0

100 1000 100 1000

0.5 0.5 2.5 2.5

IS(uM)

SIM(uM)

150

100

50

0.5

0.5 2.5

2.5

200

E-selectin protein
(% of control)

E-selectin protein
(% of control)

200

100 1000 100 1000 100 1000

150

100

50

Fig. 6. Western blots of indoxyl sulfate treatment on E-selectin of endothelial cells with or without simvastatin pre-incubation. (* indicates p b 0.05 compared with control).

C.-T. Lee et al. / Life Sciences 90 (2012) 4753

48hrs

16hrs

AT1R

AT1R

actin

actin
IS(uM)

SIM(uM)

100 1000 100 1000 100 1000

0

0.5

0.5

2.5

2.5

IS(uM)

SIM(uM)

50

AT1R protein
(% of control)

100

100 1000 100 1000 100 1000

0

0.5

0.5

2.5

2.5

150

150

AT1R protein
(% of control)

53

100

50

Fig. 7. Western blots of indoxyl sulfate treatment on AT1R of endothelial cells with or without simvastatin pre-incubation. (* indicates p b 0.05 compared with control).

Conclusion
Our study has clearly demonstrated that IS, a uremic retention
compound, exerts toxic effects on endothelial cells. Adhesion molecules expressed in endothelial cells: ICAM-1, VCAM-1 was increased
following treatment with IS and the incremental expression of
ICAM- and VCAM-1 was dependent on concentration and treatment
duration. Local RAS was not inuenced by IS. Pre-treatment with simvastatin did not have any inuence on IS-induced endothelial injury.
Conict of interest statement
No competing interest.

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