ANTIOXIDANT ACTIVITY OF RENIN-ANGIOTENSIN SYSTEM INHIBITORS IN VASCULAR
ENDOTHELIAL CELLS
Toru Maruyama1, Daisuke Kadowaki2, Makoto Anraku2, Moe Sakaya2 and Masaki Otagiri2
1
Department of Clinical Pharmaceutics, School of Pharmacy and 2Department of Biopharmaceutics,
Graduate School of Pharmaceutical Sciences, Kumamoto University, 5-1 Oe-Honmachi, Kumamoto
862-0973, Japan.
The renin-angiotensin system (RAS) plays an important role in regulating blood pressure. Angiotensin II
type 1 receptor blockers (ARBs) and angiotensin-converting enzyme inhibitors (ACEIs) inhibit the RAS and
have been shown to be effective treatments for hypertension 1. Independent of their ability to lower blood
pressure, these compounds had also been reported to protect the organs, such as kidney and heart. So far,
the mechanism of those protective effects have not been extensively studied, however, some protection may
be due to their effects on the results of oxidative stress. The aim of the present study was to characterize
the antioxidant activity of olmesartan and temocaprilat in blood vessel of hemodialysis patients. First,
oxidized albumin ratios as an oxidative stress marker were determined by HPLC to evaluate the antioxidant
activities of olmesartan and temocaprilat in plasma of hemodialysis patients. In addition, using human
umbilical vein endothelial cells (HUVECs), we examined the effect of olmesartan and temocaprilat on the
oxidative stresses induced by lipopolysaccharide (LPS), indoxyl sulfate (IS), oxidized-human serum albumin
(oxi-HSA), which are known to promote the ROS production in hemodialysis patients.
High-performance liquid chromatography (HPLC) was performed to analyze the oxidative states of serum
albumin as described previously 2. From the HPLC profile, oxidized albumin ratios were estimated by the
area of human nonmercapt- and mercapt-albumin peak. To measure the intracellular ROS, cells (1×105)
were plated in individual wells of 96-well plates. After 24 hr culture, the culture medium was removed and
the cells were incubated with 5 M CM-H2DCFDA for 30 min at 37 °C in 5 % CO2. Thereafter, cells were
pretreated with drugs, olmesartan (0.5 g/mL) or temocaprilat (0.1 g/mL) and 5 min after, were stimulated
with several reagents. The fluorescence intensity of oxidized DCF was measured at 490 nm excitation and at
530 nm emission.
To evaluate the antioxidation potentials of olmesartan and temocaprilat, these drugs were added to serum
samples from healthy subjects and dialysis patients at clinical blood concentrations. The drugs were
incubated with the samples at 37°C for 1 week, and the oxidized albumin ratio was determined. Olmesartan
did not significantly alter the oxidized albumin ratio in the sera from healthy subjects but significantly
reduced the ratio in the sera from dialysis patients (Fig.1). On the other hand, temocaprilat did not act as an
antioxidant on any of the sera. Subsequently, we determined the antioxidation potentials of these drugs on
vascular endothelial cells by inducing oxidative stress in HUVEC under various conditions, and compared
their suppressive effects on ROS production. In this study, substances that produce ROS during renal
failure, i.e., LPS, IS, and oxi-HSA, were used to induce oxidative stress. All these inducers of oxidative
stress increased ROS production in HUVEC. Interestingly, these ROS productions were significantly
suppressed by olmesartan. On the other hand, temocaprilat did not have such a significant suppressive
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Oxidized albumin ratio
(HNA/HMA)
effect, though it tended to
Normal subjects
HD patients
decrease ROS.
2
Olmesartan was shown to
suppress ROS production in the
1.5
blood vessels of dialysis
*
patients at its clinical blood
1
concentrations. On the other
hand, temocaprilat seemed to
have no such effect at its
0.5
clinical condition.
Since
olmesartan is an inverse agonist
0
for angiotensin II type 1
receptor,
angiotensin
II
: p<0.05 Compared with HD control
non-dependent pathway may
Fig. 1 Effects of olmesartan and temocaprilat on oxidized albumin ratio
further play an important role
in plasma of hemodialysis (HD) patients (n=3
(n=3))
for the inhibition of ROS
production by olmesartan. Although the mechanism of the increased ROS production in HUVEC by the
inducers of oxidative stress used in the present study remains unknown, it was postulated that NADPH
oxidase was involved at various degrees. This suggests that the suppression of ROS production by
olmesartan may be due at least to the RAS-mediated inhibition of NADPH oxidase.
The findings obtained here suggest that olmesartan may suppress the onset and progression of
complications, including CVD, not only by its hypotensive effect, but also by its antioxidant action.
*
References:
1. Suzuki H. Treatment of hypertension in chronic renal insufficiency. Intern Med. 39, 773-777 (2000)
2. Anraku M, Kitamura K, Shinohara A, Adachi M, Suenga A, Maruyama T, Miyanaka K, Miyoshi T,
Shiraishi N, Nonoguchi H, Otagiri M, Tomita K. Intravenous iron administration induces oxidation of
serum albumin in hemodialysis patients. Kidney Int. 66, 841-848 (2004)
Auther Affiliation:
Toru Maruyama is a Professor of Clinical Pharmaceutics, School of Pharmacy, Kumamoto University, Japan.
He graduated from Kumamoto University with Ph.D. degree in 1990. He continued his postdoctoral research
in Pennsylvania State University in 1992-1994. He joined PharmaDaiwa as Chief of Academic Section and
Biopharmaceutics laboratory, Faculty of Pharmaceutical Sciences, Kumamoto University, Japan, as a
postdoctoral research fellow. His main interest has been in the field of plasma protein binding of drugs. He is
currently working on development of new drug carrier using albumin.