Yonago Acta medica 2009;52:27–35

Combination Therapy with Olmesartan and Temocapril
Ameliorates Renal Damage and Upregulates the klotho Genein 5/6 Nephrectomized Spontaneously Hypertensive Rats
Satoko Maeta, Chishio Munemura, Takeaki Fukui, Chihiro Ishida and Yoshikazu
MurawakiDivision of Medicine and Clinical Science, Department of Multidisciplinary Internal Medicine,
School of Medicine, Tottori University Faculty of Medicine, Yonago 683-8504 Japan

Recent studies suggest that chronic kidney disease may induce cardiovascular disease
through oxidative stress, and that the aging suppressor gene klotho reduces oxidative stressin the kidney. In this study, we examined the changes in klotho gene expression, and therenoprotective effects of olmesartan (OLM), angiotensin II receptor blocker (ARB) alone
or in combination with temocapril (TEM), angiotensin-converting enzyme inhibitor (ACEI)
in 5/6-nephrectomized (5/6-Nx) spontaneously hypertensive rats. Male 5/6-Nx spontaneously hypertensive rats were randomly assigned to 5 groups as follows: control group;
5/6-Nx group, 5/6-Nx rats; low OLM group, 5/6-Nx rats administered low-dose OLM (3mg/kg/day); high OLM group, 5/6-Nx rats administered high-dose OLM (10 mg/kg/day);
OLM+TEM group, 5/6-Nx rats administered high-dose OLM and TEM (10 mg/kg/dayeach). These drugs were administered for 12 weeks. Systolic blood pressure, glomerularsclerosis and transforming growth factor beta 1 mRNA in high OLM and OLM+TEM
groups were significantly lower than that in the 5/6-Nx group. Only the OLM+TEM group
showed improvement of serum creatinine and urinary 8-hydroxy-2'-deoxyguanosine. Expression of klotho mRNA, which was downregulated in the 5/6-Nx group, was upregulated in the high OLM and OLM+TEM groups. OLM dose-dependently prevented klotho
mRNA downregulation in 5/6-Nx rats, thus confirming a renoprotective effect. In addition, combination therapy of OLM and TEM was more effective than OLM alone. In
conclusion, the combination of OLM and TEM inhibits the progression of renal damage in
5/6-Nx rats through the upregulation of klotho gene.
Key words: angiotensin II receptor blocker; angiotensin-converting enzyme; cardiovascular disease; chronic kidney disease; inhibitor; klotho gene

In chronic kidney disease, in addition to end-stage
renal disease, mild renal failure can cause cardiovascular disease as a result of oxidative stress (Descamps-Latscha et al., 2005). Angiotensin II, throughthe angiotensin II type 1 receptor, increases theexpression of p47phox, which is a component protein of NADPH oxidase, and leads to overproduc-tion of free radicals (Tojo et al., 2002). Angiotensin
receptor blocker (ARB) and angiotensin-converting
enzyme inhibitor (ACEI) inhibit the action of angiotensin II, which results in the reduction of oxidative
stress (Dohi et al., 2003; Fliser et al., 2005).
The aging suppressor gene klotho, which is
predominantly expressed in the kidney, prevents

Abbreviations: ACEI, angiotensin-converting enzyme inhibitor; ARB, angiotensin II receptor blocker; ELISA, enzymelinked immunosorbent assay; IGF1, insulin-like growth factor 1; 5/6-Nx, 5/6-nephrectomized; 8-OHdG, 8-hydroxy-2'deoxyguanosine; OLM, olmesartan; TEM, temocapril

27

S. Maeta et al.

excision of approximately 2/3 of the renal cortexof the left kidney was performed. One week later,
the right kidney was removed. At 1 week after
surgery, baseline measurement of body weight,
blood pressure, urine volume and urinary protein
was performed. Blood pressure was measured
in conscious rats using the tail-cuff method witha sphygmomanometer (Softron, Tokyo, Japan).
Urine was collected from individual rats housed
in metabolic cages for 24 h. Administered drugs
were olmesartan (OLM) as ARB and temocapril(TEM) as ACEI. Rats were divided into 5 experimental groups: control group, non-nephrectomized
rats (n = 8); 5/6-Nx group, 5/6-Nx rats (n = 8); low
OLM group, 5/6-Nx rats administered low-dose
OLM (3 mg/kg/day); high OLM group, 5/6-Nx
rats administered high-dose OLM (10 mg/kg/day);and OLM+TEM group, 5/6-Nx rats administeredhigh-dose OLM and high-dose TEM (10 mg/kg/
day each). Dosage of each drug was selected basedon earlier reports so that they would exhibit comparable antihypertensive effects (Kanazawa et al.,
2002; Fan et al., 2006). Drugs were administeredonce a day for 12 weeks beginning 1 week after
nephrectomy. Every 4 weeks, we measured bodyweight, blood pressure, urine volume and urinary
protein in each group.
After 12 weeks of administration, rats were
killed under pentobarbital anesthesia. Blood wascollected from the heart. Serum samples were frozen and stored at –80°C, and serum creatinine wasmeasured. Remnant kidneys were removed andfixed in 10% buffered formalin and embedded inparaffin for histological analysis.

insulin and insulin-like growth factor 1 (IGF1)
signals to reduce oxidative stress (Kurosu et al.,2005). It has been reported that angiotensin II,
through the activation of angiotensin II type 1 receptor, downregulates the expression of the klotho
gene in the kidney, and the introduction of exogenous klotho ameliorates renal injury (Mitani et al.,2002). In addition, another study showed that therenal expression of klotho gene was downregulated
in patients with chronic renal failure (Koh et al.,2001). Furthermore, in chronic kidney disease,it has been suggested that the downregulation ofrenal klotho expression is involved in the onset of
cardiovascular disease. Taken together, it appears
that angiotensin II, oxidative stress and the klotho
gene are closely related to one another, and contribute to the progression of renal failure and onset of
cardiovascular disease.
In this study, we examined the therapeutic effects of ARB and ACEI on renal damage in 5/6-nephrectomized (5/6-Nx) spontaneously hypertensive
rats. In addition, we determined the changes in urinary 8-hydroxy-2'-deoxyguanosine (8-OHdG) andklotho mRNA.

Materials and Methods
Animals
Male spontaneously hypertensive rats of the Izumo
strain were purchased from Japan SLC (Shizuoka,
Japan), and were maintained at a temperature of 24± 2°C with a 12-h light-dark cycle. Animals were
given standard pellet chow and tap water. Anesthesia was performed by intraperitoneally injecting
pentobarbital (Dainippon Pharmaceutical, Osaka,
Japan) at dose of 50 mg/kg. All experiments werecarried out in accordance with the Animal Experimentation Guidelines of Tottori University.

RNA extraction and reverse transcription
PCR analysis
Tissue samples were homogenized and total RNA
was extracted using the RNeasy Mini Kit (Qiagen,Hilden, Germany). RNA concentration was determined by measuring absorbance at 260 nm andthe RNA quality was verified by electrophoresis
on an ethidium-bromide-stained 1% agarose gel.
About 2 μg of total RNA was reverse transcribed

Establishment of model
Male 6-week-old spontaneously hypertensive rats
underwent a 5/6-nephrectomy. First, the surgical28

Effect of ARB and ACEI on renal damage

Table 1. Primer sequences
TGF- 1klotho-actinForward primer

Reverse primer

5'-cctgcccctacatttgga-3'5'-caagaagttcataatggaaagcttaaa-3'5'-ctggctcctagcaccatga-3'5'-tggttgtagagggcaaggac-3'5'-atgcggtgtacccaatgac-3'5'-tagagccaccaatccacaca-3'

in a final volume of 11.5 μL containing 4 μL of 5×standard buffer, 2 μL of 0.1 M dTT, 1 μL of SuperScript II RNase H-reverse transcriptase (Invitrogen,
Carlsbad, CA), 2 μL of 10 mM dNTPs (Promega,
Madison, WI), 1 μL of 50 pmol/μL Random Primer (Promega), 0.5 μL of 100 pmol/μL Oligo (dt) 15
Primer (Promega) and 1 μL of 40 units/μL Ribonuclease Inhibitor (Wako Pure Chemical Industries,
Osaka). Samples were incubated at 37°C for 60
min, followed by 95°C for 5 min and cooling to
4°C for 5 min.

as follows (Fig. 1): Grade 0, no change; Grade 1,
sclerotic area less than or equal to 1/4 of the glomerulus or the presence of distinct adhesion between
the capillary tuft and Bowman’s capsule; Grade 2,
sclerosis of 1/4 to 1/2 of the total glomerular area;
Grade 3, sclerosis of 1/2 to 3/4 of the total glomerular area; Grade 4, sclerosis of more than 3/4 ofthe glomerulus. The index of glomerular sclerosis
was calculated using the following formula (Saito
et al., 1987): index of glomerular sclerosis = (1N1 + 2 N2 + 3 N3 + 4 N4)/(N0 + N1 + N2 +N3 + N4), where N is the number of glomeruli ateach grade of sclerosis.

Real-time PCR
For quantitative real-time PCR, we used 10 µL ofreverse transcribed samples: PCR grade water, 4.1
μL; Universal ProbeLibrary probe, 1 μL (Roche,
Tokyo); Forward primer (10 μM), 0.2 μL; Reverse
primer (10 μM), 0.2 μL; LightCycler TaqMan
Master, 2 μL (Roche); and cDNA sample, 2.5 μL.
mRNA levels of transforming growth factor beta
1 (TGF- 1) and klotho were assessed by real-time
PCR assays using -actin as a housekeeping gene.
The forward and reverse primer sequences used forthis study are shown in Table 1. The thermal cycler conditions were as follows: hold at 95°C for 10
min, repeat 45 cycles of 95°C for 30 s and 60°C for
1 min.

Urinary 8-OHdG measurement
Urinary 8-OHdG concentrations were measured byenzyme-linked immunosorbent assay (ELISA) using a commercially available competitive 8-OHdG
ELISA kit (Japan Institute for Control of Aging,
Shizuoka). The kit can measure 8-OHdG values
ranging from 0.5 to 200 ng/mL using a specificmonoclonal antibody, N45.1.
Statistical analysis
Statistical significance of intergroup differences inquantitative data was assessed by Student’s t-test(Stat View for Windows; SAS Institute, Cary, NC).
P < 0.05 was considered significant.

Histological analysis
Three-micrometer sections of formalin-fixed,
paraffin-embedded kidneys were stained with periodic acid-Schiff and periodic acid-methenamine
silver. To calculate focal glomerular sclerosis, 100
to 150 glomeruli from each stained specimen were
examined. The degree of sclerosis in each glomerulus was subjectively graded on a scale of 0 to 4

Results
Blood pressure
Mean systolic blood pressure in the rats during the 12-week experimental period is shown
29

S. Maeta et al.

Grade 0

Grade 1

Grade 3

Grade 4

Grade 20 No change
1 Sclerosis less than or equal to
1/4 of the glomerulus or
the presence of distinct adhesion
between the capillary tuft andBowman’s capsule2 Sclerosis of 1/4–1/2 ofthe total glomerular area
3 Sclerosis of 1/2– 3/4 ofthe total glomerular area
4 Sclerosis of more than 3/4of the glomerulus

Fig. 1. Classification of glomerular sclerosis by periodic acid-Schiff stain. Bar = 100 μm.

in Fig. 2. Systolic blood pressure after nephrectomy increased progressively throughout theexperimental period in the 5/6-Nx group. Drugadministration induced a significant decrease in
the treated groups (low OLM, high OLM and
OLM+TEM groups) when compared with in the

5/6-Nx group. At the end of the 12-week administration period, the level was significantly lower
in the treated groups than in the 5/6-Nx group:
228 ± 22 for the 5/6-Nx group, 191 ± 15 for the
low OLM group, 178 ± 27 for the high OLM
group, 153 ± 16 mmHg for the OLM+TEM group.
The OLM+TEM group showed significantly lower
levels than the low OLM group (P < 0.001).

(mmHg)

Systolic blood pressure

250

Proteinuria

200

Figure 3 shows total urinary protein excretionin each group during the 12 weeks. The control
group showed no change, while the 5/6-Nx groupexhibited progressive increase throughout the experimental period. During the period, urinary protein levels were lower in the treated groups than

150
100
50
0

0

4

8

12

(week)

After operation

Fig. 2. Course of mean systolic blood pressure in the 5 groups. The systolic blood pressure of rats is significantly decreased at 12 weeks of administration in groups of low OLM ( ), high OLM ( ) and OLM+TEM ( ) when comparedwith in the 5/6-Nx group ( ). , control group. High OLM, high-dose olmesartan; 5/6-Nx, 5/6-nephrectomized; low
OLM, low-dose OLM; OLM+TEM, high-dose OLM combined with high-dose temocapril.

30

Effect of ARB and ACEI on renal damage(mg/day)
350

**
***
*

Urinary protein

300
250

*

200

*

*
**

***
*
*
**

150
100
50
0

* P < 0.05** P < 0.01*** P < 0.001

0Control group
5/6-Nx group
Low OLM group
High OLM group
OLM+TEM group

4

8

12

(week)

After operation

Fig. 3. Effects of treatments on urinary protein in the 5 groups. High OLM, high-dose olmesartan; 5/6-Nx, 5/6-nephrectomized; low OLM, low-dose OLM; OLM+TEM, high-dose OLM combined with high-dose temocapril.

in the 5/6-Nx group: especially in the OLM+TEM
group, the level was significantly lower at 4, 8 and
12 weeks after the operation.

(mg/dL)

Serum creatinine

3

Serum creatinine
Figure 4 shows serum creatinine in the 5 groupsof rats at 12 weeks. The serum creatinine level
was significantly higher in the 5/6-Nx groupthan in the control group (P < 0.01). The treatedgroups (low OLM, high OLM and OLM+TEM
groups) showed lower levels than the 5/6-Nxgroup; especially, the difference between the
OLM+TEM and 5/6-Nx groups was significant (P< 0.05).

*

2

1

0
Control

5/6-NxLow OLM

High OLM OLM+TEM

Index of glomerular sclerosis

Fig. 4. Serum creatinine concentrations in the 5 groups at
12 weeks. Values are expressed as mean ± SD. *P < 0.05.
High OLM, high-dose olmesartan; 5/6-Nx, 5/6-nephrectomized; low OLM, low-dose OLM; OLM+TEM, high-dose
OLM combined with high-dose temocapril.

Histological findings in the kidney
In the control group, only a few sclerotic changesof glomeruli and interstitial fibrosis were observed. The values of the index of glomerularsclerosis in the treated groups were significantly
lower than in the 5/6-Nx group: P < 0.05 between
the low OLM and 5/6-Nx groups; P < 0.05 between the high OLM and 5/6-Nx groups; P < 0.01
between the OLM+TEM and 5/6-Nx groups. In
comparison among the treated groups, the index
was significantly lower in the OLM+TEM groupthan in the low OLM group (P < 0.05) (Fig. 5).

2.0

*

1.5

*

**
*

1.0

0.5

0.0

Control

5/6-NxLow OLM

High OLM OLM+TEM

Fig. 5. Index of glomerular sclerosis in the 5 groups at 12
weeks. Values are expressed as mean ± SD. *P < 0.05,**P < 0.01. High OLM, high-dose olmesartan; 5/6Nx, 5/6-nephrectomized; low OLM, low-dose OLM;
OLM+TEM, high-dose OLM combined with high-dose
temocapril.

31

S. Maeta et al.

(ng/mg creatinine)*80

Discussion

Urinary 8-OHdG

70
60

The rennin-angiotensin system plays an importantrole in the development of hypertension and theprogression of renal failure. Numerous studies
have shown that ARB or ACEI is able to preventthe progression of renal failure (Parving et al.,
2001; Nakao et al., 2003; Schmieder et al., 2007).
ARB or ACEI alleviates glomerular hypertention
by reducing the constructive effects of angiotensin
II on the efferent arterioles, which leads to inhibition of glomerular sclerosis. Angiotensin II also
activates the production of free radicals, promotes
cell growth, and increases the synthesis of inflammatory and profibrotic cytokines (Wolf et al.,
2000; Brewster and Perazella., 2004; Long et al.,2004). Furthermore, there is increasing evidence

50
40
30
20
10
0

Control

5/6-NxLow OLM

High OLM OLM+TEM

Fig. 6. Urinary 8-hydroxy-2'-deoxyguanosine (8-OHdG)/
urinary creatinine ratio in the 5 groups at 12 weeks. Values are expressed as mean ± SD. *P < 0.05. High OLM,
high-dose olmesartan; 5/6-Nx, 5/6-nephrectomized; low
OLM, low-dose OLM; OLM+TEM, high-dose OLM
combined with high-dose temocapril.

Urinary 8-OHdG/urinary creatinine ratio
Levels of urinary 8-OHdG/creatinine in the 5/6Nx group at 12 weeks were slightly higher thanin the control group: the difference was not significant. Levels of urinary 8-OHdG/creatinine in
the treated groups were lower than in the 5/6-Nxgroup: the difference between the OLM+TEM
and 5/6-Nx groups was significant (P < 0.05) (Fig.
6).

a

*

0.009
0.008

**

*

0.007
0.006
0.005
0.004
0.003
0.002
0.001

mRNA quantification of TGF- 1 and klotho

0.000

The TGF- 1 mRNA level in the 5/6-Nx group was
markedly increased when compared with in thecontrol group (Fig. 7a). This increase was significantly alleviated in the high OLM and OLM+TEM
groups, but not in the low OLM group. The levelin the OLM+TEM group was significantly lower
than that in the low OLM group (P < 0.05).
The klotho mRNA level was significantly
lower in the 5/6-Nx group than in the control group(Fig. 7b). The klotho mRNA levels in the treatedgroups were higher in the 5/6-Nx group: the highOLM and OLM+TEM groups showed significant
difference to the 5/6-Nx group (P < 0.01). Amongthe treated groups, levels were significantly higher
in the high OLM and OLM+TEM groups than in
the low OLM group (P < 0.05).

0.03

b

**

**
*

*

0.02

0.01

0.00

Control

5/6-NxLow OLM

High OLM OLM+TEM

Fig. 7. Levels of mRNA for transforming growth factor- 1 (TGF- 1) and klotho gene in renal tissue.
(a) Expression of TGF- 1 mRNA.
(b) Expression of klotho mRNA.
Values are expressed as mean ± SEM. *P < 0.05, **P <0.01. High OLM, high-dose olmesartan; 5/6-Nx, 5/6-nephrectomized; low OLM, low-dose OLM; OLM+TEM,
high-dose OLM combined with high-dose temocapril.

32

Effect of ARB and ACEI on renal damage

groups. This suggests that the reduction in TGF- 1
is closely correlated with the prevention of progressive renal damage by inhibition of the renninangiotensin system.
Defects in klotho gene expression in mice
have been reported to lead to a syndrome closely
resembling human aging, including shortened life
span, infertility, arteriosclerosis, osteoporosis andpulmonary emphysema (Kuro-o et al., 1997). The
klotho gene is expressed in limited tissues, notably the distal convoluted tubules in the kidney andthe choroid plexus in the brain.
klotho protein functions as a circulating
hormone that binds to a cell-surface receptor andrepresses the intracellular signals of insulin and
IGF1. It appears that the anti-aging effects ofklotho-induced inhibition of insulin/IGF1 signaling are associated with increased resistance tooxidative stress (Kurosu et al., 2005). It has beenshown that klotho protein inhibits the phosphorylation of FoxO forkhead transcription factors,
which induce expression of manganese superoxide
dismutase, thereby facilitating removal of reactive oxygen species and conferring resistance tooxidative stress (Yamamoto et al., 2005). Mitani
et al. have demonstrated in experimental models
that continuous administration of angiotensin II
reduces renal klotho gene expression (Mitani etal., 2002).
Downregulation of klotho mRNA in thekidney has been reported in animal hypertention models, such as spontaneously hypertensive
rats and deoxycorticosterone acetate-salt hypertensive rats (Aizawa et al., 1998). In the present
study, expression of klotho mRNA in the 5/6-Nxgroup was markedly downregulated when compared with the control group. This reduction ofklotho mRNA was ameliorated in the high OLM
and OLM+TEM groups, but not in the low OLM
group. Losartan completely blocked angiotensin
II-induced klotho mRNA downregulation, whichis independent of its blood pressure-lowering effects (Mitani et al., 2002). This is the 1st reportshowing that OLM dose-dependently prevents
klotho mRNA downregulation in 5/6-Nx sponta-

supporting a potential role for aldosterone, whichis stimulated by angiotensin II, in the pathophysiology of renal injury (Hollenberg, 2004; Remuzzi
et al., 2008).
Spontaneously hypertensive rats have beenused as a model for essential hypertension in humans, and 5/6-Nx rats have commonly been used
as an experimental model for chronic renal failure
in humans. In the present study, we combined the
2 rat models, and prepared 5/6-Nx spontaneously
hypertensive rats. Systolic blood pressure, urinary
protein and glomerular sclerosis in 5/6-Nx spontaneously hypertensive rats increased progressively
throughout the experimental period. After 12
weeks, each of the ARB-treated groups (low andhigh OLM groups) showed a significant decreasein systolic blood pressure and index of glomerularsclerosis when compared with the 5/6-Nx group.
These data indicate that OLM ameliorates renal
injury in a dose-dependent manner. Indeed, Fanet al. also reported that ultrahigh doses of OLM
showed greater renoprotective effects than typicaldoses of OLM, and that efficacy was independentof blood pressure (Fan et al., 2006).
In our study, the OLM- and TEM-administered rats showed greater renoprotective effects
than only the high OLM-administered rats did, indicating that combination therapy with ARB and
ACEI has a beneficial effect on renoprotection ascompared with ARB alone. It is known that ACE
inhibition results in reduced degradation of bradykinin (Gavras, 1992), and that bradykinin may
cause selective efferent arteriolar dilatation andstimulate endothelial NO formation. Jacobsen etal. showed that dual blockade of the rennin-angiotensin system offers additional renal protection in
type 1 diabetic patients with diabetic nephropathy(Jacobsen et al., 2003).
TGF- 1 has been shown to play a predominant role in mediating angiotensin II-induced
extracellular matrix production and promotes the
fibrosis of cardiovascular and renal tissues (Saito
et al., 2004; Dabek et al., 2006). In the present
study, renal TGF- 1 mRNA levels were markedly
lower in the high OLM group and OLM+TEM
33

S. Maeta et al.

neously hypertensive rats.
Urinary 8-OHdG is a biological marker ofin vivo oxidative DNA damage (Shigenaga et al.,1989), and the average urinary 8-OHdG excretionin the OLM+TEM group was significantly lower
than in the 5/6-Nx group: this fact indicates that
OLM- and TEM-induced klotho overexpression
reduces oxidative DNA damage. In addition to
ACEI (de Cavanagh et al., 2000, 2001), OLM(Yao et al., 2004) has been reported to improveendothelin-induced hypertension and oxidative
stress in rats. Fujimoto et al. also reported that
OLM inhibits superoxide production and oxidative stress, independent of its blood pressurelowering effects (Fujimoto et al., 2008).
A recent study reported that chronic kidneydisease is an independent risk factor for cardiovascular disease, which involves excessive oxidative stress (Descamps-Latscha et al., 2005). Furthermore, it has been reported that expression of
the klotho gene in the kidney is downregulated inchronic renal failure in vivo (Aizawa et al., 1998)
and in vitro (Koh et al., 2001). Saito et al. demonstrated that klotho gene transfer in an experimental rat model of atherosclerotic disease improved
endothelial dysfunction (Saito et al., 2000), andthat klotho protein protects the cardiovascular system through endothelium-derived NO production
by humoral pathways (Saito et al., 1998). These
data suggest that the downregulation of renal
klotho expression in patients with chronic kidneydisease is involved in the onset of cardiovasculardisease. ARB reduced the incidence of cardiovascular disease (Yusuf et al., 2000; de Zeeuw et al.,2004), which raises the relationship between ARB
and inhibition of klotho gene downregulation. In
the present study, the combination of OLM andTEM significantly decreased urinary 8-OHdG,
and showed renoprotective effects. These datasuggest that the combination therapy is more effective for cardiovascular protection.
In conclusion, the present study demonstrated that OLM ameliorates klotho gene downregulation in 5/6-Nx rats in a dose-dependent
manner, and this has a renoprotective effect. In

addition, combination therapy of OLM and TEM
was more effective than OLM alone. This suggests that klotho gene upregulation by the renninangiotensin system inhibitor leads to a reduction
in cardiovascular disease.
Acknowledgments: We are grateful to Sankyo Co., Ltd.
(Tokyo, Japan) for supplying olmesartan and temocapril.

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Received December 19, 2008; accepted January 6, 2009
Corresponding author: Satoko Maeta

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