Yonago Acta medica 2011;54:49–58

The Effects of Olmesartan and Alfacalcidol
on Renoprotection and klotho Gene Expression in
5/6 Nephrectomized Spontaneously Hypertensive Rats
Takeaki Fukui, Chishio Munemura, Satoko Maeta, 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

Recently, an angiotensin inhibitor has been shown to upregulate the klotho mRNA level inchronic renal failure. In addition, the administration of vitamin D has been reported to
improve the mortality of patients with chronic renal failure. In this study, we examinedthe effects of an angiotensin inhibitor and/or vitamin D on the progression of chronic renal
failure by using male 5/6 nephrectomized (5/6Nx) spontaneously hypertensive rats. Male
5/6Nx spontaneously hypertensive rats were assigned to 4 groups as follows: 5/6Nx group,
5/6Nx rats; Alf group, 5/6Nx rats administered alfacalcidol (0.2 μg/kg/day); Olm group,
5/6Nx rats administered olmesartan (15 mg/kg/day); Alf + Olm group, 5/6Nx rats administered alfacalcidol (0.2 μg/kg/day) and olmesartan (15 mg/kg/day). These drugs wereadministered for 12 weeks. Systolic blood pressure in the Alf, Olm and Alf + Olm groups
were significantly decreased relative to that in the 5/6Nx group during the 12-week experimental period. As a result, all treated groups showed renoprotection based on improvement of the systolic blood pressure, urinary protein excretion and histological renal fibrosis. Combination therapy of alfacalcidol and olmesartan was more effective than either alfacalcidol or olmesartan alone. Expression of klotho mRNA was significantly upregulated
in the Alf + Olm group in comparison with in the 5/6Nx group. Serum levels of fibroblast
growth factor 23 in the Alf group and the Alf + Olm group were significantly higher thanthose in the 5/6Nx group and the Olm group. In conclusion, the combination of Olm and
Alf inhibited the progression of renal damage in the 5/6Nx group through the strong antihypertensive effect as well as the upregulation of the klotho gene.
Key words: angiotensin II receptor blocker; chronic renal failure; fibroblast growth factor 23;
klotho gene; vitamin D

It was discovered approximately a decade agothat a deletion of the klotho gene results in phenotypes resembling those of human aging-associateddisorders (arteriosclerosis, osteoporosis, ectopic
calcification and skin atrophy, together with shortlife-span). Transcripts of this gene are expressed
predominantly in the kidney, choroids plexus andparathyroid gland (Kuro-o et al., 1997). The complication of chronic renal failure (CRF) closely
resembles phenotypes seen in klotho mutants.

Abbreviations: Alf group, group of alfacalcidol-administered 5/6Nx rats; Alf + Olm group, group of alfacalcidol- and olmesartan-administered 5/6Nx rats; ARB, angiotensin II receptor blocker; CRF, chronic renal failure; FGF23, fibroblast growth
factor 23; IGS, index of glomerular sclerosis; Nx, nephrectomized; Olm group, group of olmesartan-administered 5/6Nx rats;
TGF, transforming growth factor

49

T. Fukui et al.

vitamin D3 has therapeutic value for patients withchronic kidney disease.
In this study, we examined the renoprotective
effects of an ARB and/or 1,25(OH)2 vitamin D3 on
renal damage in 5/6Nx spontaneously hypertensive
rats.

Therefore, the severe reduction of klotho expression
might be related to the pathophysiology of CRF(Haruna et al., 2007). Indeed, the expression of theklotho gene in the kidney of the nephrectomized(Nx) rats, a model of CRF in humans, was significantly downregulated (Aizawa et al., 1998). In addition, it is known that the production of klotho in
the kidney of human CRF patients was markedly
reduced (Koh et al., 2001). Previously, we haveshown that a renin-angiotensin system inhibitor
such as angiotensin-converting enzyme inhibitor or
angiotensin II receptor blocker (ARB) prevented theklotho mRNA downregulation in the 5/6Nx rats,
indicating that renin-angiotensin system inhibitors
have a renoprotective effect (Maeta et al., 2009).
In klotho-deficient mice, the levels of serum
P, Ca and active vitamin D [1,25(OH)2 vitamin D3]
were elevated. Ectopic calcification and vascular
calcification in klotho-deficient mice might be due
to elevated blood levels of Ca, P and active vitamin
D. Reduction of serum 1,25(OH)2 vitamin D3 concentration by dietary restriction resulted in alleviation of most of the phenotypes (Tsujikawa et al.,2003).
In addition, there is a report that active vitamin D therapy is a possible factor in the increased
serum fibroblast growth factor 23 (FGF23) levels
in chronic kidney disease (Nakanishi et al., 2005;
Saito et al., 2005). FGF23 was identified as an
endocrine factor produced by bone with known
functions to regulate urinary P excretion, as wellas the production of 1,25(OH)2 vitamin D3 andparathyroid hormone (Kurosu et al., 2006). Serum FGF 23 levels were significantly elevated inchronic kidney disease patients in a close correlation with serum creatinine (Komba and Fukagawa,2009). Increased FGF 23 level appears to be independently associated with mortality among patients
undergoing hemodialysis (Gutiérrez et al., 2008).
By contrast, observational studies of long-term
hemodialysis patients showed that vitamin D treatment has been associated with improved survival(Teng et al., 2005). In addition, vitamin D use isassociated with lower mortality in nondialysis patients with chronic kidney disease (Shoben et al.,2008). Therefore, the administration of 1,25(OH)2

Materials and Methods
Materials
Male spontaneously hypertensive rats of the Izumo
strain were obtained from Japan SLC (Shizuoka,
Japan), and maintained in a room at controlled temperature of 24 ± 2 ˚C with a 12-h light-dark cycle.
All experiments were carried out in accordancewith the Animal Experimention Guidelines of Tottoti University.
Olmesartan was generously provided by
Daiichi-Sankyo Pharmaceutical (Tokyo, Japan),
was suspended in 0.5% of methylcellulose solution
to a given concentration. Alfacalcidol was synthesized in Chugai Pharmaceutical (Fuji-Gotemba,
Japan), was dissolved in medium-chain triglycerideand diluted to given concentration.
Establishment of the model
Anesthesia was performed by intraperitoneally injecting pentobarbital (Dainippon Pharmaceutical,
Osaka, Japan) at dose of 50 mg/kg. The 6-weekold spontaneously hypertensive rats undergone a 5/6
nephrectomy, consisting of the surgical excision of
approximately 2/3 of the renal cortex of the left kidney. One week later, the right kidney was removed.
Then, the rats were divided into 4 experimentalgroups: 5/6Nx group, 5/6Nx rats (n = 8); Alf group,
5/6Nx rats administered by gavage with 0.2 μg/kg/
day alfacalcidol (n = 8); Olm group, 5/6Nx rats administered by gavage with 15 mg/kg/day olmesartan, an ARB (n = 8); and Alf + Olm group, 5/6Nx
rats administered by gavage with 0.2 μg/kg/day alfacalcidol and 15 mg/kg/day olmesartan (n = 8).
The dose of olmesartan was selected on thebasis of an earlier study reporting that the rats would50

Active vitamin D and ARB 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'

TGF, transforming growth factor.

on an ethidium-bromide-stained 1% agarose gel.
About 2 μg of total RNA was reverse transcribedin a final volume of 11.5 μL containing 4 μL of 5
× standard buffer, 2 μL of 0.1 M dithiothreitol, 1
μL of SuperScriptII RNase H-reverse transcriptase(Life Technologies, Carlsbad, CA), 2 μL of 10 mM
dNTPs (Promega, Madison, WI), 1 μL of 50 pmol/
μL Random Primers (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, Japan). The samples were incubated at 37 ˚C for 60 min, and then 95 ˚C for 5 min
and cooling to 4 ˚C for 5 min.

exhibit comparable antihypertensive effects (Porteri
et al., 2005). The dose of alfacalcidol was examinedpreviously in an ovariectomized rat model of osteoporosis for human (Shiraishi et al., 2000).
Drugs were administered once a day beginning
1 week after the nephrectomy for 12 weeks. Every
4 weeks, we measured body weight, blood pressure,urinary volume and urinary protein of each groups.
Blood pressure was measured in conscious rats by
the tail-cuff method with a sphygmomanometer(Softron, Tokyo). Urine was collected from individual rats housed in metabolic cages for 24 h. At 12
weeks after the administration, the rats were killed
under pentobarbital anesthesia. Blood was collectedfrom their hearts, and those serum samples werefrozen and stored at –80˚C.
Concentrations of serum creatinine, Ca, P,
1,25(OH)2 vitamin D3, urinary protein and urinary
creatinine were measured by routine laboratory
methods. The level of proteinuria excretion is affected by the physique of animals. So, we measured
urinary protein levels using the ratio of urinary
protein/creatinine, in which creatinine compensates
the measurement of proteinuria. Serum FGF23
concentration was determined by using an FGF23
enzyme-linked immunosorbent assay kit from Kainos Laboratories (Tokyo). The remnant kidneys
were removed and fixed in 10% buffered formalin
and embedded in paraffin for histological analysis.

Real-time PCR
To prepare a reverse transcribed sample, we mixed
4.1 µL of PCR grade water, 1 µL of Universal
PribeLibrary probe (Roche, Tokyo), 0.2 µL of 10μM Reverse primer, 2 µL of LightCycler TaqMan
Master (Roche) and 2.5 µL of a cDNA sample, andused 10 µL of the mixture for quantitative real-time
PCR. The mRNA levels of transforming growth
factor (TGF)- 1 and klotho genes were assessedby the real-time PCR assays, using -actin as aninternal standard. The forward and reverse primer
sequences used for this 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 ˚Cfor 30 s and 60 ˚C for 1 min.

RNA extraction and reverse-transcription
PCR analysis

Histological analysis

Tissue samples were homogenized and total RNA
was extracted by using the RNeasy Mini Kit(QIAGEN, Hilden, Germany). RNA concentration
was determined by measuring absorbance at 260 nm
and the RNA quality was verified by electrophoresis

Three micrometer-thick sections of formalin-fixed,
paraffin-embedded kidneys were stained with periodic acid-Schiff and periodic acid-methenamine
silver. For calculating focal glomerular sclerosis,
100 to 150 glomeruli from each stained specimen
51

T. Fukui et al.

(Stat View for Windows; SAS Institute, Cary, NC).
P < 0.05 was considered significant.

were examined. The degree of sclerosis in eachglomerulus was subjectively graded on a scale of
0 to 4 as follows: 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 sclelosis(IGS) was calculated by using the following formula (Kanazawa et al., 2002):(1 × N1 + 2 × N2 + 3 × N3 + 4 × N4)
IGS =			 (N0 + N1 + N2 + N3 + N4),
where N is the number of glomeruli at each gradeof sclerosis.

Results
Blood pressure
The systolic blood pressure of the rats during the
12-week experimental period is shown in Fig. 1.
The systolic blood pressure after nephrectomy wasincreased progressively throughout the experimental period in the 5/6Nx group. After the initiationof treatment, the systolic blood pressures in theAlf, Olm and Alf + Olm groups were gradually
decreased. At the end of the 12-week administration period, each systolic blood pressure in thetreated groups was significantly lower than that in
the 5/6Nx group (233.0 ± 9.6 mmHg for the 5/6Nxgroup, 199.4 ± 18.6 mmHg for the Alf group,
191.6 ± 14.2 mmHg for the Olm group, 136.0 ±19.9 mmHg for the Alf + Olm group). The Alf+ Olm group showed significantly lower levels of
the systolic blood pressur than any other group did
at week 12. In addition, we found a similar trend
for diastolic blood pressure (Fig. 1). At the end of
the 12-week administration period, each diastolicblood pressure was also significantly lower thanthat in the 5/6Nx group (177.3 ± 8.8 mmHg forthe 5/6Nx group, 140.6 ± 18.3 mmHg for the Alfgroup, 134.0 ± 12.1 mmHg for the Olm group, 94.1
± 15.4 mmHg for the Alf + Olm group).

Statistical analysis
Statistical significance of intergroup differences inquantitative data was assessed by Student’s t-test

Proteinuria
Figure 2 shows urinary protein excretion at every
4 weeks for each group. During the 12 weeks, urinary protein excretion in the 5/6Nx group was increased progressively throughout the experimental
period. In all treated groups, urinary protein excretion was significantly lower than that in the 5/6Nxgroup at week 12. Especially, urinary protein
excretion in the Alf + Olm group was significantly
decreased compared with that in the Alf group andOlm group.

Fig. 1. Courses of the mean SBP (a) and DBP (b) during
the 12-week experimental period in the 4 groups of rats.
Both pressures are significantly decreased at week 12 ingroups of Alf, Olm and Alf + Olm when compared within the 5/6Nx group. 5/6Nx, group of rats subjected to 5/6
nephrectomy; Alf, group of 0.2 μg/kg/day alfacalcidoladministered 5/6Nx rats; Olm, group of 15 mg/kg/day
olmesartan-administered 5/6Nx rats; Alf + Olm, group of
0.2 μg/kg/day alfacalcidol- and 15 mg/kg/day olmesartanadministered 5/6Nx rats. DPB, diastolic blood pressure;
SBP, systolic blood pressure.

52

Active vitamin D and ARB on renal damage

Fig. 2. Mean urinary protein levels at 0, 4, 8 and 12 weeks in the 4 groups. Urinary protein excretion in the Alf + Olmgroup is significantly decreased compared with that in the 5/6Nx group, Alf group and Olm group at week 12.

lower than that in the 5/6Nx group (P < 0.01), but
not significantly lower than that in the Olm group (P= 0.33). The level of serum creatinine in the Alf +Olm group was significantly lower than that in the
5/6Nx group (P < 0.01) and in 2 other alfacalcidoltreated groups (P < 0.05) (Fig. 3a). Figure 3b
showed the creatinine clearance in the 4 groups ofrats at week 12. The creatinine clearance in theAlf + Olm group was significantly improved incomparison with the 5/6Nx group (P < 0.05).

Serum creatinine
Although olmesartan did not significantly decrease
the serum creatinine level at week 12, the serum
creatinine level in the Alf group was significantly

Serum Ca, P and 1,25(OH)2 vitamin D3
Figure 4a shows serum Ca concentrations in the 4groups of rats at week 12. The alfacalcidol-treatedgroups (Alf, Alf + Olm) had significantly higher
Ca values than the 5/6Nx and Olm groups had(P < 0.01). By contrast, the serum P values in the
4 groups did not differ significantly (Fig. 4b). We
also examined the values of serum 1,25(OH)2 vitamin D3 in the 4 groups at week 12. The alfacalcidol-treated groups (Alf, Alf + Olm) had remarkably
higher serum levels of 1,25(OH)2 vitamin D3 than
the non-treated groups (5/6Nx, Olm) had (Fig. 4c).
Fig. 3. Serum creatinine concentrations (a) and creatinine
clearance levels (b) in the 4 groups at week 12. The levelof serum creatinine in the Alf + Olm group is significantly
lower than the other groups. The creatinine clearance in
the Alf + Olm group is significantly improved in comparison with the 5/6Nx group. Values are expressed as means± SD.

Serum FGF23
Levels of serum FGF23 in the alfacalcidol-treatedgroups (Alf, Alf + Olm) at week 12 were significantly higher than those in the 5/6Nx and Olmgroups (P < 0.01) (Fig. 5).
53

T. Fukui et al.

Histological findings in the kidney
Histological examination of the kidney revealed
remarkably advanced glomerular sclerosis in the
5/6Nx group at week 12. Conversely, the Alf, Olm
and Alf + Olm groups had bettered glomerularsclerosis. Among the 3 groups, the Alf + Olmgroup markedly ameliorated glomerular sclerosis(Fig. 6a).
The IGS in all treated groups was significantly lower than that in the 5/6Nx group (P < 0.05
for the Alf group, P < 0.05 for the Olm group and
P < 0.01 for the Alf + Olm group). The IGS in
the Alf + Olm group was significantly lower thanthat in the Alf group (P < 0.05) and the Olm group(P < 0.01) (Fig. 6b).
mRNA quantification of TGF- 1 and klotho
gene
Figure 7 shows the mRNA levels of the TGF- 1
gene and klotho gene in renal tissue. The TGF- 1
mRNA level in the 5/6Nx group was increased
as compared with the levels in the other treatedgroups, significantly alleviated in the Alf and Alf+ Olm groups (P < 0.01 for the Alf group, P < 0.01
for the Alf + Olm group), but not in the Olm group(Fig. 7a).
The klotho mRNA levels of the alfacalcidoltreated groups were increased in comparison withthe level of the 5/6Nx group: the increase was
especially significant in the Alf + Olm group.
Furthermore, in comparison between the 5/6Nxgroup and either of alfacalcidol-treated groups,
the increase was significantly higher for the Alf+ Olm group than for the Alf group (P < 0.05 for
the Alf + Olm group versus the 5/6Nx group, P <0.05 for the Alf + Olm group versus the Alf group)(Fig. 7b).

Fig. 4. Serum concentrations of Ca (a), P (b) and 1,25(OH)2
vitamin D3 (c). Levels of Ca and 1,25(OH)2 vitamin D3 are
significantly higher in alfacalcidol-treated groups than in
non-treated groups. Differences in P levels are not significant among the 4 groups.

Fig. 5. Serum fibroblast growth factor 23 (FGF23) in the 4groups at week 12. Levels of serum FGF23 in alfacalcidoltreated groups at week 12 are significantly higher than thosein non-treated groups.

54

Active vitamin D and ARB on renal damageaFig. 6. Levels of glomerular sclerosis examined at week 12.
a: Histological examination of kidneys of rats in the 4 groups
by periodic acid-Schiff staining. The sclerosis remarkably
advances in the 5/6Nx group, and improves in the Alf, Olm
and Alf + Olm groups. Especially, the sclerosis markedly
ameliorates in the Alf + Olm group. Bar = 200 µm.
b: Levels of the index of glomerular sclerosis (IGS) in the 4groups at week 12. All treated groups show significantly
lower levels than the 5/6Nx group. The Alf + Olm groupshow significantly lower level than the Alf and Olm groups.

Fig. 7. Expressions of mRNA for TGF- 1 and klotho genes inrenal tissue.
a: Expression of TGF- 1 mRNA. TGF- 1 mRNA levels are
significantly alleviated in the Alf and Alf + Olm groups,
but not in the Olm group.
b: Expression of klotho mRNA. klotho mRNA levels in thetreated groups are increased in comparison with the level inthe 5/6Nx group. The level is significantly increased in theAlf + Olm group compared with that in the 5/6Nx or Alfgroup.

55

T. Fukui et al.

primarily in renal tubular cells (Kuro-o, 2009). It
has been revealed that the transmembrane form ofklotho functions as a co-receptor for FGF23.
FGF23 is secreted from osteocytes in response to high blood levels of P and 1,25(OH)2
vitamin D3 (Saito et al., 2005). FGF23 reduces 1,
25(OH)2 vitamin D3 by down-regulating the expression of the synthesized enzyme (Dusso et al.,2005). Thus, FGF23 induces a negative P balance
by functioning as a phosphaturic hormone as well
as a counter-regulatory hormone for 1,25(OH)2
vitamin D3. FGF23-deficient mice have hyperphospatemia and hypervitaminosis D associatedwith ectopic calcification. Besides the predictablephenotypes, FGF23-deficient mice develop multiple aging-like phenotypes that are almost identical with those observed in klotho-deficient mice(Larsson et al., 2003). These observations indicatethat FGF23 and klotho may function in a common
signal transduction pathway. In our present study,levels of serum FGF23 in the groups treated withalfacalcidol (Alf group, Alf + Olm group) were significantly higher than those in the 5/6Nx group andolmesartan alone treated group.
1,25(OH)2 vitamin D3 plays an important rolein controlling bone metabolism through the regulation of Ca and P homeostasis. 1,25(OH)2 vitamin
D3 deficiency is frequently recognized in patientswith chronic kidney disease, in whom it is associated with increased mortality (Ravani et al., 2009).
It was reported that 1,25(OH)2 vitamin D3 deficiency has also been involved in the progressionof renal failure (González et al., 2004). Recently
vitamin D analogues have been shown to have beneficial effects in patients with diabetic nephropathy(de Zeeuw et al., 2010). Several mechanisms could
be involved in these renoprotective effects including direct antiproteinuric effects thorough theprotection of podocyte, interactions with the reninangiotensin system, and anti-inflammatory effects(Doorenbos et al., 2009). In our present study,
we also found that the alfacalcidol administration
yielded renoprotective effects and improvementin blood pressure, proteinuria and the IGS and
TGF- 1 gene expression. These renoprotective ef-

Discussion
In the present study, we demonstrated that theadministration of alfacalcidol and/or olmesartan
improved the CRF and up-regulated the klotho
gene in the kidney. In particular, the combination
therapy of the 2 drugs provided the most effective
renoprotection. Spontaneously hypertensive rats
have been used as a model for essential hypertension in humans, and 5/6Nx rats have commonly
been used as an experimental model for CRF in
humans. In this study, we combined these 2 models, and prepared 5/6Nx spontaneously hypertensive rats. During the 12-week experimental period,
blood pressure, urinary protein and glomerularsclerosis in the 5/6Nx spontaneously hypertensive
rats increased progressively. After 12 weeks, theglomerular sclerosis was remarkable.
The renin-angiotensin system plays an important role in the development of hypertension and theprogression of CRF. TGF- 1 has been shown toplay a predominant role in mediating angiotensin IIinduced extracellular matrix production and inhibits
its degradation by increasing production of proteinase inhibitors in mesangial and tubulointerstitialcells (Border et al., 1992). Thus, TGF- 1 plays acentral role in the development glomerular sclerosis
and tubulointerstitial fibrosis. In addition, continuous administration of angiotensin II resulted in thedevelopment of CRF, and decreased klotho mRNA
expression in the rat kidney (Mitani et al., 2002).
In our previous study, we have shown that thegroup treated with olmesartan prevented the progress of CRF and the klotho mRNA downregulationin the 5/6Nx rats (Maeta et al., 2009). In this study,
the administration of olmesartan improved bloodpressure, urinary protein, glomerular index of thekidney, and up-regulated the klotho mRNA.
It is well known that klotho gene in the kidneyof CRF patients was markedly reduced (Koh et al.,2001). Recently, the roles of klotho function havebeen evaluated. Klotho protein exists in 2 forms:
one is a secreted form that circulates in the blood,and the other is a transmembrane form expressed56

Active vitamin D and ARB on renal damage

could be harmful by raising FGF23 excessively, but
lower doses might promote less elevation of FGF23(Isalova et al., 2009). In the present study, thegroups treated with alfacalcidol showed an increase
in serum FGF23, but improvement of renal failure.
In conclusion, the present study demonstrated
that alfacalcidol and/or olmesartan exhibited arenoprotective effect, throughout the antihypertensive effect, as well as upregulation of klotho gene
expression in the kidney.

fects were the strongest in combination therapy ofalfacalcidol and olmesartan. There are also some
reports of investigations on the effect of vitamin
D on blood pressure (Kristal-Boneh et al., 1997;
Witham et al., 2009; Judd et al., 2010). Higher levels of active vitamin D are associated with lower
blood pressure. Although the mechanism by which
vitamin D may regulate blood pressure is unclear,
it was suggested that vitamin D is negative regulator of renin (Li et al., 2002).
It has been reported that renal injury was reduced in klotho-overexpressing transgenic mice,
because of suppression of oxidative stress (Haruna
et al., 2007). In addition, klotho inhibited TGF- 1
signaling and suppressed renal fibrosis (Doi et al.,2011). Moreover, the administration of 1,25(OH)2
vitamin D3 induced klotho gene expression in thekidney (Tsujikawa et al., 2003). The klotho mRNAlevel of the Alf + Olm group was significantly increased compared with that of 5/6Nx group, suchthat suppression of klotho gene downregulation
might attenuate the progression of renal damage.
In the present study, we selected alfacalcidol
as a vitamin D analogue. As there have been noreports of alfacalcidol being used for uremic rats,
the dose of alfacalcidol referred to in the previous
reports of dosage in ovariectomized rats, osteoporosis model, was administered (Shiraishi et al.,2000). The values of serum Ca and 1,25(OH)2
vitamin D3 of the groups treated with alfacalcidol(Alf group, Alf + Olm group) were found to bevery high. There is a possibility that hypercalcemia causes diuresis and natriuresis, which lead todehydration and renal insufficiency (Shiraishi etal., 2003). However, the values of serum creatinine
and the IGS of alfacalcidol-treated groups was
lower than those in the 5/6Nx group in the present
study, so we thought that hypercalcemia did notcause renal injury. In our study the serum FGF23
levels in the groups treated with alfacalcidol (Alfgroup, Alf + Olm group) were significantly higher
those in the 5/6Nx group and Olm group, despite
the improvement of the IGS and klotho gene expression. There was a therapeutic window for active vitamin D therapy, whereby too high a dose

Acknowledgments: We are grateful to Chugai Pharmaceutical (Fuji-Gotemba, Japan) and Daiichi Sankyo Pharmaceutical (Tokyo, Japan) for supplying alfacalcidol andolmesartan.
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Received June 28, 2011; accepted July 19, 2011
Corresponding author: Chishio Munemura, MD

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