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2 0 1 0 B J U I N T E R N A T I O N A L | 1 0 6 , 1 5 5 0 1 5 5 4 | doi:10.1111/j.1464-410X.2010.09216.x
2010 THE AUTHORS. JOURNAL COMPILATION 2010 BJU INTERNATIONAL
Original Articles
INSULIN RESISTANCE AND URINARY STONE FORMATION IN METABOLIC SYNDROME
IBA
ET AL.

Insulin resistance increases the risk of
urinary stone formation in a rat model of
metabolic syndrome

Akinori Iba, Yasuo Kohjimoto, Takashi Mori, Tomomi Kuramoto,
Satoshi Nishizawa, Reona Fujii, Yoshihito Nanpo, Nagahide Matsumura,
Yasuyo Shintani, Takeshi Inagaki and Isao Hara

Department of Urology, Wakayama Medical University, Wakayama, Japan

Accepted for publication 10 November 2009

4 weeks. Ten-week-old male OLETF and LETO
rats were divided into three groups of nine
each and treated with vehicle or oral
administration of 3 or 10 mg/kg/day
pioglitazone, an agent that improves insulin
resistance. After 4 weeks, body weight and
serum and urinary biochemistry were
determined.

RESULTS

The OLETF rats had signicantly lower
urinary pH and citrate excretion, and higher
urinary uric acid and calcium excretion, than
the LETO rats, with increases in body weight,
serum triglyceride, glucose and insulin. The
administration of pioglitazone to the OLETF
rats for 4 weeks signicantly increased
urinary pH dose-dependently. There was no
change in the urinary excretion of citrate,
uric acid, calcium, oxalate or magnesium.

CONCLUSION

These results indicate that metabolic
syndrome causes the changes in urinary
constituents, leading to increased risk of
both uric acid and calcium stone formation.
Improvement in insulin resistance, a central
cause of metabolic syndrome, might prevent
uric acid stone formation by raising urinary
pH.

KEYWORDS

urinary stone disease, metabolic syndrome,
insulin resistance, rat, pioglitazone

OBJECTIVE

To investigate the association between
metabolic syndrome and urinary stone
disease, and whether insulin resistance
associated with adiposity affects the risk of
urinary stone formation, using a rat model of
metabolic syndrome.

MATERIALS AND METHODS

Four-week-old male Otsuka Long-Evans
Tokushima Fatty (OLETF, a model of human
type 2 diabetes and metabolic syndrome)
rats, and Long-Evans Tokushima (LETO, a
non-diabetic control) rats (10 each) were
given a standardized diet and free access to
water. Body weight and serum and urinary
biochemistry were determined every

INTRODUCTION

The prevalence of kidney stones has been
increasing in several countries, in parallel
with the growing epidemics of obesity
and type 2 diabetes [14]. In large
epidemiological studies, an increased
prevalence of kidney stones was reported
in patients with obesity [5], type 2 diabetes
[6] and hypertension [7]. These medical
conditions are now collectively referred to as
metabolic syndrome, which has received
much attention in recent years as a risk
factor for developing cardiovascular diseases
[8]. In the present study, we investigated the
association between metabolic syndrome and
urinary stone disease, and whether insulin
resistance, a central cause of metabolic
syndrome, affects the risk of urinary stone
formation, using a rat model of metabolic
syndrome [9].

MATERIALS AND METHODS

Male Otsuka Long-Evans Tokushima Fatty
(OLETF, a model of human type 2 diabetes and
metabolic syndrome) rats, and Long-Evans
Tokushima (LETO, a non-diabetic control) rats,
aged 4 weeks, were kindly provided by Otsuka
Pharmaceuticals, Japan. OLETF rats, which
were developed from a strain of Long-Evans
rat by selective breeding, are a useful model of
human type 2 diabetes and metabolic
syndrome [9]. They spontaneously develop
visceral adiposity and insulin resistance at an
early age and later have hyperglycaemia,
hyperlipidaemia and hypertension. Both sets
of rats were maintained according to the
ethical guidelines of our institution, and the
Committee on Animal Investigations of the
Wakayama Medical University approved the
experimental protocols.
In protocol 1, 4-week-old male OLETF and
LETO rats (10 each) were given standardized
diet and free access to water, and weighed
every 4 weeks. A fasting blood sample was
obtained every 4 weeks for analysis of
glucose, insulin, triglyceride and total
cholesterol. A 24-h urine sample was
collected every 4 weeks to analyse the risk of
stone disease, including pH, calcium, oxalate,
citrate, magnesium and uric acid levels.
In protocol 2, 10-week-old male OLETF and
LETO rats were divided into three groups of

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nine each and treated with vehicle or oral
administration of 3 or 10 mg/kg/day
pioglitazone (Takeda Chemical Industry Co.,
Japan), an agent that improves insulin
resistance. After 4 weeks, body weight and
serum and urinary biochemistry were
determined.
Serum glucose levels were measured using
the glucose oxidase method, and serum
insulin concentrations by radioimmunoassay
using a double-antibody method, with a
commercially available radioimmunoassay kit
(Morinaga, Japan). Serum triglyceride and
total cholesterol levels were measured by an

FIG. 1.

Time courses of body weight (

A

) and fasting serum levels of triglyceride (

B

), glucose (

C

) and insulin (

D

)
in OLETF (red line) and LETO (blue line) rats (10 each). Values are the mean (

SD

). *

P



<

0.01, **

P



<

0.05.
0
100
200
300
400
500
600
700
800
4 w 8 w 16 w 12 w 20 w 24 w 8 w 16 w 12 w 20 w 24 w
8 w 16 w 12 w 20 w 24 w 8 w 16 w 12 w 20 w 24 w
OLETF
LETO
*
*
*
*
*
*
*p < 0.01
A. Body weight, g
0
50
100
150
200
250
300
350
OLETF
LETO
*
*
*
*
*p < 0.01
C. Serum glucose, mg/dl
0
500
1000
1500
2000
OLETF
LETO
*
**
**
*p < 0.01**p < 0.05
D. Serum insulin, pg/ml
0
50
100
150
200
250
300
350
400
OLETF
LETO
*
*
*
*
*
*p < 0.01
B. Serum triglyceride, mg/dl

FIG. 2.

Time course of the HOMA-R in OLETF (red line)
and LETO (blue line) rats (10 each), expressed as the
mean (

SD

). *

P



<

0.05, **

P



<

0.01.
0
5
10
15
20
25
30
35
40
*p < 0.05 **p < 0.01
OLETF
LETO
**
*
*
8 w 16 w 12 w 20 w 24 w

FIG. 3.

The time courses of urinary pH (

A

) and 24-h urinary excretions of citrate (

B

), uric acid (

C

), calcium (

D

), oxalate (E) and magnesium (F) in OLETF (red line) and LETO
(blue line) rats (10 each). Values are the mean (

SD

). *

P



<

0.01, **

P



<

0.05.
5.5
6
6.5
7
7.5
8
OLETF
LETO
*p < 0.01
*
*
*
*
*
0
10
20
30
40
50
60
70
80
OLETF
LETO
*
* *
*
*p < 0.01
0
1
2
3
4
5
OLETF
LETO
*
*
*
*
*
*
*p < 0.01
0
1
2
3
4
OLETF
LETO
*
**
*p < 0.01
**p < 0.05
0
1
2
3
4
5
6
7
OLETF
LETO
*
**
*p < 0.01
**p < 0.05
0
0.2
0.4
0.6
0.8
1
1.2
OLETF
LETO
*
*
*p < 0.01
B. Citrate, mg/day
D. Calcium, mg/day E. Oxalate, mg/day F. Magnesium, mg/day
C. Uric acid, mg/day A. Urine pH
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enzymatic colorimetric method using
commercially available kits. The homeostasis
model assessment ratio (HOMA-R), an index
of insulin resistance, was calculated as:
(fasting immunoreactive insulin level

fasting glucose)/405 [10]. Two successive
24-h urine samples were collected in 50 mL
centrifuge tubes; the rst was collected in
liquid parafn and used for the measurement
of pH, calcium, magnesium, and uric acid; the
second was collected in concentrated HCl and
used for the oxalate and citrate measurement
via capillary electrophoresis.
All results are shown as the mean (

SD

); groups
were compared using Students

t

-test and
Dunnetts multiple comparison test; in all
statistical analyses

P



<

0.05 considered to
indicate statistical signicance.

RESULTS

In protocol 1, the OLETF rats gained weight
faster than the LETO rats (

P



<

0.01; Fig. 1A).
After 8 weeks of age, the OLETF rats had
signicantly higher serum triglyceride levels
than the LETO rats (

P



<

0.01; Fig. 1B). After
12 weeks of age, the OLETF rats had
signicantly higher serum glucose and insulin
concentrations than the LETO rats (

P



<

0.05 or

<

0.01; Fig. 1C,D). The HOMA-R values of the
OLETF rats were signicantly higher than
those of the LETO rats after 12 weeks of age
(

P



<

0.05 or

<

0.01; Fig. 2).
Urinary pH and urinary citrate excretion in the
OLETF rats gradually decreased with age and
were signicantly lower than those in the
LETO rats after 8 and 12 weeks of age,
respectively (

P



<

0.01; Fig. 3A,B). By contrast,
the urinary excretion of uric acid and calcium
in the OLETF rats gradually increased and were
signicantly higher than those in the LETO
rats after 4 and 20 weeks of age, respectively
(

P



<

0.05 or

<

0.01; Fig. 3C,D). Urinary oxalate
and magnesium showed no clear trends
(Fig. 3E,F).
In protocol 2, the administration of
pioglitazone to the OLETF rats for 4 weeks did
not affect body weight or serum glucose
(Fig. 4A,B). However, it signicantly improved
hyperinsulinaemia and hypertriglyceridaemia
to a level similar to that seen in the LETO rats,
in a dose-dependent fashion (Fig. 4C,D). In
addition, there was a dose-dependent
decreasing trend in the HOMA-R in the OLETF
rats treated with pioglitazone, although the
difference was not statistically signicant
(Fig. 5). In the pioglitazone-treated OLETF rats,
urinary pH increased signicantly and dose-
dependently. There was no signicant change
in the urinary excretion of citrate, uric acid,
calcium, oxalate or magnesium (Fig. 6). The
administration of pioglitazone to the LETO
rats for 4 weeks did not affect body weight or
serum or urinary biochemistry (data not
shown).

DISCUSSION

In the present study, we used the OLETF and
LETO rats to clarify the association between
metabolic syndrome and urinary stone
disease, and investigated whether insulin
resistance associated with adiposity affects
the risk of urinary stone formation. First,
OLETF rats had signicant decreases in urinary

FIG. 4.

The effect of pioglitazone on body weight (

A

), fasting serum levels of glucose (

B

), insulin (

C

) and
triglyceride (

E

) in OLETF rats (red column, nine). Rats were treated for 4 weeks with vehicle or oral
administration of 3 or 10 mg/kg/day pioglitazone. The blue column represents LETO rats (nine) treated with
vehicle. Columns and bars show the mean (

SD

). ns, not signicant.
0
10
20
30
40
50
60
70
LETO OLETF
LETO OLETF
p < 0.01
p < 0.01
D. Serum triglyceride, mg/dl
0
50
100
150
200
250
ns
ns
B. Serum glucose, mg/dl
0
200
400
600
800
1000
1200
1400
LETO OLETF
LETO OLETF
p = 0.31
p = 0.08
C. Serum insulin, pg/ml
A. Body weight, g
0
100
200
300
400
500
600
Vehicle Vehicle 3 mg/kg 10 mg/kg
ns
ns
Vehicle Vehicle 3 mg/kg 10 mg/kg
Vehicle Vehicle 3 mg/kg 10 mg/kg Vehicle Vehicle 3 mg/kg 10 mg/kg

FIG. 5.

The effect of pioglitazone on the HOMA-R in
OLETF rats (red column, nine). Rats were treated for
4 weeks with vehicle or oral administration of 3 or
10 mg/kg/day pioglitazone. The blue column
represents LETO rats (nine) treated with vehicle.
Columns and bars show the mean (

SD

).
0
5
10
15
p = 0.19
p < 0.77
LETO OLETF
Vehicle Vehicle 3 mg/kg 10 mg/kg
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pH and urinary citrate excretion, and
signicant increases in the urinary excretion
of uric acid and calcium compared with LETO
rats, with increases in body weight, serum
triglyceride, glucose and insulin. Therefore,
the results indicate that metabolic syndrome
is associated with an increased risk of urinary
stone formation.
In humans, West

et al.

[11] reported that
metabolic syndrome traits were associated
with a self-reported history of kidney stones
in data from the Third National Health and
Nutrition Examination Survey. This raised the
question of what kind of stone composition is
associated with metabolic syndrome. Siener

et al.

[12] showed that obesity was strongly
associated with an increased risk of stone
formation due to the increased urinary
excretion of promoters but not inhibitors of
calcium oxalate stone formation. Conversely,
Daudon

et al.

[13] found that overweight,
obesity and type 2 diabetes were associated
with uric acid but not calcium oxalate stone
formation. The results from the present study
suggest that metabolic syndrome is
associated with an elevated risk of both types
of stones, because the lower urinary pH and
increased uric acid excretion in the OLETF rats
are considered to promote uric acid stone
formation, and decreased citrate excretion
and increased excretion of uric acid and
calcium will promote calcium stone
formation.
Second, we investigated how pioglitazone,
which improves insulin resistance, affects
metabolic syndrome and urinary stone
formation. Pioglitazone belongs to the
thiazolidinedione class of drugs and binds to a
nuclear receptor called the peroxisome
proliferator-activated receptor (PPAR)

, and
increases insulin sensitivity mainly at the level
of muscle and adipose tissue. In the present
study, pioglitazone decreased serum insulin
concentration but did not affect serum
glucose. As a result, the HOMA-R showed
a trend toward improvement, but it was
not statistically signicant. However,
hyperinsulinaemia itself is generally thought
to be a marker of insulin resistance in pre-
diabetic populations [1416]. As 10-week-old
OLETF rats used in the present study had not
yet developed diabetes, the pioglitazone-
induced decrease in serum insulin indicates
an improvement in insulin resistance.
In urinary biochemistry, pioglitazone
signicantly increased the urinary pH in a
dose-dependent fashion. Although urinary
citrate excretion was increased by
pioglitazone administration, the difference
was not statistically signicant because of the
large

SD

. There were no changes in urinary uric
acid or calcium excretion. These results
indicate that insulin resistance, a central
cause of metabolic syndrome, is associated
with the regulation of urinary pH. The exact
mechanism by which insulin resistance leads

FIG. 6.

The effect of pioglitazone on urinary pH (

A

) and 24-h urinary excretions of citrate (

B

), uric acid (

C

), calcium (

D

), oxalate (

E

) and magnesium (

F

) in OLETF rats (red
column, nine). Rats were treated for 4 weeks with vehicle or oral administration of 3 or 10 mg/kg/day pioglitazone. The blue column represents LETO rats (nine) treated
with vehicle. Columns and bars show the mean (

SD

). ns, not signicant.
6
6.5
7
7.5
p = 0.07
p < 0.01
A. Urine pH
0
10
20
30
40
50
ns
ns
0
0.5
1
1.5
2
2.5
3
3.5 ns
B. Citrate, mg/day C. Uric acid, mg/day
0
0.2
0.4
0.6
0.8
1
1.2
ns
ns
D. Calcium, mg/day
0.2
0.4
0.6
0.8
1
ns
ns
E. Oxalate, mg/day
0
0.5
1
1.5
2
2.5
3
3.5
ns
ns
F. Magnesium, mg/day
ns
LETO OLETF
Vehicle Vehicle 3 mg/kg 10 mg/kg
LETO OLETF
Vehicle Vehicle 3 mg/kg 10 mg/kg
LETO OLETF
Vehicle Vehicle 3 mg/kg 10 mg/kg
LETO OLETF
Vehicle Vehicle 3 mg/kg 10 mg/kg
LETO OLETF
Vehicle Vehicle 3 mg/kg 10 mg/kg
LETO OLETF
Vehicle Vehicle 3 mg/kg 10 mg/kg
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to low urinary pH remains undetermined in
the present study. However, insulin has been
shown to promote renal ammoniagenesis
from the substrate glutamine [17,18] and to
stimulate the Na

+

/H

+

exchanger 3 in the
proximal tubule [19]. Impaired ammonium
production or excretion induced by insulin
resistance might lead to low urinary pH [20].
In the present study, pioglitazone also
signicantly decreased serum triglyceride
levels. Pioglitazone is known to affect the
PPAR

receptor, and this cross reactivity to
PPAR

explains why pioglitazone is effective
at lowering triglyceride levels [21]. Takahashi

et al.

[22] reported that the administration of
PPAR

agonists signicantly raised urinary
pH levels in patients with gout, in accordance
with a reduction in serum triglyceride level.
Considering these factors, there might be a
close relationship between increasing urinary
pH and reductions in serum triglyceride
caused by pioglitazone administration. The
present study is the rst to report a clear
causal relationship between insulin resistance
and low urinary pH using pioglitazone. We
suggest that the improvement in insulin
resistance might prevent not only disorders
related to metabolic syndrome but also
urinary stone disease, by raising urinary pH.
In conclusion, this study showed that
metabolic syndrome is associated with an
increased risk of both uric acid and calcium
stone formation through lower urinary pH,
decreased citrate excretion, and increased uric
acid and calcium excretion, and that the
improving insulin resistance by pioglitazone
decreases the risk of urinary stone formation
by increasing urinary pH. We suggest that
urinary stone disease should be considered as
a component of metabolic syndrome and that
the improvement of insulin resistance due to
dietary instruction or lifestyle guidance might
help to prevent this disorder.

ACKNOWLEDGEMENTS

We thank Fumie Saji for providing excellent
technical assistance. This work was partly
supported by 18 Wakayama Medical Award
for Young Researchers.

CONFLICT OF INTEREST

None declared.

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Correspondence: Iba Akinori, Department of
Urology, Wakayama Medical University, 811-1
Kimiidera Wakayama 641-8509, Japan.
e-mail: a-iba@ommc-hp.jp
Abbreviations: OLETF, Otsuka Long-Evans
Tokushima Fatty; LETO, OLETF control;
HOMA-R, homeostasis model assessment
ratio; PPAR, peroxisome proliferator-
activated receptor.
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