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

Authors


Iba Akinori, Department of Urology, Wakayama Medical University, 811-1 Kimiidera Wakayama 641-8509, Japan.
e-mail: a-iba@ommc-hp.jp

Abstract

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 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 significantly 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 significantly 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.

Abbreviations
OLETF

Otsuka Long-Evans Tokushima ‘Fatty’

LETO

OLETF control

HOMA-R

homeostasis model assessment ratio

PPAR

peroxisome proliferator-activated receptor

INTRODUCTION

The prevalence of kidney stones has been increasing in several countries, in parallel with the growing epidemics of obesity and type 2 diabetes [1–4]. 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 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 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 first was collected in liquid paraffin 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 Student’s t-test and Dunnett’s multiple comparison test; in all statistical analyses P < 0.05 considered to indicate statistical significance.

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 significantly higher serum triglyceride levels than the LETO rats (P < 0.01; Fig. 1B). After 12 weeks of age, the OLETF rats had significantly 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 significantly higher than those of the LETO rats after 12 weeks of age (P < 0.05 or <0.01; Fig. 2).

Figure 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.

Figure 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.

Urinary pH and urinary citrate excretion in the OLETF rats gradually decreased with age and were significantly 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 significantly 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).

Figure 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.

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 significantly 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 significant (Fig. 5). In the pioglitazone-treated OLETF rats, urinary pH increased significantly and dose-dependently. There was no significant 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).

Figure 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 significant.

Figure 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).

Figure 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 significant.

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 significant decreases in urinary pH and urinary citrate excretion, and significant 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)γ, 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 significant. However, hyperinsulinaemia itself is generally thought to be a marker of insulin resistance in pre-diabetic populations [14–16]. 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 significantly increased the urinary pH in a dose-dependent fashion. Although urinary citrate excretion was increased by pioglitazone administration, the difference was not statistically significant 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 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 significantly decreased serum triglyceride levels. Pioglitazone is known to affect the PPARα receptor, and this cross reactivity to PPARα explains why pioglitazone is effective at lowering triglyceride levels [21]. Takahashi et al.[22] reported that the administration of PPARα agonists significantly 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 first 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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