Elsevier

Metabolism

Volume 57, Issue 5, May 2008, Pages 691-697
Metabolism

Effect of pitavastatin on type 2 diabetes mellitus nephropathy in KK-Ay/Ta mice

https://doi.org/10.1016/j.metabol.2008.01.007Get rights and content

Abstract

It is generally considered that 3-hydroxy-3-methylglutaryl–coenzyme A reductase inhibitors (statins) have renoprotective effects via a pathway independent of their cholesterol-lowering cascade. In the kidneys of diabetic nephropathy, monomeric endothelial nitric oxide synthase (eNOS) is thought to be overexpressed; and its dimerization is suppressed. In the present study, we investigated the expression of eNOS and oxidative stress in type 2 diabetes mellitus KK-Ay/Ta mice treated with pitavastatin, one of the statins. The KK-Ay/Ta mice were divided into 3 groups and given pitavastatin intraperitoneally starting at 8 weeks of age for 8 weeks: pitavastatin 3 mg/(kg d) (n = 5), pitavastatin 10 mg/(kg d) (n = 5), and a control group (n = 10). The urinary albumin-creatinine ratio (ACR), urinary 8-hydroxy-2'-deoxyguanosine, body weight, fasting blood glucose, hemoglobin A1c, total cholesterol, and triglyceride were measured; and the intraperitoneal glucose tolerance test was performed. The eNOS, nitrotyrosine, and p47 phox were evaluated by immunohistochemical analyses and/or Western blot analyses. Guanosine triphosphate cyclohydrolase 1 messenger RNA expression in the kidneys was evaluated using a real-time polymerase chain reaction assay. Pitavastatin improved the levels of urinary ACR and 8-hydroxy-2'-deoxyguanosine, intraperitoneal glucose tolerance test, and hemoglobin A1c. Protein levels of monomeric eNOS, nitrotyrosine, and p47 phox in the kidneys were decreased in the pitavastatin-treated groups. Guanosine triphosphate cyclohydrolase 1 messenger RNA expression was significantly increased in the pitavastatin groups. There were no significant changes in body weight, levels of fasting blood glucose, serum total cholesterol, triglyceride, and blood pressure among all groups. Pitavastatin improved urinary ACR apparently because of suppression of eNOS uncoupling and its antioxidant effect in the kidneys of KK-Ay/Ta mice.

Introduction

The 3-hydroxy-3-methylglutaryl–coenzyme A (HMG-CoA) reductase inhibitors (statins) have pleiotropic effects on cardiovascular, cerebrovascular, and microvascular diseases independent of their cholesterol-lowering effect [1], [2], [3]. Statins also have beneficial effects on kidney disease including diabetic nephropathy. Several studies have shown that statins prevented the progression of microalbuminuria and nephropathy in diabetes [4], [5]. Although possible pathways of renoprotection, such as the Rho-dependent pathway, inhibition of macrophages, and platelet coagulation, were proposed, this effect of statins has not been fully clarified [6], [7], [8], [9].

Nitric oxide (NO), known as a vasodilator and important factor for the prevention of arteriosclerosis and hypertension, is generated from vascular endothelial cells mainly by the endothelial nitric oxide synthase (eNOS) signal in the blood vessels [10], [11]. In diabetic nephropathy, NO plays an important role in the maintenance of blood pressure by reducing renal vascular tone. Although generation of eNOS can be observed in all kinds of cells in the kidney, particularly strong expression is shown in the glomeruli. Endothelial nitric oxide synthase has 2 roles depending on whether it is in the monomeric or dimeric form. In the dimeric form, eNOS may act as NO generator. However, in the monomeric state, it produces superoxide (O2) rather than NO [12]. Previous studies demonstrated that statins increased both eNOS and its cofactor, tetrahydrobiopterin (BH4), in diabetic endothelial cells. Furthermore, activity or gene expression of guanosine triphosphate cyclohydrolase 1 (GTPCH-1), the rate-limiting enzyme in BH4 synthesis, is also up-regulated by statins [13], [14], [15]. In diabetic glomeruli, previous studies also indicated that the dimeric form of eNOS was decreased and the monomeric form was increased because of the decrease of BH4. Both NO activity and reactive oxygen species (ROS) production were increased. Such imbalance of NO/ROS may cause endothelial dysfunction of the kidney [1], [2], [3]. Nicotinamide adenine dinucleotide phosphate (NAD(P)H) oxidase is an important source of ROS production. Previous reports have noted that the components of NAD(P)H oxidase, such as p47 phox, p67 phox, and NOX4, were increased in diabetic kidneys [16], [17].

In 1969, the KK-Ay/Ta mouse was established by Nishimura [18]. This mouse was produced by the transfer of the yellow obese gene (Ay allele) into the KK/Ta mouse. Because the diabetic feature in the KK-Ay/Ta mouse is more severe than that in the KK/Ta mouse, this mouse is widely used as an experimental model for type 2 diabetes mellitus [19]. The KK-Ay/Ta mouse spontaneously exhibits type 2 diabetes mellitus associated with hyperglycemia, glucose intolerance, hyperinsulinemia, obesity, and microalbuminuria. Renal lesions in the KK-Ay/Ta mouse closely resemble those in human diabetic nephropathy. Glomeruli of the KK-Ay/Ta mouse show diffuse-type hyperplasia of mesangial areas with mesangial cell proliferation, segmental sclerosis, overexpression of transforming growth factor–β1, and advanced glycation end product accumulation at 20 weeks of age [20], [21], [22]. We have reported that the KK-Ay/Ta mouse is a suitable model for the study of type 2 diabetes mellitus nephropathy in humans [21]. Furthermore, because mice have a naturally low cholesterol level and are generally unaffected by statin therapy, they are considered as good models for studying the pleiotropic effect of statins except the cholesterol-lowering effect.

In the present study, we investigated prevention of the development of type 2 diabetes mellitus nephropathy in KK-Ay/Ta mice treated with pitavastatin. We also investigated whether the renoprotective effect of statins is caused by a decrease of monomeric eNOS and whether the NAD(P)H oxidase components are decreased by statin treatment.

Section snippets

Animals

Male diabetic KK-Ay/Ta Jcl mice (6 weeks of age) and C57BL/6J mice were purchased from CLEA Japan (Tokyo, Japan). The mice were individually housed in plastic cages with free access to food (rodent pellet diet NMF; 348 kcal/100 g, containing 5.5% crude fat) and water throughout the experimental periods. All mice were maintained in the same room under conventional conditions with a regular 12-hour light/dark cycle and temperature controlled at 24°C ± 1°C.

Reagents and treatments

Pitavastatin was kindly provided by Kowa

Biochemical characterization

As shown in Fig. 1 and Table 1, the mean levels of ACR and HbA1c at 16 weeks of age in both the 3- and 10-mg/(kg d) pitavastatin groups were significantly lower than those in the untreated group depending on the dosage of pitavastatin. The mean levels of HbA1c in both the 3- and 10-mg/(kg d) groups were significantly lower than those in the untreated group. However, there were no statistically significant changes in the levels of HbA1c between the 3- and 10-mg/(kg d) groups. There were no

Discussion

In the present study, we demonstrated that pitavastatin improves the levels of urinary ACR, urinary 8-OHdG, and insulin resistance in KK-Ay/Ta mice independent of its cholesterol-lowering effect. Furthermore, pitavastatin prevented accumulation of monomeric eNOS, nitrotyrosine, and p47 phox in the kidney tissues.

Although statins have been reported to have pleiotropic effects on cardiovascular, cerebrovascular, and microvascular disease independent of their cholesterol-lowering effects in

Acknowledgment

We thank Kowa (Tokyo, Japan) for providing pitavastatin. We also want to thank Ms T Shibata and Mr K Yoshida for their skillful technical supports.

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