YM440, a novel hypoglycemic agent, protects against nephropathy in Zucker fatty rats via plasma triglyceride reduction

European Journal of Pharmacology 549 (2006) 185 – 191 www.elsevier.com/locate/ejphar

YM440, a novel hypoglycemic agent, protects against nephropathy in Zucker fatty rats via plasma triglyceride reduction Ryosuke Nakano a,⁎, Eiji Kurosaki a , Akiyoshi Shimaya a , Satoru Kajikawa b , Masayuki Shibasaki a a

Pharmacology Research Laboratories, Drug Discovery Research, Astellas Pharma Inc., Japan Drug Safety Research Laboratories, Drug Discovery Research, Astellas Pharma Inc., Japan

b

Received 26 April 2006; received in revised form 7 August 2006; accepted 14 August 2006 Available online 26 August 2006

Abstract The novel hypoglycemic agent, YM440 ((Z)-1,4-bis{4-[(3,5-dioxo-1,2,4-oxadiazolidin-2-yl) methyl] phenoxy}but-2-ene) is a ligand of the peroxisome proliferator-activated receptor, (PPAR) γ. YM440 has been shown to counteract insulin resistance in diabetic rodent models. However, it is not clear whether this compound has a significant effect on hyperlipidemia in vivo. Hyperlipidemia has been reported to be a risk factor for the early development of renal disease. The aim of this study is to examine the effects of chronic treatment with YM440 on hyperlipidemia and renal injury in obese Zucker fatty (ZF) rats. Treatment of 8-week-old ZF rats with YM440 (100 mg/kg/day) for 16 weeks decreased plasma triglyceride and cholesterol concentrations. YM440 markedly reduced the rate of progression of both albuminuria and proteinuria. YM440 normalized urinary N-acetyl-β-D-glucosaminidase (NAG) activity, which is a marker for renal proximal tubular damage, and ameliorated the rise in systolic blood pressure compared to the vehicle control. YM440 also blocked the development of nephromegaly. Histological analyses revealed that both glomerular area expansion and tubular cast accumulation gradually lessened in YM440-treated ZF rats. Regression analyses between the plasma triglyceride levels and the renal parameters (urinary protein excretion and albumin excretion) indicated that the renal parameters correlated positively with the plasma triglyceride levels. In conclusion, the hypolipidemic effects of YM440 prevent renal injury in ZF rats. YM440 might be useful for preventing the early development of diabetic nephropathy in subjects with type 2 diabetes by ameliorating metabolic control problems. © 2006 Elsevier B.V. All rights reserved. Keywords: Insulin resistance; Diabetic nephropathy; Peroxisome proliferator-activated receptor γ; YM440; Zucker fatty rat; Hyperlipidemia

1. Introduction Type 2 diabetes mellitus is often associated with a cluster of interrelated plasma lipid and lipoprotein abnormalities, including reduced high-density lipoprotein (HDL), a predominance of small dense low-density lipoprotein (LDL) particles, and elevated triglycerides. These changes are also a feature of insulin resistance syndrome (also known as metabolic syndrome), which is the underlying cause of many cases of type 2 diabetes (Krauss, 2004; Adeli et al., 2001). A number of studies have suggested that hyperlipidemia is an independent risk factor for the early ⁎ Corresponding author. Diabetes Department, Pharmacology Research Laboratories, Drug Discovery Research, Astellas Pharma Inc., 5-2-3 Toukoudai, Tsukuba-shi, Ibaraki 300-2698 Japan. Tel.: +81 29 865 7182; fax: +81 29 847 1536. E-mail address: [email protected] (R. Nakano). 0014-2999/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2006.08.032

development of human renal disease (Stevenson and Kaysen, 1999). In recent years, type 2 diabetes has reached epidemic proportions, and diabetic nephropathy is the leading cause of end-stage renal disease. The numbers of patients with type 2 diabetes that develop end-stage renal disease and end up requiring renal replacement therapy are increasing in all countries, making this situation one of global importance (Coimbra et al., 2000; Hayden et al., 2005; and references therein). Several clinical trials have demonstrated that when hyperlipidemia is ameliorated, the rate of progression into diabetic nephropathy is reduced (Tonolo et al., 1997; Gaede et al., 1999). Currently, the amelioration of hyperlipidemia has attracted attention as an effective method for the possible prevention of diabetic nephropathy. Various animal models have been employed to gain more insight into the pathogenesis of the nephropathy associated with type 2 diabetes (Janssen et al., 1999). One of the oldest of such

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models is the Zucker fatty (ZF) rat model, which is characterized by hyperinsulinemia, hyperlipidemia, glucose intolerance, and obesity. ZF rats therefore exhibit most of the metabolic abnormalities associated with human type 2 diabetes. As they age, ZF rats spontaneously develop proteinuria and focal segmental glomerulosclerosis, which ultimately lead to renal failure (Coimbra et al., 2000; and references therein). However, these two conditions can be ameliorated by treatment with antilipemic agents (Kasiske et al., 1988), anti-hypertensive agents (Schmitz et al., 1992), or thiazolidinediones, which are all antidiabetic agents that are agonists of peroxisome proliferator-activated receptor (PPAR) γ (Yoshioka et al., 1993; Buckingham et al., 1998; Smith et al., 2000). These intervention studies suggest that the pathogenesis of renal damage in ZF rats involves a complex interrelationship of metabolic disturbances and/or hemodynamic factors. A previous study showed that YM440 ((Z)-1,4-bis{4-[(3,5dioxo-1,2,4-oxadiazolidin-2-yl)methyl] phenoxy}but-2-ene), an analog of the oxazolidinediones, bound to PPARγ (Ki = 4.0 μM) causing a unique conformational change (Kurosaki et al., 2003a,b). YM440 has little effect on adipocyte differentiation or PPARγ transactivation in vitro. In diabetic db/db mice, this agent significantly decreases blood glucose to a level comparable to that seen in thiazolidinedione-treated mice without affecting body weight (Shimaya et al., 2000). ZF rats treated with YM440 for 2 weeks showed improved hepatic glucose metabolism and hepatic insulin sensitivity (Nakano et al., 1999; Kurosaki et al., 2002, 2003a,b). However, it is not clear whether long-term treatment with YM440 would produce significant effects on hyperlipidemia in vivo. It has been reported that hepatic insulin resistance influences several factors, and may play a pivotal role in the development of diabetic dyslipidemia (Adeli et al., 2001). Therefore it was expected that YM440 would affect hyperlipidemia, which might in turn influence diabetic nephropathy. In this study, the effects of long-term treatment with YM440 on hyperlipidemia and renal function of ZF rats were investigated based on the theory that the outcome might be related to damage suffered by the organ during the clinical course of type 2 diabetes. 2. Materials and methods 2.1. Materials All reagents used in this study were of analytical grade and obtained commercially. (Z)-1,4-bis{4-[(3,5-dioxo-1,2,4-oxadiazolidin-2-yl)methyl] phenoxy}but-2-ene (YM440) was synthesized at Astellas Pharma Inc. (Tokyo, Japan). 2.2. Animals and drug treatment Male Zucker fatty rats (fa/fa) and age-matched Zucker lean rats (Fa/?) were purchased from Charles River Japan (Tokyo, Japan) and housed on a 12-h light cycle. At ages of 8 weeks, the Zucker fatty rats were separated into two weight-matched groups, YM440-treatment and control groups, comprised of 9–10 rats;

8 lean control rats were also included. YM440-treated rats were given 100 mg/kg/day of YM440 suspended in 0.5% methylcellulose at the rate of 5 mL/kg by oral gavage once a day for 16 weeks. The control rats were given 0.5% methylcellulose (vehicle) alone. The animal study was performed in accordance with the legal requirements of the Animal Use Committee of Astellas Pharma Inc. 2.3. Routine procedures Blood samples were collected every 4 weeks from the tail veins of rats and assayed for glucose, insulin, and triglyceride. Blood glucose, plasma triglyceride and cholesterol levels were measured using the glucose-CII test, triglyceride-E test, and cholesterol-C test, respectively (Wako Pure Chemical Industries, Ltd., Tokyo, Japan). Plasma insulin concentrations were measured using a radioimmunoassay kit (Amersham Pharmacia Biotech, Piscataway, NJ, USA). Systolic blood pressure was measured every 4 weeks using the indirect tail-cuff method (PS200, Riken Kaihatsu, Tokyo, Japan). The rats were placed in individual metabolic cages every 2 weeks to collect 24-hour urine samples. The urine samples were used to measure the urinary volume, total protein (Micro-TP test, Wako Pure Chemical Industries, Ltd.) and N-acetyl-β-D-glucosaminidase (NAG) activity (NAG test, Shionogi, Tokyo, Japan). Urinary Na+ and K+ were measured using a flame photometer (model 710, Hitachi, Tokyo, Japan). The urinary albumin level was measured using an in-house sandwich enzyme-linked immunosorbent assay (ELISA) system for rat albumin. Briefly, each well of a 96-well microtiter plate was coated with diluted (1:200) rabbit anti-rat albumin antibody (No. 55711, Cappel, Aurora, OH, USA) and incubated overnight at 4 °C. The plates were washed with phosphate buffered saline (PBS) buffer and then blocked with 0.1% PBS buffer containing gelatin. The diluted rat albumin standards (No. 55952, Cappel) and urine samples were dispensed into coated plates, and the plates were incubated for 1 h at room temperature. The wells were washed, and then diluted (1:200) horseradish peroxidase-labeled anti-rat albumin antibody (No. 55776, Cappel) was added. After incubating for 1.5 h at room temperature, the wells were washed, and substrate for horseradish peroxidase (tetramethybenzine peroxidase substrate system, Bio-Rad, Hercules, CA, USA) was added. Color development was done for 10 min at room temperature and stopped with 2 M H2SO4. Absorbance at 450 nm was then measured. The detection limit for this assay was 2 ng/mL. 2.4. Kidney tissue and renal function analysis At the end of the experiments, the rats were sacrificed after their body weights were measured. Kidney tissue and blood samples were collected. Blood urea nitrogen was measured with a urea-nitrogen test (Wako Pure Chemical Industries, Ltd.). The combined weight of the 2 kidneys (total kidney weight) was determined gravimetrically, with the degree of renal hypertrophy being expressed as the ratio of total kidney weight to body weight.

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Table 1 Characteristics of rats treated with YM440 (100 mg/kg/day) for 16 weeks Parameter

Zucker lean rats (ZL)

Zucker fatty rats (ZF)

ZF + YM440

(n = 8)

(n = 9)

(n = 10)

Body weight (g)

489 ± 13

730 ± 26a

772 ± 15

Blood parameters Blood urea nitrogen (mg/dL)

18.7 ± 0.6

22.9 ± 1.7b

28.1 ± 5.2

Renal function parameters Creatinine clearance (L/day) Urinary K+ excretion (Eq/day) Urinary Na+ excretion (Eq/day)

6.1 ± 0.3 3.1 ± 0.2 0.74 ± 0.07

5.5 ± 0.6 3.6 ± 0.2 1.1 ± 0.18

5.5 ± 0.5 4.0 ± 0.3 1.5 ± 0.21

NOTE. Each value represents the mean ± S.E.M. P b 0.001, bP b 0.05 vs. ZL.

a

Kidneys were preserved in 10% neutral buffered formalin, embedded in paraffin, sectioned, and stained with periodic acidSchiff for examination using light microscopy. Evaluation of the glomerular area was performed according to the methods reported by Bauer and Rosenberg (1960). Briefly, long-axis (a) and short-axis (b) measurements of each Bowman's capsule were made with an eyepiece micrometer at × 400 magnification. The glomerular area (S) was calculated using the following equation: S = 1 / 4 × πab. The measurements from 25 glomeruli were used to calculate the mean glomerular area for each tissue specimen. These estimates of mean glomerular area were used to compare the relative differences in glomerular size between experimental groups. The number of tubules in the inner stripe of the outer medulla that contained urinary cast were determined at × 100 magnification. Renal function was evaluated by measuring the creatinine clearance with plasma- and urinary-creatinine levels determined

Fig. 1. Effects of YM440 on blood parameters in ZF rats. Blood samples were taken from the tail vein monthly. Blood glucose (A) and plasma insulin (B) were measured as described in Materials and methods. Each point represents the mean ± S.E.M. (N = 8–10). *P b 0.05, **P b 0.01, ***P b 0.001 vs. ZL,†P b 0.05 vs. ZF.

Fig. 2. Effects of YM440 on plasma triglyceride (TG) and cholesterol (TC) levels in ZF rats. Blood samples were taken from the tail vein monthly. Plasma TG (A) and TC (B) were measured as described in Materials and methods. Each point represents the mean ± S.E.M. (N = 8–10). **P b 0.01, ***P b 0.001 vs. ZL, †P b 0.05, †††P b 0.001 vs. ZF.

using a creatinine test kit (Wako Pure Chemical Industries, Ltd.). 2.5. Statistical analysis Comparisons between the experimental groups were made using the one-way analysis of variance (ANOVA) followed by Fisher's least significant difference test. Differences were considered significant at P levels less than 0.05. The correlative relationships between plasma triglyceride levels and renal parameters (urinary protein excretion and

Fig. 3. Effects of YM440 on urinary protein and albumin excretion in ZF rats. Urine samples were taken every 2 weeks using individual metabolic cages. Urinary protein excretion (A) and albumin excretion (B) were measured as described in Materials and methods. Each point represents the mean ± S.E.M. (N = 8–10). *P b 0.05, **P b 0.01 vs. ZL, †P b 0.05, ††P b 0.01 vs. ZF.

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Fig. 4. Effects of YM440 on urinary N-acetyl-β-D-glucosaminidase (NAG) activity in ZF rats. Urine samples were taken every 2 weeks using individual metabolic cages. Urinary NAG activity was measured as described in Materials and methods. Each point represents the mean ± S.E.M. (N = 8–10). *P b 0.05, **P b 0.01 vs. ZL, †P b 0.05, ††P b 0.01, †††P b 0.001vs. ZF.

albumin excretion) were examined using linear regression analysis. To test the regression for significance, the slope of the regression line obtained by plotting these parameters was assessed using the null hypothesis “slope = 0” and the t-test. 3. Results 3.1. Effect of YM440 on body weight, blood glucose, blood urea nitrogen, plasma insulin, triglyceride and cholesterol The body weights of ZF rats were higher than those of ZL rats. YM440 did not influence the body weight of ZF rats (Table 1). Blood glucose at 16 weeks was slightly, though significantly, higher in the fatty controls (158.9 ± 21.7 mg/dL) than in the lean controls (99.0 ± 7.5 mg/dL). In the YM440-treated groups, the values (133.2 ± 14.5 mg/dL) were between those for ZF and ZL rats, but were not significantly lower than those of the ZF group (Fig. 1A). The ZF rats were hyperinsulinemic compared to the ZL rats (Fig. 1B). In control ZF rats, the insulin level increased until week 4 of the study, then it began to decrease gradually. Although YM440 treatment reduced the insulin levels until week 8, YM440-treated rats had higher plasma insulin levels than untreated rats after 12 weeks (Fig. 1B). At the end of study, the plasma insulin concentrations for the ZL, ZF, and YM440-treated ZF groups were 6.8± 1.3 ng/mL, 92.4 ± 18.8 ng/mL, and 132.0± 17.8 ng/mL, respectively. ZF rats also exhibited hyperlipidemia (Fig. 2A and B) during the study period. Treatment with YM440 significantly reduced

Fig. 5. Effects of YM440 on systolic blood pressure in ZF rats. Systolic blood pressure was measured monthly using the indirect tail-cuff method as described in Materials and methods. Each point represents the mean ± S.E.M. (N = 8–10). †P b 0.05, ††P b 0.01 vs. ZF.

Fig. 6. Effects of YM440 on renal hypertrophy in ZF rats. Kidney tissues were collected from rats after 16 weeks of treatment with YM440. The combined weight of 2 kidneys is shown as the total kidney weight (A). The ratio of the total kidney weight to body weight is shown as the kidney weight percentage (B). Each point represents the mean ± S.E.M. (N = 8–10). ⁎⁎⁎P b 0.001 vs. ZL, †P b 0.05 vs. ZF.

the plasma triglyceride concentrations to levels similar to those of the lean controls (Fig. 2A). Treatment with YM440 also reduced plasma cholesterol level (Fig. 2B). The level of blood urea nitrogen in ZF rats was significantly higher than that in ZL rats (Table 1). YM440 did not change the blood urea nitrogen level. 3.2. Effect of YM440 on urinary protein and albumin excretion There was no obvious evidence of proteinuria in ZF rats at the beginning of the experiment (ZL group, 17.1 mg/day and ZF group, 19.7 mg/day). Proteinuria did not become a persistent problem until the rats reached the age of approximately 12–14 weeks, at which time there was a substantial escalation in urinary protein excretion (Fig. 3A). YM440 markedly retarded the rate of proteinuria progression once it was established (week 4) (Fig. 3A). At the end of the study, the urinary protein excretion values for the ZL, ZF, and YM440-treated ZF groups were 81.2 ± 40.7 mg/day, 351.1 ± 65.6 mg/day, and 143.0 ± 15.8 mg/day, respectively. The urinary albumin data essentially mimicked the proteinuria results (Fig. 3B). However the onset was sooner than that of the proteinuria. In the beginning of the study, ZF rats exhibited albuminuria more distinctly than ZL rats (ZL, ZF, and YM440treated ZF groups: 0.3 ± 0.0 mg/day, 5.7 ± 1.1 mg/day, 5.4 ± 1.0 mg/day, respectively). YM440 delayed the rate of progression of albuminuria (Fig. 3B). At the end of study, the urinary albumin excretion values for the ZL, ZF, and YM440-treated ZF groups were, 57.3 ± 36.1 mg/day, 272.0 ± 55.9 mg/day, and 102.4 ± 11.6 mg/day, respectively. 3.3. Effect of YM440 on urinary N-acetyl-β-D-glucosaminidase activity Urinary NAG activity, a marker for renal proximal tubular damage, was significantly elevated in ZF rats, compared with

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increased by a factor of 4.6 compared with ZL rats. Treatment with YM440 reduced protein cast accumulation by half (Fig. 7B). 3.7. Relationships between the plasma triglyceride and renal parameters We analyzed the correlation between the plasma triglyceride levels and the renal parameters (urinary protein excretion and urinary albumin excretion) using linear regression analysis. The plasma triglyceride level in each rat correlated significantly with these renal parameters (Fig. 8A and B). However, no correlation was observed between the blood glucose level and these renal parameters in the experimental groups (data not shown). 4. Discussion Fig. 7. Effects of YM440 on glomerular area hypertrophy and protein cast accumulation in ZF rats. Kidney tissues were collected from rats after 16 weeks of treatment with YM440. Glomerular area (A) and the number of tubules in the inner stripe of the outer medulla that contained urinary cast (B) were measured as described in the Materials and methods section. Each point represents the mean ± S.E.M. (N = 8–10). ⁎⁎⁎P b 0.001 vs. ZL, †P b 0.05, ††P b 0.01 vs. ZF.

ZL rats, from 6 weeks on (Fig. 4). In the YM440-treated ZF group, NAG activity significantly fell to a level similar to that of the ZL rats from the second week on (Fig. 4).

This study demonstrates that the novel hypoglycemic agent YM440 protects against renal injury and ameliorates hyperlipidemia in ZF rats. The earliest markers of impending nephropathy in ZF rats were increases in the urinary albumin levels (Fig. 3B) and NAG (an enzyme found in the lysosomes of renal proximal tubular cells) activity (Fig. 4). In the YM440-treated group in this study,

3.4. Effect of YM440 on systolic blood pressure Initially, the systolic blood pressure of ZF rats was below that of the lean controls. However, it began to increase slowly, and after 12 weeks it was slightly, but not significantly, elevated (Fig. 5). YM440 slowed the rate at which the systolic blood pressure increased starting at 4 weeks. At 16 weeks, the systolic blood pressure values for the ZL, ZF, and YM440-treated ZF groups were 125.1 ± 2.5 mm Hg, 130.0 ± 2.2 mm Hg, and 115.9 ± 2.7 mm Hg, respectively. 3.5. Effect of YM440 on kidney weights and renal function parameters The kidney mass in ZF rats was greater than that in ZL rats (Fig. 6A), and the kidney weight to body weight percentage was slightly higher than that of the ZL rats (Fig. 6B). YM440 blocked the development of nephromegaly from the aspects of both kidney weight and percent of body weight (Fig. 6A and B). The creatinine clearance did not differ between groups (Table 1). The urinary sodium and potassium excretion rates were not altered in ZF rats compared with ZL rats (Table 1). YM440 did not change the excretion rates of urinary sodium or potassium. 3.6. Pathological analysis Histopathological changes occurred in the kidneys. The mean glomerular area was significantly greater (30%) in ZF rats compared with ZL rats. YM440 significantly reduced 15% of the glomerular area in ZF rats (Fig. 7A). Protein cast accumulation in the inner stripe of the outer medulla in ZF rats was

Fig. 8. The relationship between the plasma triglyceride (TG) concentration and the urinary protein concentration (A) and urinary albumin excretion (B) in experimental Zucker lean and fatty rats. The correlation between TG and these renal parameters was analyzed using regression analysis. To test these regressions for significance, the slope of the regression line obtained by the method mentioned above was assessed using the null hypothesis “slope = 0” and the t-test.

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there was early normalization of urinary NAG activity. Even when proteinuria/albuminuria was established in the YM440treated group, it progressed more gradually than in the ZF control rats. Our observations on renal pathology in ZF rats confirm published observations with respect to nephromegaly, glomerular hypertrophy, and intratubular cast formation (Kasiske et al., 1985, 1989). In the YM440-treated group, kidney weight, glomerular area, and the amount of tubular cast decreased significantly (Figs. 6A, B; 7A, B). These urinary parameters and histological analysis results suggest that YM440 would prevent the progression of renal damage in ZF rats. Chronic treatment with YM440 also reduced the plasma triglyceride and cholesterol levels dramatically (Fig. 2A and B). The ZF rats exhibited mild hyperglycemia at the age of 24 weeks, thus YM440 did not reduce the blood glucose level significantly. However, the changes in the blood glucose levels in YM440treated rats were lower than those in the ZF rats during the examination period (Fig. 1A). In other words, YM440 controlled the blood glucose levels in ZF rats well during the study. In this study, the level of plasma insulin in ZF rats gradually increased and then declined (Fig. 1B). These results agree with those of Larsson et al. (1977) and Buckingham et al. (1998). YM440 treatment decreased the plasma insulin level until the eighth week of the study, and then increased it (compared to untreated group) from weeks 12 to 16 (Fig. 1B). Although the YM440-treated rats showed a higher insulin level than the ZF control rats, the blood glucose level of the YM440-treated rats was still lower than that of the ZF control rats during this period. Similar changes in the plasma insulin levels were reported in a study of ZF rats treated with rosiglitazone for 9 months (rosiglitazone ameliorates or prevents pancreatic morphological abnormalities in ZF rats) (Buckingham et al., 1998). In Zucker diabetic fatty (ZDF) rats, the PPARα/γ dual agonist JTT-501 was also shown to protect against the reduction of the plasma insulin level and insulin content in the pancreatic islets (Shibata et al., 2000). It was previously reported that chronic treatment with YM440 improves glucose intolerance in ZF rats and insulin resistance in other diabetic rodent models (Nakano et al., 1999; Shimaya et al., 2000; Kurosaki et al., 2002, 2003b). These reports, along with observations made in this study, lead to the speculation that the increase in the plasma insulin level seen in the latter part of this study resulted from a protective effect exerted on the pancreas by YM440. Although the data generated by this study has not led to the identification of the mechanism responsible for YM440's renal protective action, there are two possibilities. First, the correlation between the plasma triglyceride levels and renal parameters (urinary protein excretion and albumin excretion) were examined in our study. Regression analysis results showed that the renal parameters positively correlated with the plasma triglyceride level (Fig. 8A and B), but not with the blood glucose level (data not shown). Hyperlipidemia is known as a risk factor for the early development of renal disease in both humans and diabetic rats (Stevenson and Kaysen, 1999). Kasiske et al. (1988) reported that hyperlipidemia treated with the cholesterol synthesis inhibitor, mevinolin, reduced glomerular injury in ZF rats. The results of this study lead to the

conclusion that the hypolipidemic effects of YM440 result in protection against renal injury in ZF rats. The question remains, though, as to how YM440 improves hyperlipidemia in ZF rats. In a previous report, the glucose clamp technique was used to show that YM440 ameliorates hepatic, but not peripheral insulin resistance in ZF rats (Nakano et al., 1999). YM440 also ameliorates abnormalities in hepatic glycogen metabolism and hepatic gluconeogenesis (Kurosaki et al., 2002, 2003a,b). These previous and current studies strongly suggest that the main target organ of YM440 is the liver. Insulin resistance in the liver influences several factors and may play a pivotal role in the development of diabetic dyslipidemia which is defined by increases in triglyceride and cholesterol levels, along with a decrease in HDL cholesterol. For example, hepatic insulin resistance causes hypertriglycemia via increases in very low-density lipoprotein (VLDL) secretion from the liver (Adeli et al., 2001). Insulin resistance also increases hepatic lipase activity, which is responsible for the hydrolysis of phospholipids in LDL and HDL particles. This, in turn, leads to smaller and denser LDL particles and decreases in the numbers of HDL2 particles (Krauss, 2004). This suggests that the improvement of hepatic insulin resistance by YM440 ameliorates hyperlipidemia, which then prevents renal injury in ZF rats. Second, as reported previously, YM440 is a high-affinity ligand for PPARγ (Ki = 4.0 μM; Kurosaki et al., 2003a,b). Low, but significant, levels of PPARγ expression have also been reported in many tissues besides adipose, including the kidney and vasculature (Braissant et al., 1996). This suggests that PPARγ may play an important role in renal physiology and blood pressure regulation (Izzedine et al., 2005; Zhang and Guan, 2005; Guan, 2004). Several groups have reported that the treatment of diabetic rodent models with PPARγ agonists such as thiazolidinediones results in a marked reduction of proteinuria/ albuminuria and improves renal function (Buckingham et al., 1998; Fujiwara et al., 2000; Smith et al., 2000; Shibata et al., 2000). Isshiki et al. (2000) reported that the thiazolidinediones troglitazone and pioglitazone prevent the development of renal disease in diabetic rats by inhibiting the activation of the diacylglycerol-protein kinase C pathway, independent of their insulin-sensitizing activities. Because of these reports, we cannot exclude the possibility that YM440 acts as a renal protector via a direct effect on this receptor in the kidney. During the study, there was no hypertension in the ZF rats compared with the ZL rats (Fig. 5). Several groups reported noticeable hypertension in ZF rats that were 24 weeks of age or more (Buckingham et al., 1998; Yoshioka et al., 1993; Coimbra et al., 2000). Our observations agreed with these reports. However, YM440 significantly reduced the systolic blood pressure level compared to that of ZF control rats. These data indicate that YM440 has a significant effect on renal injury and hemodynamics in ZF rats. Two theories as to why YM440 would reduce systolic blood pressure level are suggested by this data. First, high concentrations of plasma triglyceride and cholesterol are also determinants of hypertension (Chen et al., 1991; Reaven, 1991), and the current findings show that YM440 dramatically reduces plasma triglyceride and cholesterol levels

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in ZF rats (Fig. 2A and B). Second, Diep et al. (2002) reported that the PPARγ activators rosiglitazone and pioglitazone suppress the elevation of systolic blood pressure in angiotensin II-infused rats. These data suggest that PPARγ agonist inhibits activation of the renin–angiotensin system. As noted above, YM440 is a high-affinity ligand of PPARγ. Therefore, it is possible that the anti-hypertensive action of YM440 may be at least partly due to the inhibition of the renin–angiotensin system activation via PPARγ signaling. The data herein lead to the conclusion that chronic treatment of ZF rats with YM440 improves hyperlipidemia and renal injury. The results of correlative analysis suggest that lipidemic control by YM440 prevents diabetic nephropathy in ZF rats. Thus, by improving metabolic control, YM440 might be used for preventing the development and progression of diabetic nephropathy in subjects with type 2 diabetes. Acknowledgements The authors thank Dr. Hisataka Shikama for his helpful advice and useful discussion during the course of these studies, and Dr. Atsuo Tahara for constructing a sandwich ELISA system for rat albumin. References Adeli, K., Taghibiglou, C., Van Iderstine, S.C., Lewis, G.F., 2001. Mechanisms of hepatic very low-density lipoprotein overproduction in insulin resistance. Trends Cardiovasc. Med. 11, 170–176. Bauer, W.C., Rosenberg, B.F., 1960. A quantitative study of glomerular enlargement in children with tetralogy of Fallot. A condition of glomerular enlargement without an increase in renal mass. Am. J. Pathol. 37, 695–712. Braissant, O., Foufelle, F., Scotto, C., Dauca, M., Wahli, W., 1996. Differential expression of peroxisome proliferator-activated receptors (PPARs): tissue distribution of PPAR-alpha,-beta, and-gamma in the adult rat. Endocrinology 137, 354–366. Buckingham, R.E., Al-Barazanji, K.A., Toseland, C.D., Slaughter, M., Connor, S.C., West, A., Bond, B., Turner, N.C., Clapham, J.C., 1998. Peroxisome proliferator-activated receptor-gamma agonist, rosiglitazone, protects against nephropathy and pancreatic islet abnormalities in Zucker fatty rats. Diabetes 47, 1326–1334. Chen, Y.D., Sheu, W.H., Swislocki, A.L., Reaven, G.M., 1991. High density lipoprotein turnover in patients with hypertension. Hypertension 17, 386–393. Coimbra, T.M., Janssen, U., Grone, H.J., Ostendorf, T., Kunter, U., Schmidt, H., Brabant, G., Floege, J., 2000. Early events leading to renal injury in obese Zucker (fatty) rats with type II diabetes. Kidney Int. 57, 167–182. Diep, Q.N., Mabrouk, M.E., Cohn, J.S., Endemann, D., Amiri, F., Virdis, A., Neves, M.F., Schiffrin, E.L., 2002. Structure, endothelial function, cell growth, and inflammation in blood vessels of angiotensin II-infused rats: role of peroxisome proliferator-activated receptor-gamma. Circulation 105, 2296–2302. Fujiwara, K., Hayashi, K., Ozawa, Y., Tokuyama, H., Nakamura, A., Saruta, T., 2000. Renal protective effect of troglitazone in Wistar fatty rats. Metabolism 49, 1361–1364. Gaede, P., Vedel, P., Parving, H.H., Pedersen, O., 1999. Intensified multifactorial intervention in patients with type 2 diabetes mellitus and microalbuminuria: the Steno type 2 randomised study. Lancet 353, 617–622. Guan, Y., 2004. Peroxisome proliferator-activated receptor family and its relationship to renal complications of the metabolic syndrome. J. Am. Soc. Nephrol. 15, 2801–2815. Hayden, M.R., Whaley-Connell, A., Sowers, J.R., 2005. Renal redox stress and remodeling in metabolic syndrome, type 2 diabetes mellitus, and diabetic nephropathy: paying homage to the podocyte. Am. J. Nephrol. 25, 553–569.

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