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Sep 20, 2006 - blockade of Ang II receptor ameliorates adipocytokine ... KEYWORDS: angiotensin II type1 receptor blocker; adiponectin; adipocyto-.
original article

http://www.kidney-international.org & 2006 International Society of Nephrology

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Blockade of Angiotensin II type-1 receptor reduces oxidative stress in adipose tissue and ameliorates adipocytokine dysregulation A Kurata1, H Nishizawa1, S Kihara1, N Maeda1, M Sonoda1, T Okada1, K Ohashi1, T Hibuse1, K Fujita1, A Yasui1, A Hiuge1, M Kumada1, H Kuriyama1, I Shimomura1 and T Funahashi1 1

Department of Metabolic Medicine, Graduate School of Medicine, Osaka University, Suita, Japan

Dysregulated production of adipocytokines may be involved in the development of atherosclerotic cardiovascular disease in metabolic syndrome and chronic kidney disease (CKD) associated with metabolic syndrome. The aim of this study was to determine the effects of treatment with angiotensin II (Ang II) type-1 receptor blocker (ARB) on the regulation of adipocytokines. Olmesartan, an ARB, significantly blunted the age- and body weight-associated falls in plasma adiponectin both in genetically and diet-induced obese mice, without affecting body weight, but had no effect on plasma adiponectin levels in lean mice. Olmesartan also ameliorated dysregulation of adipocytokines in obesity, such as tumor necrosis factor-a, plasminogen activator inhibitor-1, monocyte chemotactic protein-1, and serum amyloid A3. Olmesartan significantly reduced reactive oxygen species originating from accumulated fat and attenuated the expression of nicotinamide adenine dinucleotide phospho hydrogenase oxidase subunits in adipose tissue. In cultured adipocytes, olmesartan acted as an antioxidant and improved adipocytokine dysregulation. Our results indicate that blockade of Ang II receptor ameliorates adipocytokine dysregulation and that such action is mediated, at least in part, by targeting oxidative stress in obese adipose tissue. Ang II signaling and subsequent oxidative stress in adipose tissue may be potential targets for the prevention of atherosclerotic cardiovascular disease in metabolic syndrome and also in metabolic syndrome-based CKD. Kidney International (2006) 70, 1717–1724. doi:10.1038/sj.ki.5001810; published online 20 September 2006 KEYWORDS: angiotensin II type1 receptor blocker; adiponectin; adipocytokine; oxidative stress; NADPH oxidase

Correspondence: H Nishizawa, Department of Metabolic Medicine, Graduate School of Medicine, Osaka University, 2-2-B5 Yamadaoka, Suita 565-0871, Japan. E-mail: [email protected] Received 20 February 2006; revised 13 July 2006; accepted 18 July 2006; published online 20 September 2006 Kidney International (2006) 70, 1717–1724

The metabolic syndrome is a cluster of insulin resistance, hypertension, dyslipidemia, and visceral obesity.1 Recent studies demonstrated that the metabolic syndrome is also strongly associated with chronic kidney disease (CKD).2,3 In addition to the effects of elevated blood pressure and blood glucose, there is evidence to suggest that visceral obesity per se can cause vascular endothelial dysfunction resulting in renal damage (termed obesity-related glomerulopathy).4,5 Cardiovascular diseases are major causes of mortality and morbidity in the metabolic syndrome and also in CKD.6 Several biomarkers such as microalbuminuria and plasma homocysteine, and C-reactive protein levels, have been investigated as potential risk factors for cardiovascular disease in CKD.7–12 Adipose tissue was previously considered as an energy-storage organ, but recent research has demonstrated that this tissue produces and secretes a variety of biologically active molecules, conceptualized as adipocytokines.13–18 Increased production of plasminogen activator inhibitor-1 (PAI-1) from accumulated visceral fat leads to thrombotic tendency, and high levels of plasma tumor necrosis factor-a (TNF-a) result in inflammation.15,16,19 Adiponectin, which is an adipose-specific endocrine factor, exhibits anti-atherogenic, anti-diabetic, and anti-inflammatory properties,20–23 but its plasma level is low in visceral obesity.18 Hypoadiponectinemia is associated with coronary artery disease, type 2 diabetes, essential hypertension, and metabolic syndrome.24–28 Interestingly, it was demonstrated that plasma adiponectin level could be a negative predictor of cardiovascular outcomes among patients with end-stage renal disease.29,30 Considered together, dysregulation of adipocytokine may be also involved in the development of cardiovascular diseases in CKD associated with metabolic syndrome. Regimens designed to increase plasma adiponectin levels and to ameliorate adipocytokine dysregulation might prevent the development of cardiovascular diseases. The renin– angiotensin system (RAS), a hormonal cascade that includes angiotensinogen, renin, angiotensin-converting enzyme (ACE), angiotensin (Ang), and its receptors, is involved in the maintenance of systemic blood pressure. In pathological 1717

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A Kurata et al.: ARB augments plasma adiponectin in obesity

state, Ang II also functions as a local biologically active mediator in the progression of cardiovascular remodeling and nephropathy through Ang II type-1 (AT1) receptor.31,32 Excessive production of local Ang II in the kidney increases intraglomerular pressure resulting in microalbuminuria, and enhances synthesis of extracellular matrix.32 Therefore, AT1 receptor blockers (ARBs) are considered to have cardio- and reno-protective effects beyond their anti-hypertensive effects.33–35 Moreover, several reports demonstrated that ARBs reduced the risk of new-onset diabetes in humans.36 Recently, RAS blockers were reported to increase plasma adiponectin concentrations in patients with essential hypertension.37 However, the effect of ARB on adipose-function, that is, adipocytokine production, has not been defined in experimental study. In the present study, we investigated the effects of olmesartan, an ARB, on the dysregulation of adipocytokines, with a special focus on adiponectin.

(Table 1). Olmesartan significantly prevented the decline in plasma adiponectin in obese KKAy mice (Figure 1a, Table 1). In contrast, olmesartan treatment did not significantly change plasma adiponectin concentrations in lean C57BL/ 6J mice (Figure 1b, Table 1). To confirm the effect of olmesartan in preventing the reduction in plasma adiponectin concentrations during the development of obesity, we repeated the experiments in genetically obese db/db and diet-induced obesity (DIO) mice, and investigated the changes in plasma adiponectin concentrations. Olmesartan treatment blunted the reduction of plasma adiponectin both in db/db and diet-induced obesity mice (Figure 1c and d). Furthermore, adiponectin messenger RNA (mRNA) levels in adipose tissue were significantly higher in olmesartan-treated mice compared with control KKAy mice (Figure 1e). Insulin tolerance tests showed improved insulin sensitivity in olmesartan-treated KKAy mice (Figure 1f), compared with control KKAy mice.

RESULTS Effects of olmesartan on plasma adiponectin levels

Effects of olmesartan on adipocytokine expression

We treated lean C57BL/6J and obese KKAy mice with olmesartan and investigated the changes in plasma adiponectin levels. We analyzed KKAy mice as an obese mouse model because KKAy mice exhibit phenotypes of the metabolic syndrome including obesity, diabetes, dyslipidemia, fatty liver, hypertension and hypoadiponectinemia.38 Plasma adiponectin concentrations were similar in 8- and 20week-old C57BL/6J mice (34.371.4 and 38.171.2 mg/ml, respectively). In contrast, plasma adiponectin concentrations in obese KKAy mice decreased with age, in parallel with the increase in body weight (8-week-old: 25.271.3, 20-week-old: 16.570.9 mg/ml). Lean C57BL/6J and obese KKAy mice (21-week-old) were treated with olmesartan for 2 weeks. Olmesartan treatment resulted in a significant fall in blood pressure level but did not affect the body weights of both C57BL/6J and KKAy mice, compared with control mice

To test whether olmesartan directly affects the adipose tissue to improve the obesity-related low plasma adiponectin level, we investigated the expression of AT1a receptor mRNA in various tissues of mice. The expression level of AT1a receptor in adipose tissue was abundant, compared with heart, kidney, and skeletal muscle, which are considered as the major target organs of ARB,39,40 but there was no significant difference in AT1a receptor mRNA levels of adipose tissues between lean C57BL/6J and obese KKAy mice (Figure 2a). These results suggest that the adipose tissue is one of the major target organs of ARB in vivo. We also investigated the effect of ARB on the dysregulated expressions of other adipocytokines. The mRNA expression levels of TNF-a, PAI-1, monocyte chemotactic protein-1 (MCP-1), and serum amyloid A3 (SAA3), which is an acute phase reactant in adipose tissue like C-reactive protein,41,42 were overexpressed in the obese

Table 1 | Body weight, SBP, plasma glucose, insulin, and plasma adiponectin concentrations in C57BL/6 and KKAy mice before and after 2-week treatment with olmesartan Control (n=4)

C57BL/6 mice Body weight (g) SBP (mmHg) Glucose (mmol/l) Insulin (nmol/l) Adiponectin (mg/ml ) KKAy mice Body weight (g) SBP (mmHg) Glucose (mmol/l) Insulin (nmol/l) Adiponectin (mg/ml)

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22.070.7 100.372.9 10.370.5 0.0670.01 37.772.6

24.271.3z 105.571.3 10.870.2 0.0570.01 38.073.3

22.570.4 106.773.0 9.970.5 0.0770.01 38.871.0

24.570.3z 86.074.1y,* 10.571.2 0.0570.01 37.872.9

51.271.8 116.473.3 26.372.7 13.970.7 15.671.1

51.071.6 119.873.9 27.472.8 17.671.0z 12.670.4 z

50.971.1 110.972.4 26.672.2 13.071.1 15.171.7

51.770.8 80.072.9y,* 24.173.0 14.672.3 18.171.6*

SBP, systolic blood pressure. Values are mean7s.e.m. *Po0.01, compared with control mice. z Po0.05, yPo0.01, compared with the basal value (before olmesartan treatment).

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A Kurata et al.: ARB augments plasma adiponectin in obesity

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Figure 1 | Effects of olmesartan on plasma concentrations of adiponectin in mice. (a and b) Twenty-one-week-old KKAy and C57BL/6J mice were treated with olmesartan (Olme), or no drug (Control) for 2 weeks (n ¼ 4, each). Plasma adiponectin concentrations were measured once a week. (c) Sixteen-week-old db/db mice were treated with olmesartan (Olme), or no drug (Control) (n ¼ 9, each) for 2 weeks. (d) Eight-week-old C57BL/6J mice were fed high fat/high sucrose. At 21 weeks of age, they were fed the same diet mixed with either olmesartan (Olme) or no drug (Control) (n ¼ 5, each) for 3 weeks. Plasma adiponectin concentrations were presented as percentage change relative to their levels before olmesartan treatment. (e) Adiponectin mRNA expression in adipose tissues. The values were normalized to the level of cyclophilin mRNA. (f) Insulin sensitivity of olmesartan-treated KKAy mice. Glucose curves under the insulin tolerance test. Plasma glucose levels were normalized to those at 0 min (100%). In sections a–d and f, open circles: control group, solid circles: olmesartan group. Values are mean7s.e.m., *Po0.05 compared with control mice. Similar results were obtained in another independent experiment.

KKAy mice compared with lean C57BL/6J mice. Figure 2b clearly shows that treatment with olmesartan attenuated the enhanced mRNA expressions of TNF-a, PAI-1, SAA3, and MCP-1 in obese KKAy mice, but not in lean C57BL/6J mice. These findings indicate that olmesartan does not affect the expression of adipocytokines in the lean mouse, but improves the dysregulation of adipocytokines in the obese mouse. In the next step, we examined the molecular mechanism of the action of ARB on adipocytokines in obese mice. Kidney International (2006) 70, 1717–1724

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Figure 2 | Effects of olmesartan on mRNA expression of adipocytokines in mice. (a) Expression levels of AT1a receptor mRNA in various mouse tissues. Values are normalized to the level of 36B4 mRNA. (b) The mRNA expression levels of TNF-a, PAI-1, SAA3, and MCP-1 in adipose tissue of C57BL/6J and KKAy mice treated with olmesartan (Olme), or no drug (Cont) (n ¼ 4, each). The values were normalized to the level of cyclophilin mRNA. Data displayed in A and B are expressed as mean7s.e.m. *Po0.05, **Po0.01.

Effect of olmesartan on reactive oxygen species in adipose tissue

It has been reported that ARBs improve oxidative stress in blood vessels and skeletal muscles.40,43 Recently, Furukawa et al.38 reported that reactive oxygen species (ROS) are increased in obesity before the rise in plasma glucose concentrations. They demonstrated that actively produced ROS originated from accumulated fat, which subsequently caused dysregulation of various adipocytokines, such as adiponectin, PAI-1, and TNF-a. ARBs might ameliorate adipocytokine dysregulation through a reduction of oxidative stress in adipose tissue. To test this hypothesis, we measured the concentration of adipose thiobarbituric acid reactive substance (TBARS), a marker of lipid peroxidation (LPO), in C57BL/6J and KKAy mice. Adipose TBARS concentrations were higher in KKAy mice than in C57BL/6J mice, in agreement with previous data.38 Treatment with olmesartan markedly attenuated adipose TBARS concentrations in obese KKAy mice, but no such effect was observed in lean C57BL/6J mice (Figure 3a). Moreover, plasma levels of H2O2, a hazardous ROS against tissues and cells, were also attenuated 1719

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Figure 3 | (a) Effects of olmesartan on LPO levels in adipose tissues of C57BL/6J and KKAy mice treated for 2 weeks with olmesartan (Olme), or no drug (Cont) (n ¼ 4, each). The level of LPO in adipose tissue homogenates was measured as TBARS. (b) Effects of olmesartan on plasma H2O2 concentrations in mice. Data are mean7s.e.m. *Po0.05, **Po0.01. MDA, malondialdehyde.

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Figure 4 | Effects of olmesartan on mRNA expression levels of NADPH oxidase subunits, PU.1, Cu, Zn-superoxide dismutases and catalase in adipose tissues of C57BL/6J and KKAy mice treated for 2 weeks with olmesartan (Olme), or no drug (Cont) (n ¼ 4, each). Values are normalized to the level of cyclophilin mRNA and are expressed as mean7s.e.m. *Po0.05, **Po0.01.

by olmesartan treatment in obese KKAy mice but not in lean C57BL/6J mice (Figure 3b).

elimination system, were decreased in obese KKAy mice, but olmesartan did not affect the expression of these genes.

Effects of olmesartan on production and removal system of fat ROS

Effects of olmesartan on adipocytokines in adipocytes

Next, we investigated the expression of genes related to the production and removal of ROS in adipose tissue and the effects of olmesartan on their expression levels. Nicotinamide adenine dinucleotide phospho hydrogenase (NADPH) oxidase complex is a major source of ROS in various organs especially in accumulated fat.38,44 The NADPH oxidase complex consists of membrane-associated flavocytochrome b558 family of proteins, which includes gp91phox and p22phox as well as cytosolic components p47phox p67phox, and p40phox The expression levels of all subunits were markedly augmented in adipose tissue of obese KKAy mice (Figure 4), as described previously.38 In obese KKAy mice, we also found increased expression of transcriptional factor PU.1, which is known to upregulate the transcription of NADPH oxidase subunits.45 Olmesartan clearly reduced the expression of NADPH oxidase subunits and PU.1 in the adipose tissue of obese KKAy mice, but had no effect on the expression of these genes in lean C57BL/6 mice. The expression levels of CU, Zn-superoxide dismutases, and catalase, the ROS 1720

In the above experiments, we demonstrated that ARB reduced ROS production in obese adipose tissue. Recently, RAS inhibitors were reported to prevent oxidative stressinduced apoptosis of vascular endothelial cells.46 Therefore, to investigate whether ARB directly affects downstream ROSmediated adipocytokine dysregulation, we conducted in vitro experiments using primary adipocytes. We used differentiated adipocytes derived from stromal vascular cells (SVCs) of mouse adipose tissues. The secretion of adiponectin from adipocytes was diminished by incubation with H2O2 in a dose-dependent manner (Figure 5a). Antioxidant N-acetyl cysteine canceled the H2O2-induced inhibition of adiponectin secretion, as described previously.38 These results validated the experimental procedure and H2O2 treatment in this study. In subsequent experiments, olmesartan resulted in a significant amelioration in the inhibitory effect of H2O2 on adiponectin secretion, whereas it had no effect on basal adiponectin secretion (Figure 5b). ACE inhibitor, captpril, significantly but slightly ameliorated oxidative stress-induced impairment of adiponectin production (Figure 5c). The Kidney International (2006) 70, 1717–1724

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Figure 5 | Effects of olmesartan on production of adipocytokines in adipocytes. (a) The secretion of adiponectin from adipocytes exposed to ROS, with or without antioxidant N-acetyl cysteine. Differentiated adipocytes derived from SVCs (day 7) were exposed to the indicated concentrations of H2O2 and N-acetyl cysteine (n ¼ 3, each). (b) Effects of olmesartan on adiponectin secretion by adipocytes exposed to ROS. Differentiated adipocytes were treated with the indicated concentrations of H2O2 and olmesartan (Olme) (n ¼ 3, each). Adiponectin secretion in media was analyzed by adiponectin enzyme-linked immunosorbent assay. The values of vehicle-treated adipocytes were arbitrarily set as 1.0. Values are expressed mean7s.e.m. *Po0.05, **Po0.01. Similar results were obtained in three other independent experiments. (c) Effects of captopril on adiponectin secretion by adipocytes exposed to ROS. Differentiated adipocytes were treated with the indicated concentrations of H2O2 and captpril (Capt) (n ¼ 3, each). Similar results were obtained in two other independent experiments. (d) Effects of olmesartan on mRNA expressions of various adipocytokines. Values are expressed as mean7s.e.m. after normalization to the level of cyclophilin mRNA. *Po0.05, **Po0.01.

effect of olmesartan on adiponectin production was observed at mRNA expression level (Figure 5d). The mRNA expression levels of PAI-1 and MCP-1 were elevated in H2O2-treated adipocytes. Treatment with olmesartan suppressed the elevation of mRNA expressions of PAI-1 and MCP-1 in H2O2-treated adipocytes but not in untreated adipocytes. These results suggest that amelioration of adipocytokine dysregulation by ARB is mediated, at least in part, via its antioxidative effect, in addition to reduction of ROS production. DISCUSSION

In the present study, we demonstrated for the first time that blockade of AT1 receptor ameliorates adipocytokine Kidney International (2006) 70, 1717–1724

original article

dysregulation including hypoadiponectinemia using various obese mouse models. Furthermore, ARB ameliorated adipocytokine dysregulation in an obesity-specific manner. We also demonstrated that such action is mediated, at least in part, by targeting oxidative stress in obese adipose tissue. We found that the ARB, olmesartan, prevented the obesity-related decline in plasma adiponectin concentration. Olmesartan also suppressed the expression of other adipocytokines such as TNF-a, PAI-1, SAA3, and MCP-1, which were upregulated in obesity. These results suggest that olmesartan can improve adipocytokine dysregulation in an obesity-specific manner. Recently, it was reported that activation of RAS by Ang II infusion elicited hypoadiponectinemia.47 What is (are) the mechanism(s) by which ARB improve adipocytokine dysregulation in adipose tissue? Several studies showed that Ang II promotes and ARBs prevent ROS generation by modulation of NADPH oxidase in atherosclerotic blood vessels and diabetic kidney.43,48–50 In the present study, we demonstrated that ARB ameliorated TBARS in obese adipose tissues, a marker of LPO, and attenuated the expression levels of all subunits of NADPH oxidase and their upstream transcriptional factor, PU.1, which was upregulated in the obese adipose tissue (Figure 3b and ref.38). Inhibition of ROS generation via NADPH oxidase in adipose tissue may be one of the mechanisms to ameliorate of adipocytokine dysregulation by ARB. We also investigated whether ARB directly affects downstream ROS-mediated adipocytokine dysregulation using cultured adipocytes. The results showed that ARB ameliorated H2O2-mediated adipocytokine dysregulation (Figure 5). It was reported that oxidative stress modulates RAS in vascular endothelial cells.51 Recently, Akishita et al.46 demonstrated that RAS inhibitors, including olmesartan, inhibited H2O2-induced apoptosis of endothelial cells. Captopril, an ACE inhibitor, slightly but significantly ameliorated the inhibitory effect of H2O2 on adiponectin secretion (Figure 5c), suggesting that RAS inhibition at the level of ACE may ameliorate ROS-mediated impairment of adiponectin secretion. However, captopril had much less effective than ARB, also suggesting that direct antioxidant property of this ARB besides AT1 receptor blockade and/or activation of AT2 under the presence of an ARB showed inhibition against the ROS cascade. In our in vitro experiments, sarile ([Sar1, Ile8] angiotensin II), a peptide Ang II receptor antagonist did not show significant effects (data not shown). This data might support the above interpretation. Therefore, the entire molecular mechanism(s) of oxidative stress-related activation of RAS remains unclear. Considered together, amelioration of adipocytokine dysregulation in obesity by ARB is mediated, at least in part, by its anti-oxidative effect, in addition to reduction of ROS production. Peroxisome proliferator-activated receptor (PPAR)-g-agonists, thiazolidinediones, were reported to improve adipocytokine dysregulation including adiponectin.52,53 In vitro studies demonstrated that some ARBs, such as telmisartan and irbesartan, had PPAR-g agonistic effect and enhanced the 1721

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production of adiponectin in 3T3-L1 adipocytes.54–56 Olmesartan might ameliorate adipocytokine dysregulation through PPAR-g activation. However, this is unlikely because it was reported that olmesartan did not have PPAR-g agonistic effect in vitro.54 In our present in vivo and in vitro studies, we could not observe significant changes in mRNA expression levels of PPAR-g target genes, such as aP2 and CD36, following olmesartan treatment (data not shown). It is also possible that amelioration of adipocytokine dysregulation involves other pathways downstream of ROS such as mitogen-activated protein kinase, and janus kinase-signal transducer, and activator of transcription pathways.57,58 The effect of AT1 blocker may not be entirely owing to the blockade of AT1. When AT1 is blocked, increase Ang II may act on AT2 receptor. AT2 stimulation by ARB is reported to result in renal production of nitric oxide.59 It should be also considered that nitric oxide production via AT2 stimulation by ARB may improve adypocytokine dysregulation. Further studies are needed to elucidate the entire mechanism(s) by which ARB improves adipocytokine dysregulation in adipose tissue. The present study clearly demonstrated that blockade of AT1 receptor ameliorated adipocytokine dysregulation in adipose tissue of obesity. It should be clinically relevant that ARB, which is widely used for the treatment of hypertension, ameliorate adipocytokine dysregulation in obesity, and metabolic syndrome. These results suggest that Ang II signaling and subsequent oxidative stress in adipose tissue may be potential targets for the prevention of atherosclerotic cardiovascular disease in metabolic syndrome and also in metabolic syndrome-based CKD. MATERIALS AND METHODS Animals and experimental protocol All animals were obtained from Clea Japan (Tokyo, Japan). In C57BL/6J and KKAy mice studies, 21-week-old female mice were treated with or without olmesartan for 2 weeks (n ¼ 4, each). Mice of the olmesartan group were fed regular chow containing 0.0015% olmesartan medoxomil (kindly provided by Sankyo Co., Tokyo, Japan). We selected the dose of the drug in this study so that it produces a sufficient fall in blood pressure without affecting food intake or body weight. In db/db mice studies, 16-week-old female db/db mice were treated with or without olmesartan (0.003% olmesartan medoxomil) for 2 weeks (n ¼ 9 per group). In dietinduced obesity mice studies, 8-week-old female C57BL/6J mice were fed high-fat/high-sucrose (HF/HS) diet (Oriental Yeast). At the age of 21 weeks, the mice were treated with or without olmesartan (0.003% olmesartan medoxomil) for 3 weeks (n ¼ 5 per group). Subcutaneous adipose tissues (inguinal region) were analyzed in these studies. All experimental protocols described in this report were approved by the Ethics Review Committee for Animal Experimentation of Osaka University. Blood analysis All blood samples were collected after 6-h fast. Plasma concentrations of adiponectin, glucose, insulin, and H2O2 were measured by the adiponectin enzyme-linked immunosorbent assay kit (Otsuka, Tokushima, Japan), the glucose CII-test (Wako Pure Chemical, 1722

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Osaka, Japan), the insulin measurement enzyme-linked immunosorbent assay kit (Morinaga, Yokohama, Japan), and the Amplex Red hydrogen peroxide assay kit (Molecular Probe Inc., Leiden, The Netherlands), respectively. Isolation, culture, and differentiation of SVCs Subcutaneous adipose tissues (inguinal region) were removed from 8-week-old C57BL/6J mice, and SVCs were obtained by using a collagenase digestion procedure.60 Adipose tissues were incubated with digestion medium (Dulbecco’s modified Eagle’s medium containing 1 mg/ml type II collagenase (Sigma-Aldrich Co., St Louis, MO, USA, No. C6885)) for 60 min in a shaking water bath at 371C. After filtration and centrifugation, the pellet SVCs were seeded in 12-well plates, and cultured until confluence in the culture medium (Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum and 200 mM ascorbic acid). Two days after confluence (day 0), the medium was switched to differentiation medium (culture medium plus 5 mM troglitazone (kindly provided by Sankyo Co.), 0.25 nM dexamethasone, 0.5 mM 3-isobutyl-1-metyl-xanthin, and 10 mg/ml insulin). Two days later, the differentiation medium was switched to maintenance medium (culture medium plus 10 mg/ml insulin). At day 7, the SVCs were differentiated into adipocytes. Effect of olmesartan on ROS and adipocytokine production in adipocytes Differentiated adipocytes derived from SVCs (day 7) were incubated with serum-free culture medium containing the indicated reagents or vehicle for 12 h. The medium was replaced with freshly prepared medium containing the same reagents, and further incubated for 12 h. Then, the medium was collected to measure adiponectin secretion using the aforementioned adiponectin enzyme-linked immunosorbent assay kit, and the cells were harvested for RNA analysis. Because olmesartan medoxomil is a prodrug, we performed in vitro experiments using RNH-6270, which is the active form of olmesartan medoxomil (kindly provided by Sankyo Co.). Captopril, an ACE inhibitor, was kindly provided by Sankyo Co. Blood pressure measurement Systolic blood pressure was measured with an automatic sphygmomanometer (BP98A; Softron, Tokyo, Japan) at the tail artery whereas the animals were restrained. LPO measurement The level of TBARS, representing the extent of LPO, was measured in tissue homogenates using the LPO test (Wako Pure Chemical), as described previously.43 Insulin tolerance test After 6-h fast, insulin (3 mU/g body weight) was injected intraperitoneally into KKAy mice.22 Blood samples were obtained at the indicated time intervals. Insulin sensitivity was estimated by percentage changes in plasma glucose concentrations. RNA extraction and quantitative reverse transcriptasepolymerase chain reaction Isolated total RNA was subjected to cDNA synthesis using ThermoScript reverse transcriptase-polymerase chain reaction System (Invitrogen, San Diego, CA, USA). Real-time polymerase chain reaction was performed on an ABI PRISM 7700 using the Kidney International (2006) 70, 1717–1724

original article

A Kurata et al.: ARB augments plasma adiponectin in obesity

SYBR GREEN polymerase chain reaction Master Mix (Applied Biosystems, Warrington, UK). The sequences of primers were designed for cyclophilin, adiponectin, TNF-a, PAI-1, MCP-1, gp91phox, p22phox, p67phox, p47phox, p40phox, Cu, Zn-superoxide dismutases, catalase,43 AT1a receptor,61 and SAA3,40 as reported previously. The polymerase chain reaction primers for PU.1 and 36B4 were as follows; PU.1 50 -CCGCACACCATGTCCACAAC-30 (forward), 50 -GGACGTGCATCTGTTCCAGC-30 (reverse), and 36B4 50 -AAGCGCGTCCTGGCATTGTCT-30 (forward) 50 -CCGCAGGGG CAGCAGTGGT-30 (reverse). Statistical analysis Data were expressed as mean7s.e.m. of n separate experiments. Differences between groups were examined for statistical significance using Student’s t-test or analysis of variance followed by Fisher’s protected least significant test for comparisons between two groups or among multiple groups, respectively. A P-value less than 0.05 denoted the presence a statistically significant difference. ACKNOWLEDGMENTS

We thank Toshiyuki Takagi, Takuya Fujita, Hideki Kobayashi, Hironori Kobayashi, Shigetada Furukawa, and all members of the Funahashi Adiposcience Laboratory for the helpful discussion and comments. This work was supported in part by Grants-in-Aid for Scientific Research (B) no. 17390271 (to TF), and (C) no. 17590960 (to SK), Health and Labour Science Research Grants (to IS and TF), Grant-inAid for Scientific Research on Priority Area no. 15081208 (to SK) and no. 15081209 (to IS), the Research Fellowships of the Japan Society for the promotion of Science for Young Scientists no. 7829 (to NM) and no. 9340 (to HN), the Research Grant for Longevity Sciences (15–8) from the Ministry of Health, Labour and Welfare, Mitsubishi Pharma Research Foundation (to NM), Yamanouchi Foundation for Research on Metabolic Disorders (to NM and HN), Japan Heart Foundation Grant for Research on Arteriosclerosis Update (to NM), Japan Heart Foundation/Novartis Grant for Research Award on Molecular and Cellular Cardiology (to NM), Takada Science Foundation (to HN and TF), Suzuken Memorial Foundation (to HN), Senri Life Science Foundation (to HN), The Mochida Memorial Foundation for Medical and Pharmaceutical Research (to HN), and Smoking Research Foundation (to TF). REFERENCES 1. Isomaa B, Almgren P, Tuomi T et al. Cardiovascular morbidity and mortality associated with the metabolic syndrome. Diabetes Care 2001; 24: 683–689. 2. Chen J, Muntner P, Hamm LL et al. The metabolic syndrome and chronic kidney disease in US adults. Ann Intern Med 2004; 140: 167–174. 3. Palaniappan L, Carnethon M, Fortmann SP. Association between microalbuminuria and the metabolic syndrome: NHANES III. Am J Hypertens 2003; 16: 952–958. 4. Kambham N, Markowitz GS, Valeri AM et al. Obesity-related glomerulopathy: an emerging epidemic. Kidney Int 2001; 59: 1498–1509. 5. Wiecek A, Kokot F, Chudek J, Adamczak M. The adipose tissue – a novel endocrine organ of interest to the nephrologist. Nephrol Dial Transplant 2002; 17: 191–195. 6. Berl T, Hunsicker LG, Lewis JB et al. Impact of achieved blood pressure on cardiovascular outcomes in the irbesartan diabetic nephropathy trial. J Am Soc Nephrol 2005; 16: 2170–2179. 7. Pontremoli R, Leoncini G, Ravera M et al. Microalbuminuria, cardiovascular, and renal risk in primary hypertension. J Am Soc Nephrol 2002; 13: S169–S172. 8. Anavekar NS, Gans DJ, Berl T et al. Predictors of cardiovascular events in patients with type 2 diabetic nephropathy and hypertension: a case for albuminuria. Kidney Int Suppl 2004; 92: S50–S55. Kidney International (2006) 70, 1717–1724

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Kidney International (2006) 70, 1717–1724