Research Article iMedPub Journals Journal of Clinical & Experimental Nephrology 2016 http://www.imedpub.com/ Vol.1 No.4:24 ISSN 2472-5056 DOI: 10.21767/2472-5056.100024 Topiroxostat Showed Renoprotection via Various Mechanisms Including Suppression of Xanthine Oxidoreductase-Induced-Oxidative Stress and Inflammation in db/db Mice Takashi Nakamura1*, Goshi Nagao1, Mai Nampei1, Takayo Murase2, Nobutaka Morimoto1, Naoki Ashizawa3, Takashi Iwanaga3 and Ryusuke Sakamoto4 1Pharmacological Study Group, Pharmaceutical Research Laboratories, Sanwa Kagaku Kenkyusho, Mie, Japan 2Radioisotope and Chemical Analysis Center, Sanwa Kagaku Kenkyusho, Mie, Japan 3Biological Research Group, Research Department, Medical R&D Division, Fuji Yakuhin, Saitama, Japan 4Project Management Department, Sanwa Kagaku Kenkyusho, Aichi, Japan *Corresponding author: Takashi Nakamura, Pharmacological Study Group, Pharmaceutical Research Laboratories, Sanwa Kagaku Kenkyusho, Mie, 5110406, Japan, Tel: +81594726221; E-mail: [email protected] Received date: December 06, 2016; Accepted date: December 20, 2016; Published date: December 28, 2016 Citation: Nakamura T, Nagao G, Nampei M, Murase T, Morimoto N, et al. (2016) Topiroxostat Showed Renoprotection via Various Mechanisms Including Suppression of Xanthine Oxidoreductase-Induced-Oxidative Stress and Inflammation in db/db Mice. J Clin Exp Nephrol 1: 24. DOI: 10.21767/2472-5056.100024 Copyright: © 2016 Nakamura T, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. relative to the vehicle. Moreover, febuxostat upregulated the gene expression of renin 1 (Ren1) and downregulated Abstract that of adenine phosphoribosyltransferase (APRT) and hypoxanthine guanine phosphoribosyltransferase (HPRT) Background: Xanthine oxidoreductase (XOR) produces compared with that of the diabetic control. superoxide along with uric acid. This reactive oxygen species induces organ damage. Therefore, we aimed to investigate Conclusion: Topiroxostat and febuxostat exhibited whether XOR-induced superoxide is associated with renal renoprotective effects by suppressing oxidative stress and abnormality in diabetic mice. inflammation, and enhancing the salvage pathway via XOR inhibition. Methods: Db/db mice (9-week-old) were fed standard diets with or without topiroxostat (1, 3, and 10 mg/kg/day) or febuxostat (3, 10, 30, and 45 mg/kg/day) for 4 weeks. Keywords: Topiroxostat; Xanthine oxidoreductase; Furthermore, topiroxostat (1 and 3 mg/kg/day) and Diabetic nephropathy; Albuminuria; Salvage pathway; Oxidative febuxostat (3 mg/kg/day) were administered for 7 days or 8 stress weeks. The levels of urinary albumin excretion (UAE), monocyte chemoattractant protein-1 (MCP-1), and angiotensin II were determined. For renal pathological or Introduction mechanistic analysis, periodic acid-Schiff (PAS), nitrotyrosine, and MCP-1 staining or renal gene expression, Xanthine oxidoreductase (XOR) is the rate-limiting enzyme XOR activity, and purine body levels were evaluated. that catalyzes the hydroxylation step in the conversion of hypoxanthine to xanthine and xanthine to uric acid (UA) in Results: Both topiroxostat and febuxostat suppressed UAE : purine metabolism [1,2]. Furthermore, this process can topiroxostat 3 mg/kg/day, 11.5 ± 33 μg/day versus vehicle simultaneously produce UA and reactive oxygen species (ROS) in 253.7 ± 69 μg/day, p<0.05; febuxostat 3 mg/kg/day, 255 ± 92 μg/day versus vehicle 634 ± 101 μg/day, p=0.05, for 4 vivo [1,2]. ROS-induced oxidative stress is considered a common weeks, respectively, and reduced immunohistological pathologic factor, leading to microvascular complications such as staining of enhanced oxidative stress and inflammation diabetic nephropathy, atherosclerosis, and hypertension [3-6], markers without affecting glycated hemoglobin. which cause albuminuria via vascular endothelial cell Topiroxostat but not febuxostat decreased urinary MCP-1 dysfunction [7]. Increased urinary albumin indicates a risk of and angiotensin II levels and attenuated morphological nephropathy progression in patients with diabetes [8] or hypertrophy in the glomerulus and epithelial cell cardiovascular events in the prognosis of those with acute enlargement (diameter and height) in the proximal tubules. In the early stage, topiroxostat and febuxostat decreased myocardial infarction [9]. renal XOR activity in response to increased purine body XOR inhibitors such as allopurinol, febuxostat, and levels (topiroxostat 3 mg/kg/day, 2.25 ± 0.19 mg/g tissue, p=0.07; febuxostat 3 mg/kg/day, 2.34 ± 0.09 mg/g tissue, topiroxostat are well-known medicines for lowering urate levels. p<0.05 to vehicle 2.01 ± 0.12 mg/g tissue, for 7 days) Several studies have reported that allopurinol reduced © Under License of Creative Commons Attribution 3.0 License | This article is available from: http://dx.doi.org/10.21767/2472-5056.100024 1 Journal of Clinical & Experimental Nephrology 2016 ISSN 2472-5056 Vol.1 No.4:24 tubulointerstitial injury by lowering UA [10] and febuxostat and detected using high-performance liquid chromatography (Tosoh, topiroxostat had a renoprotective effect by attenuating Tokyo, Japan). Urinary creatinine, albumin, monocyte inflammatory and oxidative stress in an animal model of chemotactic protein-1 (MCP-1), vascular endothelial growth diabetes [11] and of adenine-induced renal injury [12], factor (VEGF), and angiotensin II were measured using creatinine respectively. Compared with that reported for placebo, M L-type Wako (Wako Pure Chemical Industries, Ltd., Osaka, topiroxostat has been shown to decrease the urinary albumin- Japan), mouse albumin enzyme-linked immunosorbent assay to-creatinine ratio (ACR) in patients with chronic kidney disease (ELISA) kit (Bethyl Laboratory, Montgomery, TX, USA), mouse C-C (stage 3) with hyperuricemia in clinical trial [13] and an animal motif chemokine ligand 2 (CCL2)/JE/MCP-1 Quantikine ELISA kit, model of diabetic nephropathy [14]. However, there are a few mouse VEGF Quantikine ELISA kit (both R&D Systems, reports that topiroxostat has protective effects on the kidneys. Minneapolis, MN, USA), and angiotensin II ELISA kit (Peninsula Therefore, in the present study, we aimed to investigate Laboratories, LLC, San Carlos, CA, USA), respectively. whether topiroxostat has renoprotective effects and provided insights into the difference in the effect of topiroxostat and Liver and renal XOR activity assay febuxostat. The XOR activity was measured according to the procedure reported previously [15]. In brief, the liver and kidney Material and Methods homogenates were centrifuged for 60 min at 105,000 ×g, 4°C. Topiroxostat, febuxostat, and [13C , 15N ]-UA were synthesized The cytosolic or plasma fractions were added to the reaction 2 2 15 in our laboratory while [15N ]-xanthine and [15N ]-UA were mixtures containing [ N2]-xanthine (60 nmol), nicotinamide 2 2 + purchased from Cambridge Isotope Laboratories (Tewksbury, adenine dinucleotide (NAD , 150 nmol), and oxonate (2.0 nmol). MA, USA). All other chemicals used were of molecular biology Tris-hydrochloride (HCl) buffer (pH 8.5) was then added to adjust grade. the total volume to 150 μL for each reaction mixture. These mixtures were incubated at 37°C for 30 min and then 50 μL 200 13 15 Animal studies μmol/L [ C2, N2]-UA was added as the internal standard (ISTD). Subsequently, the mixture was heated for 5 min at 95°C All the experimental protocols were approved by the to stop the reaction. The resulting suspensions were centrifuged Committee on Animal Care of Sanwa Kagaku Kenkyusho. Male for 10 min at 15,000 × g at 4°C. The supernatants were filtered db/db and their littermate db/lean (db/m) mice obtained from through an ultrafiltration membrane (Amicon® ultra-0.5 Charles River Japan (Osaka, Japan) at 8 weeks of age were fed centrifugal filter devices, 3K, Millipore) and the production levels 15 laboratory standard powder chow, CRF-1 (Oriental Yeast Co., of [ N2]-UA were determined using liquid chromatography/ Ltd., Tokyo, Japan). The mice were allowed ad libitum access to mass spectrometry (LC/MS, linear trap quadrupole [LTQ]- food and water and were maintained in plastic cages at a Orbitrap, Thermo Fisher Scientific GmbH, Bremen, Germany). 15 constant temperature of 23 ± 2°C under a 12-h light-dark cycle Each activity was expressed as [ N2]-UA nmol. (lights on from 7:00 am to 7:00 pm). At 9 weeks of age, the diabetic mice were divided into four or five groups with matched Renal purine bodies concentrations body mass, food intake, urinary albumin excretion (UAE), and glycated hemoglobin (HbA1c) and treated as indicated in the The kidney tissue samples were homogenized in phosphate- following studies: study A, vehicle and topiroxostat treatment (1, buffered saline (PBS, pH 7.4) containing a protease inhibitor 3, and 10 mg/kg/day) for 4 weeks; study B, vehicle and cocktail (Roche, Basel, Switzerland) and centrifuged at 20,000 × febuxostat treatment (3, 10, 30, and 45 mg/kg/day) for 4 weeks; g at 4°C for 20 min. For the determination of hypoxanthine and and study C, vehicle and either drug (topiroxostat 1 and 3 xanthine, the homogenate was added to Tris buffer (pH 8.5) 15 15 mg/kg/day and febuxostat 3 mg/kg/day) for 7 days or 8 weeks. containing sodium chloride (NaCl), [ N2]-xanthine, and [ N2]- UA as the ISTD and then heated immediately at 95°C for 5 min. The