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Potassium Oxonate Induces Acute Hyperuricemia in the Tree Shrew (Tupaia Belangeri Chinensis)

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Potassium Oxonate Induces Acute Hyperuricemia in the Tree Shrew (Tupaia Belangeri Chinensis) Exp. Anim. 66(3), 209–216, 2017 —Original— Potassium oxonate induces acute hyperuricemia in the tree shrew (tupaia belangeri chinensis) Dong-Hong TANG1)*, You-Song YE1)*, Chen-Yun WANG1), Zhe-Li LI1), Hong ZHENG2), and Kai-Li MA1) 1) Medical Primate Research Center of China, Institute of Medical Biology, Chinese Academy of Medical Sciences/ Peking Union Medical College, No. 935, Jiaoling Road, Kunming, Yunnan 650118, P.R. China 2) Kunming Medical University, 1168 West Chunrong Road, Yuhua Avenue, Chenggong District, Kunming, Yunnan 650504, P.R. China Abstract: Potassium oxonate, a selectively competitive uricase inhibitor, produced hyperuricemia (HUA) in rodents in a previous study. In this study, we employed the tree shrew as an animal model to study potassium oxonate-induced HUA. The effect of allopurinol (ALLO), a uric acid reducer, was also examined in this model. Potassium oxonate at doses of 5, 20, 40, 60, 80, 100, and 1,000 mg/ kg was given intraperitoneally to tree shrews. The results showed that potassium oxonate can effectively increase the levels of uric acid in tree shrews at doses ranging from 40 to 100 mg/kg. Semiquantitative RT-PCR showed that the xanthine dehydrogenase/oxidase (XDH/XO) mRNA expression level was significantly higher in the liver tissue of tree shrews with high levels of uric acid. There were no changes in serum urea nitrogen, or serum creatinine values. ALLO can significantly decrease serum uric acid levels (P<0.01) and raise XDH/XO mRNA expression levels in the liver tissue of tree shrews with HUA. XDH/XO mRNA expression levels did not change in untreated tree shrews. Studies on acute toxicity in the tree shrew did not show any significantly abnormal signs. There were no adverse effects at the macroscopic level up to doses ≤100 mg/kg. Potassium oxonate induced acute HUA in tree shrews at lower doses compared with other animal models. Potassium oxonate-treated tree shrews may be a potential animal model for studying pathogenic mechanism and evaluating a new therapeutic agent for treatment of HUA in humans. Key words: allopurinol, hyperuricemia, potassium oxonate, tree shrew, xanthine dehydrogenase/ oxidase (XDH/XO) Introduction disorder caused by the deposition of monosodium urate crystals in the joints and other tissues because of extra- Uric acid, the end product of purine metabolism in the cellular urate supersaturation [7]. HUA is defined as a human body, is produced mainly in the liver by xanthine serum urate concentration that allows urate saturation in dehydrogenase/oxidase (XDH/XO) and excreted through body fluids (>7.0 mg/dl, equal to 416.5 µmol/l). The the kidney. Hyperuricemia (HUA) is characterized by incidence of gout and HUA in humans has been increas- an elevated level of uric acid in the blood. Gout is a ing in recent times [13]. Currently, China has about 120 (Received 1 November 2016 / Accepted 21 February 2017 / Published online in J-STAGE 16 March 2017) Addresses corresponding: D.-H. Tang, Medical Primate Research Center of China, Institute of Medical Biology, Chinese Academy of Medical Sci- ences/Peking Union Medical College, No. 935, Jiaoling Road, Kunming, Yunnan 650118, P.R. China K.-L. Ma, Center for Drug Safety Evaluation and Research, Institute of Medical Biology, Chinese Academy of Medical Sciences/Peking Union Medi- cal College No. 935, Jiaoling Road, Kunming, Yunnan 650118, P.R. China *Dong-Hong Tang and You-Song Ye contributed equally. ©2017 Japanese Association for Laboratory Animal Science 210 D.-H. TANG, ET AL. million patients with HUA, accounting for approxi- able and simple to handle, as opposed to primates. Re- mately 10% of the total population. Middle-aged or cent studies using whole genomic sequences have sug- older men and postmenopausal women usually have a gested that tree shrews are more closely related to higher incidence of HUA. However, in recent years, the primates than to rodents [4]. In an earlier study, we onset age is getting lower. HUA is also associated with prepared cDNA from fresh livers of tree shrews and the pathogenesis of hypertension and metabolic syn- determined the sequence of an XDH/XO nucleotide frag- drome. According to clinical reports, the key causative ment. Sequence alignment studies showed that the hu- agent, uric acid, is associated not only with an increased man and tree shrew nucleotide fragment sequences of risk of gout, but also with an increased risk of cardio- XDH/XO were 87.04% identical (manuscript submitted). vascular disorders, nephrolithiasis, diabetes, obesity, and Based on these findings, we hypothesized that the tree dyslipidemia [1, 5]. Therefore, there is an urgent need shrew might be a potential animal model for studying for a reasonable widely recognized, reasonable animal the pathogenesis of HUA. In this study, we employed model for HUA studies not only for drug screenings, but the tree shrew as the animal model to study potassium also for pathogenesis research. oxonate-induced HUA. The effect of allopurinol, a uric Potassium oxonate, a selectively competitive uricase acid reducer, was also examined in this model. The serum inhibitor, blocks the effect of hepatic uricase and pro- urate levels, XDH/XO mRNA expression level in liver duces HUA in rodents [14, 16, 18, 19]. There are sev- tissue, serum urea nitrogen, and serum creatinine (Cr) eral limitations to the rodent (rat and mouse) HUA values associated with acute toxicity were investigated. models, which are most commonly used at present [12, 14, 16, 18, 19]. Studies using rodent models [12, 14] Materials and Methods have indicated a need for another animal model for studying drug sensitivity and stability of potassium ox- Reagents onate. Mice and rats have a relatively low sensitivity to Potassium oxonate was purchased from Sigma-Al- drugs. Furthermore, despite the fact that mice, rats, and drich Corporation Co. (lot#STBC6418V). ALLO was humans are all mammals, mice and rats are taxonomi- purchased from Nanjing Dolai Biological Co., Ltd. So- cally far from that of humans. Mice and rats also possess dium carboxymethyl cellulose (CMC-Na) was purchased anatomical and physiological characteristics different from Chengyue Technology Co. Uric acid (UA) assay from those of humans. Thus, the parameters derived from kit, Cr and blood urea nitrogen (BUN) assay kits were working with these animal models are of limited use as purchased from Nanjing Jiancheng Biological Engineer- reference values. Therefore, establishing and developing ing Institute. A Transcriptor First Strand cDNA Synthe- new HUA animal models with serum uric acid metabo- sis Kit (cat# 04897030001) was purchased from Roche lism that is similar to that of humans and performing an Co. Premix Taq™ (code No. RR003A) and RNAiso Plus in-depth study on them is an urgent need in the field of (Lot#AK8504) were purchased from Takara Biotechnol- HUA research. ogy (Dalian) Co., Ltd. The tree shrew (Tupaia belangeri chinensis) is a non- rodent mammal, phylogenetically located between in- Animals and ethics statement sectivores and primates. Currently, the tree shrew is A total of two hundred 2- to 3-year-old adult male and classified within its own order Scandentia. Tree shrews female Chinese tree shrews (Tupaia belangeri chinensis) are small squirrel-like animals, found mainly in South were used for this study. Body weight ranged from 110 Asia, Southeast Asia, and Southern China [2, 4, 17]. The to 140 g. The tree shrews were from the colony of the tree shrew (T. belangeri chinensis) is a new experimen- Medical Primate Research Center of the Institute of tal animal that is being used widely in medical and bio- Medical Biology (Certificate No. is SCXK (Dian) logical research, because of its unique characteristics K2013-0001). All animals were fed ad libitum with such as its small size, ease of feeding, convenient man- regular mixed soft food. The feed formula, which was agement, low price, and its high degree of evolution. Its based on the nutritional needs of the experimental tree metabolism and gross anatomy are closer to those of shrews, was≥200 g crude protein,≥50 g crude fat, ≤30 g humans compared with other animals such as dogs, rats, crude fiber, ≤60 g crude ash, 10–15 g calcium, and 6–8 and mice [11]. Tree shrews are relatively easy to obtain- g phosphorus, per kilogram of the diet, with a Ca/P ratio TREE SHREW AS MODEL OF HYPERURICEMIA 211 of 1.2:1–1.4:1; the tree shrews also received fresh fruits. Serum uric acid levels were measured using commer- The animals were maintained individually in cages (100 cially available UA assay kits. Serum Cr levels and BUN cm length, 50 cm width, 40 cm height) at a temperature levels were also measured using assay kits. between 18 and 27°C (the difference in temperature be- tween day and night was ≤4°C), at a relative humidity Dose-dependent effectiveness of potassium oxonate in of approximately 60%, and with a 12 h light/dark cycle. inducing acute HUA in tree shrews The air change frequency was≥10 times/h. Other condi- Tree shrews were randomly assigned to 6 groups of 6 tions included an ammonia concentration of ≤14 mg/m3, each. The control group was injected intraperitoneally noise level ≤60 dB (A), and minimum illuminance of with 1% CMC-Na, whereas the others were injected 150 lx (the level of illumination in a conventional ex- intraperitoneally with potassium oxonate suspension in perimental animal environment is 15–20 lx). In this facil- 1% CMC-Na at doses of 5, 20, 40, 80, or 100 mg/kg. ity, a single corridor with buffer rooms separates the Blood samples were collected 0, 1, 2, 4, and 8 h after streams of people, objects, and animals from each other. administration.
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