Establishment and optimization of a hyperuricemic nephropathy mouse model / 药学学报

The aim of this study was to establish an efficient and stable mouse model of hyperuricemic nephropathy (HN) by testing different modes of administration of potassium oxonate (PO) combined with hypoxanthine (Hx). Animal welfare and experimental procedures were in accordance with the regulations of the Animal Ethics Committee of Guangdong Pharmaceutical University. Male C57BL/6 mice were randomly divided into a control group, a PO+Hx group (i.g.; 100 mg·kg-1·d-1 and 500 mg·kg-1·d-1, respectively), and a PO+Hx group (i.p.; 100 mg·kg-1·d-1, and 500 mg·kg-1·d-1). This HN model was induced by combination of PO and Hx administration once daily for 21 days. The results of serum biochemistry showed that the levels of serum creatinine and 24 h albuminuria were increased compared with the normal group in intragastric administration of PO combined with Hx (P < 0.05), but there was no significant difference in serum uric acid and hepatic levels of xanthine oxidase. The maximum value of serum uric acid and creatinine was 349.3 μmol·L-1 and 26.4 μmol·L-1, respectively, in mice injected with PO combined with Hx. The levels of liver xanthine oxidase and 24 h albuminuria were significantly increased in mice injected with PO combined with Hx (P < 0.01). Pathological data showed that renal tubules were dilated, the epithelial cells of renal tubules were disordered, and the production of collagen fibers, reactive oxygen species (ROS) and lipid peroxidase 4-hydroxynonenal (4-HNE) were slightly increased after intragastric administration of PO combined with Hx mice. Obvious infiltration of inflammatory cells and large area of collagen deposition, with a large amount of ROS and the lipid peroxide 4-HNE were produced in mice injected with PO combined with Hx. Western blot analysis showed that the expression of fibronectin (FN) and urate transporter 1 (URAT1) was increased after intragastric administration of PO combined with Hx in mice and further increased in mice injected with PO combined with Hx. This study demonstrates that injection with 100 mg·kg-1 potassium oxonate combined with 500 mg·kg-1 hypoxanthine establishes a stable and efficient mouse HN model.

Role of 3-methyladenine in uric acid-induced apoptosis of renal tubular epithelial cells / 中华肾脏病杂志

Objective:To investigate the effect of autophagy inhibitor 3-methyladenine (3-MA) on uric acid (UA)-induced apoptosis of renal tubular epithelial cells.Methods:(1) Totally 24 SD rats were randomly divided into 4 groups: control group, treatment with 3-MA group, hyperuricemic nephropathy (HN) group, and HN+3-MA group, with 6 rats in each group. According to the body weight of the rats, adenine (100 mg/kg) and potassium oxonate (1 500 mg/kg) were mixed with distilled water to make a suspension, and the rats were given intragastrically once daily for consecutive 21 days to establish HN rat model. The control group and the 3-MA treatment group were fed an equivalent amount of distilled water. At the same time, the 3-MA treatment group and the HN+3-MA group were intraperitoneally injected with 3-MA (15 mg/kg), and the control group and HN group were intraperitoneally injected with an equal volume of saline once daily for 21 consecutive days. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay (TUNEL) was used to observe renal cell apoptosis. Western blotting was used to detect the expression levels of cleaved caspase-3 and Bax, and immunofluorescence staining was used to detect the expression and localization of cleaved caspase-3 in renal tissue. (2) Human renal tubular epithelial cells (HK-2) were stimulated with UA (800 μmol/L), and cells were administrated with different concentrations of 3-MA or Beclin-1 small interfering RNA (siRNA). The apoptosis of renal tubular epithelial cells was detected by Western blotting and immunofluorescence staining.Results:Compared with the normal rats, the apoptosis of renal tubular epithelial cells in the HN group was significantly increased ( P<0.01), and the expression levels of cleaved caspase-3 and Bax were significantly up-regulated (both P<0.05). Compared with the HN group, the apoptosis of renal tubular epithelial cells in the HN+3-MA group was significantly decreased ( P<0.01). In addition, high level of uric acid could significantly increase the levels of apoptosis associated proteins in HK-2 cells (all P<0.05), and using different concentrations of 3-MA or transfecting with Beclin-1 siRNA could significantly reduce the expression of cleaved caspase-3 and Bax (all P<0.05). Conclusion:Autophagy plays an important role in uric acid-induced apoptosis of renal tubular epithelial cells. Inhibiting the excessive activation of autophagy may be a new strategy to prevent the progression of HN.

Dioscin attenuates inflammatory injury in uric acid-induced renal tubular epithelial cells by suppression of NF-κB signaling pathway / 中国临床药理学与治疗学

AIM: To observe the effect of Dioscin treatment on NF-κB signaling pathway and cellular inflammatory injury and explore its potential mechanism in uric acid-induced mouse tubular epithelial cells (mTECs). METHODS: After 1.2 mol/L uric acid induced mTECs, Dioscin and NF-κB P65 inhibitor BAY11-7082 were given to intervene respectively. IκB-α, NF-κB P65, PP65, NLRP3, IL-1β and β-actin were detected by Western Blot, immunofluorescence staining and real-time PCR. RESULTS: Western Blot, immunofluorescence staining and real-time PCR analysis showed that expression levels of PP65, NLRP3 and IL-1β were significantly downregulated in the uric acid-induced mTECs with Dioscin and BAY11-7082 treatment. CONCLUSION: Dioscin attenuates uric acid-induced cellular inflammatory damage by suppression NF-κB signaling pathway.

A Novel UMOD Mutation (c.187T>C) in a Korean Family with Juvenile Hyperuricemic Nephropathy

Familial juvenile hyperuricemic nephropathy (FJHN; OMIM 162000) is an autosomal dominant disorder characterized by hyperuricemia and gouty arthritis due to reduced kidney excretion of uric acid and progressive renal failure. Gradual progressive interstitial renal disease, with basement membrane thickening and glomerulosclerosis resulting from fibrosis, starts in early life. In most cases of FJHN, uromodulin gene (UMOD) is responsible for the disease; however, there has been only one report of a genetically confirmed FJHN family in Korea. Here we report another Korean family with FJHN, in which three male members. a father and 2 sons.developed gout and progressive renal insufficiency. The clinical, laboratory, and radiological findings were consistent with FJHN, and renal biopsy showed chronic parenchymal damage, which can be found in FJHN but is not specific to this disease. In order to confirm the diagnosis, sequence analysis of the UMOD was performed, and a novel heterozygous missense variant (c.187T>C; p.Cys63Arg) in exon 3 was identified. We assume that this variant is likely to be the causative mutation in this family, as the variant segregated with the disease. In addition, approximately two-thirds of the known mutations lead to a cysteine amino acid change in uromodulin, and all such variants have been shown to cause UMOD-associated kidney disease. In summary, we report a Korean FJHN family with three affected members by genetic analysis of the UMOD, and provide the first report of a novel heterozygous missense mutation.