Materials and reagents
Yokuininto (K-25) Kampo medicine was donated from Kracie (Seoul, Korea). The dried extract powder of K-25 (Lot No. 15071713, Kracie) was used in this present study. The components and indications of K-25, a Kampo medicine, are described in details [13, 15, 16]. Stock solution was diluted in DMSO at 50 mg/mL and stored at − 20 °C. IL-1α recombinant was purchased from Thermo scientific (Rockford, IL, USA). Allopurinol (A8003), LPS (L6529) and potassium oxonate (PO, 156124) were purchased from Sigma-Aldrich (St. Louis, MO, USA).
Cell culture
We purchased four cell lines from American Type Culture Collection cell line (ATCC, MD, USA) and their ATCC® Numbers were Raw 264.7 (TIB-71™), LLC-PK1 (CL-101™), HEK293 (CCL-1573™) and MDCK (CCL-34™). All the four cell lines, i.e., Raw 264.7, LLC-PK1, HEK293, and MDCK cells, were kept in DMEM (Gibco, MD, USA) supplemented with 10% FBS, 1% penicillin/streptomycin and cultured in a humidified incubator at 37 °C in 5% CO2.
Measurement of cytotoxicity
Raw 264.7 and LLC-PK1 cells were seeded into 96-well plates and then the cells were treated with K-25 in media for 24 h. Cell viabilities were determined using the Ez-cytox assay kit (Dogen, Republic of Korea) according to the manufacturer’s instructions.
Immunoblot analysis
The protein samples were prepared in RIPA lysis buffer (89,900, Thermo Fisher Scientific). Samples were separated on 12% SDS-polyacrylamide gels and then, transferred to polyvinylidenefluoride (PVDF) membranes, which were blocked with 5% dried milk in PBS containing 0.5% Tween-20. The blots were incubated with the appropriate antibodies at a dilution of 1:1000. The antibodies were those against OAT1 (TA322017, OriGene), OAT3 (TA321636, OriGene), ABCG2 (sc-58,222, Santa cruz), URAT1 (LS-C335533, LsBio), GLUT9 (NBP1–06271, novusbio), KIM-1 (ab47635, abcam), NGAL (ab63929, abcam) enzymes. Images of the blotted membranes were obtained using a LAS-4000 lumino-image analyzer (GE Healthcare Life Sciences, Republic of Korea).
Cytokine antibody array
At the end of each treatment, the culture medium was collected in eppendorf tubes. After centrifugation at 10,000 × g for 10 min, the supernatants were assayed for secreted mediators using RayBio C-Series Mouse Cytokine Antibody Array C2000 (AAM-CYT-2000-8, RayBiotech) according to the manufacturer’s instructions.
Measurement of nitric oxide
The nitrite in culture supernatants was measured as an indicator of nitric oxide (NO) production. An aliquot of the culture supernatant was mixed with a volume of Griess reagent (G2930, Promega), and the absorbance at 570 nm was determined using a microplate reader.
NGAL, KIM-1 and transporters measurements
NGAL and KIM-1 were determined by an ELISA assay kit (Cloud-clone, USA) according to the manufacturer’s instructions. LLC-PK1 cells (1 × 106 cells/6 well) were treated with various concentrations of IL-1α (0–100 ng/ml) for 24 h. After incubation, the medium was collected, and centrifuged at 3000 rpm. The supernatants were carefully transferred into fresh tubes. Supernatants (50 μl) were added to 50 μl of a 1/1 mixture of 0. 01 mol/L PBS (pH 7.0). Plates were incubated at 4 °C for overnight. Transporters were measured using an ELISA kit (Mybiosource, USA) according to the manufacturer’s recommendation.
Transporter uptake assays
Transporter uptake assays were performed as described previously [17]. In brief, the transporter expressing HEK293 cells were seeded on BD poly-D-lysine 24 well microplates with a density of approximately 1 × 105 cells/wells in DMEM supplemented with 10% FBS. Uptake of [3H]para-aminohippuric acid (PAH) for OAT1, [3H]estrone sulfate (ES) for OAT3 and [3H]1-methyl-4-phenylpyridinium (MPP+) for OCT2 were assayed at 37 °C in Ringer’s solution (130 mM NaCl, 4 mM KCl, 1 mM CaCl2, 1 mM MgSO4, 20 mM HEPES, 1 mM NaH2PO4, 18 mM glucose, pH 7.4) for 5 min in the absence or presence of Yokuininto. The uptake was terminated by three washes with 0.5 ml of ice-cold Ringer’s solution. Cells were then solubilized in 0.5 ml of 1 N NaOH. After neutralization with 0.5 mL of 1 N HCl, their hydrogen isotype [3H] content was assayed by Packard Tri-Carb 2700TR liquid scintillation counter. MDCKII-BCRP transporter assay was determined by the Cihalova method [18]. The Xenopus oocytes system was measured as previously reported [19].
Drug administration
Male Institute for Cancer Research (ICR) mice (7 weeks) were obtained from Daehan Bio Link company (DBL, Eumseong, Korea). The animals were kept in a clean animal room under specific pathogen free conditions. Mice were allowed to adapt to the environment for a week before being used for the experiment. All animals were housed under 12 h light–12 h dark cycle, lights at 10:00 am. Housing room temperature was maintained at 20–24 °C and humidity at 40–60%. Prior to experimental testing, they were housed in groups of four in standard cages containing a supply of food pellets and water. Mice were assigned as seven per each group to explore the feasibility: (1) control group (n = 7; intraperitoneally vehicle injected plus vehicle treated group), (2) PO 400 mg/kg/day group (n = 7; intraperitoneally PO injected plus vehicle treated group), (3) Allopurinol 50 mg/kg/day group (n = 7; intraperitoneally allopurinol injected plus vehicle treated group), and (4) PO + K-25 300 mg/kg/day group (n = 7; intraperitoneally PO plus K-25 treated group). Mice were treated with 400 mg/mL PO for 3 days, and then K-25 was administered intraperitoneally once a day for 3 days. All the procedures were randomly ordered between the groups. At the end of the experiment, mice were sacrificed by CO2 inhalation. Animal maintenance and treatment were conducted in compliance with the Principles of Laboratory Animal Care. All animal procedures were approved by the institutional animal care and use committee of Korea Institute of Oriental Medicine (Approval No. KIOM #16–069).
Immunofluorescence analysis
Cells were fixed through incubation with 4% paraformaldehyde at room temperature for 30 min. Fixed cells were rinsed in PBS, treated with 0.5% BSA for 30 min, and then incubated overnight at 4 °C with the rabbit anti-GLUT9 and anti-OAT3 antibodies. They were then incubated for 2 h with an Alexa 488 Fluor-conjugated secondary antibody. The cells were finally washed in PBS and mounted using Vectashield Mounting Medium containing DAPI. Immunofluorescent images were captured using microscope (Olympus Microscope System BX51; Olympus).
Xanthine oxidase inhibition (XOI) activity and uric acid assay
XO catalyzes the conversion of hypoxanthine to xanthine and then uric acid as a final product in the presence of molecular oxygen to yield superoxide anion [20]. Inhibitory effects on xanthine oxidase activity were measured by a decrease in uric acid formation. Urine and serum level of uric acid were determined by the uric acid detection kit (ab65344, abcam). The experimental process performed according to manufacturer’s instructions.
Statistical analysis
The variables including primary ones, serum and urinary uric acid concentrations were expressed as the mean ± standard error of the mean (S.E.M.). The statistical variables were analyzed using a one-way analysis of variance (ANOVA) and post hoc multiple mean comparisons (Bonferroni test). All variables were analyzed using the GraphPad Prism 5.10 software (GraphPad Software Inc., USA).