Chemicals and Kits
Potassium Oxonate (PO), agarose, ALP and ethidium bromide were purchased from Sigma-Aldrich (St. Louis, MO, USA). ZUR was purchased from Ironwood Pharmaceuticals, Inc., Cambridge, MA02142. 100 bp DNA ladder and reverse transcriptase enzymes were from MBI (Fermentas, Thermo Fisher Scientific, USA). Qiazol and Oligo dT were from QIAGEN (Valencia, CA, USA). The kits for catalase, MDA and Glutathione Peroxidase (GPx) were purchased from Biodiagnostic Co. (Dokki, Giza, Egypt). While the kits of GPT, GOT, BUN and uric acid were from BIO-MED Diagnostics and EGY- CHEM for lab technology, Badr City, Industrial Area Piece 170, 250 Fadan In East of Elrubaki, Egypt. Xanthine Oxidase kit (Catalog No: E-BC-K024), mouse IL-1 beta (Catalog No: E-EL-M0037) and mouse TNF-alpha (Catalog NO: E-EL-M0049) were obtained from Elabscience Biotechnology Inc. USA.
Experimental Animals and Design
The Scientific Deans of Taif University, Saudi Arabia, along with its Ethical Committee, approved all procedures and in vivo animal usage in this study for the project #1–439–6099, based on the NIH Guide for the Care and Use of Laboratory Animal. The Swiss mice used in this experiment consisted of a total of forty-two male mice aged ten weeks (weight 30-35g), originating from the College of Pharmacy, King Abdel-Aziz University, Jeddah, Saudi Arabia. The mice were handled manually for seven days in order to overcome stress. The animals were kept in a 12/12 hrs day-dark cycle and were given free access to food and water. Seven groups of mice were employed, each consisting of six individuals.
Group 1 was the negative control (CNT), with the mice given free access to food and water. Group 2 was the positive hyperuricemic group, in which the mice received PO intraperitoneally (250 mg/kg bw, single dose every day at 8:00 am). The dosages of PO and timing were determined as stated above . Group 3 received ALP orally (ALP; 5 mg/kg body weight daily one hour after PO administration) for seven days . Group 4 was administered ZUR, accompanied by an orally administered dose of 80 mg/kg, in accordance with Wu et al. (2017) . For seven days, groups 5 and 6 were administered PO at 8.00 am followed by ALP for Group 5 and ZUR for Group 6 one hour later (9.am). For seven consecutive days, Group 7 was administered PO first at 8:00 am, followed by a combination of ALP and ZUR at 9:00 am. After two weeks and at the end of the design, the mice inhaled dimethyl ether and were decapitated after overnight fasting. Serum was extracted and stored at –20 °C until the chemistry measurements took place. Kidney and liver tissues were soaked in Qiazol for RNA extraction and in Bowan’s solution for histology and cellular immunohistochemistry.
Serum biochemistry, antioxidants, and cytokines assessments
The serum levels of the antioxidants, liver and kidney biomarkers and cytokines were assayed calorimetrically, using specific commercial kits based on the manufacturer’s instruction manual.
Xanthine Oxidase activity
The kit was used for the determination of XOD activity in serum, plasma, erythrocyte and tissue. XOD catalyzes hypoxanthine, to form xanthine and superoxide anion free radicals, resulting in a purplish red substance in the presence of electronic receptors and a chromogenic agent. XOD activity can be calculated by measuring the OD value of the purplish red substance at 530 nm. For liver tissues, following homogenization in normal saline on ice, in a ratio of one liver tissue and nine for normal saline, homogenate is centrifuged for ten minutes and supernatant used for assay. The XOD unites for serum values are U/l, and for liver homogenates are U/g protein tissue. The protocol employed was partially modified in accordance with the method of Haidari et al. (2011) .
RNA extraction, cDNA synthesis and quantitative real time PCR (qRT-PCR)
Total RNA was, as described elsewhere , extracted from the kidney tissues and frozen samples homogenized in a homogenizer. Chloroform (200 µL) was added to the homogenate, then centrifuged at 4 °C at 12000 rpm for ten minutes. The supernatant was separated and an equal volume of isopropyl alcohol was added. The pellets of RNA were dissolved in Diethylpyrocarbonate (DEPC) water after washing and drying. The integrity of RNA was confirmed  and 3 µg of total RNA and 0.5 ng oligo dT (Qiagen Valencia, CA, USA) was denatured following incubation in a Bio-Rad T100TM Thermal Cycle at 70 °C for five minutes. Denatured RNA was reverse transcribed after the addition of 2 µL of 10 mM dNTPs, 100 U of M-MuLV (SibEnzyme, Ak, Novosibirsk, Russia) and 2 µL of 10X RT-buffer, before being incubated in a Bio-Rad T100TM Thermal Cycler for one hour at 37 °C, then for ten minutes at 90 °C, to ensure enzyme inactivation. For quantitative real time PCR analysis (qRT-PCR), primers for the examined genes (Table 1) were designed using GenScript Real-time PCR (TaqMan) Primer Design (https://www.genscript.com/tools/real-time-pcr-taqman-primer-design-tool). Each PCR reaction consisted of 1.5 μl of 1μg/μl cDNA, 10 μl SYBR Green PCR Master Mix (Quanti Tect SYBR Green PCR Kit, Qiagen), along with 1 μM of forward and reverse primer for each examined gene and nuclease free H2O to a final volume of 20 μl. Reactions were run and analysed in Applied Biosystem 7500 Fast Real time PCR Detection system. qRT-PCR conditions are: 95°C for ten minutes (first denaturation) and forty cycles of 95°C for fifteen seconds (second denaturation stage) followed by 60°C for one minute (annealing and extension stage). The critical threshold (Ct) of the target gene was normalized with quantities (Ct) of the housekeeping gene (β-actin), using the formula x = 2−ΔΔCt, where there is x = fold difference relative to the control.
Histological and immunohistochemistry analyses of kidney
The kidney tissue was dehydrated and embedded in paraffin, then sectioned at 5µm. The slides were subsequently stained with hematoxylin and eosin (H&E) and the morphological changes examined using a microscope (Eclipse 80i, Nikon, Japan), with images being captured by a digital camera (Fuij, Sapporo, Japan).
For immunohistochemistry, the paraffin-embedded renal sections were deparaffinized, rehydrated and immersed in H2O2 (3%) for ten minutes, in order to block any peroxidase activity. Following this, the slides were washed in phosphate buffer saline. Nonspecific binding sites were blocked by bovine serum albumin (5%) prior to the addition of TGF-1β polyclonal antibody in a dilution of 1:300 overnight at 4 ₒC. The slides were then washed in PBS and incubated with a secondary antibody, developed with 3.3’-diaminobezidine tetrahydrochloride and counterstained with hematoxyline.
Data are means ± standard error of six values collected from six different mice per each treatment. Data were analyzed using one-way ANOVA (analysis of variance) setting the probability level P<0.05, with the individual comparisons obtained by Duncan's multiple range tests for SPSS software version 11.5 for Windows (SPSS, IBM, Chicago, IL, USA). The probability level P<0.05 was considered statistically significant.