This study was approved by the Ben-Gurion University of the Negev Animal Use and Care Committee, protocol number IL-39-07-2018. All protocols comply with the NIH Guidelines and are reported in accordance with ARRIVE guidelines (https://arriveguidelines.org). Animals were housed in standard laboratory cages. Food and water were given ad libitum. Eight weeks old male C57BL/6 mice (Harlan Laboratories Inc. Rehovot, Israel) were divided into 4 groups (n=6-10 in each group): C, C-col, CKD and CKD-col. CKD was induced by adenine diet . First an 0.3% adenine, 0.9% phosphorus and 75 ppm iron diet was given for 10 days, switched then to a 0.2% adenine diet and unchanged phosphorus and iron, for additional 11 days. Control groups were fed with a control diet (0.3% phosphorus, ~75 ppm iron). All diets were purchased from Envigo Teklad, (Huntingdon, UK). Colchicine )Sigma, Missouri, USA( was diluted in saline to a 10 mg/ml solution and given at a dose of 30 mg/kg intraperitoneally (ip) to C-Col and CKD-Col mice seven days a week, while C and CKD groups were ip injected with saline. Mice were sacrificed after 21 days, using anesthesia with ketamine and xylazine, collecting: blood, liver and kidney. Serum creatinine levels were analyzed using the AU2700 analyzer (Beckman-Coulter, CA, USA).
RNA extraction and real-time PCR
Assays were performed with power SYBR green PCR master mix (Applied Biosystems, Foster City, CA, USA) as previously described , using the ABI Prism 7300 Sequence Detection System (Applied Biosystems, Foster City, CA, USA). Primers for quantification (Sigma-Aldrich, Rehovot, Israel) are summarized in Table 1.
Quantitative analysis of free and polymerized tubulin
Analysis of free and polymerized tubulin was done as described by Ishibashi et al . Briefly, 50mg of kidney tissue was homogenized (polytron, Kinematica, Littau, Switzerland) in microtubule stabilizing buffer (50% glycerol, 5% dimethyl sulfoxide, 10 mmol/L sodium phosphate, 0.5 mmol/L MgSO4, 0.5mmol/L EGTA). The homogenate was centrifuged and the supernatant containing the free tubulin was kept. The pellet was re-suspended in microtubule depolymerizing buffer (0.25 M sucrose, 10 mmol/L sodium phosphate, and 0.5 mmol/L MgSO4), centrifuged and the supernatant that contains the polymerized tubulin kept at -80°C for further analysis.
Western immunoblot analysis
The following antibodies were used for evaluation of the kidney extracts: β-Tubulin (Santa Cruz Biotechnology Inc, Dallas TX USA); Caspase 1 (Novus Biologicals, CO, USA); STAT3, p-STAT3 (polyclonal, Cell Signaling Technology Inc. Danvers, MA); GAPDH (Proteintech Group, IL , USA) and β-actin (Clone C4, MP Biomedical Solon, OH, USA), as previously described .
Kidney segments were fixed in 4% formalin for 48 hours, then embedded in paraffin and cut. Kidney sections were deparaffinized, rehydrated and stained with Masson’s trichrome (Bio-Optica, Milano, Italy). Fibrotic area quantification of Masson’s trichrome staining was performed as described by Chen et al. using an ImageJ software .
Immunohistochemistry staining was performed as previously described 9. Primary antibodies against F4/80 (BM-8) (diluted 1:30; Santa Cruz biotechnology Inc., Dallas Texas, USA) and Myeloperoxidase (MPO) (diluted 1:50; Abcam, Cambridge, UK) were used for the immunohistochemistry staining. For image processing, Cellsense Entry software (MATIMOP, Tel Aviv, Israel) was used.
Nonparametric Mann-Whitney U test was used to determine significant differences. The null hypotheses were rejected at the 5% level. Values along the manuscript are presented as means ± standard errors. Asterisk indicate a significant difference between the indicated and the control group, where hash marks indicate a significant difference between the indicated and the CKD group.