AKI model construction and treatments
C57BL/6 mice (8–12 wk) mice purchased from Cavens (Changzhou, China) were divided into four groups: control (saline), CDDP only (CP, 20 mg/kg; MCE, United States), CP (20 mg/kg) + ADAMTS13 (0.1nmol/kg) and CP (20 mg/kg) + ADAMTS13 (0.3nmol/kg). 0.1 and 0.3nmol/kg rhADAMTS13 were injected into the caudal vein daily for 3 days after surgery. All animal experiments comply with the ethical requirements of the animal council. The experiment was approved by the Ethics Committee of The Fourth Affiliated Hospital of Guangxi Medical University. To further examine the role of ferroptosis in it. Mice were randomly divided into five groups by ferroptosis inhibitor Fer-1 and iron supplement ferric citrate (Fe): Control group, CP group, CP (20 mg/kg) + ADAMTS13 (0.3nmol/kg) group, Fer-1 + CP + ADAMTS13 group and Fe + CP + ADAMTS13 group. The Fer-1 + CP + ADAMTS13 group was injected with 1.5mg/kg Fer-1 (HY-100579, MCE, United States) through caudal vein before injection of CP, and the Fe + CP + ADAMTS13 group was injected with 15mg/kg ferric citrate (HY-B1645, MCE, United States) through caudal vein before injection of CP.
Measurement of BUN and Scr
BUN (C013-2, Nanjing Jiancheng Bioengineering Institute, China) and Scr (ab65340, Abcam) levels in serum were measured using the corresponding detection kits in accordance with the manufacturer’s instructions.
After the mice were sacrificed, part of the kidney tissues was taken and fixed with 10% formaldehyde quickly, dehydrated, embedded into paraffin sections, and stained with HE. The pathological morphology of the kidney tissues was observed under light microscope.
The kidney was embedded in paraffin block with 4µm sections. Paraffin sections were deparaffinised and rehydrated using serial xylene and alcohol. Specimens were then stained with Periodic Acid-Schiff (PAS) to determine tubular injury.
0.1g of renal tissue was added with 1 mL of RIPA lysate (Beyotime Institute of Biotechnology) and placed on ice, and homogenized thoroughly by a homogenizer. The protein concentration was then detected using a BCA kit (Bio-Rad Laboratories, Inc.). The denatured proteins (30μg) were separated by SDS-PAGE gel electrophoresis. The isolated proteins were transferred to PVDF membrane and sealed with 5% skimmed milk powder for 2h. The proteins were incubated overnight in primary antibody at 4 ℃. On the second day, PVDF membrane was incubated in the secondary antibody for 2h and developed with enhanced chemiluminescence kit(GE Healthcare) and Image J software (version 146; National Institutes of Health, Bethesda, MD, USA) was used to analyze the fold-changes of protein levels. The primary antibody information is as follows:anti- KIM1 (ab66062, Abcam, UK), anti-NGAL (ab23477, Abcam, UK), anti- SLC7A11 (ab175186, Abcam, UK), anti- GPX4 (ab125006, Abcam, UK), anti- FTH1 (ab75972, Abcam, UK), anti- FPN1 (ab78066, Abcam, UK), anti- Nrf2 (ab62352, Abcam, UK), anti- HO-1 (ab68477, Abcam, UK), anti- Lamin B (ab232731, Abcam, UK), anti-GAPDH (ab8245, Abcam, UK).
The iron content in tissue sections was determined using an iron stain kit (ab150674; Cambridge, UK). Histological sections were deparaffinized and rehydrated. Slides containing tissue sections were incubated in the Perl's solution(5% potassium ferrocyanide and hydrochloric acid solutions) for 3 min, washed with distilled water, stained using 3,3‐diaminobenzidine (DAB; Vector Laboratories, Burlingame, USA), and hematoxylin (Sigma‐Aldrich, St Louis, MO, USA) for 5 min, and then washed with distilled water. Finally, the sections were dehydrated in 95% ethanol, followed by absolute ethanol. Three sections per animal were viewed and photographed under a microscope (Nikon TE300; Nikon).
Iron concentration detection
To detect iron concentration in rental tissue, an iron assay kit (MAK025, Sigma-Aldrich) was used according to the manufacturer's instructions.
To detect Fe2+ content in the cells, an iron assay kit (ab83366, Abcam, UK) and presented as nanogram Fe2+ per milligram of protein according to the manufacturer's instructions.
TNF-α (H052), IL-1β (H002), IL-6 (H007-1-1) and MPO (A044-1-1) levels in renal tissues were measured using the corresponding ELISA kits purchased form Nanjing Jiancheng Bioengineering Institute in accordance with the manufacturer’s instructions.
Measurement of Oxidative stress levels
SOD (A001-3), GSH (A006-2), MDA (A003-1) and ROS (E004) levels in renal tissues were measured using the corresponding detection kits purchased form Nanjing Jiancheng Bioengineering Institute in accordance with the manufacturer’s instructions.
The data are presented as the mean ± standard deviation. All experiments were performed in triplicate. Statistical significance in the study was analyzed by one-way ANOVA followed by Tukey's post hoc test and Bonferroni's correction. P<0.05 was considered to indicate a statistically significant difference. The analyses were performed using SPSS 17.0 software (SPSS, Inc.).