A total of 60 ICR female mice (20 ± 2g, 5 mice/group) were divided into 12 groups (control and 10 furosine treatment groups). Furosine was dissolved in distilled water and a dose of 0.24 g/kg b.w. was administered by gavage or tail vein injection (0.2 mL volume per mouse) once at the beginning. This dose was chosen based on the median lethal dose (LD50) determined in previous acute toxicity experiments, in which the LD50 of furosine was 1.6 g/kg b.w. Mice were fasted for 4 h prior to dosing; animals were sacrificed at 0 (controls), 0.25, 0.5, 1, 2, 3, 4, 6, 8, 10, 12 h after administration, and kidney tissue was dissected and blood samples were collected.

Male Sprague-Dawley (SD) rats (5 weeks old, weighting 180-220 g) were purchased from Shanghai SLAC Laboratory Animal Co., Ltd. SD rats were anesthetized with an intraperitoneal (i.p.) injection of pentobarbital sodium (25 mg/kg) and placed on a surgical thermostator. Then the rats were subjected to an abdominal incision, and the right kidney was carefully liberated from surrounding tissue, and nephrectomy was performed. The left kidney was exposed after a midline incision, and the renal artery was clamped with non-traumatic clamps for 45 min, followed by restoring of the renal blood flow. The kidneys were harvested and the serum was collected 48 h after the surgery. The rats in sham group were subjected to an abdominal incision without clamping the renal artery.

C57BL/6 male mice, 6 to 8 weeks old, were purchased from Liaoning Changsheng Biotechnology Co. (Liaoning, China). The animal experiment was conducted in three parts. In the first part, mice were randomly divided into 4 groups (n = 12/group): (1) control group that received an intraperitoneal injection of saline, (2) FG-4592 group that received intraperitoneal injection of FG-4592 once (10 mg/kg, dissolved in DMSO at 50 mg/ml and then further diluted in sterile phosphate-buffered saline to 1 mg/ml), (3) FA group that received intraperitoneal injection of a single dose of FA (250 mg/kg, dissolved in 0.3 M sodium bicarbonate), and (4) FA + FG-4592 group that received FG-4592 two days prior to FA single-dose injection. Kidney specimens and blood samples were collected on the second day (n = 6/group) and the fourteenth day (n = 6/group) after FA injection for further examination. In the second part, mice were treated with a ferroptosis inhibitor (Fer-1). In the third part, mice were treated with a PI3K inhibitor (wortmannin).

All specific pathogen free (SPF) grade Balb/c mice (6-8 weeks) were purchased from the Experimental Animal Center of Baiqiuem Medical College, Jilin University (China). The mice were reared under the conditions of 12 h of light and 12 h of darkness, supplemented with sufficient feed and free drinking water. The mice were treated according to a modified model as previous mentioned. In brief, a total of 15 mice were subjected to different doses of Cd (0, 2.5 and 5 mg/kg body weight/d) for 3 consecutive days intraperitoneally, and control mice were treated with 0.9% physiological saline. The concentrations of Cd were selected based on previous studies. At the day 4, the kidney tissue was collected and stored at -80 until detection.

Male Sprague-Dawley (SD) rats (5 weeks old, weighting 180-220 g) were purchased from Shanghai SLAC Laboratory Animal Co., Ltd. SD rats were anesthetized with an intraperitoneal (i.p.) injection of pentobarbital sodium (25 mg/kg) and placed on a surgical thermostator. Then the rats were subjected to an abdominal incision, and the right kidney was carefully liberated from surrounding tissue, and nephrectomy was performed. The left kidney was exposed after a midline incision, and the renal artery was clamped with non-traumatic clamps for 45 min, followed by restoring of the renal blood flow. The kidneys were harvested and the serum was collected 48 h after the surgery. The rats in sham group were subjected to an abdominal incision without clamping the renal artery.