Lip 1

The HK2 cells were purchased from Procell Life Science & Technology Co., Ltd. (Wuhan, China). The cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Gibco, China) supplemented with 10% fetal bovine serum (FBS; CellMax, Australia) and 1% penicillin/streptomycin in a humidified atmosphere of 5% CO₂. Human kidney fibroblasts were also purchased from Procell Life Science & Technology Co. The fibroblasts were resuspended in DMEM/F-12 medium (Hyclone, USA) containing 10% FBS (CellMax, Australia) and 1% penicillin/streptomycin and cultured in a humidified atmosphere of 5% CO₂.
HK2 cells were seeded into dishes and grown to 80–90% confluence. Cells were then treated with 0.5 μM Lip-1 and/or 1.0 μM RSL3 (MedChemExpress, USA) for 24 h. After treating cells with RSL3/Lip-1 for 24 h, we cultured the cells with a new medium for collecting factors for another 24 h. All the medium was collected and used for enzyme-linked immunosorbent assay (ELISA) analysis. The remaining HK2-conditioned medium (HK2-CM) was used in co-culture experiments with the fibroblasts.

The surgical procedure was performed as described in our previous study [18 (link)]. Briefly, the left ureter was exposed and ligated with 4-0 silk sutures, and the ligatures were dissected to prevent retrograde urinary tract infections. The sham operation was executed in a similar manner, but without ureter ligation. Twenty-four C57BL/6 mice were randomly divided into the following four groups: (1) sham group, (2) Lip-1 (>98% purity; MW 340.85, MedChemExpress, USA) group, (3) UUO group, and (4) UUO + Lip-1 group. The mice in the Lip-1 and UUO + Lip-1 groups were intraperitoneally injected with 200 μL Lip-1 (10 mg/kg/d, dissolved in DMSO, and then diluted with 0.9% NaCl) for 14 consecutive days after surgery. The mice in the sham and UUO groups were intraperitoneally injected with equal volumes of 0.9% NaCl solution. After the last treatment, all animals were anesthetized with sodium pentobarbital and sacrificed. Blood was collected from the ophthalmic arteries by retro-orbital bleeding. Part of the left kidney was fixed in a 4% paraformaldehyde solution and the remaining portion was snap-frozen in liquid nitrogen and stored at −80 °C for further analysis.

Human umbilical vein endothelial cells (HUVECs) were obtained from ScienCell Research Laboratories (San Diego, CA, USA) and cultured in plates pre-coated with 0.2% gelatin in endothelial cell medium supplemented with 5% fetal bovine serum, 1% penicillin/streptomycin, and 1% endothelial cell growth supplement (ScienCell) at 37 °C with 5% CO₂.
For each experiment using cultured HUVECs, cells were seeded in the incubator and cultured to 70–80% confluence, then exposed to L-glutamic acid (GLU, 20 mM, Cat#: G8415, Sigma-Aldrich, St. Louis, MO, USA) or N-Methyl-D-aspartic acid (NMDA, 1 mM, Cat#: HY-17551, MedChemExpress, Shanghai, China) for 24 h in the absence or presence of Ferrostatin-1 (Fer-1, 10 μM, Cat#: HY-100579, MedChemExpress), Liproxstatin-1 (Lip-1, 2 μM, Cat#: HY-12726, MedChemExpress), LB-100 (5 μM, Cat#: S7537, Sellechchem, Houston, TX, USA), Acadesine (AUCAR, 2 μM, Cat#: HY-13417, MedChemExpress), Deferoxamine (DFO, 10 μM, Cat#: HY-B0988, MedChemExpress), and Erastin (5 μM, Cat#: HY-15763, MedChemExpress) for 24 h. These plating conditions were used in all experiments, unless mentioned otherwise.