A5512

LLC-PK1 cells (ATCC) were maintained in minimum essential medium (MEM) supplemented with 3% FBS. Mouse renal PTCs were developed as previously described (54 (link)). Cells were maintained at 33°C in DMEM/F-12 containing 2.5% FBS and IFN-γ and then transferred to 37°C for differentiation before use (54 (link)). The HEK293T (ATCC) cells were maintained in DMEM supplemented with 10% FBS. Cells were treated with AA (2.5–5 μg/mL; Sigma-Aldrich, A5512), PAC (10 μM; Sigma-Aldrich, T7402), or respective vehicles in complete medium for 48 hours. Primary PTCs were isolated from WT mice or CG1-KO mice as described previously (55 (link)). In brief, kidney cortex was minced, digested with collagenase (Worthington Biochemical Corporation) and trypsin inhibitor (Worthington Biochemical Corporation), and centrifuged in 32% Percoll medium to purify PTCs. Primary PTCs were then plated in collagen-coated dishes and maintained in DMEM/F-12 medium supplemented with insulin, transferrin, selenium, 0.05 μM hydrocortisone, and 50 μM vitamin C. To avoid changes in PTC phenotype due to repeated passages, these cells were plated as soon as they became confluent for experiments. Primary PTCs were treated with AA at a concentration of 5 to 10 μg/mL for the times indicated.

Male 6-week-old C57BL/6 mice (weighing 20 ± 2 g) were provided by the Guangdong Medical Laboratory Animal Center (SCXK (YUE) 2018-0002, Foshan, China). The mice were housed under controlled conditions of light (12 h light/dark cycle), temperature (24°C ± 2°C) and humidity (50%–60%) and had adequate food and tap water to ad libitum. The mice were divided into four groups randomly after 1 week of acclimatization: C, control group (n = 6); L, low-dose AAI group (n = 6); M, medium-dose AAI group (n = 6); H, high-dose AAI group (n = 6). Mice in the AAI groups were administered by intraperitoneal injection of AAI (A5512, Sigma-Aldrich, St Louis, MO, United States) at the dose of 1.25 (L), 2.5 (M), and 5 mg/kg/d (H), respectively, for 5 days. Control mice were injected intraperitoneally with PBS solution containing 5% DMSO as vehicle for 5 days. Twenty-4 hours after the last dose of intraperitoneal injection of AAI or vehicle, all mice were euthanized. Blood and kidney samples were collected immediately for further experiments. All animal experiments were carried out in accordance with the National Research Council’s Guide for the Care and Use of Laboratory Animals and approved by the Ethics Committee of Shenzhen Top Biotech Co., Ltd (approved ID: TOP-IACUC-2021-0137).

AA (A5512, Sigma-Aldrich, St. Louis, Missouri, USA) was dissolved in PBS and administered intraperitoneally to the mice (5 mg/ kg). Fourteen days after AA administration, mice were euthanized or received RenCa cells inoculation.