Hgecs

The protocol of this study was approved by the independent Ethical Committee of Wuhan Hospital of Traditional Chinese Medicine. Human renal glomerular endothelial cells (hGECs) (ScienCell Research Laboratories, USA) were maintained in DMEM/F12 (Gibco, USA) containing 10% fetal bovine serum (FBS) (Gibco). The hGECs were grown in humidified air with 5% CO₂ at 37°C. Cells were exposed to high glucose (30 mM) condition, treated with or without additional cabozantinib (5 or 10 μM) for 24 h. The hGECs were transduced with Ad-viral Egr-1, followed by stimulation with high glucose (30 mM) and cabozantinib (10 μM) for 24 h.

Isolated monocytes were resuspended at 1 × 10⁶ cells/mL in RPMI 1640 medium (Gibco, Invitrogen, Carlsbad, CA) containing 10% heat-inactivated fetal bovine serum (FBS, Gibco). Monocytes were cultured in 6-well plates with or without 100 ng/mL LPS (Peprotech), MPO-ANCA, or control IgG at a final concentration of 250 μg/mL. The cells were incubated at 37°C for 24 h, after which cells were collected and stained with an antibody mix containing Human TruStain FcX™, Zombie Aqua™ Fixable Viability Kit, anti-human CD45, CD16, CD14, CCR2, and CX3CR1 (BioLegend), followed by flow cytometry. Data acquisition was performed using a BD FACS Canto II flow cytometer. Data were analyzed using FlowJo software (version 10.0).
Human glomerular endothelial cells (HGECs, ScienCell, San Diego, CA, USA) were cultured in endothelial cell medium (ECM, ScienCell) supplemented with 10% FBS, 1% penicillin/streptomycin, and 1% endothelial cell growth factor. HGECs were cultured in 12-well plates with or without 10ng/mL TNF-α (Peprotech), MPO-ANCA, or control IgG at a final concentration of 500 μg/mL. The cells were incubated at 37°C for 24h, after which total RNA of HGECs was extracted for polymerase chain reaction (PCR) amplification, and supernatants were collected and stored at -80°C for subsequent ELISA detection.

Primary human glomerular endothelial cells (HGECs, ScienCell Company, Beijing Yuhengfeng Agency, China) were cultured in Endothelial Cell Medium containing 10% fetal bovine serum (Endothelial Cell Medium and serum were from ScienCell Company, Beijing Yuhengfeng Agency, China). The cells were used within six passages. Cells in the high insulin concentration group were treated with medium containing different concentrations of insulin: 7.14 ul, 14.28ul, 35.7ul, 71.4ul of Novolin insulin solution (Novo Nordisk, Copenhagen, Denmark) was added to 2 ml ECM medium separately(The ECM medium did not contain any insulin) to form 5 ng/ml, 10ng/ml, 25ng/ml, 50ng/ml high concentration insulin medium. Cells in the high glucose concentration group were treated with medium containing anhydrous glucose (Solarbio, Beijing, China) at 8.3, 11.1, 16.7, or 33.3 mmol/L. D-Mannitol (Solarbio, Beijing, China) was used as a control for osmolarity. HGECs were transfected with miR-21 mimics or miR-21 inhibitor (GenePharma, Shanghai, China) using the Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA). The transfection was performed under strictly aseptic conditions according to the manufacturer’s instructions. The effectiveness of miR-21 mimics and inhibitor was verified by PCR after transfection (Figures 4A, E).