Cell culture and treatment

HK-2 human renal proximal TECs (American Type Culture Collection, Manassas, VA, USA) were cultured in Dulbecco's modified Eagle's medium and Ham's F-12 medium (DMEM/F12, Gibco) supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 mg/mL streptomycin. HK-2 cells at logarithmic growth phase were digested with trypsin (Solarbio, T1300-100), seeded in a 6-well plate (3×10⁵/well), and then cultured for another 8 h before treatment.

For TREM-1 depletion studies, TREM-1-specific siRNA (Santa Cruz, Cat# sc-42999), control (CON) shRNA plasmid (Santa Cruz, Cat# sc-108060), TREM-1 cDNA ORF clone (Sino Bio, Cat# HG10511-UT), or negative control vector (Sino Bio, Cat# CV011) was transfected into HK-2 cells using Lipofectamine 3000 (Thermo Fisher). Cells were grown to a subconfluent density at 37°C with 5% CO₂ and stimulated with LPS (100 ng/mL).