Hk 2 cells

The HK-2 cell line was obtained from American Type Culture Collection (ATCC, Manassas, VA, USA). The HK-2 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen, CA, USA) with 10% fetal bovine serum (FBS) and maintained in a humidified incubator filled with 5% CO₂ and 95% air at 37°C. PM was obtained from Sigma-Aldrich (St. Louis, MO, USA). After confirming that the HK-2 cells were seeded at 80% confluence, they were cultured in 2% FBS DMEM. After fasting for 24 hours, the HK-2 cells were exposed to DMEM-containing 5.5 mM glucose (the normal glucose group or NG group) or 30 mM glucose (the high glucose group or HG group) and in the presence or absence of PM for 48 hours. After all treatments, the cells were washed 3 times with ice-cold 1×phosphate-buffered saline (PBS) and harvested.

Human renal proximal tubular epithelial cell line (HK-2 cell line) was provided by China Center for Type Culture Collection (CCTCC, China). The HK-2 cells were incubated in plates and cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen, USA) containing 5% fetal bovine serum, nonessential amino acids, 50 ng/mL human recombinant epidermal growth factor, 50ng/mL bovine pituitary extract, 100μg/mL streptomycin, 100 U/mL penicillin. The parameters of the incubator were set to 5% CO2, 95% air and 37°C.

HK-2 cells were purchased from AmericanType Culture Collection (Rockville, MD, USA). The cells were cultured in a medium consisting of DMEM/F12 (Gibco) supplemented with 10% FBS at intervals of 3-4 days to continuously passaged. HK-2 cells were cultured in DMEM/F12 medium for 16 h and cultured in the presence or absence of adipolin (300 ng/ml) for 1 h, followed by stimulation with Ang (1 μmol/L) or vehicle for 24 h. Knockdown of PPARα, HMGCS2, AdipoR1, or AdipoR2 was achieved by siRNA transduction at 10 nM with Lipofectamine RNAiMAX (Invitrogen) 24 h before experiments. Lipofectamine RNAiMAX and siRNAs were dissolved in Opti-MEM (Gibco). The ON-TARGETplus siRNA SMART pools targeting each gene were purchased from Horizon Discovery. Control cultures were transfected with an unrelated scrambled siRNA (ON-TARGET plus Control Non-Targeting Pool, Horizon Discovery).
Conditionally immortalized murine MPC-5 podocyte clonal cells (University of Bristol) were maintained in RPMI1640 medium containing 10% fetal bovine serum, 10 IU/ml recombinant murine interferon-γ (Wako) at 33°C for proliferating cells. To induce differentiation, MPC-5 cells were incubated at 5% CO2 at 37°C without interferon-γ for 14 days.