Rab0308 1kt

The measurement of the IL-6 and TNF-α levels was performed using commercial ELISA kits (RAB0308-1KT and RAB0477-1KT (Sigma Chemical Co., St. Louis, MO, United States), respectively) according to the manufacturer’s instructions. Briefly, samples and standards were added to antibody-coated 96 wells and were incubated for 2.5 h at room temperature. After incubation, wells were washed with wash buffer four times, and then detection antibody was added and incubated for another 1 h at room temperature. After washing, 3,3′,5,5′-tetramethylbenzidine (TMB) substrate was added and incubated in the dark for 30 min at room temperature followed by adding stop solution and reading absorbance at 450 nm immediately.

The ROS (#DRE901Mu, ShangHaiLianShuo Biological Co., Ltd.), SOD (R&D Systems, #DYC3419-2) and MDA (#EU2577, Wuhan Fine Biotech Co., Ltd.) levels in mice renal tissues and α-klotho (R&D Systems, #AF1819), TNF-α (R&D Systems, #MTA00B) and IL-6 (Sigma, #RAB0308-1KT) levels in mice serum were detected by ELISA kit. In addition, ROS (#NG-EA691, ShangHaiYuanmin Biotechnology Co., Ltd.), TNF-α (R&D Systems, #DTA00D) and IL-6 (Sigma, #RAB0306-1KT) levels in HRGECs cellular supernatant were detected by ELISA kit.

Differentiated BMDMs were seeded at a density of 0.9 × 10⁶ cells per well in 6-well plates in 2 mL of DMEM complete medium overnight before the media was either replaced with AE147 CM or was supplemented with 200 μM KIV, 200 μM KMV, 200 μM KIV or BCKA pool. After 24 h, cell-free supernatants were collected. The levels of IL-6 (Sigma, RAB0308-1KT), TNF-α (Sigma, RAB0477-1KT), TGF-β (Invitrogen, BMS608-4) and VEGF (Sigma, RAB0509-1KT) in the supernatants were measured by ELISA kits according to the manufacturer’s instructions. The experiment was performed in 3 biological replicates.