Figure 4

HMGB1-induced MCP-1 secretion and its signaling pathways in rat RTECs in vitro. (a) MCP-1 was measured by ELISA in supernatants from NRK-52E RTECs on stimulation with 250 ng/ml HMGB1 for 0, 6, 12, 24, 36, or 48 h. The MCP-1 levels increased significantly with time, peaking at four-fold above the control levels after 36 h. Each figure is representative of three separate experiments. n=3; ^**P<0.01. (b) Phosphorylation of PI3K/Akt and three different MAPKs in rat RTECs incubated with HMGB1 and determined by western blotting. Phosphorylation peaked at different times for the four kinases after HMGB1 addition. Each figure is representative of three separate experiments. (c–f) MCP-1 was measured by ELISA in supernatants from rat RTECs incubated with or without the protein kinase inhibitors LY294002 (which inhibits PI3K/Akt), U0126 (which inhibits ERK), SP600125 (which inhibits JNK), and SB203580 (which inhibits p38). The inhibitors partially but significantly reduced the HMGB1-induced MCP-1 secretion in rat RTEC. Each figure is representative of three separate experiments. ^**P<0.01 compared with HMGB1 stimulation alone.