Figure 5.

S1P₁R agonists attenuate LPS-induced injury in cultured kidney epithelial cells through MAPK and Akt pathways and knockdown of S1P₁R expression abolishes protection by S1P₁R agonist. Necrotic and/or apoptotic cell death measured by FACS analysis of Annexin V and 7-AAD labeling 24 hours after treatment of TKPTS cells. (A) TKPTS cells were treated with SEW2871 (1 μM) and/or with VPC44116 (10 μM) 1 hour before treatment with or without LPS (1 μg/ml); n = 3 to 4. †††P < 0.001, †P < 0.05 relative to vehicle; ***P < 0.001 relative to LPS. (B) Cells were pretreated with the ERK inhibitor PD098059 (5 μM), Akt inhibitor LY294002 (5 μM), or pertussis toxin (PTX; 100 ng/ml) 1 hour before SEW2871 and LPS. †††P < 0.001, ††P < 0.01, and †P < 0.05 relative to vehicle; *P < 0.05 relative to LPS; ##P < 0.01, #P < 0.05 relative to LPS+SEW2871. Results are from a representative experiment that was performed three times with n = 3 to 4 replicates of each treatment. (C) Relative mRNA levels of S1P₁R (reduced by approximately 50%) and S1P₃R (unchanged) after transfection of TKPTS cells with 25 nM scrambled sequence or S1P₁R siRNA; n = 3. Inset shows RT-PCR products for S1P₁R, S1P₃R, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from a representative gel. **P < 0.01 relative to siRNA-scramble transfected cells. Experiment was performed three times. (D) Apoptotic cell death in siRNA-transfected cells after LPS treatment (1 μg/ml) for 24 hours with and without 1 hour of pretreatment with SEW2871 (1 μM). ††P < 0.01, †P < 0.05 relative to vehicle; **P < 0.01 relative to LPS; ±P < 0.05 relative to LPS in scramble.