Regulation of P-Glycoprotein in Renal Proximal Tubule Epithelial Cells by LPS and TNF-α
Figure 1
P-gp activity and expression after exposure to TNF and/or LPS in GERP cells. (a)–(c) P-gp transport activity in GERP cells after exposure to different concentrations of TNF-α ((a) ) or LPS ((b) ) for 24 hours compared to control (cells exposed to culture medium). ((c) ) Optimal concentrations of both sepsis mediators were used for exposure to GERP cells for 24 hours. P-gp activity was determined using the calcein accumulation assay. ((d) ) Abcb1b expression in GERP cells (control; open bars) after exposure to either 10 g/mL LPS (closed bars), 10 ng/mL TNF-α (striped bars) or both 10 g/mL LPS and 10 ng/mL TNF-α (chequered bars) for 2, 6 or 24 hours. mRNA levels were determined with RQ-PCR and expression was normalized for the GAPDH value (15.9 ± 1.0). ((e) ) P-gp protein expression in GERP cells (control; lanes 1, 2, 7 and 8) after exposure to either 10 g/mL LPS (lanes 3 and 4), 10 ng/mL TNF-α (lanes 5 and 6) or to both 10 g/mL LPS and 10 ng/mL TNF-α (lanes 9 and 10) for 24 hours. Total cell lysate fractions of GERP cells were used and expression of P-gp was determined by Western blotting. Relative pixel intensities (ratio P-gp/-actin) were determined through image analysis. Data are expressed as mean SEM. Significantly different compared to control cells (: , : ).
