Up-regulation of multidrug resistance P-glycoprotein via nuclear factor- κB activation protects kidney proximal tubule cells from cadmium- and reactive oxygen species-induced apoptosis

Abstract

Cadmium-mediated toxicity of cultured proximal tubule (PT) cells is associated with increased production of reactive oxygen species (ROS) and apoptosis. We found that cadmium-dependent apoptosis (Hoechst 33342 and annexin V assays) decreased with prolonged CdC12 (10 μM) application (controls: 2.4 ± 1.6%; 5 h: +5.1 ± 2.3%, 20 h: +5.7 ± 2.5%, 48 h: +3.3 ± 1.0% and 72 h: +2.1 ± 0.4% above controls), while cell proliferation was not affected. Reduction of apoptosis correlated with a time-dependent up- regulation of the drug efflux pump multidrug resistance P-glycoprotein (mdr1) in cadmium-treated cells (≃4-fold after 72 h), as determined by immunoblotting with the monoclonal antibody C219 and measurement of intracellular accumulation of the fluorescent probe calcein ± the mdr1 inhibitor PSC833 (0.5 μM). When mdr1 inhibitors (PSC833, cyclosporine A, verapamil) were transiently added to cells with mdr1 up-regulation by pretreatment for 72 h with cadmium, cadmium-induced apoptosis increased significantly and to a percentage similar to that obtained in cells with no mdr1 up-regulation (72-h cadmium: 5.2 ± 0.9% versus 72-h cadmium + 1-h PSC833: 7.2 ± 1.4%; p ≤ 0.001). Cadmium-induced apoptosis and mdr1 up- regulation depended on ROS, since co-incubation with the ROS scavengers N- acetylcysteine (15 mM) or pyrrolidine dithiocarbamate (0.1 mM) abolished both responses. Moreover, cadmium- and ROS-associated mdr1 up-regulation was linked to activation of the transcription factor NF-κB; N-acetylcysteine, pyrrolidine dithiocarbamate, and the IκB-α kinase inhibitor Bay 11-7082 (20 μM) prevented both, mdr1 overexpression and degradation of the inhibitory NF-κB subunit, IκB-α, induced by cadmium. The data show that 1) cadmium- mediated apoptosis in PT cells is associated with ROS production, 2) ROS increase mdr1 expression by a process involving NF-κB activation, and 3) mdr1 overexpression protects PT cells against cadmium-mediated apoptosis. These data suggest that mdr1 up-regulation, at least in part, provides anti- apoptotic protection for PT cells against cadmium-mediated stress.