Up-regulation of multidrug resistance P-glycoprotein via nuclear factor-kappaB activation protects kidney proximal tubule cells from cadmium- and reactive oxygen species-induced apoptosis.
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 CdCl(2) (10 microM) 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 ( approximately 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 microM). 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 < or = 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-kappaB; N-acetylcysteine, pyrrolidine dithiocarbamate, and the IkappaB-alpha kinase inhibitor Bay 11-7082 (20 microM) prevented both, mdr1 overexpression and degradation of the inhibitory NF-kappaB subunit, IkappaB-alpha, 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-kappaB 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.
Thévenod
F
F
Department of Physiology II, University of Saarland, 66421 Homburg, Germany. frank.thevenod@med-rz.uni-sb.de
Friedmann
J M
JM
Katsen
A D
AD
Hauser
I A
IA
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D017304:Annexin A5 / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects* / Q000235:genetics; D002104:Cadmium / Q000494:pharmacology*; D019256:Cadmium Chloride / Q000494:pharmacology; D002121:Calcium Channel Blockers / Q000494:pharmacology; D002460:Cell Line; D016572:Cyclosporine / Q000494:pharmacology; D003524:Cyclosporins / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005452:Fluoresceins / Q000378:metabolism; D007687:Kidney Tubules, Proximal / Q000378:metabolism* / Q000473:pathology; D007700:Kinetics; D016328:NF-kappa B / Q000494:pharmacology*; D009336:Necrosis; D018384:Oxidative Stress; D051381:Rats; D011921:Rats, Inbred WKY; D017382:Reactive Oxygen Species; D013997:Time Factors; D015854:Up-Regulation; D014700:Verapamil / Q000494:pharmacology
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D017304:Annexin A5; D002121:Calcium Channel Blockers; D003524:Cyclosporins; D004791:Enzyme Inhibitors; D005452:Fluoresceins; D016328:NF-kappa B; D017382:Reactive Oxygen Species; D002104:Cadmium; D016572:Cyclosporine; D014700:Verapamil; D019256:Cadmium Chloride; C070272:valspodar; C007740:fluorexon