Cadmium-mediated oxidative stress in kidney proximal tubule cells induces degradation of Na+/K(+)-ATPase through proteasomal and endo-/lysosomal proteolytic pathways.
The mechanisms of cadmium (Cd)-dependent nephrotoxicity were studied in a rat proximal tubule (PT) cell line. CdCl(2) (5 microM) increased the production of reactive oxygen species (ROS), as determined by oxidation of dihydrorhodamine 123 to fluorescent rhodamine 123. The levels of ubiquitin-conjugated cellular proteins were increased by Cd in a time-dependent fashion (maximum at 24-48 h). This was prevented by coincubation with the thiol antioxidant N-acetylcysteine (NAC, 15 mM). Cd also increased apoptosis (controls: 2.4+/-1.6%; Cd: 8.1+/-1.9%), but not necrosis (controls: 0.5 +/- 0.3%; Cd: 1.4+/- 2.5%). Exposure of PT cells with Cd decreased protein levels of the catalytic subunit (alpha1) of Na+/K(+)-ATPase, a long-lived membrane protein (t(1/2)>48 h) that drives reabsorption of ions and nutrients through Na(+)-dependent transporters in PT. Incubation of PT cells for 48 h with Cd decreased Na+/K(+)-ATPase alpha1-subunit, as determined by immunoblotting, by approximately 50%, and NAC largely prevented this effect. Inhibitors of the proteasome such as MG-132 (20 microM) or lactacystin (10 microM), as well as lysosomotropic weak bases such as chloroquine (0.2 mM) or NH(4)Cl (30 mM), significantly reduced the decrease of Na(+)/K(+)-ATPase alpha1-subunit induced by Cd, and in combination abolished the effect of Cd on Na+/K(+)-ATPase. Immunofluorescence labeling of Na+/K(+)-ATPase showed a reduced expression of the protein in the plasma membrane of Cd-exposed cells. After addition of lactacystin and chloroquine to Cd-exposed PT cells, immunoreactive material accumulated into intracellular vesicles. The data indicate that micromolar concentrations of Cd can increase ROS production and exert a toxic effect on PT cells. Oxidative damage increases the degradation of Na+/K(+)-ATPase through both the proteasomal and endo-/lysosomal proteolytic pathways. Degradation of oxidatively damaged Na+/K(+)-ATPase may contribute to the 'Fanconi syndrome'-like Na(+)-dependent transport defects associated with Cd-nephrotoxicity.
Thévenod
F
F
II. Department of Physiology, University of Saarland, D-66421 Homburg, Germany. frank.thevenod@med-rz.uni-sb.de
Friedmann
J M
JM
D000643:Ammonium Chloride / Q000494:pharmacology; D000818:Animals; D001692:Biological Transport; D002104:Cadmium / Q000633:toxicity*; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology; D002738:Chloroquine / Q000494:pharmacology; D003546:Cysteine Endopeptidases; D011992:Endosomes; D004795:Enzyme Stability; D007687:Kidney Tubules, Proximal / Q000166:cytology / Q000187:drug effects*; D007976:Leupeptins / Q000494:pharmacology; D008247:Lysosomes; D008954:Models, Biological; D009097:Multienzyme Complexes; D018384:Oxidative Stress / Q000502:physiology*; D046988:Proteasome Endopeptidase Complex; D051381:Rats; D011921:Rats, Inbred WKY; D017382:Reactive Oxygen Species / Q000378:metabolism; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*; D014452:Ubiquitins / Q000378:metabolism
D007976:Leupeptins; D009097:Multienzyme Complexes; D017382:Reactive Oxygen Species; D015842:Serine Proteinase Inhibitors; D014452:Ubiquitins; D002104:Cadmium; D000643:Ammonium Chloride; D002738:Chloroquine; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D000254:Sodium-Potassium-Exchanging ATPase; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde