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. CdCl2 (5 mu M) increased the production of reactive oxygen species (ROS), as determined by oxidation of dihydrorho damine 123 to fluorescent rhodamine 123. The levels of ubiquitin-conjugated cellular proteins were increased by Cd in a time-dependent fashion (maximu m at 24-48 h). This was prevented by coincubation with the thiol antioxidan t 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 cat alytic subunit (alpha 1) of Na+/ K+-ATPase, a long-lived membrane protein ( t(1/2) >48 h) that drives reabsorption of ions and nutrients through Na+-de pendent transporters in PT, Incubation of PT cells for 48 h with Cd decreas ed Na+/ K+-ATPase alpha 1-subunit, as determined by immunoblotting, by appr oximate to 50%, and NAC largely prevented this effect, Inhibitors of the pr oteasome such as MG-132 (20 mu M) or lactacystin (10 mu M), as well as lyso somotropic weak bases such as chloroquine (0.2 mM) or NH4Cl (30 mM), signif icantly reduced the decrease of Na+/K+-ATPase alpha 1-subunit induced by Cd , and in combination abolished the effect of Cd on Na+/ K+-ATPase. Immunofl uorescence labeling of Na+/ K+-ATPase showed a reduced expression of the pr otein in the plasma membrane of Cd-exposed cells. After addition of lactacy stin and chloroquine to Cd-exposed PT cells, immunoreactive material accumu lated into intracellular vesicles. The data indicate that micromolar concen trations of Cd can increase ROS production and exert a toxic effect on PT c ells. Oxidative damage increases the degradation of Na+/K+-ATPase through b oth the proteasomal and endo-/lysosomal proteolytic pathways. Degradation o f oxidatively damaged Na+/K+-ATPase may contribute to the 'Fanconi syndrome '-like Nat-dependent transport defects associated with Cd-nephrotoxicity. - Thevenod, F., Friedmann, J. M. Cadmium-mediated oxidative stress in kidney proximal tubule cells induces degradation of Na+/K+-ATPase through proteaso mal and endo-/lysosomal proteolytic pathways.