Apoptosis by Cd2+ or CdMT in proximal tubule cells:: different uptake routes and permissive role of endo/lysosomal CdMT uptake

The mechanisms of cadmium-metallothionein ( CdMT) uptake and toxicity in proximal tubule ( PT) cells are not well understood. The effects of 10 muM CdCl2 or Cd7MT- 1 (MT-1 saturated with 10 muM CdCl2) on Cd-109(2+) uptake, viability, and MT levels of cultured rat PT cells were investigated. Apical Cd-109(2+) uptake was measured in confluent monolayers, apoptosis was assessed with Hoechst 33342, and intracellular MT levels were monitored by immunofluorescence and quantitative morphometry. Cd-109(2+) uptake into PTC increased over time and plateaued at 24 h. (Cd7MT)-Cd-109-1 uptake was delayed but reached a similar magnitude after 40 h. With Cd2+, apoptosis occurred within 4 h, peaked at 24 h, and declined at 48 - 72 h. Cd7MT-1 induced apoptosis after 24 - 36 h, reaching similar levels as with Cd2+ after 48 h. Cd2+ and Cd7MT-1 significantly increased intracellular MT immunoreactivity after 20 and 4 h, respectively. The weak base chloroquine and the inhibitor of phosphatidylinositol 3- kinases, LY-294002, selectively inhibited the effects of Cd7MT-1 on MT immunoreactivity and apoptosis. PT cells accumulated (Cd7MT)-Cd-109-1 in membrane vesicles associated with the late endo/lysosomal marker LAMP1 but less with the early endosomal marker Rab5a, which was abolished by chloroquine or LY-294002. Thus development of apoptosis followed the uptake kinetics of Cd2+ and Cd7MT-1. Endo/ lysosomal inhibitors prevented uptake of Cd7MT-1 into endo/ lysosomes and apoptosis but had no effect on these parameters with Cd2+, suggesting that apoptosis of PT cells is triggered by free cytosolic Cd2+, either by direct apical transport or by translocation of free Cd2+ from endo/ lysosomes after endocytosis of Cd7MT-1.