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 microM CdCl2 or Cd7MT-1 (MT-1 saturated with 10 microM CdCl2) on 109Cd2+ uptake, viability, and MT levels of cultured rat PT cells were investigated. Apical 109Cd2+ uptake was measured in confluent monolayers, apoptosis was assessed with Hoechst 33342, and intracellular MT levels were monitored by immunofluorescence and quantitative morphometry. 109Cd2+ uptake into PTC increased over time and plateaued at 24 h. 109Cd7MT-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 109Cd7MT-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.
Erfurt
Cornelia
C
Dept. of Physiology & Pathophysiology, Univ. of Witten/Herdecke, D-58448 Witten, Germany.
Roussa
Eleni
E
Thévenod
Frank
F
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D002104:Cadmium / Q000494:pharmacology*; D002478:Cells, Cultured; D004705:Endocytosis / Q000502:physiology; D011992:Endosomes / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D007424:Intracellular Fluid / Q000737:chemistry; D007687:Kidney Tubules, Proximal / Q000187:drug effects* / Q000378:metabolism* / Q000473:pathology; D008247:Lysosomes / Q000378:metabolism; D008668:Metallothionein / Q000494:pharmacology*; D051381:Rats; D013997:Time Factors
C116509:cadmium-metallothionein complex; D002104:Cadmium; D008668:Metallothionein