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1 Physiology, University of the Saarland, D-66421 Homburg, Germany
2 Neuroanatomy, University of Goettingen, D-37075 Goettingen, Germany
3 Physiology, University of the Saarland, D-66421 Homburg, Germany; Physiology & Pathophysiology, University of Witten/Herdecke, D-58448 Witten, Germany
* To whom correspondence should be addressed. E-mail: frank.thevenod{at}uni-wh.de.
The S1-segment of the kidney proximal tubule (PT) is a major target of chronic cadmium (Cd2+ toxicity. Cd2+ complexed to the high-affinity metal-binding protein metallothionein (MT) is the major form by which Cd2+ is delivered to the kidneys in vivo. The mechanisms of CdMT uptake and the molecular processes underlying CdMT toxicity in PT cells (PTC) are not well understood. This study compares the effects of 10µM CdCl2 and Cd7MT-1 (MT-1 saturated with 10µM CdCl2) on 109Cd2+ uptake, viability and MT levels of cultured rat PTC. Apical transport of 109Cd2+ was measured in confluent monolayers, apoptosis and necrosis were monitored with Hoechst 33342 and ethidium bromide, respectively. Intracellular MT levels were assessed by immunofluorescence labeling of PTC and quantitative morphometry. 109Cd2+ uptake into PTC increased linearly with time reaching a plateau after 24 h. 109Cd7MT-1 uptake was initially delayed but reached a plateau of similar magnitude after 40 h. Cd2+ caused apoptosis, but not necrosis, within 4 h, which reached a maximum at 24 h and declined at 48-72 h. Cd7MT-1 also caused apoptosis, which developed after 36 h, reaching a similar magnitude as with Cd2+ after 48 h. Both Cd2+ and Cd7MT-1 exposure significantly increased intracellular MT immunoreactivity after 20 h and 4 h, respectively. The lysosomotropic weak base chloroquine (0.1mM) and the inhibitor of phosphatidyl 3-kinases LY294002 (5µM) prevented the increase of MT immunoreactivity in the presence of Cd7MT-1 and subsequent apoptosis, but had no effect on these parameters in PTC exposed to Cd2+. PTC exposed to 109Cd7MT-1 for 24 h accumulated 109Cd7MT-1 in membrane vesicles, which were enriched in the late endo-/lysosomal marker Lamp1 and to a lesser extent in the early endosomal marker Rab5a. Co-incubation of PTC with chloroquine or LY294002 abolished 109Cd7MT-1 accumulation into membrane vesicles. Thus, both Cd2+ and Cd7MT-1 induced apoptosis of cultured PTC, but with different kinetics. Development of apoptosis followed the uptake kinetics of Cd2+ and Cd7MT-1. Inhibitors of the endo-/lysosomal trafficking pathway prevented uptake of Cd7MT-1 into endo-/lysosomes and apoptosis, but had no effect on Cd2+-mediated apoptosis suggesting that apoptosis is triggered by the free Cd2+ ion in the cytosol of PTC, either by direct apical transport or by translocation of free Cd2+ from endo-/lysosomes subsequent to endocytosis of Cd7MT-1.
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