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

Abstract

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.

Caspase-dependent and -independent pathways for cadmium-induced apoptosis in cultured kidney proximal tubule cells

“…The activation of caspases after 24-h exposure to 10 M Cd 2ϩ occurs concomitantly with the observation of cytochrome c release (Fig. 2) and correlates with the kinetics of accumulation of 109 Cd 2ϩ by PT cells (11). We have recently shown that direct application of 2-10 M Cd 2ϩ to isolated kidney cortex mitochondria triggers mitochondrial swelling and the release of cytochrome c (20).…”

Cd2+-induced cytochromecrelease in apoptotic proximal tubule cells: role of mitochondrial permeability transition pore and Ca2+uniporter

“…Here, we demonstrate that 10 M Cd 2ϩ induces apoptosis of rat kidney PT cells at 6 and 24 h whereas Cyt c release is observed after 24 h only, when intracellular Cd 2ϩ concentrations may be high enough to interact directly with mitochondria (16). Cyt c release may be partially mediated by direct effects of Cd 2ϩ on mitochondrial permeability pathways; Cd 2ϩ induces swelling (EC 50 ϳ 9 M) and release of Cyt c from energized mitochondria and mitoplasts isolated from rat kidney cortex.…”

“…After degradation of GSH by ␥-glutamyltranspeptidase bound to the apical plasma membrane of PT cells (10), Cd 2ϩ may also be taken up as a free ion through yet unknown apical transport pathways. In a cell line derived from the S1 segment of rat PT, we have previously demonstrated that Cd 2ϩ (10 M) is taken up by apical pathways, which operate with slow kinetics and saturate at 24 h (16).…”

Lipocalin-2 (24p3/Neutrophil Gelatinase-associated Lipocalin (NGAL)) Receptor Is Expressed in Distal Nephron and Mediates Protein Endocytosis

“…Therefore we used the property of MT as a high-affinity Cd 2ϩ -binding protein to further characterize RME via 24p3R. In PT cells, endocytosed Cd 2ϩ 7 -MT is degraded in lysosomes and free Cd 2ϩ transported via DMT-1 into the cytosol, which induces cell death (39,58). Indeed, a similar mechanism was operative in 24p3R-transfected CHO cells and mDCT209 cells and Cd 2ϩ 7 -MT-induced cell death could be prevented by co-incubation with 24p3 (Fig.…”