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Initial Autophagic Protection Switches to Disruption of Autophagic Flux by Lysosomal Instability during Cadmium Stress Accrual in Renal NRK‐52E Cells
Author(s) -
Thevenod Frank,
Lee WingKee,
SantoyoSánchez Mitzi P.,
Probst Stephanie,
Kerek Evan,
Prenner Elmar J
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.470.8
Subject(s) - autophagy , viability assay , microbiology and biotechnology , programmed cell death , bafilomycin , atg5 , lysosome , unfolded protein response , apoptosis , lamp1 , thapsigargin , autophagosome , endoplasmic reticulum , atf6 , biology , chemistry , biochemistry , enzyme
The renal proximal tubule (PT) is the major target of cadmium (Cd 2+ ) toxicity where Cd 2+ causes stress and apoptosis. Autophagy is induced by cell stress, e.g. endoplasmic reticulum (ER) stress, and may contribute to cell survival or death. The role of autophagy in Cd 2+ ‐induced nephrotoxicity remains unsettled due to contradictory results and lack of evidence for autophagic machinery damage by Cd 2+ . Cd 2+ ‐induced autophagy in the rat kidney PT cell line NRK‐52E and its role in cell death was investigated. Autophagy markers LC3‐II and p62 indicate rapid induction of autophagic flux by Cd 2+ (5–10μM) after 1h, accompanied by ER stress (increased p‐PERK, p‐eIF2α, CHOP). Cd 2+ exposure exceeding 3h results in p62/LC3‐II accumulation, but diminished effect of lysosomal inhibitors (bafilomycin A1, pepstatin A+E64d) on p62/LC3‐II levels, indicating decreased autophagic flux and cargo degradation. At 24h exposure, Cd 2+ (5–25μM) activates intrinsic apoptotic pathways (Bax/Bcl‐2, PARP1), which are not evident earlier (≤ 6h) despite decreased cell viability by MTT assay. Autophagy inducer rapamycin (100nM) does not overcome autophagy inhibition or Cd 2+ ‐induced cell viability loss. The autophagosome‐lysosome fusion inhibitor liensinine (5μM) increases CHOP and Bax/Bcl‐2 dependent apoptosis by low Cd 2+ stress, but not by high Cd 2+ . Lysosomal instability by Cd 2+ (5μM; 6h) is indicated by increased cellular sphingomyelin and membrane fluidity and decreased cathepsins and LAMP1. The data suggest dual and temporal impact of Cd 2+ on autophagy: Low Cd 2+ stress rapidly activates autophagy counteracting damage but Cd 2+ stress accrual disrupts autophagic flux and lysosomal stability, possibly resulting in lysosomal cell death. Support or Funding Information Center for Biomedical Training and Research (ZBAF) of the University of Witten/Herdecke (F.T.), Deutsche Forschungsgemeinschaft (TH345/11‐1 to F.T.), Max Kade Foundation (W.‐K.L.), CONACYT (Consejo Nacional de Ciencia y Tecnología, Mexico) (235537 to M.P.S.‐S.), and NSERC Discovery grant (E.J.P.)