Initial autophagic protection switches to disruption of autophagic flux by lysosomal instability during cadmium stress accrual in renal NRK-52E cells.
The renal proximal tubule (PT) is the major target of cadmium (Cd2+) toxicity where Cd2+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 Cd2+-induced nephrotoxicity remains unsettled due to contradictory results and lack of evidence for autophagic machinery damage by Cd2+. Cd2+-induced autophagy in rat kidney PT cell line NRK-52E and its role in cell death was investigated. Increased LC3-II and decreased p62 as autophagy markers indicate rapid induction of autophagic flux by Cd2+(5-10 µM) after 1 h, accompanied by ER stress (increased p-PERK, p-eIF2α, CHOP). Cd2+exposure exceeding 3 h results in p62/LC3-II accumulation, but diminished effect of lysosomal inhibitors (bafilomycin A1, pepstatin A +E-64d) on p62/LC3-II levels, indicating decreased autophagic flux and cargo degradation. At 24 h exposure, Cd2+(5-25 µM) activates intrinsic apoptotic pathways (Bax/Bcl-2, PARP-1), which is not evident earlier (≤6 h) although cell viability by MTT assay is decreased. Autophagy inducer rapamycin (100 nM) does not overcome autophagy inhibition or Cd2+-induced cell viability loss. The autophagosome-lysosome fusion inhibitor liensinine (5 μM) increases CHOP and Bax/Bcl-2-dependent apoptosis by low Cd2+stress, but not by high Cd2+. Lysosomal instability by Cd2+(5 μM; 6 h) is indicated by increases in cellular sphingomyelin and membrane fluidity and decreases in cathepsins and LAMP1. The data suggest dual and temporal impact of Cd2+on autophagy: Low Cd2+stress rapidly activates autophagy counteracting damage but Cd2+stress accrual disrupts autophagic flux and lysosomal stability, possibly resulting in lysosomal cell death.
Department of Physiology, Pathophysiology and Toxicology, Centre for Biomedical Education and Research (ZBAF), Witten/Herdecke University, Witten, Germany. wing-kee.lee@uni-wh.de.
W-K
WK
Lee
Department of Physiology, Pathophysiology and Toxicology, Centre for Biomedical Education and Research (ZBAF), Witten/Herdecke University, Witten, Germany.
S
S
Probst
Department of Physiology, Pathophysiology and Toxicology, Centre for Biomedical Education and Research (ZBAF), Witten/Herdecke University, Witten, Germany.|Department of Toxicology, Cinvestav-IPN, México D.F., Mexico.
M P
MP
Santoyo-Sánchez
Department of Physiology, Pathophysiology and Toxicology, Centre for Biomedical Education and Research (ZBAF), Witten/Herdecke University, Witten, Germany.
W
W
Al-Hamdani
Department of Physiology, Pathophysiology and Toxicology, Centre for Biomedical Education and Research (ZBAF), Witten/Herdecke University, Witten, Germany.
I
I
Diebels
Department of Physiology, Pathophysiology and Toxicology, Centre for Biomedical Education and Research (ZBAF), Witten/Herdecke University, Witten, Germany.
J-K
JK
von Sivers
Department of Biological Sciences, University of Calgary, Calgary, AB, T2N 1N4, Canada.
E
E
Kerek
Department of Biological Sciences, University of Calgary, Calgary, AB, T2N 1N4, Canada.
E J
EJ
Prenner
Department of Physiology, Pathophysiology and Toxicology, Centre for Biomedical Education and Research (ZBAF), Witten/Herdecke University, Witten, Germany. frank.thevenod@uni-wh.de.
F
F
Thévenod
D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology; D001343:Autophagy / Q000187:drug effects*; D002104:Cadmium / Q000008:administration & dosage / Q000633:toxicity*; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D059865:Endoplasmic Reticulum Stress / Q000187:drug effects; D007546:Isoquinolines / Q000494:pharmacology; D007687:Kidney Tubules, Proximal / Q000166:cytology; D051907:Lysosomal Membrane Proteins / Q000378:metabolism; D008247:Lysosomes / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D010636:Phenols / Q000494:pharmacology; D051381:Rats; D020123:Sirolimus / Q000494:pharmacology; D056811:Unfolded Protein Response / Q000187:drug effects; D052118:Lysosomal-Associated Membrane Protein 1
D002104:Cadmium; D007546:Isoquinolines; D051907:Lysosomal Membrane Proteins; D008869:Microtubule-Associated Proteins; D010636:Phenols; D020123:Sirolimus; C526109:Anxa3 protein, rat; C489117:Lamp1 protein, rat; D052118:Lysosomal-Associated Membrane Protein 1; C080095:liensinine
Laurdan; Lysenin; Lysosomal membrane permeabilization; Transition metal toxicity; Unfolded protein response