Initial autophagic protection switches to disruption of autophagic flux by lysosomal instability during cadmium stress accrual in renal NRK-52E cells

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Abstract
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.
Funding Information
  • Deutsche Forschungsgemeinschaft (TH345/11-1)
  • MAX-KADE Foundation
  • Secretaría de Ciencia, Humanidades, Tecnología e Innovación (235537)
  • Canadian Network for Research and Innovation in Machining Technology, Natural Sciences and Engineering Research Council of Canada (CA)
  • PhD Program Universität Witten/Herdecke

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