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

Abstract

The renal proximal tubule (PT) is the major target of cadmium (Cd) toxicity where Cd 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-induced nephrotoxicity remains unsettled due to contradictory results and lack of evidence for autophagic machinery damage by Cd. Cd-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 Cd (5-10 µM) after 1 h, accompanied by ER stress (increased p-PERK, p-eIF2α, CHOP). Cd 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, Cd (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 Cd-induced cell viability loss. The autophagosome-lysosome fusion inhibitor liensinine (5 μM) increases CHOP and Bax/Bcl-2-dependent apoptosis by low Cd stress, but not by high Cd. Lysosomal instability by Cd (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 Cd on autophagy: Low Cd stress rapidly activates autophagy counteracting damage but Cd stress accrual disrupts autophagic flux and lysosomal stability, possibly resulting in lysosomal cell death.

Activation of Ca2+-sensing receptor as a protective pathway to reduce Cadmium-induced cytotoxicity in renal proximal tubular cells

“…S2c , it showed these two reagents reduced Cd-induced ROS generation but aggravated Cd-induced cell death. The rate of autophagic flux can be approximated by the amount of LC3-II with degradation of p62, whereas both of them are elevated when autophagic flux was impaired 6 . Cd increased the expressions of LC3-II and p62, which was aggravated by CQ (Fig.…”

“…In kidney, the renal proximal tubule is the first opportunistic site of Cd reabsorption following plasma filtration in the glomerulus 4 , 5 . Therefore, the renal proximal tubular cells are excellent model to study Cd-induced cytotoxicity and renoprotective strategies 6 , 7 . Exposure to Cd could induce various cellular responses such as carcinogenesis, necrosis, apoptosis, proliferation and autophagy 8 – 10 .…”

“…Additionally, intracellular Ca 2+ signaling pathway also mediated Cd-induced autophagy 17 , which played a renoprotective role in both acute kidney injury and chronic kidney diseases 31 , and was indicated as a protective way against Cd-induced apoptosis in lung epithelial fibroblast cells WI38 32 , pheochromocytoma cell line PC-12 33 , and rat renal tubular cells 34 . However, initial autophagic protection would switch to disruption of autophagic flux and result in cell death during Cd stress accrual in renal NRK-52E cells 6 . Therefore, it is important to understand the roles of intracellular Ca 2+ signaling pathways in Cd-induced apoptosis and autophagy, and their relationship in renal tubular cells.…”

Trehalose protects against cadmium-induced cytotoxicity in primary rat proximal tubular cells via inhibiting apoptosis and restoring autophagic flux

“… 45 Immunoblot analysis of LC3 and p62, two autophagy marker proteins, has been widely used to monitor autophagic flux. 46 It is a consensus that enhanced p62 protein level has been regarded as an indicator for blockage of autophagic flux, 46 , 47 and the net amount of cytosolic LC3-II is a critical hallmark for monitoring autophagy in mammalian cells. 46 We previously proved that Cd-induced inhibition of autophagic flux results from overall autophagic degradation rather than autophagosome formation.…”

Stem cell-based test methods

“…Recently, Agapios Sachinidis and colleagues from the University of Cologne published a review about possibilities and limitations of stem cell-based test methods in pharmacology and toxicology (Sachinidis et al, 2019[ 18 ]). In recent years, much progress has been achieved concerning in vitro techniques of liver (Godoy et al, 2013[ 7 ]; Grinberg et al, 2014[ 10 ]; Leist et al, 2017[ 15 ]; Ghallab et al, 2016[ 6 ]), kidney (Sjögren et al, 2018[ 24 ]; Jiang et al, 2018[ 11 ]; Su et al, 2016[ 25 ]; Valente et al, 2012[ 26 ]; Lee et al, 2017[ 14 ]), neuronal (Keil et al, 2018[ 12 ]; Yang et al, 2018[ 28 ]; Colaianna et al, 2017[ 5 ]; Sisnaiske et al, 2014[ 23 ]) and developmental toxicity (Adam et al, 2019[ 2 ]; Bridges et al, 2019[ 3 ]; Abbott, 2019[ 1 ]). Particularly, in developmental and reproductive toxicity testing, large numbers of animals are needed for analysis of a single compound (Krug et al, 2013[ 13 ]).…”