Molybdenum and cadmium co-exposure induces CaMKKβ/AMPK/mTOR pathway mediated-autophagy by subcellular calcium redistribution in duck renal tubular epithelial cells

Excessive molybdenum (Mo) and cadmium (Cd) are toxic environmental pollutants. Our previous research confirmed excessive Mo and Cd co-induced calcium homeostasis disorder and autophagy in duck kidneys, but how calcium ion (Ca2+) regulates autophagy is unclear. The results revealed that the Mo- and/or Cd-induced cytosolic Ca2+ concentration ([Ca2+]c) increase mainly came from intracellular calcium stores. Mo and/or Cd caused mitochondrial Ca2+ content ([Ca2+]mit) and [Ca2+]c increase with endoplasmic reticulum (ER) Ca2+ content ([Ca2+]ER) decrease and upregulated calcium homeostasis-related factor expression levels, but 2-Aminoethoxydiphenyl borate (2-APB) reversed subcellular Ca2+ redistribution. Increased Phospholipase C (PLC) and inositol 1,4,5-trisphosphate (IP3) activities and inositol 1,4,5-trisphosphate receptor (IP3R) expression level were observed in Mo- and/or Cd-treated cells, which was reversed by the PLC inhibitor U-73122. 2-APB and 1,2-Bis (2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester (BAPTA-AM) addition mitigated [Ca2+]c and autophagy (variations in microtubule-associated protein light chain 3 (LC3), LC3B-II/LC3B-I, autophagy related 5 (ATG5), sequestosome-1(P62), programmed cell death-1 (Beclin-1) and Dynein expression levels, LC3 puncta, autophagosomes and acid vesicle organelles) under Mo and/or Cd treatment, respectively, while thapsigargin (TG) had the opposite impacts. Additionally, the calmodulin-dependent protein kinase kinase β (CaMKKβ) inhibitor STO-609 reversed the increased CaMKKβ, adenosine 5'-monophosphate-activated protein kinase (AMPK), Beclin-1, and LC3B-II/LC3B-I protein expression levels and reduced mammalian target of rapamycin (mTOR) and P62 protein expression levels in Mo- and/or Cd-exposed cells. Collectively, the results confirmed that [Ca2+]c overload resulted from PLC/IP3/IP3R pathway-mediated ER Ca2+ release, and then activated autophagy by the CaMKKβ/AMPK/mTOR pathway in Mo- and/or Cd-treated duck renal tubular epithelial cells.