title: Cadmium disrupts autophagic flux by inhibiting cytosolic Ca2+-dependent autophagosome-lysosome fusion in primary rat proximal tubular cells.
abstract: Previous studies have shown that subcellular Ca2+redistribution is involved in Cd-induced autophagy inhibition in primary rat proximal tubular (rPT) cells, but the mechanism remains unclear. In this study, the status of autophagic flux was monitored by the GFP and RFP tandemly tagged LC3 method. Pharmacological inhibition of cytosolic Ca2+concentration ([Ca2+]c) with 2-APB or BAPTA-AM significantly alleviated Cd-elevated yellow puncta formation and restored Cd-inhibited red puncta formation, while thapsigargin (TG) had the opposite regulatory effect, demonstrating that Cd-induced [Ca2+]celevation inhibited the autophagic flux in rPT cells. Resultantly, Cd-induced autophagosomes accumulation was obviously modulated by 2-APB, BAPTA-AM and TG, respectively. Meanwhile, blockage of autophagosome-lysosome fusion and decreased recruitment of Rab7 to autophagosomes by Cd exposure was noticeably restored by 2-APB or BAPTA-AM, but co-treatment with Cd and TG further impaired Cd-induced autophagy arrest. Moreover, Cd-induced oxidative stress intimately correlated with cytosolic Ca2+mobilization, and N-acetylcysteine (NAC) markedly rescued Cd-blocked autophagosome-lysosome fusion and recruitment of Rab7 to autophagosomes in rPT cells, implying that Cd-induced autophagy inhibition was due to [Ca2+]celevation-triggered oxidative stress. In summary, these results suggest that Cd-mediated autophagy inhibition in rPT cells is dependent on cytosolic Ca2+overload. Elevation of [Ca2+]cinhibited the autophagosome-lysosome fusion to block the degradation of autophagosomes, which aggravated Cd-induced cytotoxicity in rPT cells.
authors.affiliation: College of Animal Science and Veterinary Medicine, Shandong Agricultural University, Daizong Road No. 61, Tai'an 271018, People's Republic of China.
authors.forename: Fei
authors.initials: F
authors.lastname: Liu
authors.affiliation: College of Animal Science and Veterinary Medicine, Shandong Agricultural University, Daizong Road No. 61, Tai'an 271018, People's Republic of China.
authors.forename: Xin-Yu
authors.initials: XY
authors.lastname: Wang
authors.affiliation: Beijing University of Agriculture, Beijing 102206, People's Republic of China.
authors.forename: Xu-Ping
authors.initials: XP
authors.lastname: Zhou
authors.affiliation: College of Veterinary Medicine, Yangzhou University, 12 East Wenhui Road, Yangzhou 225009, People's Republic of China. Electronic address: liuzongping@yzu.edu.cn.
authors.forename: Zong-Ping
authors.initials: ZP
authors.lastname: Liu
authors.affiliation: College of Animal Science and Veterinary Medicine, Shandong Agricultural University, Daizong Road No. 61, Tai'an 271018, People's Republic of China.
authors.forename: Xiang-Bin
authors.initials: XB
authors.lastname: Song
authors.affiliation: College of Animal Science and Veterinary Medicine, Shandong Agricultural University, Daizong Road No. 61, Tai'an 271018, People's Republic of China.
authors.forename: Zhen-Yong
authors.initials: ZY
authors.lastname: Wang
authors.affiliation: College of Animal Science and Veterinary Medicine, Shandong Agricultural University, Daizong Road No. 61, Tai'an 271018, People's Republic of China. Electronic address: wanglin2013@sdau.edu.cn.
authors.forename: Lin
authors.initials: L
authors.lastname: Wang
mesh_terms: D000818:Animals; D000071182:Autophagosomes / Q000187:drug effects; D001343:Autophagy / Q000187:drug effects*; D002104:Cadmium / Q000633:toxicity*; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D003600:Cytosol / Q000378:metabolism; D007687:Kidney Tubules, Proximal / Q000166:cytology*; D008247:Lysosomes / Q000187:drug effects; D008297:Male; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D020691:rab GTP-Binding Proteins / Q000378:metabolism; D000090802:rab7 GTP-Binding Proteins
chemical_list: D002104:Cadmium; D002118:Calcium; D008869:Microtubule-Associated Proteins; D016601:RNA-Binding Proteins; D020691:rab GTP-Binding Proteins; D000090802:rab7 GTP-Binding Proteins; C526109:Anxa3 protein, rat; C000716466:rab7 GTP-binding proteins, rat
keywords: Autophagosome-lysosome fusion; Autophagy; Cadmium; Cytosolic calcium overload; Oxidative stress; Proximal tubular cells