title: Alpha-Actinin-4 is a Possible Target Protein for Aristolochic Acid I in Human Kidney Cells In Vitro.
abstract: Aristolochic acid I (AA-I) is a strong nephrotoxin, carcinogen, and mutagen found in plants such as the Aristolochia species. The mechanisms underlying AA-I toxicity in the kidneys are poorly understood. In this study, we aimed to gain insight into the mechanism of AA-I nephrotoxicity by analyzing the uptake, subcellular distribution, and intracellular targets of AA-I in the human kidney cell line HK-2 using immunocytochemistry, immunoprecipitation, and LC-MS/MS. In HK-2 cells incubated with 20[Formula: see text][Formula: see text]g/mL AA-I for different periods of time (up to 12[Formula: see text]h), AA-I was detected by a specific monoclonal antibody (MAb) against AA-I, both in the cytoplasm and nuclei. Nuclear localization depended on the exposure time. A protein with the molecular weight of 100 kDa was immunoprecipitated with the anti-AA-I MAb from the AA-I-treated cell lysates and was identified by LC-MS/MS as [Formula: see text]-actinin-4 after digestion of the protein, and was confirmed by immunoblotting with a specific anti-[Formula: see text]-actinin-4 MAb. This evidence shows, for the first time, that [Formula: see text]-actinin-4 is a protein targeted by AA-I in kidney cells. Our findings strongly suggest an association between [Formula: see text]-actinin-4 and AA-I nephrotoxic activity.
authors.affiliation: * State Key Laboratory of Natural and Biomimetic Drugs.|† Department of Chemical Biology, School of Pharmaceutical Sciences, Peking University, Beijing 100191, P.R. China.
authors.forename: Dan
authors.initials: D
authors.lastname: Wang
authors.affiliation: * State Key Laboratory of Natural and Biomimetic Drugs.|† Department of Chemical Biology, School of Pharmaceutical Sciences, Peking University, Beijing 100191, P.R. China.
authors.forename: Xiao-Wei
authors.initials: XW
authors.lastname: Li
authors.affiliation: † Department of Chemical Biology, School of Pharmaceutical Sciences, Peking University, Beijing 100191, P.R. China.
authors.forename: Xuan
authors.initials: X
authors.lastname: Wang
authors.affiliation: ‡ Department of Pharmacology, Peking University, Health Science Center, Beijing 100191, P.R. China.
authors.forename: Huan-Ran
authors.initials: HR
authors.lastname: Tan
authors.affiliation: § Renal Division, Department of Medicine, Peking University First Hospital, Beijing 100034, P.R. China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Jia
authors.affiliation: § Renal Division, Department of Medicine, Peking University First Hospital, Beijing 100034, P.R. China.
authors.forename: Li
authors.initials: L
authors.lastname: Yang
authors.affiliation: § Renal Division, Department of Medicine, Peking University First Hospital, Beijing 100034, P.R. China.
authors.forename: Xiao-Mei
authors.initials: XM
authors.lastname: Li
authors.affiliation: * State Key Laboratory of Natural and Biomimetic Drugs.
authors.forename: Ming-Ying
authors.initials: MY
authors.lastname: Shang
authors.affiliation: * State Key Laboratory of Natural and Biomimetic Drugs.
authors.forename: Feng
authors.initials: F
authors.lastname: Xu
authors.affiliation: * State Key Laboratory of Natural and Biomimetic Drugs.
authors.forename: Xing-Xin
authors.initials: XX
authors.lastname: Yang
authors.affiliation: ¶ Department of Pharmacognosy, Faculty of Pharmaceutical Sciences, Nagasaki International University, Sasebo, Nagasaki 859-3298, Japan.
authors.forename: Yukihiro
authors.initials: Y
authors.lastname: Shoyama
authors.affiliation: * State Key Laboratory of Natural and Biomimetic Drugs.
authors.forename: Shao-Qing
authors.initials: SQ
authors.lastname: Cai
mesh_terms: D000185:Actinin / Q000378:metabolism*; D034341:Aristolochic Acids / Q000378:metabolism* / Q000633:toxicity*; D002478:Cells, Cultured; D002853:Chromatography, Liquid; D006801:Humans; D007150:Immunohistochemistry; D047468:Immunoprecipitation; D007668:Kidney / Q000166:cytology / Q000378:metabolism*; D053719:Tandem Mass Spectrometry
chemical_list: C111453:ACTN4 protein, human; D034341:Aristolochic Acids; D000185:Actinin; C000228:aristolochic acid I
keywords: -Actinin-4; Aristolochic Acid; HK-2 Cells; IP-MS/MS; Immunocytochemistry; Immunoprecipitation (IP); Monoclonal Antibody