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- Title
Alpha-Actinin-4 is a Possible Target Protein for Aristolochic Acid I in Human Kidney Cells In Vitro.
- Authors
Wang, Dan; Li, Xiao-Wei; Wang, Xuan; Tan, Huan-Ran; Jia, Yan; Yang, Li; Li, Xiao-Mei; Shang, Ming-Ying; Xu, Feng; Yang, Xing-Xin; Shoyama, Yukihiro; Cai, Shao-Qing
- 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 20g/mL AA-I for different periods of time (up to 12h), 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 -actinin-4 after digestion of the protein, and was confirmed by immunoblotting with a specific anti--actinin-4 MAb. This evidence shows, for the first time, that -actinin-4 is a protein targeted by AA-I in kidney cells. Our findings strongly suggest an association between -actinin-4 and AA-I nephrotoxic activity.
- Subjects
CARCINOGENS; CELL culture; ELECTROPHORESIS; HIGH performance liquid chromatography; IMMUNOHISTOCHEMISTRY; KIDNEYS; LIQUID chromatography; MASS spectrometry; MOLECULAR structure; MONOCLONAL antibodies; MUSCLE proteins; NEPHROTOXICOLOGY; PROBABILITY theory; RESEARCH funding; WESTERN immunoblotting; DATA analysis software; DESCRIPTIVE statistics; PRECIPITIN tests; IN vitro studies
- Publication
American Journal of Chinese Medicine, 2016, Vol 44, Issue 2, p291
- ISSN
0192-415X
- Publication type
Article
- DOI
10.1142/S0192415X16500178