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Alpha-Actinin-4 is a Possible Target Protein for Aristolochic Acid I in Human Kidney Cells In Vitro.

Authors
  • Wang, Dan1, 2
  • Li, Xiao-Wei1, 2
  • Wang, Xuan2
  • Tan, Huan-Ran3
  • Jia, Yan4
  • Yang, Li4
  • Li, Xiao-Mei4
  • Shang, Ming-Ying1
  • Xu, Feng1
  • Yang, Xing-Xin1
  • Shoyama, Yukihiro5
  • Cai, Shao-Qing1
  • 1 * State Key Laboratory of Natural and Biomimetic Drugs.
  • 2 † Department of Chemical Biology, School of Pharmaceutical Sciences, Peking University, Beijing 100191, P.R. China. , (China)
  • 3 ‡ Department of Pharmacology, Peking University, Health Science Center, Beijing 100191, P.R. China. , (China)
  • 4 § Renal Division, Department of Medicine, Peking University First Hospital, Beijing 100034, P.R. China. , (China)
  • 5 ¶ Department of Pharmacognosy, Faculty of Pharmaceutical Sciences, Nagasaki International University, Sasebo, Nagasaki 859-3298, Japan. , (Japan)
Type
Published Article
Journal
The American journal of Chinese medicine
Publication Date
Jan 01, 2016
Volume
44
Issue
2
Pages
291–304
Identifiers
DOI: 10.1142/S0192415X16500178
PMID: 27080942
Source
Medline
Keywords
License
Unknown

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.

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