title: Disruption of gap junctions attenuates aminoglycoside-elicited renal tubular cell injury.
abstract: OBJECTIVE
  Gap junctions play important roles in the regulation of cell phenotype and in determining cell survival after various insults. Here, we investigated the role of gap junctions in aminoglycoside-induced injury to renal tubular cells.
  
  
  METHODS
  Two tubular epithelial cell lines NRK-E52 and LLC-PK1 were compared for gap junction protein expression and function by immunofluorescent staining, Western blot and dye transfer assay. Cell viability after exposure to aminoglycosides was evaluated by WST assay. Gap junctions were modulated by transfection of the gap junction protein, connexin 43 (Cx43), use of Cx43 siRNA and gap junction inhibitors.
  
  
  RESULTS
  NRK-E52 cells expressed abundant Cx43 and were functionally coupled by gap junctional intercellular communication (GJIC). Exposure of NRK-E52 cells to aminoglycosides, G418 and hygromycin, increased Cx43 phosphorylation and GJIC. The aminoglycosides also decreased cell viability that was prevented by gap junction inhibitors and Cx43 siRNA. LLC-PK1 cells were gap junction-deficient and resistant to aminoglycoside-induced cytotoxicity. Over-expression of a wild-type Cx43 converted LLC-PK1 cells to a drug-sensitive phenotype. The gap junction inhibitor alpha-glycyrrhetinic acid (alpha-GA) activated Akt in NRK-E52 cells. Inhibition of the Akt pathway enhanced cell toxicity to G418 and abolished the protective effects of alpha-GA. In addition, gentamycin-elicited cytotoxicity in NRK-E52 cells was also significantly attenuated by alpha-GA.
  
  
  CONCLUSIONS
  Gap junctions contributed to the cytotoxic effects of aminoglycosides. Modulation of gap junctions could be a promising approach for prevention and treatment of aminoglycoside-induced renal tubular cell injury.
authors.affiliation: Department of Molecular Signaling, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, Chuo, Yamanashi, Japan. yao@yamanashi.ac.jp
authors.forename: Jian
authors.initials: J
authors.lastname: Yao
authors.forename: Tao
authors.initials: T
authors.lastname: Huang
authors.forename: Xin
authors.initials: X
authors.lastname: Fang
authors.forename: Yuan
authors.initials: Y
authors.lastname: Chi
authors.forename: Ying
authors.initials: Y
authors.lastname: Zhu
authors.forename: Yigang
authors.initials: Y
authors.lastname: Wan
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Matsue
authors.forename: Masanori
authors.initials: M
authors.lastname: Kitamura
mesh_terms: D000617:Aminoglycosides / Q000633:toxicity*; D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D015153:Blotting, Western; D002450:Cell Communication / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002934:Cinnamates / Q000633:toxicity; D018031:Connexin 43 / Q000235:genetics / Q000378:metabolism; D019610:Cytoprotection; D004305:Dose-Response Relationship, Drug; D004847:Epithelial Cells / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D005455:Fluorescent Antibody Technique; D017629:Gap Junctions / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D005839:Gentamicins / Q000633:toxicity; D006034:Glycyrrhetinic Acid / Q000494:pharmacology*; D006921:Hygromycin B / Q000031:analogs & derivatives / Q000633:toxicity; D007684:Kidney Tubules / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D018374:LLC-PK1 Cells; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D034622:RNA Interference; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013552:Swine; D014162:Transfection; D000077191:Wortmannin
chemical_list: D000617:Aminoglycosides; D000730:Androstadienes; D002934:Cinnamates; D018031:Connexin 43; D005839:Gentamicins; D047428:Protein Kinase Inhibitors; D011993:Recombinant Fusion Proteins; D006921:Hygromycin B; C026273:hygromycin A; C010680:antibiotic G 418; D051057:Proto-Oncogene Proteins c-akt; D006034:Glycyrrhetinic Acid; D000077191:Wortmannin