Activation of protein kinase C isozymes protects LLCPK1 cells from H2O2 induced necrotic cell death

Am J Nephrol. Nov-Dec 2003;23(6):380-9. doi: 10.1159/000073984. Epub 2003 Oct 9.

Abstract

Background/aims: We have previously reported that ischemia/reperfusion injury (IRI) to the kidney leads to induced expression of RACK1 and changes in the level of expression and subcellular distribution of PKC isozymes alpha, betaII and zeta. In order to further define the role of PKC isozymes in IRI we investigated the effect of activation or inhibition of the isozymes on cytotoxicity mediated by H(2)O(2) in LLCPK(1) cells.

Methods: Cytotoxicity was analyzed by Trypan blue assay and LDH release assay. Translocation of PKC isozymes postinjury in LLCPK1 cells was analyzed by immunostaining and Western blot analysis.

Results: Western blot analysis showed that the expression of PKC-alpha was up-regulated in a triphasic pattern with the initial induction within the first 10 min of injury followed by higher levels of expression at 2 and 24 h postinjury. The expression of PKC-zeta was highly induced within the first 15 min of injury but its expression was down-regulated to that of normal levels by 30 min postinjury. Immunocytochemistry showed that both PKC-alpha and PKC-zeta translocated to the nucleus and perinuclear region during H(2)O(2) treatment. Following injury, PKC-alpha expression was localized to the nuclear membrane at earlier time points but a translocation to the nucleus occurred at later time points. PKC-zeta translocated to nucleus at 30 minutes post injury and relocated back to the nuclear membrane at later time points.

Conclusion: These data suggest that activation of PKC-alpha and PKC-zeta is involved in the H(2)O(2) induced injury of LLCPK1 cells.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Blotting, Western
  • Cell Death
  • Cytoprotection
  • Enzyme Activation
  • Enzyme Inhibitors / pharmacology
  • Hydrogen Peroxide / toxicity
  • Isoenzymes / metabolism
  • LLC-PK1 Cells / drug effects
  • LLC-PK1 Cells / enzymology*
  • LLC-PK1 Cells / pathology
  • Necrosis
  • Oxidants / toxicity
  • Protein Kinase C / antagonists & inhibitors
  • Protein Kinase C / metabolism*
  • Swine

Substances

  • Enzyme Inhibitors
  • Isoenzymes
  • Oxidants
  • Hydrogen Peroxide
  • Protein Kinase C