title: Heat shock-induced protection of renal proximal tubular epithelial cells from cold storage and rewarming injury.
abstract: Cold storage and reperfusion injury to transplanted kidneys contributes to increased incidence of delayed graft function and may have a negative impact on graft survival. This study examined the mechanisms by which previous heat shock protects against cell death in an in vitro model of kidney storage. Cold storage is mimicked by incubating human renal proximal tubular epithelial (HK-2) cells in University of Wisconsin solution at 4 degrees C with and without subsequent rewarming. Heat shock was induced by incubation of cells at 42 degrees C for 1 h. Altered protein expression was measured by Western blot, and cell viability and apoptosis were measured by propidium iodide DNA staining using flow cytometry. The specific role of heat-shock protein 70 (HSP-70) was determined both by siRNA knockdown and by stable overexpression approaches. Cold storage and rewarming-induced cell death was associated with decreased expression of HSP-70, HSP-90, HSP-27, and Bcl-2. Previous heat shock significantly reduced HK-2 cell death after cold storage and rewarming and was associated with the maintenance of HSP-70, HSP-27, and Bcl-2 protein levels. Blocking heat stress-induced HSP-70 with siRNA did not significantly block the protective effect of heat stress against cold storage and rewarming cell death; however, overexpression of HSP-70 protected HK-2 cells from this stress. It is concluded that previous heat shock protects HK-2 cells from cold storage and rewarming injury. siRNA inhibition of HSP-70 induction did not block the protective effect of heat shock, indicating that HSP-70 is not essential to the heat stress-induced protective effect reported in this study.
authors.affiliation: UCD School of Medicine and Medical Science, UCD Conway Institute of Biomolecular and Biomedical Research, Mater Misericordiae University Hospital, University College Dublin, Belfield, Dublin 4, Ireland.
authors.forename: Declan A
authors.initials: DA
authors.lastname: Healy
authors.forename: Padraig J
authors.initials: PJ
authors.lastname: Daly
authors.forename: Neil G
authors.initials: NG
authors.lastname: Docherty
authors.forename: Madeline
authors.initials: M
authors.lastname: Murphy
authors.forename: John M
authors.initials: JM
authors.lastname: Fitzpatrick
authors.forename: R William G
authors.initials: RW
authors.lastname: Watson
mesh_terms: D015153:Blotting, Western; D016923:Cell Death; D002478:Cells, Cultured; D015925:Cryopreservation / Q000379:methods*; D004847:Epithelial Cells / Q000473:pathology*; D050884:HSP72 Heat-Shock Proteins / Q000378:metabolism; D006360:Heat-Shock Proteins / Q000378:metabolism*; D018869:Heat-Shock Response; D006801:Humans; D007687:Kidney Tubules, Proximal / Q000166:cytology*; D009336:Necrosis / Q000473:pathology; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D012313:RNA / Q000032:analysis; D015427:Reperfusion Injury / Q000517:prevention & control*; D017694:Rewarming; D012680:Sensitivity and Specificity
chemical_list: D050884:HSP72 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D012313:RNA