Apoptosis versus necrosis during cold storage and rewarming of human renalproximal tubular cells.

Background. A recent clinical study demonstrated that in renal allografts p reserved in the cold apoptosis occurred soon after reperfusion. The mode of cell death during cold storage is generally considered necrotic. Whether a poptosis occurs as a part of cold storage is uncertain. The objective was t o determine in human renal tubular cells whether apoptosis is specific for rewarming or it also occurs during cold storage and whether it could be mod ified. Methods and Results. Cold storage (4 degreesC) of primary human renal proxi mal tubular epithelial (RPTE) in University of Wisconsin (UW) solution up t o 48 hr caused a time-dependent increase in cell death measured by lactic d ehydrogenase (LDH) release and vital dye exclusion methods. Transmission el ectron microscopy (TEM) demonstrated that cell death in the cold was necrot ic, involving considerable mitochondrial disruption, and was not apoptotic. The TUNEL assay that provides a specific, quantitative measure for apoptos is showed no increase in TUNEL-positivity during flow cytometry of cells st ored in cold: 37 degreesC, 0.23 +/-0.14%; 24 hr cold, 0.23 +/-0.1%; 48 hr c old, 1.79 +/-0.58%. Annexin-V staining, a sensitive method for detecting ea rly apoptosis, similarly showed no increase in positively stained cells dur ing cold storage. Addition of antioxidants 2-methyl aminochroman and defero xamine to UW solution inhibited necrotic cell death and preserved mitochond rial structure. In contrast to cold storage alone, rewarming (37 degreesC f or 24 hr) of cold stored cells, however, resulted in significant apoptosis (TUNEL positive: 48 hr cold: 2 +/-0.6%, 48 hr cold and 24 hr rewarming: 54 +/- 17%), which was confirmed by the TEM based on typical apoptotic feature s. Addition of 2-MAC and DFO significantly inhibited rewarming-induced apop totic cell death (plus 2-MAC: 3 +/-1%, plus DFO: 3 +/-2%). Conclusion. Our study in human tubular cells provides evidence that cold st orage per se does not result in apoptosis, but is primarily necrotic. Howev er, rewarming is associated with significant apoptosis in the presence of o ngoing necrosis, speculatively due to the activation of the apoptotic enzym ic process of sublethally injured cells. Inclusion of antioxidants in the s torage solution confers protection against both cold storage and rewarming- induced necrosis and apoptosis.