Background.

Cold storage of tissues induces reactive oxygen species (ROS), which contribute to cell injury. We have compared different antioxidants in protection of renal tubular cells against hypothermia injury and studied their effect on cold-induced mitogen-activated protein (MAP) kinase activation.

Methods.

Cultured renal tubular epithelial cells (LLC-PK1) were stored in University of Wisconsin solution supplemented with compounds tested for 16 hr at 4 C. Release of lactate dehydrogenase and cellular adenosine triphosphate were measured. Activation of MAP kinases was determined by Western blotting. Intracellular ROS were monitored with a fluorescent probe.

Results.

Cold storage resulted in a substantial loss of cell viability. The simple phenol butylated hydroxyanisol (BHA) most effectively prevented hypothermia-induced cell injury, whereas about 100-fold higher concentration of the polyphenol epigallocatechin gallate (EGCG) was needed, although EGCG most effectively scavenged intracellular ROS elicited by serum withdrawal. The MEK inhibitor U0126 and reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor diphenyleneiodonium effectively protected the cells against hypothermia injury. ERK1/2 was rapidly activated during chilling of the cells and this was inhibited by BHA but not by EGCG.

Conclusion.

The results suggest that chilling of renal epithelial cells induces ROS generation by NADPH oxidase, which leads to rapid activation of the MEK-ERK1/2 cascade and initiation of cell injury. This can be prevented by antioxidants.