High glucose initiates calpain-induced necrosis before apoptosis in LLC-PK1 cells

Harwood, S. M.; Allen, D. A.; Raftery, M. J.; Yaqoob, M. M.
April 2007
Kidney International;Apr2007, Vol. 71 Issue 7, p655
Academic Journal
Cells exposed to high ambient glucose concentrations are subject to increases in intracellular calcium ([Ca2+]i). We therefore considered it likely that the calcium-dependent cysteine protease calpain would play a role in the development of high glucose-induced cell injury. After 3 and 24 h, high glucose concentrations (25 mM D-glucose) produced almost identical increases in the degree of necrotic cell death in kidney proximal tubular epithelial cells (LLC-PK1) compared to cells treated with control glucose (5 mM D-glucose). Necrotic cell death could be restricted by inhibiting the activity of calpain. High glucose-treated LLC-PK1 cells were found to have significantly elevated [Ca2+]i concentrations within 1 h, and elevated calpain activity within 2 h compared to control treated cells. The DNA nick sensor poly(ADP-ribose) polymerase (PARP) has previously been shown to be an important driver of high glucose-induced cell death, but here we found that although PARP activity was increased after 24 h, it was unaltered after 3 h. Furthermore, PARP inhibition with PJ-34 did not restrict early high glucose-induced necrosis. Using a gene knockdown strategy with small interference RNA, we found that silencing calpain was effective in reducing the degree of early high glucose-induced necrosis. We conclude that high glucose concentrations evoke an early, calpain-mediated necrosis in cultured proximal tubular cells that is PARP-independent, and precedes the previously recognized activation of apoptosis.Kidney International (2007) 71, 655–663. doi:10.1038/sj.ki.5002106; published online 7 February 2007


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