High glucose initiates calpain-induced necrosis before apoptosis in LLC-PK1 cells.
Cells exposed to high ambient glucose concentrations are subject to increases in intracellular calcium ([Ca(2+)](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-PK(1)) 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-PK(1) cells were found to have significantly elevated [Ca(2+)](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.
Harwood
S M
SM
Centre for Experimental Medicine, Nephrology and Critical Care, Queen Mary, University of London, William Harvey Research Institute, London, UK. s.m.harwood@qmul.ac.uk
Allen
D A
DA
Raftery
M J
MJ
Yaqoob
M M
MM
D000818:Animals; D017209:Apoptosis; D002154:Calpain / Q000494:pharmacology*; D004847:Epithelial Cells / Q000473:pathology*; D005947:Glucose / Q000008:administration & dosage*; D007687:Kidney Tubules, Proximal / Q000473:pathology*; D018374:LLC-PK1 Cells / Q000473:pathology*; D009336:Necrosis / Q000139:chemically induced; D013552:Swine
D002154:Calpain; D005947:Glucose