Gentamicin-induced apoptosis in renal cell lines and embryonic rat fibroblasts

Gentamicin, an aminoglycoside antibiotic, induces apoptosis in the proximal tubule epithelium of rats treated at low, therapeutically relevant doses ( El Mouedden et al., Antimicrob. Agents Chemother. 44, 665-675, 2000). Renal cell Lines (LLC-PK1 and MDCK-cells) have been used to further characterize and quantitate this process (electron microscopy; terminal deoxynucleotidy l transferase-mediated dUTP-biotin nick-end labeling of fragmented DNA [TUN EL]; and DNA size analysis [oligonucleosomal laddering]). Cells were expose d for up to 4 days to gentamicin concentrations of up to 3 mM. Apoptosis de veloped, almost linearly, with time and drug concentration, and was (i) pre ventable within the time-frame of the experiments by overexpression of the anti-apoptotic protein Bcl-2, and by co-incubation with cycloheximide (MDKC but not LLC-PK1 cells); (ii) associated with an increased activity of casp ases (MDCK cells; bcl-2 transfectants showed no increase of caspase activit ies and Z-VAD.fmk afforded full protection). Gentamicin-induced apoptosis a lso developed to a similar extent in embryonic fibroblasts cultured under t he same conditions. In the 3 cell types, apoptosis (measured after 4 days) was directly correlated with cell gentamicin content (apoptotic index I sim ilar to 10 to 18% of TUNEL (+) cells for a content of 20 mu g of gentamicin /mg protein; kidney cortex of rats showing apoptosis in proximal tubule epi thelium typically contains similar to 10 mu g of gentamicin/mg protein). Th us, gentamicin has an intrinsic capability of inducing apoptosis in eucaryo tic cells. Development of apoptosis in proximal tubules of kidney cortex in vivo after gentamicin systemic administration is therefore probably relate d to its capacity to concentrate in this epithelium after systemic administ ration.