Aminoglycoside-induced apoptosis in cultured renal (LLC-PK1) and non-renal (J774 macrophages) cells: comparison between gentamicin and amikacin

Abstract number: 1732_276

Denamur S., Van Bambeke F., Mingeot-Leclercq M.-P., Tulkens P.

Objectives: Apoptosis is now recognized as an early, and probably critical determinant in gentamicin (GEN)-induced nephrotoxicity in animals (Antimicrob Agents Chemother. 2000; 44: 665–75) as well as in renal cultured cells (Toxicol Sci. 2000; 56: 229–39). Models using electroporated cells also show that direct delivery of GEN in the cytosol of cultured renal cells enhances at least 30-fold its capacity to induce apoptosis (Antimicrob Agents Chemother. 2006; 50: 1213–21). Our aims were (i) to examine whether the capacity of GEN to induce apoptosis is restricted to renal cells; (ii) to compare amikacin (AMK) to GEN in this context, since AMK is generally considered to be less nephrotoxic than GEN (Antimicrob Agents Chemother. 1999; 43: 1003–12).

Methods: We used non-confluent murine J774 macrophages and porcine LLC-PK1 renal cells grown to 80% of confluency. Electroporation was performed on trypsinised LLC-PK1 cells (8 square wave pulses; 800 v/cm; 1 ms) as previously described (Antimicrob Agents Chemother. 2006;50:1213–21). Cell viability was checked by measurement of LDH release (only cultures with <10% release were used for evaluation). Apoptotic cells were enumerated after DAPI staining by observers unaware of the experimental conditions, and expressed as percentage of all visible cells.

Results: The Table shows the extent of apoptosis observed in controls (no aminoglycoside), in cells exposed to GEN concentrations known from previous studies to induce marked apoptosis or to AMK equimolar concentrations. For GEN, apoptosis developed on a concentration-dependent manner from an extracellular concentration of 1 mM for incubated cells and from 32 mM for electroporated cells. For AMK, no significant increase of apoptosis was seen at concentrations tested.

Conclusions: Apoptosis develops in both renal and non-renal cells upon incubation with GEN. The lack of apoptosis observed with AMK with both incubated (renal and non-renal) and electroporated (renal) cells support the concept that this aminoglycoside is intrinsically less toxic than GEN.