Gentamicin Causes Apoptosis at Low Concentrations in Renal LLC-PK ₁ Cells Subjected to Electroporation

Abstract

Gentamicin accumulates in the lysosomes of kidney proximal tubular cells and causes apoptosis at clinically relevant doses. Gentamicin-induced apoptosis can be reproduced with cultured renal cells, but only at high extracellular concentrations (1 to 3 mM; 0.4 to 1.2 g/liter) because of its low level of uptake. We recently showed that gentamicin-induced apoptosis in LLC-PK 1 cells involves a rapid (2-h) permeabilization of lysosomes and activation of the mitochondrial pathway of apoptosis (10 h). We now examine whether the delivery of gentamicin to the cytosol by electroporation would sensitize LLC-PK 1 cells to apoptosis. Cells were subjected to eight pulses (1 ms) at 800 V/cm (square waves) in the presence of gentamicin (3 M to 3 mM; 1.2 mg/liter to 1.2 g/liter); returned to gentamicin-free medium; and examined at 8 h for their Bax (a marker of mitochondrial pathway activation) contents by Western blotting and competitive reverse transcriptase PCR and at 24 h for apoptosis by 4,6-diamidino-2-phenylindole staining (confirmed by electron microscopy) and for necrosis (by determination of lactate dehydrogenase release). Nonelectroporated cells were incubated with gentamicin for 8 and 24 h. Significant increases in Bax levels (8 h) and apoptosis (24 h) were detected with 0.03 mM (13.2 mg/liter) gentamicin in electroporated cells compared with those achieved with 2 mM (928 mg/liter) in incubated cells. The increase in the Bax level was not associated with an increase in the level of its mRNA but was associated with the accumulation of ubiquitinated forms (probably as a result of impairment of its degradation by the proteasome). Assay of cell-associated gentamicin showed a marked, immediate, but transient accumulation in electroporated cells, whereas a slow, steady uptake was detected in incubated cells. The data indicate that cytosolic gentamicin triggers apoptosis. Sequestration of gentamicin in lysosomes would, to some extent, protect against apoptosis.

Combination Therapy for Treatment of Infections with Gram-Negative Bacteria

“…They bind to glycoproteins on the brush borders of renal tubular cells, which is necessary for internalization of the drug (181). When there is significant accumulation of the drug in the cytosol, aminoglycosides activate apoptosis, causing cell death (226). These findings have also been demonstrated clinically.…”

Ameliorative effect of berberine against gentamicin-induced nephrotoxicity in rats via attenuation of oxidative stress, inflammation, apoptosis and mitochondrial dysfunction

“…70 Apoptosis is a programmed cell death and a key mechanism in the GM-induced renal injury. 71 Thus, apoptosis inhibition is very important for the maintenance of renal integrity and Bcl-2 proteins play a central role in the GM-induced renal apoptosis through its antiapoptotic action. 69,[72][73][74] Bax, a pro-apoptotic protein endorse cytochrome c release from mitochondria in response to toxic stimuli and thereby indulge in apoptosis.…”

Identification of genes involved in gentamicin-induced nephrotoxicity in rats – A toxicogenomic investigation

“…Within one week a replicative rate increase in the proximal tubule is observed. In addition, apoptosis plays an important role in gentamicin toxicity which could also contribute to clear off damaged cells and to control the compensatory proliferative response (Servais et al, 2006). Finally, there is evidence for oxidative stress in gentamicin-induced nephrotoxicity.…”