Intracellular mechanisms of apoptosis induced by aminoglycoside antibiotics

In a context of resurgence in use of aminoglycosides for their effectiveness against bacterial strains resistant to other classes of antibiotics, as well as for their activities in the treatment of genetic diseases or viral affections, the nephrotoxicity induced by these antibiotics remains so far a barrier against their use. Aminoglycosides are eliminated by glomerular filtration and picked up, mainly by receptor-mediated endocytosis, by proximal tubular epithelial cells that can poison. The tubular toxicity involves the death of epithelial cells, and apoptosis has been observed in rats and cultured cells treated with therapeutically relevant doses. The present study was therefore designed to gain further knowledge about the intracellular mechanisms underlying gentamicin-induced apoptosis in LLC-PK1 proximal tubular cells. In the first part of this work, we evaluated the role and the mechanisms of lysosomal membrane permeabilization induced by gentamicin. Direct evidence for gentamicin-induced permeabilization of lysosomal membrane was provided by vital imaging, and we showed an involvement of ROS by an iron-dependent mechanism in this process. In a second part of this work, we focused our interest on the role that can play cytosolic aminoglycosides. We observed that electroporation of more nephrotoxic aminoglycosides (gentamicin and neomycin B) induced more apoptosis than low toxic ones (amikacin and isepamicin). The low concentrations needed to observe induction of apoptosis by nephrotoxic drugs after electroporation highlights the high apoptogenic potential of cytosolic aminoglycosides, and the important consequences that could have their release from lysosomes into the cytosol. Finally, we explored several pathways that could link lysosomal membrane permeabilization and activation of mitochondrial intrinsic pathway of apoptosis. The partial protective effect of the p53 inhibitor pifithrin α on gentamicin-induced apoptosis led us to suggest an implication of p53 signaling pathway in this process, while inhibition of the proteasome by gentamicin did not appear to be key mechanism of gentamicin-induced apoptosis in LLC-PK1 cells. Our work helps to gain a comprehensive view about intracellular mechanisms implicated in gentamicin-induced apoptosis. These observations highlight the important role of lysosomes in aminoglycoside-induced apoptosis, and it would now be very interesting to investigate their implication in vivo in rats or human models.