A second area where the interactions between drugs and lipids are critical is the cross-talks between cellular organelles. A better knowledge of these interaction pathways could allow a better knowledge of the molecular mechanism induced by drugs. We focused on the nephrotoxicity induced by aminoglycosides, and particularly gentamicin, by using a combination of biochemical, morphological and biophysical techniques.
At therapeutic concentration, gentamicin accumulates in lysosomes of proximal tubular cells after endocytosis and induces apoptosis via mitochondrial intrinsic pathway, with release of cytochrome c and activation of caspases 9 and 3, but the event cascade between lysosomal accumulation and mitochondria activation remained unclear. In gentamicin-treated LLC-PK1 cells, lysosomal membrane permeabilization, precedes the apoptotic cascade. We clearly evidenced gentamicin-induced lysosomal membrane permeabilization by showing the release to the cytosol of Lucifer yellow, a membrane-impermeant endocytic tracer with a comparable molecular weight as gentamicin, and found by vital confocal imaging that gentamicin induced lysosomal reactive oxygen species (ROS) production prior to acridine orange release from lysosomes and apoptosis. ROS antioxidant or scavenger, catalase and N-acetylcysteine largely prevent these events. We also evidenced the implication of iron in these phenomenon suggested by the protective effect afforded by the iron chelator deferoxamine.
We are further interested in the consequences of lysosomal membrane permeabilization and gentamicin release to the cytosol on the other cellular organelles as proteasome and endoplasmic reticulum (ER), and consequences in terms of apoptosis induction. We assessed chymotrypsin-, trypsin- and caspase-like activities of proteasome in cellular lysates incubated with gentamicin, and showed an inhibition of both trypsin- and caspase-like activities, as an accumulation of ubiquitinated proteins in LLC-PK1 cells incubated with gentamicin. Besides proteasomal inhibition, we evidence p53-pathway implication in gentamicin induced apoptosis, suggested by the protective effect afforded by the p53-inhibitor pifithrin α. As consequence of p53 activation, we evidenced increase in p21 cellular level, phenomenon probably amplified due to proteasome inhibition, also responsible for p27 accumulation and phosphorylation of eIF2α. Cell cycle analysis of LLC-PK1 cells treated with gentamicin tend to show a slight increase in the percentage of cells in phase G2 accompanied by a corresponding decrease in G1 cells. No ER stress, evaluated by GRP78, GRP94 cellular levels and caspase 12 activation, was observed in our conditions.
Finally, to confirm the importance of the role played by cytosolic aminoglycoside in apoptosis induction and nephrotoxicity, we examined whether aminoglycosides from different nephrotoxic potential could be differentiated for apoptosis induction using incubated and electroporated cells.
Studies aiming to decipher the link between the lysosomal permeabilisation, the mitochondrial activation, and the caspase activity leading to apoptosis can be schemed as follows (Figure 4)
Alterations of membranes of intracellular organelles like lysosomes and mitochondria are therefore at the heart of the apoptotic process induced by aminoglycoside antibiotics. The sub-micrometric domains of membranes of intracellular organelles could also play a critical role in cellular toxicology and in the delicate balance between pro-and anti-apoptotic stimuli.