Impairment of lysosome-pinocytic vesicle fusion in rat kidney proximal tubules after treatment with gentamicin at low doses

Gentamicin, an aminoglycoside antibiotic, is known to accumulate within the kidney cortex and to elicit nephrotoxic reactions due to the necrosis of proximal tubules. Female Sprague-Dawley rats, treated for 9 days with gentamicin at a low dose (10 mg/kg ip, once a day), were used to determine the fate of the antibiotic within the proximal tubular cells and its effects on the functional properties of the lysosomes. The analysis of the lysosomes by isopyknic equilibration in sucrose gradient (density 1.10-1.24 g/ml) revealed that gentamicin remains associated with these organelles (marker enzymes sulfatase B and cathepsin B) throughout the treatment duration. Gentamicin treatment markedly decreased the buoyant density of the lysosomes. As was shown by electron microscopic examination of the subcellular fractions collected from the sucrose gradient, the shift of the lysosomes toward lower densities was a result of overloading with undegraded phospholipids (myelin-like figures). The effect of gentamicin on the functional properties of the lysosomes was examined by using horseradish peroxidase (HRP) as a marker of endocytic activity and of the processing by tubular cells of exogenous proteins. Treatment with gentamicin did not significantly modify the intracortical accumulation of HRP, which was estimated to be 2.2% of the amount injected. HRP was shown by isopyknic equilibration to be mostly associated with the lysosomes. This was confirmed by electron microscopic examination of proximal tubular cells after cytochemical demonstration of HRP with diaminobenzidine and H2O2. In rats not exposed to gentamicin, more than half of the lysosomes contained HRP activity. In animals treated with gentamicin, one-third of the lysosomes that retained a normal appearance exhibited HRP activity. In contrast, lysosomes overloaded with phospholipids (identified by the presence of myelin-like figures) were very seldom labeled with HRP activity. Taken altogether, the present observations suggest that the alterations induced by gentamicin treatment impair their ability to fuse with incoming endocytic vesicles