Effect of gentamicin on the lysosomal system of cultured human proximal tubular cells. Endocytotic activity, lysosomal pH and membrane fragility.

Gentamicin treatment results in significant changes in lysosomal morphology and enzyme activity in renal tubular epithelium both in vivo and in vitro. In this study, cultured human proximal tubular cells (PTC) were treated with gentamicin (0, 0.01, 0.1, and 1.0 mg/ml) for 3, 7, 10 and 14 days, and the endocytotic activity, pH, and membrane fragility of the lysosomal system were examined. Fluorescein isothiocyanate-labeled dextran (FITC-dextran) was used to estimate endocytotic activity and intralysosomal pH. The fragility of isolated lysosomes was estimated by the release of N-acetyl-beta-glucosaminidase (NAG, EC3.2.1.30) into the medium. Gentamicin content was measured and correlated with the changes seen in lysosomal function. Gentamicin treatment caused a slight decrease in the rate with which human PTC accumulated FITC-dextran and a slight increase in intralysosomal pH. Treatment of human PTC with NH4Cl, a lysosomotropic compound, significantly increased the lysosomal pH; the NH4Cl-induced increase in the lysosomal pH of gentamicin-treated PTC, however, was not significantly different from control (0 mg gentamicin/ml). Lysosomes isolated from human PTC cultures released NAG upon incubation for 60 min at 37 degrees. There was no significant effect on the fragility of lysosomes isolated from cultures exposed to gentamicin for less than or equal to 7 days. Significantly increased fragility was seen, however, after 10 days of treatment with 1.0 mg gentamicin/ml and especially after a 14-day exposure to 0.01, 0.1, and 1.0 mg gentamicin/ml. Human PTC accumulated 0.47, 2.05 and 10.30 micrograms gentamicin/mg protein with 10 days of exposure to 0.01, 0.1 and 1.0 mg gentamicin/ml medium respectively. Gentamicin treatment associated with increased numbers of morphologically altered lysosomes, i.e. myeloid bodies, did not affect significantly the endocytotic activity and pH of lysosomes in cultured human PTC. Prolonged exposure (14 days) of human PTC to gentamicin, however, did increase the fragility of lysosomes after isolation. The increased numbers of morphologically altered lysosomes with increased fragility were not associated with any significant in vitro cell death. Therefore, it would appear that these lysosomal alterations are not directly responsible for the in vivo nephrotoxicity.