CELLULAR MECHANISM OF AMINOGLYCOSIDE TOLERANCE IN LONG-TERM GENTAMICIN TREATMENT

In the rat, nephrotoxicity results from uptake of gentamicin at the ap ical membrane of proximal tubule (PT) cells. However, during continuou s gentamicin treatment, the PT epithelium has been shown to recover. T he mechanism(s) of cellular recovery and development of tolerance rema ins unknown. Therefore, we undertook studies designed to characterize cellular adaptations that occur during long-term gentamicin (LTG) trea tment. After 19 days of gentamicin treatment, electron microscopy morp hological evaluation revealed cellular recovery with an apparent mild decrease in height and number of microvilli. Enzymatic analysis of LTG PT membranes showed that apical and basolateral membranes had essenti ally returned to normal. Analysis of apical membrane lipid content rev ealed persistent statistically significant (P < 0.01) elevations in ph osphatidylinositol (PI). In vivo immunogold morphological studies and biochemical studies in LTG rats revealed that endocytosis of gentamici n was selectively reduced, whereas the markers of fluid-phase (horsera dish peroxidase) and receptor-mediated (beta(2)-microglobulin) endocyt oses were unaffected or increased. Biochemical analysis showed that, a lthough gentamicin binding to apical membranes isolated from LTG rats increased greater than twofold (P < 0.05) over membranes from untreate d rats, in vivo cellular uptake, quantified with [H-3]gentamicin, was reduced. Western blot analysis of LTG apical membranes and immunofluor escent staining of perfusion-fixed LTG kidneys showed no change in meg alin levels or its apical membrane localization. These data imply that recovery of PT cells from and tolerance to LTG treatment involve a se lective inhibition of gentamicin uptake across the apical membrane. Th ey indicate that the mediators of gentamicin endocytosis were affected differently: PI levels increased, whereas megalin levels did not chan ge. We conclude that selective inhibition of gentamicin uptake during LTG treatment is not affected by a reduction in PI or megalin levels. We postulate that trafficking of gentamicin and/or gentamicin-containi ng endocytic structures is reduced in LTG rats, allowing cells to deve lop tolerance to gentamicin.