Cellular mechanism of aminoglycoside tolerance in long-term
gentamicin treatment.
Sundin, David P., Chris Meyer, Rolf Dahl, Alison Geerdes, Ruben
Sandoval, and Bruce A. Molitoris.
Renal Epithelial Biology Experimental Laboratories, Department of
Medicine, Indiana University Medical Center, Indianapolis, Indiana,
University of Colorado Health Sciences Center and the Veteran Affairs
Medical Centers in Denver, CO and Indianapolis, IN
APStracts 3:0347C, 1996.
In the rat, nephrotoxicity results from uptake of gentamicin at the
apical membrane of proximal tubule (PT) cells. However, during
continuous gentamicin treatment, the PT epithelium has been shown to
recover. The mechanism(s) of cellular recovery and development of
tolerance remain unknown. Therefore, we undertook studies designed to
characterize cellular adaptations that occur during long-term
gentamicin (LTG) treatment. After 19 days of gentamicin treatment, EM
morphological evaluation revealed cellular recovery with an apparent
mild decrease in height and number of microvilli. Enzymatic analysis
of LTG PT membranes showed apical and basolateral membranes had
essentially returned to normal. Analysis of apical membrane lipid
content revealed persistent statistically signficant (P<0.01)
elevations in phosphatidylinositol (PI). In vivo immunogold
morphological studies and biochemical studies in LTG rats revealed
endocytosis of gentamicin was selectively reduced while the markers
of fluid-phase (horseradish peroxidase, HRP) and receptor-mediated
([beta]2-microglobulin) endocytosis were unaffected or increased.
Biochemical analysis showed that while gentamicin binding to apical
membranes isolated from LTG rats increased >2-fold
(P<0.05) over membranes from untreated rats, in vivo cellular
uptake, quantified using 3H-gentamicin, was reduced. Western blot
analysis of LTG apical membranes and immunofluorescent staining of
perfused-fixed LTG kidneys showed no change in megalin levels or its
apical membrane localization. These data imply that recovery of PT
cells from and tolerance to LTG treatment involves a selective
inhibition of gentamicin uptake across the apical membrane. They
indicate the mediators of gentamicin endocytosis were effected
differently: PI levels increased while megalin levels did not change.
We conclude that selective inhibition of gentamicin uptake during LTG
treatment is not effected by a reduction in PI or megalin levels. We
postulate that trafficking of gentamicin and/or gentamicin-containing
endocytic structures is reduced in LTG rats allowing cells to develop
tolerance to gentamicin.
Received 6 August 1996; accepted in final form 23 October 1996.
APS Manuscript Number C445-6.
Article publication pending Am. J. Physiol. (Cell Physiology).
ISSN 1080-4757 Copyright 1996 The American Physiological Society.
Published in APStracts on 13 November 1996