The long term objectives of this research proposal are to define the mechanism by which aminoglycosides are transported and accumulated by renal proximal tubular cells and to elucidate the metabolic pathways by which these drugs induce cellular injury with the ultimate goal of devising strategies to obviate the development of aminoglycoside toxicity.
The specific aims are to test the hypothesis that the transport and accumulation of aminoglycosides by proximal tubular cells involves binding of drug to phosphatidylinositol (PI) of the brush border membrane followed by endocytosis of the drugreceptor complex and subsequent translocation and sequestration within lysosomes. Our first goal will be to study the renal tubular transport of aminoglycosides using clearance and micropuncture techniques in the rat with streptozotocin-induced diabetes mellitus to ascertain why drug is not accumulated by proximal tubular cells in this animal model. To assess the role of PI as the receptor for aminoglycosides, brush border and basolateral membrane vesicles will be harvested from diabetic and control rats and the PI content will be measured and correlated with the capacity of these membranes to bind 3H-netilmicn. In other studies membrane vesicles will be exposed to a PI-specific phospholipase C to determine whether a decrease in membrane PI results in a predictable reduction of drug binding. The role of endocytosis in the proximal tubular transport of aminoglycosides will be assessed by determining the effects of pharmacological inhibition of endocytosis on the renal accumulation of 3H-netilmicin in vivo. Pulse labeling techniques will be used in vivo to determine the effects of aminoglycosides on membrane phospholipid synthesis and degradation with the aim of testing the hypothesis that myeloid body is derived from undegraded components of brush border membrane. Finally, we will examine the effects of aminoglycosides on membrane permeability to potassium as well as try to ascertain why potassium depletion augments aminoglycoside toxicity.
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