Aminoglycoside antibiotics remain main line therapy for gram negative infections and are used in conjunction with other antibiotics for certain gram positive organisms. Unfortunately, they still have an unacceptably high rate of causing acute neprhotoxicity especially in the elderly and in patients with chronic kidney disease, heart and liver disorders. We have previously identified a nonnephrotoxic, yet bactericidal, congener of gentamicin that we now propose to use to determine the intracellular determinants of toxicity in proximal tubule cells in vivo in the rat model of aminoglycoside toxicity. We will build on our previous data where we identified Arf1 as a major upstream cytosolic target of gentamicin leading to intracellular trafficking defects using the power of yeast genetics. Using complimentary yeast and rat studies of gentamicin toxicity we will determine the biochemical, molecular and cell biologic mechanisms of Arf1 dysregulation by gentamicin and the protective benefit of the nontoxic gentamicin congener.
Three specific aims will directly test the following hypothesis: We hypothesize that aminoglycoside antibiotics, following endocytic uptake and cytosolic release, interact directly with cytoplasmic proteins such as Arf1 to mediate cellular toxicity. We further hypothesize that these aminoglycoside-protein interactions rapidly alter intracellular trafficking processes in renal proximal tubule cells (PTCs) by altering the properties of identifiable molecular targets and that this in turn results in PTC dysfunction and ultimately nephrotoxicity. The nontoxic congener will allow us to directly compare and contrast intracellular differences between toxic and nontoxic forms thereby leading to greater understanding of the factors mediating the cytotoxic process. Finally, we will use siRNA to directly probe target molecules as we have previously demonstrated with p53 in proximal tubule cells using siRNA in rats.
to VA statement: Aminoglycoside antibiotics remain the main line therapy for gram negative infections and are used inconjunction with other antibiotics for certain gram positive organisms. Unfortunately, they still have an unacceptably high rate of causing acute neprhotoxicity especially in the elderly and in patients with chronic kidney disease, heart and liver disorders. The present studies use a unique approach to determine the intracellular mediators of animoglycoside toxicity by directly comparing nephrotoxic and nonnephrotoxic forms of gentamicin in yeast, rat, proteomic and genomic studies. A specific target protein, Arf1, has been identified as a major upstream mediator of cellular toxicity and the specific biochemical, molecular and cellular mechanisms will be determined. These studies will help direct future therapeutic approaches.