Mechanisms and Key Molecular Target of Gentamacin Toxicity

描述（由申请人提供）： 摘要：氨基糖苷类抗生素仍然是革兰氏阴性菌感染的主要治疗药物，并与其他抗生素联合用于某些革兰氏阳性菌。不幸的是，它们仍然具有不可接受的高比率引起急性肾毒性，特别是在老年人和患有慢性肾脏疾病、心脏和肝脏疾病的患者中。我们以前已经确定了一个非肾毒性，但杀菌，庆大霉素的同源物，我们现在建议使用，以确定在近端小管细胞内的毒性决定因素在体内的大鼠模型中的氨基糖苷类毒性。我们将建立在我们以前的数据，其中我们确定Arf 1作为庆大霉素的主要上游胞质靶点，使用酵母遗传学的力量导致细胞内运输缺陷。使用庆大霉素毒性的互补酵母和大鼠研究，我们将确定Arf 1失调的生化，分子和细胞生物学机制，由庆大霉素和无毒庆大霉素同源物的保护效益。三个具体的目标将直接测试以下假设：我们假设，氨基糖苷类抗生素，内吞摄取和胞质释放后，直接与细胞质蛋白，如Arf 1介导的细胞毒性。我们进一步假设，这些氨基糖苷类-蛋白质相互作用通过改变可识别的分子靶点的性质迅速改变肾近端小管细胞（PTC）的细胞内运输过程，这反过来又导致PTC功能障碍并最终导致肾毒性。无毒同系物将使我们能够直接比较和对比细胞内的差异之间的有毒和无毒的形式，从而导致更好地了解介导的细胞毒性过程的因素。最后，我们将使用siRNA直接探测靶分子，正如我们先前在大鼠近端小管细胞中使用siRNA证明的那样。

DESCRIPTION (provided by applicant): Abstract: 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.