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治疗革兰氏阴性菌感染的主要药物，并与其他抗生素联合应用于某些革兰氏阳性菌。不幸的是，它们仍有令人无法接受的高急性脑磷脂毒性，特别是在老年人和慢性肾脏疾病、心脏和肝脏疾病患者中。我们以前已经确定了庆大霉素的一种无肾毒性但具有杀菌作用的同系物，现在我们建议使用它来确定氨基糖苷类毒性大鼠体内近端小管细胞内毒性决定因素。我们将建立在我们之前的数据的基础上，我们确定Arf1是庆大霉素的主要上游胞浆靶标，利用酵母遗传学的力量导致细胞内转运缺陷。利用免费的酵母和大鼠庆大霉素毒性研究，我们将确定庆大霉素引起Arf1失调的生化、分子和细胞生物学机制，以及无毒的庆大霉素同系物的保护作用。三个特定的目标将直接检验以下假设：我们假设氨基糖苷类抗生素在胞内摄取和胞内释放后，直接与Arf1等细胞质蛋白相互作用，介导细胞毒性。我们进一步假设，这些氨基糖苷类与蛋白质的相互作用通过改变可识别的分子靶标的性质，迅速改变了肾近端小管细胞(PTCs)的细胞内转运过程，进而导致PTC功能障碍，最终导致肾毒性。无毒的同系物将使我们能够直接比较和对比有毒和无毒形式之间的细胞内差异，从而使我们更好地理解介导细胞毒性过程的因素。最后，我们将使用siRNA直接探测靶分子，就像我们之前在大鼠近端小管细胞中使用siRNA对p53进行演示一样。

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.