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疗法，并与其他抗生素联合使用以治疗某些革兰氏阳性菌。不幸的是，它们引起急性肾毒性的几率仍然高得令人无法接受，尤其是在老年人和患有慢性肾病、心脏和肝脏疾病的患者中。我们之前已经鉴定出一种无肾毒性但具有杀菌作用的庆大霉素同系物，现在我们建议用它来确定氨基糖苷类毒性大鼠模型体内近端小管细胞毒性的细胞内决定因素。我们将基于之前的数据，利用酵母遗传学的力量，我们确定 Arf1 是庆大霉素的主要上游胞质靶标，导致细胞内运输缺陷。通过对庆大霉素毒性的补充酵母和大鼠研究，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庆大霉素同系物的保护作用。三个具体目标将直接检验以下假设：我们假设氨基糖苷类抗生素在内吞摄取和胞质释放后，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-蛋白质相互作用通过改变可识别分子靶标的特性，快速改变肾近端小管细胞（PTC）的细胞内运输过程，这反过来会导致 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有毒和无毒形式之间的细胞内差异，从而更好地了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，正如我们之前在大鼠中使用 siRNA 在近曲小管细胞中使用 p53 所证明的那样。

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.