Ameliorating systemic gentamicin uptake by sensory hair cells

描述（由申请人提供）：氨基糖苷类抗生素是治疗危及生命的细菌性败血症所必需的，但会引起急性肾毒性和永久性耳聋/平衡障碍。在美国，每年有多达12万人受到这些有害副作用的影响。本研究的目的是确定氨基糖苷通过血迷宫屏障进入耳蜗液和感觉毛细胞诱导细胞毒性和听力损失的分子机制。长期目标是保护耳蜗感觉毛细胞免受药物诱导的耳毒性，并维持终身听力功能。我们发表的数据表明，在毛细胞摄取之前，系统递送的庆大霉素从纹状脉管系统穿过血管纹进入内淋巴。我们已经确定了一个候选氨基糖苷转运蛋白，抑制该转运蛋白可减少耳蜗对荧光标记氨基糖苷的摄取。毛细胞对氨基糖苷的摄取也会因先前的噪音创伤而增强，这意味着除了机电转导通道外，还涉及到其他庆大霉素渗透的阳离子通道。该项目的具体目的是：首先，确定候选氨基糖苷转运体是否需要庆大霉素的摄取、耳蜗运输和体内耳毒性的诱导（目的1）。其次，测试毛细胞表达的其他非选择性阳离子通道（除了MET通道）是否被庆大霉素渗透并诱导细胞毒性（目的2）。第三，由于噪音创伤和氨基糖苷会触发毛细胞的氧化应激，我们将确定氧化应激是否激活庆大霉素渗透的阳离子通道并增强毛细胞对庆大霉素的体内摄取（目的3）。确定氨基糖苷通过BLB转运并进入毛细胞的分子机制对于合理制定新的临床策略至关重要，这些策略可以在挽救生命的氨基糖苷治疗期间保护耳蜗功能。例如，可以对参与这些机制的基因进行单核多态性（SNP）筛选，这些多态性会在已鉴定的庆大霉素渗透通道和转运体中诱导功能获得活性。这将使临床医生能够更明智地使用氨基糖苷类药物，并在治疗危及生命的细菌性败血症、结核病和早产儿预防以及严重烧伤和爆炸伤伤员之前针对个别病例进行个性化庆大霉素治疗。

DESCRIPTION (provided by applicant): Aminoglycoside antibiotics are essential for treating life-threatening bacterial sepsis, yet induce acute nephrotoxicity and permanent deafness/balance disorders. These noxious side-effects affect as many as 120,000 individuals each year in the US. The goal of this proposal is to identify the molecular mechanisms that traffic aminoglycosides across the blood-labyrinth barrier into the cochlear fluids and sensory hair cells to induce cytotoxicity and hearing loss. The long-term goal is to protect the cochlear sensory hair cells from drug-induced ototoxicity, and maintain life-long hearing function. Our published data indicate that systemically-delivered gentamicin is trafficked from the strial vasculature, across the stria vascularis, into endolymph prior to hair-cell uptake. We have identified a candidate aminoglycoside transporter, and inhibition of this transporter reduces cochlear uptake of fluorescently-tagged aminoglycosides. Aminoglycoside uptake by hair cells is also potentiated by prior noise trauma, implicating the involvement of additional gentamicin-permeant cation channels besides the mechanoelectrical transduction channel. The specific aims of this project are: First, to determine if the candidate aminoglycoside transporter is required for gentamicin uptake, cochlear trafficking and induction of ototoxicity in vivo (Aim 1). Second, to test if other non-selective cation channels (besides the MET channel) expressed by hair cells are gentamicin-permeant and induce cytotoxicity (Aim 2). And, third, since noise trauma and aminoglycosides trigger oxidative stress in hair cells, we will determine if oxidative stress activates gentamicin-permeant cation channels and enhances hair cell uptake of gentamicin in vivo (Aim 3). Identifying the molecular mechanisms of aminoglycoside trafficking across the BLB and entry into hair cells is crucial to rational development of new clinical strategies that protect cochlear function during life-saving aminoglycoside therapy. For example, the genes involved in these mechanisms can then be screened for single nuclear polymorphisms (SNP) that induce gain-of-function activity in identified gentamicin-permeant channels and transporters. This will allow clinicians to use aminoglycosides more judiciously and personalize gentamicin therapy for individual cases prior to treatment for life-threatening bacterial sepsis, tuberculosis and for prophylaxis in premature babies, and casualties with severe burns and blast injuries.