Ameliorating systemic gentamicin uptake by sensory hair cells

描述（由申请方提供）：氨基糖苷类抗生素对于治疗危及生命的细菌性脓毒症至关重要，但会诱导急性肾毒性和永久性耳聋/平衡障碍。这些有害的副作用每年在美国影响多达120，000人。本提案的目标是确定氨基糖苷类药物穿过血迷路屏障进入耳蜗液和感觉毛细胞以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。我们已发表的数据表明，全身递送的庆大霉素在毛细胞摄取之前从血管系统穿过血管纹进入内淋巴。我们已经确定了一个候选的氨基糖苷转运蛋白，抑制这种转运蛋白减少耳蜗摄取荧光标记的氨基糖苷类。先前的噪声创伤也增强了毛细胞对氨基糖苷类药物的摄取，这意味着除了机械电转导通道之外，还涉及了其他庆大霉素渗透阳离子通道。本项目的具体目的是：首先，确定候选氨基糖苷转运蛋白是否是体内庆大霉素摄取、耳蜗运输和诱导耳毒性所必需的（目的1）。其次，检测毛细胞表达的其他非选择性阳离子通道（除MET通道外）是否具有庆大霉素渗透性并诱导细胞毒性（目的2）。第三，由于噪声创伤和氨基糖苷类药物引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）。确定氨基糖苷类药物通过BLB运输并进入毛细胞的分子机制，对于合理开发在挽救生命的氨基糖苷类药物治疗期间保护耳蜗功能的新临床策略至关重要。例如，然后可以筛选参与这些机制的基因的单核多态性（SNP），其在鉴定的庆大霉素渗透通道和转运蛋白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抗生素，并在治疗危及生命的细菌性败血症、结核病和预防早产儿以及严重烧伤和爆炸伤的伤员之前，对个体病例进行个性化庆大霉素治疗。 公共卫生相关性：本项目将测试：（i）候选转运蛋白是将耳毒性庆大霉素运输到内淋巴和耳蜗毛细胞中所必需的;（ii）其它阳离子通道也是庆大霉素渗透性的并且有助于细胞毒性;以及（iii）噪声或药物诱导的氧化应激是否增加毛细胞对庆大霉素的摄取。拟定的实验将检测在（a）药物治疗和（B）协同增强氨基糖苷类耳毒性的噪声诱导阈值偏移期间全身庆大霉素转运至毛细胞的特定机制。了解这些病理生理学分子机制对于开发新的药理学或遗传学策略以预防细菌性脓毒症、结核病和心内膜炎以及许多其他适应症治疗期间庆大霉素诱导的耳毒性至关重要。

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. PUBLIC HEALTH RELEVANCE: This project will test if: (i) a candidate transporter is required for trafficking ototoxic gentamicin into endolymph and cochlear hair cells; (ii) other cation channels are also gentamicin-permeant and contribute to cytotoxicity; and (iii) whether noise or drug-induced oxidative stress increases hair cell uptake of gentamicin. The proposed experiments will test specific mechanisms for systemic gentamicin trafficking to hair cells during (a) drug therapy and (b) noise-induced threshold shifts that synergistically potentiate aminoglycoside ototoxicity. Understanding these pathophysiological molecular mechanisms is crucial to developing new pharmacological or genetic strategies to prevent gentamicin-induced ototoxicity during treatment for bacterial sepsis, tuberculosis and endocarditis, among many others indications.