New mouse model of cisplatin-induced AKI and development of prevention therapy

摘要 顺铂是目前应用最广泛的抗实体瘤药物之一，但它会对肾脏造成严重损害。 肾小管间质组织，病理表现为肾小球滤过率降低，炒作- 尿酸血症、低钾血症、继发性肾小球硬化和肾小球缺血加速。结局就是- 结果是急性肾损伤(AKI)。顺铂可诱发急性心肌梗死，25%接受顺铂治疗的癌症患者会患上急性KI 全世界。除了极少数例外，目前尚无有效的预防或暴露后治疗顺铂的方法。 Aki(C-AKI)。氧化应激和线粒体损伤是AKI相关病理的驱动因素；然而， 调节这些事件的分子途径还没有明确的定义。用小鼠C-AKI模型肾和双肾移植 我们最近从C-AKI患者的眼球样本中发现，氧化应激和线粒体损伤都是可能的。 AGE与肾无嘌呤/脱嘧啶核酸内切酶2(APE2)表达上调有关。在初审前- 我们发现，顺铂体内暴露增加了小鼠APE2的表达，并抑制了 肌球蛋白重链9(Myh9)的作用，它是肾功能的关键调节分子。这些Altera- 感染转化为对肾小管间质组织的影响。我们的总体工作假设是顺铂改变了 肾脏APE2信号，导致Myh9抑制并伴随肾小管间质损伤。在这里我们支持- 姿势，以更准确地阐明APE2在AKI发展中的目标角色，特别是假设 (I)APE2的高表达影响线粒体DNA的完整性和Myh9/肌动蛋白中间的线粒体分裂和 (Ii)这反过来通过触发细胞死亡途径而促进肾脏损伤。为了解决这些双重问题，我们 提出了利用APE2转基因和基因敲除(KO)小鼠模型来追求这两个具体目标的具体方法 下面。在目标1中，我们将验证APE2基因工程小鼠模型是否与临床直接相关 致C-AKI。我们将全面定义APE2小鼠的表型，并将其与C-AKI进行比较 小鼠模型和人类C-AKI分子病理生理学特征 水平使用全基因组、转录和免疫组学方法。我们将定义的结合部位 APE2/Myh9及其对进一步翻译研究的功能意义。在目标2中，我们将进行证明- APE2靶向治疗预防C-AKI的概念研究此外，我们将定义一部有针对性的小说 APE2缺口反义寡核苷酸或小分子靶向给药预防肾毒性 选择性抑制APE2转基因和C-AKI小鼠近曲小管细胞APE2的表达。 影响：实现这些具体目标将是构建时间机制的第一步 顺铂暴露时发生的事件图，转化为肾小管间质性肾炎，并可能最终- 使AKI病新的靶向治疗方法的开发成为可能。

SUMMARY Cisplatin is among the most widely used drugs against solid tumors, but it can cause severe damage of renal tubulointerstitial tissues, manifesting itself pathologically in the form of glomerular filtration rate reduction, hype- ruricemia, hypokalemia, and accelerated secondary glomerulosclerosis and glomerular ischemia. The end re- sult is acute kidney injury (AKI). Cisplatin induces AKI, which afflicts 25% of cisplatin-treated cancer patients worldwide. With few exceptions, there is no effective preventive or post-exposure therapy for cisplatin-induced AKI (C-AKI). Oxidative stress and mitochondrial damage are drivers of AKI-associated pathology; however, the molecular pathways that mediate these events are poorly defined. Using murine C-AKI model kidney and bi- opsy samples from C-AKI patients, we have recently found that both oxidative stress and mitochondrial dam- age are associated with upregulation of renal apurinic/apyrimidinic endonuclease 2 (APE2). In preliminary ex- periments, we discovered that cisplatin exposure in vivo increases expression of APE2 in mice and inhibits the action of the protein myosin heavy chain 9 (MYH9), a key regulatory molecule of kidney function. These altera- tions translate into impact on tubulointerstitial tissue. Our overall working hypothesis is that cisplatin alters kidney APE2 signaling, leading to inhibition of MYH9 with concomitant tubulointerstitial damage. Here we pro- pose to more precisely illuminate the target role of APE2 in AKI development, and specifically hypothesize (i) that highly expressed APE2 affects mtDNA integrity and MYH9/actin intermediate mitochondrial fission and (ii) that this in turn promotes renal injury by triggering cell-death pathways. To address these dual issues, we propose using APE2 transgenic and knockout (KO) mouse models to pursue the two Specific Aims detailed below. In Aim 1, we will validate a genetically engineered murine model of APE2 with direct clinical relevance to C-AKI. We will comprehensively define the APE2 mouse phenotype and compare it with that of the C-AKI mouse model and the human C-AKI signature with respect to pathophysiological features at the molecular level using whole genomic, transcriptional, and immunomic approaches. We will define the binding site of APE2/MYH9 and its functional significance for further translational studies. In Aim 2, we will conduct proof-of- concept studies on APE2 targeted therapy for prevention of C-AKI. Additionally, we will define a novel targeted therapy to prevent nephrotoxicity by delivering APE2 gapmer antisense oligonucleotides or small-molecule in- hibitors to selectively suppress APE2 expression in proximal tubule cells in APE2 transgenic and C-AKI mice. Impact: Accomplishing these Specific Aims will be the first step toward constructing a temporal-mechanistic map of events that occur upon cisplatin exposure and translate into tubulointerstitial nephritis and may ulti- mately enable development of novel targeted therapy for AKI disease.