High content in vivo screening for acute kidney injury ameliorating drugs

摘要 在这项应用中，我们将执行高通量、高含量、筛选改进的小分子 急性肾损伤(AKI)后肾脏再生。Aki提出了一个可怕的未得到满足的医疗需求，因为它 患病率高，对健康有长期不利影响，并有危及生命的后遗症。死亡率很高，而且 唯一有效的治疗方法是肾脏替代疗法。触发事件的开始时间是 不可预测，在许多情况下，一旦病人被送进医院，伤害就已经发生了。 因此，改善伤情恢复提供了一个有吸引力的干预机会，但到目前为止，还没有 如果在受伤后给予治疗是有效的，治疗是可用的。脊椎动物的肾脏有一种先天的能力 再生并遵循明确的细胞机制，包括存活的去分化 肾小管细胞，由此产生的祖细胞的增殖，以及裸露的小管的再生。此序列 与它们各自的分子标记一起，在人类、小鼠和 斑马鱼。在再生过程中，转录因子在器官发生过程中正常表达(例如，lhx1a， Pax2和Pax8)被重新激活。我们之前证明了小分子介导的增强 内源性Lhx1a的表达可改善斑马鱼和小鼠AKI模型的恢复。把这些放在一起 数据支持的总体假设是，驱动细胞程序的小分子增强 损伤后的肾脏修复是治疗急性肾损伤及其相关疾病的一种新的药理学方法 后遗症。利用表达Lhx1a-EGFP的转基因斑马鱼品系，我们开发了一种人造 以智力为基础的高含量分析，以量化活胚胎中lhx1a的表达。使用多变量 分析，该检测符合公认的HTS检测性能标准，并在三天变异性中进行了验证 书房和一个小小的飞行员图书馆屏幕。我们将执行来自MLPCN的50,000种化合物的初级HTS 收集。优先命中将接受完全实施的、严格的二次分析范例 包括肾器官发育、代谢稳定性、体内疗效和活动性 病理生理学相关的AKI模型。在这些研究结束时，我们将在功能和 体内化学探针的机械特征，以研究肾脏损伤的生物学和 再生，其中一些预计将具有使其适合开发为 临床前线索。

ABSTRACT In this application we will perform high throughput, high content, screening for small molecules that improve kidney regeneration after acute kidney injury (AKI). AKI presents a dire unmet medical need because of its high prevalence with long-term adverse health effects and life-threatening sequelae. Mortality is high and the only effective treatments are renal replacement therapies. The onset of the precipitating event is unpredictable, and in many instances once patients are admitted to the hospital, injury has already occurred. Improving recovery from injury therefore presents an attractive opportunity for intervention, but to date, no therapies are available that are effective if administered post injury. The vertebrate kidney has an innate ability to regenerate and follows a well-defined cellular mechanism that encompasses dedifferentiation of surviving renal tubule cells, proliferation of resulting progenitors, and repopulation of the denuded tubule. This sequence of events, together with their respective molecular markers, is conserved between humans, mouse, and zebrafish. During regeneration, transcription factors normally expressed during organogenesis (e.g.,lhx1a, pax2, and pax8) are reactivated. We previously demonstrated that small molecule-mediated augmentation of endogenous Lhx1a expression can ameliorate recovery in zebrafish and mouse models of AKI. Together these data support the overall hypothesis that augmentation by small molecules of cellular programs that drive kidney repair after injury represents a novel pharmacologic approach for the treatment of AKI and associated sequelae. Using a transgenic zebrafish line that expresses Lhx1a-EGFP we have developed an artificial intelligence-based, high-content assay to quantify lhx1a expression in the living embryo. Using multivariate analysis, the assay met accepted HTS assay performance standards and was validated in three-day variability studies and a small pilot library screen. We will perform a primary HTS of 50,000 compounds from the MLPCN collection. Prioritized hits will be subjected to a fully implemented, rigorous secondary assay paradigm encompassing kidney organ development, metabolic stability, in vivo efficacy, and activity profiling in a pathophysiological relevant AKI model. At the end of these studies we will have identified functionally and mechanistically characterized in vivo chemical probes to investigate the biology of kidney injury and regeneration, some of which are expected to have features that make them suitable for development into preclinical leads.