High content in vivo screening for acute kidney injury ameliorating drugs

摘要 在这个应用中，我们将进行高通量，高含量，筛选小分子，改善 急性肾损伤（AKI）后的肾脏再生。AKI提出了一个可怕的未满足的医疗需求，因为它 流行率高，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， 唯一有效的治疗方法是肾脏替代疗法。突发事件的发生是 这是不可预测的，而且在许多情况下，一旦病人入院，伤害已经发生。 因此，改善受伤后的恢复是一个有吸引力的干预机会，但迄今为止， 如果在损伤后施用，则可获得有效的治疗。脊椎动物的肾脏有一种天生的能力 再生，并遵循明确的细胞机制，包括生存的去分化， 肾小管细胞、所得祖细胞的增殖和裸露小管的再增殖。该序列 事件，连同它们各自的分子标记，在人类、小鼠和 斑马鱼在再生过程中，通常在器官发生过程中表达的转录因子（例如，lhx1a， PAX2和PAX8）被重新激活。我们以前证明，小分子介导的增强， 内源性Lhx1a表达可以改善AKI的斑马鱼和小鼠模型的恢复。综合这些 数据支持这一总体假设，即通过小分子增强细胞程序， 损伤后的肾修复代表了治疗AKI和相关疾病的新的药理学方法。 后遗症使用表达Lhx1a-EGFP的转基因斑马鱼系，我们开发了一种人工的 智能为基础的，高含量的测定，以量化LHX1A在活胚胎中的表达。使用多变量 分析中，该检测试剂盒符合公认的HTS检测试剂盒性能标准，并在3天变异性中得到验证 研究和一个小型试点图书馆屏幕。我们将对MLPCN中的50，000种化合物进行初步HTS 收藏.优先命中将受到一个完全实施，严格的二次测定范例 包括肾器官发育、代谢稳定性、体内功效和活性分析， 病理生理学相关AKI模型。在这些研究的最后，我们将在功能上确定， 机械表征的体内化学探针，以研究肾损伤的生物学， 再生，其中一些预计将有特点，使他们适合发展成 临床前电极导线。

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.