High-throughput Flow Culture of 3D Human PKD Models for Therapeutic Screening

常染色体显性遗传性多囊肾病（ADPKD）是一种遗传性多器官系统疾病，其特征是在肾脏中形成充满液体的囊肿和肾小管间纤维化。这导致肾功能逐渐下降，最终导致肾衰竭。重要的是，心血管并发症是ADPKD患者发病和死亡的主要原因。这是肾素-血管紧张素-醛固酮系统过度活化的直接结果，其导致高血压和冠心病、心律失常和心力衰竭的风险增加。不幸的是，有有限的治疗选择和透析和肾移植仍然是唯一可行的选择，以取代在疾病的晚期肾功能下降。 多年来，囊肿形成和进展的分子机制已得到更好的理解。然而，这并没有被新的治疗选择的发展所取代。虽然潜在的原因是多方面的，但需要进行战略转变以加速ADPKD的治疗开发。因此，我们假设，使用成人肾小管/集合管细胞的基于人的离体系统，暴露于流体流动并具有高通量能力，将最好地解决这一巨大的未满足的需求。为了开发这样一种新的高通量筛选方法，我们提出了以下两个目标：在目标1中，我们将开发利用基因工程的原代人尿源性肾小管，一个96孔的流动室和一个自动化的分析管道。基于人的模型的开发将具有广泛的应用，所述模型由在流动条件下的不同肾单位区段和集合管的成人完全分化的肾上皮细胞组成并且以高通量筛选方式操作。虽然短期目标是将其用于开发ADPKD治疗方法，但设置和数据分析管道可用于研究成人肾细胞的任何方法。因此，从长远来看，它适用于广泛的筛选方法，包括例如，基于Crispr/CAS9的基因编辑筛选。

Autosomal Dominant Polycystic Kidney Disease (ADPKD) is a genetically inherited, multi-organ-system disease that is characterized by the formation of fluid filled cysts and intertubular fibrosis in the kidney. This results in a gradual decline in kidney function ultimately leading to kidney failure. Importantly, cardiovascular complications are the main cause of morbidity and death in ADPKD patients. This is a direct result of the over- activation of renin-angiotensin-aldosterone system, which causes hypertension and an increased risk for coronary heart disease, arrhythmias and cardiac failure. Unfortunately, there are limited therapeutic options and dialysis and kidney transplantation are still the only viable options to substitute for declining kidney function at late stage of the disease. Over the years the molecular mechanisms underlying cyst formation and progression have become better understood. Yet, this has not been paralleled by the development of novel therapeutic options. While the underlying reasons are multifold, a strategic shift is needed to accelerate therapeutics development for ADPKD. As such we hypothesize that a human-based ex vivo system using adult kidney tubule/collecting duct cells, exposed to fluid flow and with high throughput abilities will best tackle this large unmet need. To develop such a novel high-throughput screening approach we propose the following two Aims: In Aim 1 we will develop utilizing genetically engineered primary human urinary-derived kidney tubuloids, a 96-well based flow chamber and an automated analysis pipeline. The development of a human-based model that consists of adult fully, differentiated kidney epithelial cells of the different nephron segments and the collecting duct under flow conditions and operational in a high-throughput screening manner will have wide ranging applications. While the short-term aims are to utilize it towards developing ADPKD therapeutics, the setup and the data analysis pipeline can be used for any approach studying adult human kidney cells. Thus, in the long-term it is applicable towards a wide range of screening approaches including e.g., Crispr/CAS9-based genetic editing screens.