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)是一种遗传的多器官系统疾病，其特征是肾脏内形成充满液体的囊性病变和肾小管间质纤维化。这会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-醛固酮系统过度激活的直接结果，肾素-血管紧张素-醛固酮系统会导致高血压，增加患冠心病、心律失常和心力衰竭的风险。不幸的是，治疗选择有限，透析和肾移植仍然是唯一可行的选择，以替代疾病晚期肾功能下降。 多年来，人们对包囊形成和发展的分子机制有了更好的了解。然而，这并不能与新治疗方案的开发相媲美。虽然潜在的原因是多方面的，但需要进行战略转变，以加快ADPKD的治疗开发。因此，我们假设，使用成人肾小管/收集管细胞、暴露于流体中并具有高吞吐能力的基于人类的体外系统将最好地满足这一巨大的未得到满足的需求。为了开发这样一种新的高通量筛查方法，我们提出了以下两个目标：在目标1中，我们将利用基因工程的原代人类尿液来源肾小管、基于96孔的流动室和自动分析管道来开发。以人为基础的模型的开发将具有广泛的应用前景，该模型由不同肾单位段的成人完全分化的肾上皮细胞和流动条件下的集合管组成，并以高通量筛选的方式操作。虽然短期目标是利用它来开发ADPKD疗法，但该设置和数据分析管道可以用于任何研究成人人类肾脏细胞的方法。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的基因编辑屏幕。

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.