Molecular Regulators of Renal Collecting Duct Differentiation and Maintenance

摘要 在美国，大约有10.1万人在等待肾脏移植，而只有 2013年，大约有1.7万例肾移植手术。这种可移植肾脏的短缺是可以克服的 通过从患者来源的诱导多能干细胞分化肾脏来提高END的存活率 肾脏疾病(ESRD)期患者。由于肾脏由输尿管芽(UB)和后肾发育而来 间充质细胞(MM)，我们需要使不同类型的细胞区别于UB和MM以工程 培养中的肾脏。我们的目标是了解调节肾脏集合管的分子机制。 分化为主细胞(PC)和间质细胞(ICs)。这些知识将被用来建立 方法在体外培养条件下将UB细胞转化为PC和ICs。我们假设PC促进信号(信号-P) 与Notch合作激活ELF5表达和其他PC谱系转录因子(PCF)以促进 PC分化(图1)。激活的Notch1的异位表达启动了Hes1和ELF5等 候选PCF在体内发育CD中诱导性PC早熟分化，但不能打开ELF5 或培养的未成熟UB细胞中的其他PC特异性基因。Notch信号抑制Foxi1(一种重要的IC 因子)、其他候选IC转录因子(ICF)以及候选信号通路的组件 (信号-I)促进IC分化。Aim1中集合管分化的早期调节因子 在ELF5上，将确定早期PC标记和Foxi1，早期嵌合细胞(IC)标记。AIM2焦点 从Foxi1、ELF5的贡献确定PC和IC分化的中间调节因子 和Hes1利用转基因小鼠、UB细胞和成熟的PC和IC收集导管分化 台词。在Aim3中，我们将确定如何维护成熟的PC。我们有证据表明诺奇 在维持成熟PC细胞状态中的信号转导。这将通过有条件地停用Notch来验证 成人CD中的信号和Notch介导的PC维护的机制将被确定。一只老鼠 获得性肾源性尿崩症(ANDI)模型，其中成熟PC减少，可能是由于 细胞命运转换，将被用来确定哪些CD分化因素参与PC的维护。 通过血统追溯，我们将最终确定PC到IC的命运是否在安迪期间发生变化。这些 研究不仅是渐进的，而且为我们对集合管的理解提供了一个变革性的步骤 开发和维护。

Summary Approximately 101,000 people are waitlisted for a kidney transplant in the United States, while only about 17,000 kidney transplants took place in 2013. This shortage of transplantable kidneys can be overcome by differentiating kidneys from patient derived induced pluripotent-stem cells to improve survival rates of end stage renal disease (ESRD) patients. Since kidneys develop from the ureteric bud (UB) and metanephric mesenchyme (MM), we need to make the different cell types that differentiate from the UB and MM to engineer kidneys in culture. Our goal is to understand the molecular mechanisms regulating kidney collecting duct differentiation into principal cells (PCs) and intercalated cells (ICs). This knowledge will be used to establish methods to convert UB cells into PCs and ICs in culture. We hypothesize that PC promoting signals (Signal-P) co-operate with Notch to activate Elf5 expression and other PC lineage transcription factors (PCFs) to promote PC differentiation (Fig.1). Ectopic expression of activated Notch1 turns on Hes1 and Elf5 among other candidate PCFs to induce precocious PC differentiation in developing CDs in vivo, but is unable to turn on Elf5 or other PC specific genes in cultured immature UB cells. Notch signaling suppresses Foxi1 (an essential IC factor), other candidate IC transcription factors (ICFs), and components of candidate signaling pathways (Signal-I) that promote IC differentiation. In Aim1 the early regulators of collecting duct differentiation that turn on Elf5, an early PC marker, and Foxi1, an early intercalated cell (IC) marker, will be determined. Aim2 focuses on determining intermediate regulators of PC and IC differentiation by examining the contribution of Foxi1, Elf5 and Hes1 to collecting duct differentiation using genetically modified mice, UB cells, and mature PC and IC lines. In Aim3 we will determine how mature PCs are maintained. We have evidence implicating Notch signaling in maintenance of mature PC cell state. This will be verified by conditional inactivation of Notch signaling in adult CDs and the mechanisms of Notch mediated PC maintenance will be determined. A mouse model of acquired Nephrogenic Diabetes Insipidus (aNDI) in which mature PCs are reduced, potentially due to cell fate switching, will be used to determine which CD differentiation factors are involved in PC maintenance. By lineage tracing we will definitively determine whether PC to IC fate change occurs during aNDI. These studies are not merely incremental but provide a transformative step in our understanding of collecting duct development and maintenance.