Molecular Regulators of Renal Collecting Duct Differentiation and Maintenance

肾集合管分化和维持的分子调节剂

• 批准号: 9305081
• 负责人: Kameswaran Surendran
• 金额: $ 36.34万
• 依托单位: SANFORD RESEARCH/USD
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-07-01 至 2020-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9305081
• 关键词: Adult; Biomedical Engineering; Cell Culture Techniques; Cell Differentiation process; Cell Line; Cell Lineage; Cell Maintenance; Cells; Development; Differentiated Gene; Disease; Duct (organ) structure; Ectopic Expression; Electrolytes; End stage renal failure; Engineering; F Factor; Gene Expression Profiling; Genes; Goals; In Vitro; Intercalated Cell; Kidney; Kidney Transplantation; Knowledge; Maintenance; Mediating; Mesenchyme; Metanephric Diverticulum; Methods; Modeling; Molecular; Mus; Patients; Positioning Attribute; Publishing; Signal Pathway; Signal Transduction; Stem cells; Survival Rate; Testing; United States; Water; activating transcription factor; base; cell type; collecting tubule structure; design; improved; in vivo; induced pluripotent stem cell; mouse model; notch protein; pH Homeostasis; promoter; regenerative; transcription factor; transcriptome; vasopressin resistant diabetes insipidus

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.

总结 在美国，大约有101，000人在等待肾脏移植，而只有 2013年进行了约17 000例肾脏移植手术。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， 晚期肾病（ESRD）患者。由于肾脏是从输尿管芽（UB）和后肾发育而来， 为了构建间充质细胞（MM），我们需要制造从UB和MM分化出来的不同细胞类型， 肾脏在文化。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）和闰细胞（IC）。这些知识将用于建立 在培养中将UB细胞转化为PC和IC的方法。我们假设PC促进信号（Signal-P） 与Notch合作激活Elf 5表达和其他PC谱系转录因子（PCF），以促进 PC分化（图1）。激活的Notch 1的异位表达开启Hes 1和Elf 5等 在体内发育CD中诱导早熟PC分化的候选PCF，但不能开启Elf 5 或其它PC特异性基因。Notch信号转导抑制Foxi 1（一种重要的IC 因子）、其他候选IC转录因子（ICF）和候选信号传导途径的组分 （信号-I），促进IC分化。在Aim 1中，集合管分化的早期调节因子， 将测定早期PC标志物Elf 5和早期嵌入细胞（IC）标志物Foxi 1。AIM 2聚焦 通过检查Foxi 1、Elf 5的贡献来确定PC和IC分化的中间调节物 和Hes 1对使用遗传修饰小鼠、UB细胞和成熟PC和IC的集合管分化的影响 线在目标3中，我们将确定如何维护成熟的PC。我们有证据表明 维持成熟PC细胞状态的信号传导。这将通过Notch的条件性灭活进行验证 将确定成年CD中的信号传导和Notch介导的PC维持的机制。鼠标 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，将用于确定哪些CD分化因子参与PC维持。 通过谱系追踪，我们将明确确定在aNDI期间是否发生PC到IC的命运改变。这些 研究不仅是增量的，而且为我们理解集合管提供了变革性的一步 开发和维护。