Molecular Regulators of Renal Collecting Duct Differentiation and Maintenance

肾集合管分化和维护的分子调节剂

• 批准号: 9173773
• 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/9173773
• 关键词: Adult; Biomedical Engineering; Cell Culture Techniques; Cell Differentiation process; Cell Line; Cell Lineage; Cell Maintenance; Cells; Development; Disease; Duct (organ) structure; Ectopic Expression; Electrolytes; End stage renal failure; Engineering; Factor X; 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; Staging; 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.

概括 在美国，大约有 101,000 人被列入肾脏移植的候补名单，而只有 2013年进行了约17,000例肾移植。可移植肾的短缺是可以克服的 通过将肾脏与患者来源的诱导多能干细胞区分开来提高最终的存活率 阶段肾病（ESRD）患者。由于肾脏是从输尿管芽 (UB) 和后肾发育而来 间充质（MM），我们需要制造从 UB 和 MM 中分化出来的不同细胞类型来进行工程设计 文化中的肾脏。我们的目标是了解调节肾集合管的分子机制 分化为主细胞（PC）和闰细胞（IC）。这些知识将用于建立 将 UB 细胞转化为培养中的 PC 和 IC 的方法。我们假设 PC 促进信号（Signal-P） 与Notch配合激活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 的异位表达会开启 Hes1 和 Elf5 等 候选 PCF 在体内发育 CD 时诱导早熟 PC 分化，但无法启动 Elf5 或培养的未成熟UB细胞中的其他PC特异性基因。 Notch 信号传导抑制 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成部分 (信号-I)促进IC差异化。在 Aim1 中，集合管分化的早期调节因子将 将确定早期 PC 标记 Elf5 和早期嵌入细胞 (IC) 标记 Foxi1。目标2聚焦 通过检查 Foxi1、Elf5 的贡献来确定 PC 和 IC 分化的中间调节因子 使用转基因小鼠、UB 细胞以及成熟的 PC 和 IC 将 Hes1 和 Hes1 进行集合管分化 线。在 Aim3 中，我们将确定如何维护成熟的 PC。我们有证据表明Notch 维持成熟 PC 细胞状态的信号传导。这将通过 Notch 的条件失活来验证 成人 CD 中的信号传导和 Notch 介导的 PC 维持机制将得到确定。一只老鼠 获得性肾性尿崩症 (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 维护。 通过谱系追踪，我们将明确确定 aNDI 期间是否发生 PC 到 IC 的命运变化。这些 研究不仅是渐进式的，而且为我们对集合管的理解提供了变革性的一步 开发和维护。