Regulation of nephron numbers

肾单位数量的调节

• 批准号: 7061694
• 负责人: SANJAY K NIGAM
• 金额: $ 35.78万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-05-01 至 2008-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7061694
• 关键词: embryo /fetus tissue /cell culture; familial hypertension; growth inhibitors; laboratory mouse; laboratory rat; laser capture microdissection; mammalian embryology; microarray technology; nephrogenesis; renal tubule; transforming growth factors

The long term goal of this proposal is to understand the biology of negative regulators of branching during kidney organogenesis. The collecting system of the kidney arises from iterative branching of an embryonic structure known as the ureteric bud (UB). Branching morphogenesis determines nephron number, which is believed by some to be an important factor in the development of hypertension and the progression of renal injury. Investigators in the field of renal development have generally tended to focus on stimulators of branching, rather than those that might act as negative regulators or "stop signals." Here we argue that there is no reason, a priori, that the study of these negative regulators should be of secondary importance, since it is the balance of positive and neqative factors that ultimately determines the deqree of arborization of the developing kidney's collecting system (as well as nephron number). We also arque that studies of total RNA levels during branching morphogenesis are of limited value without similar information on spatiotemporal expression at tips, branch points and stalks for understanding how the arborization pattern of the developing tree becomes established. Our lab has devised a number of in vitro model systems for analyzing branching morphogenesis of the ureteric bud. These include the isolated UB culture model and cell culture-based models (UB and IMCD) of branching morphogenesis. We have amassed a considerable amount of preliminary data that demonstrates the robustness of these model systems and how they can be employed, together and separately, to understand regulatory pathways involved in branching morphogenesis, tn particular, we have shown that the branching pattern is negatively modulated by a number of soluble growth factors, including members of the TGF-beta superfamily, and an as yet unidentified activity elaborated by the conditioned medium of a metanephric mesenchyme cell line. In this proposal, we aim to: a) utilize the aforementioned model systems to better understand how negative modulators of branching exert their inhibitory effects by examining spatiotemporal expression patterns on tips, branch points and stalks under conditions of: 1) branching, 2) inhibition of branching AND proliferation, and 3) inhibition of branching WITHOUT major effects on proliferationofollowed by functional perturbation experiments (SA1: cell and organ culture, in vitro functional assays, immunocytochemistry, arrays, laser microdissection, knockout animals); and b) purify an apparently novel activity that inhibits branching of the cultured isolated UB (SA2: column chromatography, immunoblotting, microsequencing). In the preliminary data, we provide examples of our expertise in all the techniques required to successfully complete the aims of this project.

这项建议的长期目标是了解肾脏器官发生过程中分支的负调控因素的生物学。肾脏的收集系统起源于一种被称为输尿管芽(UB)的胚胎结构的迭代分支。分支形态发生决定了肾单位的数量，一些人认为这是高血压发生和肾损伤进展的一个重要因素。肾脏发育领域的研究人员通常倾向于关注分支的刺激因素，而不是那些可能起到负面调节或“停止信号”作用的因素。在这里，我们争辩说，没有理由，先验地，这些负面调节器的研究应该是次要的，因为它 是积极因素和消极因素的平衡，最终决定了发育中的肾脏收集系统的分支程度(以及肾单位数)。我们还指出，在没有关于枝梢、分枝点和茎的时空表达的相似信息的情况下，对分枝形态发生过程中总RNA水平的研究价值有限，以了解发育中的树木的分枝模式是如何建立的。我们实验室已经设计了一些体外模型系统来分析输尿管芽的分枝形态发生。这些模型包括分离的UB培养模型和基于细胞培养的分支形态发生模型(UB和IMCD)。我们已经积累了相当数量的 初步数据表明了这些模型系统的稳定性，以及它们如何一起和单独地用于理解涉及分支形态发生的调控途径，特别是，我们已经表明，分支模式受到一些可溶性生长因子的负面调节，包括转化生长因子-β超家族的成员，以及由后肾间充质细胞系的条件培养液阐述的迄今未知的活动。在该方案中，我们的目标是：a)利用上述模型系统，通过检测在1)分枝，2)分枝和增殖抑制，3)分枝抑制条件下，茎尖、分支点和茎上的时空表达模式，更好地理解分枝负调控因子如何发挥其抑制作用 在对UB的增殖没有重大影响的情况下，进一步进行功能扰动实验(SA1：细胞和器官培养、体外功能分析、免疫细胞化学、阵列、激光显微切割、基因敲除动物)；以及b)纯化一种明显抑制培养的UB分支的新活性(SA2：柱层析、免疫印迹、微测序)。在初步数据中，我们提供了我们在成功完成该项目目标所需的所有技术方面的专业知识的例子。