Regulation of nephron numbers

肾单位数量的调节

• 批准号: 6735484
• 负责人: SANJAY K NIGAM
• 金额: $ 22.29万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-05-01 至 2008-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6735484
• 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在肾器官发生过程中分支的负调节因子的生物学。肾脏的集合系统起源于称为输尿管芽（UB）的胚胎结构的反复分支。分支形态发生决定了肾单位的数量，这被一些人认为是高血压发展和肾损伤进展的重要因素。肾脏发育领域的研究者通常倾向于关注分支的刺激物，而不是那些可能作为负调节剂或“停止信号”的刺激物。“在这里，我们认为，没有理由，先验，这些负调节器的研究应该是次要的，因为它 是正性和负性因素的平衡，其最终决定发育中的肾集合系统的分支程度（以及肾单位数目）。我们还arque，总RNA水平在分支形态发生的研究是有限的价值，没有类似的信息时空表达的提示，分支点和茎了解如何发展树的树枝化模式成为建立。我们的实验室已经设计了一些体外模型系统，用于分析输尿管芽的分支形态发生。这些包括分离的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 初步数据表明这些模型系统的稳健性，以及它们如何可以一起或单独使用，以理解涉及分支形态发生的调节途径。特别地，我们已经表明分支模式受到许多可溶性生长因子的负调节，包括TGF-β超家族的成员，和一种尚未鉴定的由后肾间充质细胞系的条件培养基产生的活性。在这个提议中，我们的目标是：a）利用上述模型系统，通过检查在以下条件下尖端、分支点和茎的时空表达模式来更好地理解分支的负调节剂如何发挥其抑制作用：1）分支，2）抑制分支和增殖，和3）抑制分支 对增殖无重大影响，随后进行功能干扰实验（SA 1：细胞和器官培养、体外功能测定、免疫细胞化学、阵列、激光显微切割、敲除动物）;和B）纯化抑制培养的分离UB分支的明显新活性（SA 2：柱层析、免疫印迹、微测序）。在初步数据中，我们提供了成功完成该项目目标所需的所有技术的专业知识的例子。