Regulation of nephron numbers

肾单位数量的调节

• 批准号: 7393844
• 负责人: SANJAY K NIGAM
• 金额: $ 59.02万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-05-01 至 2008-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7393844
• 关键词: 3-Dimensional; Biological; Biological Assay; Biological Models; Biology; Budgets; Cell Line; Cell model; Cells; Cellular biology; Column Chromatography; Computer Systems Development; Condition; Conditioned Culture Media; Cultured Cells; D Cells; Data; Development; Embryo; Embryonic Structures; Epithelial; Equilibrium; Essential Hypertension; Funding; Gene Expression; Genes; Glean; Goals; Growth Factor; Hypertension; Immunoblotting; In Vitro; Individual; Injury; Kidney; Laboratories; Lasers; Lead; Lung; Mesenchyme; Metanephric Diverticulum; Microdissection; Modeling; Molecular; Morphogenesis; Nephrons; Numbers; Organ; Organ Culture Techniques; Organogenesis; Outcome; Pancreas; Pattern; Physiological; Purpose; RNA; Rat Strains; Regulation; Regulatory Pathway; Research; Research Personnel; Right kidney; Role; Signal Transduction; Study models; System; Systems Analysis; Techniques; Tissues; Transforming Growth Factor beta; Trees; Work; base; immunocytochemistry; in vitro Model; insight; knockout animal; member; nephrogenesis; novel; programs; research study; spatiotemporal

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.

该提案的长期目标是了解肾脏器官发生过程中分支负调节因子的生物学。肾脏的集合系统源自称为输尿管芽 (UB) 的胚胎结构的迭代分支。分支形态发生决定肾单位数量，一些人认为肾单位数量是高血压发生和肾损伤进展的重要因素。肾脏发育领域的研究人员通常倾向于关注分支刺激剂，而不是那些可能充当负调节剂或“停止信号”的刺激剂。在这里，我们认为，先验地没有理由认为对这些负调节因子的研究应该是次要的，因为它 是正负因素的平衡，最终决定发育中的肾脏集合系统的树枝化程度（以及肾单位数量）。我们还认为，如果没有关于尖端、分支点和茎的时空表达的类似信息，对于理解发育中的树木的分枝模式是如何建立的，对分枝形态发生过程中总 RNA 水平的研究价值有限。我们的实验室设计了许多体外模型系统来分析输尿管芽的分支形态发生。其中包括分离的 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 初步数据证明了这些模型系统的稳健性以及如何一起和单独使用它们来了解分支形态发生中涉及的调控途径，特别是，我们已经表明分支模式受到许多可溶性生长因子（包括TGF-β超家族成员）的负调节，以及后肾间充质细胞系的条件培养基所阐述的尚未鉴定的活性。在本提案中，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、分支点和茎上的时空表达模式，更好地理解分支负调节剂如何发挥其抑制作用：1）分支，2）分支和增殖的抑制，3）分支的抑制 对增殖没有重大影响，随后进行功能扰动实验（SA1：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、基因敲除动物）； b) 纯化一种明显新颖的活性，抑制培养的分离UB的分支（SA2：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示例。