Gene Expression Changes in Early 3D Renal Tubulogenesis

描述(申请人提供)：许多上皮性器官，如肾脏，主要由分支管状结构组成。我们的目标是了解一般的肾小管发生的生物学，特别是肾小管的发生。小管形成是一个涉及多种因素和受体的鲜为人知的过程。由于器官发生的复杂性和暂时性，在体内研究小管发生是非常困难的。我们和其他人已经成功地使用二维(2D)体外细胞培养(生长在塑料或可渗透过滤器上的细胞)来研究肾小管上皮细胞生长因子依赖的调节。然而，越来越多的证据表明，上皮细胞根据细胞微环境的不同而受到不同的调控，强烈支持使用合适的三维(3D)模型来研究小管形成，例如研究得很好的实验，涉及Madin-Darby犬肾(MDCK)细胞在3D胶原基质中生长直到囊肿期，以及用肝细胞生长因子(HGF)诱导小管形成。我们已经使用这个系统来鉴定对小管发生至关重要的蛋白质。我们的假设是，3D培养中的MDCK细胞在HGF诱导后的基因表达变化比在HGF刺激之前在2D培养中生长的MDCK细胞中的基因表达变化更具生理性，因此与肾小管的形成有关。为了通过一种无偏见的方法确定参与MDCK细胞小管形成的新基因和新途径，我们提出了一种系统，该系统利用3D MDCK/HGF分析与新推出的犬类DNA微阵列相结合，我们最近描述并验证了这一系统(Aim 1.1)。我们将具体确定HGF刺激的肾小管MDCK细胞中基因表达的差异，这取决于微环境(即2D和3D培养)(Aim 1.2)。最有希望的候选“小管生成”将根据信使核糖核酸的折叠变化、途径的位置和机制的似然性来选择(目标2.1)。这些候选基因将在体外肾小管形成的不同阶段通过其蛋白产物的定位和突变分析来表征，以确定它们在肾小管形成中的作用(目标2.2)。随后将提出进一步的研究，包括建立体内模型来测试假定的小管生成因子在肾脏发育中的作用，最终目标是调节这些小管生成因子和修复小管形成过程中的缺陷的途径。

DESCRIPTION (provided by applicant): Many epithelial organs, such as the kidney, are composed largely of branching tubular structures. Our goal is to understand the biology of tubulogenesis in general and renal tubulogenesis in particular. Tubule formation is a poorly understood process involving multiple factors and receptors. Due to the complexity and transitory nature of organogenesis, it is exceedingly difficult to study tubulogenesis in vivo. We and others have successfully used two-dimensional (2D) in vitro cell culture (cells grown on plastic or permeable filters) to study growth factor dependent regulation of tubular epithelial cells. However, a growing body of evidence indicates that epithelial cells are differentially regulated depending upon the cellular microenvironment, arguing strongly for the study of tubulogenesis using an appropriate three-dimensional (3D) model, such as the well-studied assay involving growth of Madin-Darby canine kidney (MDCK) cells in a 3D collagen matrix until the cyst stage and induction of tubule formation with hepatocyte growth factor (HGF). We have used this system to identify proteins critical for tubulogenesis. Our underlying hypothesis is that gene expression changes following HGF induction in MDCK cells grown in 3D culture is more physiologic than gene expression changes in MDCK cells grown in 2D culture prior to stimulation with HGF, and, therefore, is relevant to renal tubulogenesis. To define novel genes and pathways involved in tubulogenesis of MDCK cells by an unbiased approach, we propose a system utilizing the 3D MDCK/HGF assay in combination with a newly available canine DNA microarray, which we recently described and validated (Aim 1.1). We will specifically determine how gene expression differs in renal tubular MDCK cells stimulated with HGF depending on the microenvironment (i.e. in 2D versus 3D culture) (Aim 1.2). The most promising candidate "tubulogenes" will be selected based on mRNA fold change, pathway placement, and mechanistic plausibility (Aim 2.1). These candidate genes will be characterized during the different stages of in vitro renal tubule formation by localization of their protein products and mutational analysis, to define their role in tubulogenesis (Aim 2.2). Further studies will then be proposed that involve generation of in vivo models to test the role of the putative tubulogenes in renal development, with the ultimate goal of modulating these tubulogenes and pathways to repair defects in tubule formation.