Mechanisms of Renal Tubulogenesis

描述(申请人提供)：肾脏，像许多上皮性器官一样，主要由单层极化上皮细胞排列的小管组成。虽然我们已经了解了很多控制肾脏发育的基因，但我们对细胞重新排列成小管的机制知之甚少。我们使用一个简化的模型系统的Madin-Darby犬肾(MDCK)细胞生长在三维(3D)胶原胶。单个MDCK细胞生长形成内衬单层上皮细胞的中空包囊。当这些囊被肝细胞生长因子(HGF)刺激时，它们在~3天内形成小管。这是一个很好的研究肾小管形成的模型系统，也可以提供几个病理生理过程的信息，如急性肾小管坏死、肾纤维化、多囊肾病的恢复，以及可能的干细胞再生肾脏。我们将研究细胞对HGF作出反应的信号通路。ERK的激活动力学表明，它是由涉及B-Raf的MAPK级联激活的，而不是更知名的Raf-1。我们将通过研究B-Raf途径组件的激活以及使用显性负离子和RNAi来验证这一假设。我们将测试Rho家族GTPase、Rho Kinase和脂筏在小管形成的早期阶段，特别是从囊内细胞的基底外侧表面形成延伸的过程中的参与。我们将使用时间推移共聚焦显微镜来观察扰动这些分子组分对延伸形成的影响。我们将测试磷脂酰肌醇(3，4，5)P3[PI(3，4，5)P3]在伸展形成中的作用。我们已经发现外源PI(3，4，5)P3诱导延伸形成，我们将测试这些延伸是否与HGF产生的延伸相同。我们将分析PI(3，4，5)P3可能的效应器。肾脏疾病是主要的健康问题，包括发育缺陷、肾脏损伤和在肾脏中形成多个囊肿的遗传条件。肾脏由许多排列着细胞的狭窄的管子组成。我们正在使用一个简单的模型系统，其中肾脏细胞在培养中生长并产生这样的管，以研究管的形成以及我们如何影响它来治疗和预防肾脏疾病。

DESCRIPTION (provided by applicant): The kidney, like many epithelial organs, consists mainly of tubules lined by a monolayer of polarized epithelial cells. Though we have learned a great deal about the genes that control kidney development, we know much less about the mechanisms by which cells rearrange themselves into tubules. We use a simplified model system of Madin-Darby canine kidney (MDCK) cells grown in a 3 dimensional (3D) collagen gel. Single MDCK cells grow to form hollow cysts lined by a monolayer of epithelial cells. When these cysts are stimulated with Hepatocyte Growth Factor (HGF), they form tubules over a period of ~3 days. This is a good model system for studying renal tubule formation, and can also provide information on several pathophysiological processes, such as recovery from acute tubular necrosis, renal fibrosis, polycystic kidney disease and perhaps renal regeneration from stem cells. We will study the signaling pathways by which cells respond to HGF. The kinetics of activation of ERK suggest that it is activated by a MAPK cascade involving B-Raf, rather than the better known Raf-1. We will test this hypothesis by studying activation of components of the B-Raf pathway and by using dominant negatives and RNAi. We will test the involvement of Rho family GTPases, Rho Kinase and lipid rafts in the early stages of tubulogenesis, especially the formation of extensions from the basolateral surface of cells in cysts. We will use time lapse confocal microscopy to observe the effects of perturbing these molecular components on extension formation. We will test the involvement of phosphatidyl inositol (3,4,5)P3 [PI(3,4,5)P3] in extension formation. We have found that exogenous PI(3,4,5)P3 induces extension formation and we will test if these extensions are identical to those produced by HGF. We will analyze possible effectors of PI(3,4,5)P3. Kidney diseases, including developmental defects, kidney injury and genetic conditions that form multiple cysts in the kidney, are major health problems. The kidney consists of many narrow tubes lined by cells. We are using a simple model system where kidney cells are grown in culture and produce such tubes, to study tube formation and how we can influence this to treat and prevent kidney diseases.