Mechanisms of Renal Tubulogenesis

描述（由申请方提供）：与许多上皮器官一样，肾脏主要由排列着单层极化上皮细胞的小管组成。虽然我们已经对控制肾脏发育的基因有了很多了解，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）胶原凝胶。单个MDCK细胞生长形成由单层上皮细胞内衬的中空囊肿。当这些囊肿用肝细胞生长因子（HGF）刺激时，它们在约3天的时间内形成小管。这是一个很好的模型系统，用于研究肾小管形成，也可以提供一些病理生理过程的信息，如从急性肾小管坏死，肾纤维化，多囊肾病，也许从干细胞肾再生的恢复。我们将研究细胞对HGF反应的信号通路。ERK激活的动力学表明，它是由涉及B-Raf的MAPK级联激活的，而不是更好的已知Raf-1。我们将通过研究B-Raf通路组分的激活以及使用显性负性和RNAi来验证这一假设。我们将测试Rho家族GTP酶，Rho激酶和脂筏在小管形成的早期阶段的参与，特别是从囊肿中细胞的基底外侧表面延伸的形成。我们将使用时间推移共聚焦显微镜观察的影响，扰动这些分子组件上的扩展形成。我们将测试磷脂酰肌醇（3，4，5）P3 [PI（3，4，5）P3]在延伸形成中的参与。我们发现外源性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的可能效应子。肾脏疾病，包括发育缺陷、肾损伤和在肾脏中形成多个囊肿的遗传条件，是主要的健康问题。肾脏由许多排列着细胞的细管组成。我们正在使用一个简单的模型系统，其中肾细胞在培养物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.