Mechanisms of Renal Tubulogenesis

肾脏，像许多上皮器官一样，主要由肾小管组成，肾小管由单层极化的 上皮细胞虽然我们已经了解了很多关于控制肾脏发育的基因， 对细胞重新排列成小管的机制知之甚少。Weuse a Madin-Darby犬肾（MDCK）细胞在三维胶原中生长的简化模型系统 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，它们在约3天的时间内形成小管。这是一 研究肾小管形成的良好模型系统，也可以提供一些信息 病理生理过程，如从急性肾小管坏死、肾纤维化、多囊肾 我们将研究干细胞的信号通路， 对HGF有反应ERK激活的动力学表明它是由MAPK级联激活的， B-Raf，而不是更知名的Raf-1。我们将通过研究这些成分的激活来验证这一假设 的B-Raf途径，并通过使用显性负和RNAi。我们将测试罗是否参与 在微管形成的早期阶段，尤其是在微管形成的早期阶段， 从囊肿中细胞的基底外侧表面延伸。我们将使用时间推移共聚焦显微镜， 观察扰动这些分子成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]参与延伸形成。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那些相同， 由HGF生产。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，肾损伤和遗传条件，形成多种 肾囊肿是一种主要的健康问题。肾脏由许多排列着细胞的细管组成。我们 他们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， 以及我们如何影响它来治疗和预防肾脏疾病。

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. Weuse a simplified model system of Madin-Darbycanine kidney (MDCK) cells grown in a 3 dimensionalcollagen gel. Single MDCK cellsgrow 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 informationon several pathophysiological processes, such as recovery from acute tubular necrosis, renal fibrosis, polycystickidney 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 ERKsuggest that it is activated by a MAPK cascadeinvolving 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.