Mechanisms of Renal Tubulogenesis

像许多上皮性器官一样，肾脏主要由排列着单层极化的小管组成。 上皮细胞。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。我们使用的是 MDCK细胞在三维胶原膜中生长的简化模型体系 凝胶。单个MDCK细胞生长形成由单层上皮细胞组成的中空囊泡。当这些囊泡 在肝细胞生长因子(HGF)的刺激下，它们在~3天内形成小管。这是一个 用于研究肾小管形成的良好模型系统，还可以提供关于几个 病理生理过程，如急性肾小管坏死、肾纤维化、多囊肾的恢复 疾病，也许还有来自干细胞的肾脏再生。我们将研究细胞通过哪些信号通路 对HGF作出回应。ERK的激活动力学表明它是由一个MAPK级联激活的 B-Raf，而不是更知名的Raf-1。我们将通过研究组件的激活来检验这一假设 B-Raf途径，并使用显性负性和RNAi。我们将测试Rho的参与 肾小管形成早期的家族GTP酶、Rho激酶和脂筏，尤其是 从包囊细胞的基侧表面延伸而来。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]参与伸展形成。我们发现， 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那些相同 由HGF制作。我们将分析PI(3，4，5)P3可能的效应器。 肾脏疾病，包括发育缺陷、肾脏损伤和形成多发性 肾脏中的囊肿是主要的健康问题。肾脏由许多排列着细胞的狭窄的管子组成。我们 正在使用一种简单的模型系统，在其中肾脏细胞在培养中生长并产生这样的管，以研究 管子的形成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.