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.
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