Although members of the Ras GTPase family control cell growth, differentiation, proliferation, and migration, their role in development of the collecting system of the kidney is unexplored. The Ras gene family has been found to be activated in approximately 30% of human cancers, including myeloproliferative disorders and leukemias. The LZRS-GFP empty vector or carrying the different Ras family members was transfected into the packaging cell line Phoenix 293 using FuGENE 6 transfection reagent (Roche Diagnostics, Indianapolis, IN). The G-domain, which catalyzes GTP hydrolysis and mediates downstream signaling, is 95% conserved between the Ras isoforms. Original magnification, 200×.To determine the signaling molecules involved in UB branching morphogenesis, cells were cultured as indicated above in the presence or absence of 1 μM Y27632 (a Rho kinase inhibitor), 10 μg/ml C3 (a Rho inhibitor), 1 μM LY294002 (a PI3-K inhibitor), 20 nM wortmannin (a PI3-K inhibitor), 5 μM PD98059 (an ERK inhibitor), and 0.5 μM PD169316 (a p38 inhibitor) (Calbiochem, San Diego, CA), which were added to the medium at the start of the cultures.

DMEM supplemented with 10% FCS was added to the top and bottom of the transwells. Kidneys were subsequently incubated with anti-E-cadherin antibodies (1:100 in 2% goat serum in PBST) for 4 h at 4°C. The kidneys were then washed in PBST four times for 1 h and incubated with a fluorescein isothiocyanate-conjugated secondary antibody (1:100 in 2% goat serum in PBST) for 3 h at room temperature. Transfection of 80% of cells is usually achieved 48 h after transfection as assessed by the percentage of GFP-positive Phoenix cells. Mouse kidneys at different stages of development were stained with antibodies to H-Ras, R-Ras, and TC21. 10.1128/MCB.21.5.1444-1452.2001 [PMC free article] [Google Scholar] These differences in branching morphogenesis are mediated by differential regulation/activation of the Rho family of GTPases and mitogen-activated protein kinases.

Stable cell populations of UB cells expressing equal GFP levels were selected using an FACStar Plus cell sorter (BD Biosciences, Franklin Lakes, NJ). Polyclonal cell populations from the top one-third of gated cells were used in experiments presented in this article. In contrast, ERK and p38 play a significant role in determining tubule length and branching of both wild-type and Ras-expressing UB cells. For the inhibitor experiments, 20 μM Y27632, 50 μM LY294002, 100 μM PD98059, and 100 μM PD169316 were added to the medium. Using murine UB cells expressing activated forms of H-Ras, R-Ras, and TC21, we show that H-Ras inhibits the ability of UB cells to branch in three-dimensional (3D) cultures, but it does not alter their ability to form tubular structures. The G-domain, which catalyzes GTP hydrolysis and mediates downstream signaling, is 95% conserved between the Ras isoforms. Ras genes, encoding three highly homologous 21-kDa proteins, K-Ras, H-Ras and N-Ras, regulate a variety of biological processes including cell growth, differentiation, proliferation and apoptosis. The arrowheads indicate UB-derived structures in the embryonic kidneys. Because most branching of the UB occurs early in development, it is conceivable that R-Ras and TC-21 play a role in facilitating branching and growth in early UB development, whereas H-Ras might favor cell migration and elongation of tubules, events that occur later in development.This work was supported by National Cancer Institute/National Institutes of Health Grant R01-CA94849 (to A. P.), National Institutes of Health/National Institute of Diabetes and Digestive and Kidney Diseases Grant R01-DK074359 (to A. P.), National Institutes of Health/National Institute of Diabetes and Digestive and Kidney Diseases Grant R01-DK 69921 (to R. Because of their very high sequence identity, biochemical studies done on H-Ras …

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