Volume 77, Issue 6, Pages 509-518 (March 2010) Deletion of H-Ras decreases renal fibrosis and myofibroblast activation following ureteral obstruction in mice  M. Teresa Grande, Isabel Fuentes-Calvo, Miguel Arévalo, Fabiana Heredia, Eugenio Santos, Carlos Martínez-Salgado, Diego Rodríguez-Puyol, M. Angela Nieto, José M. López-Novoa  Kidney International  Volume 77, Issue 6, Pages 509-518 (March 2010) DOI: 10.1038/ki.2009.498 Copyright © 2010 International Society of Nephrology Terms and Conditions

Figure 1 Effects of deficiency of H-Ras in interstitial fibrosis after unilateral ureteral obstruction. (a) Hematoxylin–eosin (HE-EO) staining, immunohistochemistry for fibronectin, Sirius red staining in nonobstructed (NO) and obstructed (O) kidneys from H-ras+/+ and H-ras-/- mice. Bar=100 μm. (b) Western blot analysis of α-smooth-muscle actin (α-SMA) and vimentin protein expression from NO and O kidneys from H-ras+/+ and H-ras-/- mice. Data represent the average±s.e.m. of the optical density. #P<0.01 vs corresponding NO kidneys. *P<0.01 vs O kidneys from H-ras+/+ mice. □ H-ras+/+ mice, ▪ H-ras-/- mice. Kidney International 2010 77, 509-518DOI: (10.1038/ki.2009.498) Copyright © 2010 International Society of Nephrology Terms and Conditions

Figure 2 Effects of deficiency of H-Ras in epithelial–mesenchymal transition markers after unilateral ureteral obstruction. (a) Immunohistochemistry for α-smooth-muscle actin (α-SMA) and vimentin in nonobstructed (NO) and obstructed (O) kidneys from H-ras+/+ and H-ras-/- mice. Bar=100 μm. (b) Western blot analysis of α-smooth-muscle actin (α-SMA), and vimentin protein expression from NO and O kidneys from H-ras+/+ and H-ras-/- mice. Data represent the average ±s.e.m. of the optical density. #P<0.01 vs corresponding NO kidneys. *P<0.05 vs O kidneys from H-ras+/+ mice. □ H-ras+/+ mice, ▪ H-ras-/- mice. Kidney International 2010 77, 509-518DOI: (10.1038/ki.2009.498) Copyright © 2010 International Society of Nephrology Terms and Conditions

Figure 3 Effects of deficiency of H-Ras in Ras, extracellular signal-regulated kinase (ERK) and Akt activation after unilateral ureteral obstruction (UUO). (a) Ras activation in nonobstructed (NO) and obstructed (O) kidneys of H-ras+/+and H-ras-/- mice. H-RasGTP levels were determined by enzyme-linked immunosorbent assay (ELISA). Data represent the average±s.e.m. of relative light units. #P<0.01 vs corresponding NO kidneys. *P<0.05 vs NO and O kidneys from H-ras+/+ mice. □ H-ras+/+ mice, ▪ H-ras-/- mice. (b) Representative images of western blot analysis of ERK activation and Akt activation in NO and O kidneys from H-ras+/+and H-ras-/- mice. (c) Representative staining of NO and O kidneys from H-ras+/+and H-ras-/- mice 15 days after UUO. Bar=100 μm. Kidney International 2010 77, 509-518DOI: (10.1038/ki.2009.498) Copyright © 2010 International Society of Nephrology Terms and Conditions

Figure 4 Effects of deficiency of H-Ras in proliferation after unilateral ureteral obstruction (UUO). (a) Representative staining of Ki-67 in nonobstructed (NO) and obstructed (O) kidneys from H-ras+/+and H-ras-/- mice 15 days after UUO. Histogram represents number of Ki-67-positive interstitial and tubular cells in NO and O kidneys from H-ras+/+and H-ras-/- mice 15 days after UUO. #P<0.01 vs corresponding NO kidneys. *P<0.01 vs O kidney from H-ras+/+ mice. □ H-ras+/+ mice, ▪ H-ras-/- mice. (b) Double immunofluorescence of α-smooth-muscle actin (α-SMA) (green) and Ki-67 (red) in NO and O kidneys from H-ras+/+ and H-ras-/- mice. Arrows: tubular proliferating cells, arrowheads: interstitial, fibroblast shaped, α-SMA-expressing proliferating cells. Kidney International 2010 77, 509-518DOI: (10.1038/ki.2009.498) Copyright © 2010 International Society of Nephrology Terms and Conditions

Figure 5 Effects of deficiency of H-Ras on epithelial–mesenchymal transition after unilateral ureteral obstruction. Quantitative real-time RT-PCR for transforming growth factor-β (TGF-β), Snail1, Snail2, vimentin, E-cadherin, Cadherin-16 and collagen type I. In nonobstructed (NO) and obstructed (O) kidneys from H-ras+/+ and H-ras-/- mice. #P<0.01 vs corresponding NO kidneys. *P<0.01 vs O kidneys from H-ras+/+ mice. +P<0.05 vs O kidneys from H-ras+/+ mice. □ H-ras+/+ mice, ▪ H-ras-/- mice. Kidney International 2010 77, 509-518DOI: (10.1038/ki.2009.498) Copyright © 2010 International Society of Nephrology Terms and Conditions

Figure 6 Effects of the absence of H-Ras isoform in proliferation on embrionary fibroblasts. (a) Effect of transforming growth factor-β1 (TGF-β1) treatment (1 ng/ml) on cell proliferation in H-ras+/+ and H-ras-/- fibroblasts evaluated by Ki-67 immunostaining (and by crystal violet staining). Left panel shows representative pictures of Ki-67 immunostaining of different experiments, performed in similar conditions, histogram represents the mean±s.e.m. of 8–16 experiments, expressed as % of Ki-67 stained cells/Hoechst stained cells over basal values (H-ras+/+ and H-ras-/- fibroblasts in 0% fetal calf serum (FCS), 100%). *P<0.01 vs H-ras+/+ fibroblasts in basal conditions; +P<0.05 vs H-ras-/- fibroblasts in basal conditions; #P<0.01 vs H-ras+/+ fibroblasts treated with 1 ng/ml TGF-β1. (b) Effect of TGF-β1 treatment (1 ng/ml) on cell proliferation in H-ras+/+ and H-ras-/- fibroblasts evaluated by crystal violet staining. Histogram represents the mean±s.e.m. of 80 (H-ras+/+) and 14 experiments (H-ras-/-) performed in triplicate and expressed as % over basal values (WT fibroblasts in 0% FCS, 100%). *P<0.01 vs H-ras+/+ fibroblasts in basal conditions; +P<0.05 vs H-ras-/- fibroblasts in basal conditions; #P<0.01 vs H-ras+/+ fibroblasts treated with 1 ng/ml TGF-β1. (c) Percentage of WT and H-ras-/- fibroblasts in each phase of the cell cycle, evaluated by flow cytometry (see Materials and Methods section). G0: quiescent cells; G1, G2: cell-cycle phases 1 and 2; S: synthesis phase; M: mitosis. *P<0.01 vs H-ras+/+ fibroblasts. □ H-ras+/+ fibroblasts, ▪ H-ras-/- fibroblasts. Kidney International 2010 77, 509-518DOI: (10.1038/ki.2009.498) Copyright © 2010 International Society of Nephrology Terms and Conditions

Figure 7 Effects of the absence of H-Ras isoform on epithelial–mesenchymal transition marker expression and migration on embrionary fibroblasts. (a) Quantitative real-time RT-PCR for Snail1, Snail2, vimentin, transforming growth factor-β (TGF-β) and collagen type I in H-ras+/+ and H-ras-/- fibroblasts with and without TGF-β1 treatment (1 ng/ml). #P<0.01 vs fibroblasts in basal conditions. *P<0.01 vs H-ras+/+ fibroblasts (treated with 1ng/ml TGF-β1. (b) H-ras+/+ and H-ras-/- fibroblast migration evaluated by the analysis of wounds half-closure time. Lines in the micrographs represent the migration front after wound. (c) H-ras+/+ and H-ras-/- migration evaluated by the analysis of migration through trans-wells. Histogram represents the mean±s.e.m. of three experiments quantifying the number of crystal violet stained cells that go through the migration chamber, expressed in absorbance units. *P<0.01 vs H-ras+/+ fibroblasts after 12 h. #P<0.01 vs H-ras+/+ fibroblasts after 24 h. □ H-ras+/+ fibroblasts, ▪ H-ras-/- fibroblasts. Kidney International 2010 77, 509-518DOI: (10.1038/ki.2009.498) Copyright © 2010 International Society of Nephrology Terms and Conditions