1. Heterozygous disruption of activin receptor–like kinase 1 is associated with increased renal fibrosis in a mouse model of obstructive nephropathy 
José M. Muñoz-Félix, José M. López-Novoa, Carlos Martínez-Salgado 
Kidney International 
Volume 85, Issue 2, Pages (January 2014)
DOI: /ki
Copyright © 2014 International Society of Nephrology Terms and Conditions

2. Figure 1
Effects of activin receptor–like kinase 1 (ALK1) haploinsufficiency in interstitial fibrosis after unilateral ureteral obstruction. (a) Hematoxylin–eosin staining in sham-operated (SO), nonobstructed (NO), and obstructed (O) kidneys in ALK1+/+ and ALK1+/- mice. Long arrows mark tubular dilatation, short arrows mark tubular atrophy, and arrowheads mark hypercellularity. (b) Masson’s trichrome staining in SO, NO, and O kidneys from ALK1+/+ and ALK1+/- mice. (c1) Sirius red–stained area in SO, NO, and O kidneys from ALK1+/+ and ALK1+/- mice, and (c2) quantification by Image-Pro Plus in NO (n=6 different areas evaluated) and O kidneys (n=6) from ALK1+/+ and ALK1+/- mice. Western blot analysis of collagen type I (d1) and fibronectin (d2) protein expression from SO (n=5), NO (n=5), and O kidneys (n=5) from ALK1+/+ and ALK1+/- mice. Data represent the average±s.e.m. of the optical density. *P<0.01 vs. SO kidneys from ALK1+/+ mice. #P<0.01 vs. SO kidneys from ALK1+/- mice. +P<0.05 vs. O kidneys from ALK1+/+ mice. Original magnification × 400.
Kidney International  , DOI: ( /ki )
Copyright © 2014 International Society of Nephrology Terms and Conditions

3. Figure 2
Effect of activin receptor–like kinase 1 (ALK1) haploinsufficiency in myofibroblasts number following ureteral obstruction. (a) Immunohistochemistry for α-smooth muscle actin (α-SMA) in sham-operated (SO), nonobstructed (NO), and obstructed (O) kidneys from ALK1+/+ and ALK1+/- mice. (b) Immunohistochemistry for S100A4 in SO, NO, and O kidneys from ALK1+/+ and ALK1+/- mice. Quantification of positive-stained cells was carried out in 6 different areas in sham kidneys, 6 areas in NO kidneys, and in 12 areas in O kidneys. (c) Western blot analysis of α-SMA and S100A4 protein expression from SO, NO, and O kidneys from ALK1+/+ and ALK1+/- mice. Data represent the average±s.e.m. of the optical density of five different sample groups analyzed in the same conditions. *P<0.01 vs. SO kidneys from ALK1+/+ mice. #P<0.01 vs. SO kidneys from ALK1+/- mice. Original magnification × 400.
Kidney International  , DOI: ( /ki )
Copyright © 2014 International Society of Nephrology Terms and Conditions

4. Figure 3
Effect of activin receptor–like kinase 1 (ALK1) haploinsufficiency in cellular proliferation following ureteral obstruction. (a) Immunohistochemistry for Ki67 in sham-operated (SO), nonobstructed (NO), and obstructed (O) kidneys from ALK1+/+ and ALK1+/- mice. Quantification of positive-stained cells was carried out in 6 different areas in SO kidneys, 6 areas in NO kidneys, and in 12 areas in O kidneys. (b) Bright-field and immunofluorescent double staining of Ki67 and α-smooth muscle actin (α-SMA) in NO and O kidneys from ALK1+/+ and ALK1+/- mice. Arrows in pictures mark tubular cells, and arrowheads mark interstitial cells. (c) Western blot analysis of proliferation cellular nuclear antigen (PCNA) expression from SO, NO, and O kidneys from ALK1+/+ and ALK1+/- mice. Data represent the average±s.e.m. of the optical density of five different sample groups analyzed in the same conditions. *P<0.01 vs. SO kidneys from ALK1+/+ mice. #P<0.01 vs. SO kidneys from ALK1+/- mice. +P<0.05 vs. O kidneys from ALK1+/+ mice. Original magnification × 400 (visible) and × 630 (fluorescence).
Kidney International  , DOI: ( /ki )
Copyright © 2014 International Society of Nephrology Terms and Conditions

5. Figure 4
Effect of activin receptor–like kinase 1 (ALK1) haploinsufficiency in Smad signaling following ureteral obstruction. Western blot analysis of phospho-Smad3, phospho-Smad2, phospho-Smad1, and Smad2/3 and Smad1 protein expression from sham-operated (SO), nonobstructed (NO), and obstructed (O) kidneys from ALK1+/+ and ALK1+/- mice. Data represent the average±s.e.m. of the optical density of four different sample groups analyzed in the same conditions. *P<0.01 vs. SO kidneys from ALK1+/+ vs. SO kidneys from ALK1+/+ mice. #P<0.01 vs. SO kidneys from ALK1+/- mice. +P<0.05 vs. O kidneys from ALK1+/+ mice.
Kidney International  , DOI: ( /ki )
Copyright © 2014 International Society of Nephrology Terms and Conditions

6. Figure 5
Immunofluorescence staining of phospho-Smad2/3 and phospho-Smad1/5/8 in nonobstructed (NO) and obstructed (O) kidneys from ALK1+/+ and ALK1+/- mice. (a) Immunofluorescence staining of phospho-Smad2/3 and bright-field, original magnification × (b) Phospho-Smad2/3 and α-SMA double immunofluorescence in NO and O kidneys from ALK1+/- mice. Original magnification × 630. (c) Phospho-Smad1/5/8 immunostaining in NO and O kidneys from ALK1+/+ and ALK1+/- mice. Original magnification × 400. Arrows in pictures mark tubular cells, and arrowheads mark interstitial cells (a, b). Quantification of tubular and interstitial positive-stained cells (b) and total positive cells (c) was carried out in 6 different areas in sham kidneys, 6 areas in NO kidneys, and in 12 areas in O kidneys.
Kidney International  , DOI: ( /ki )
Copyright © 2014 International Society of Nephrology Terms and Conditions

7. Figure 6
Effect of activin receptor–like kinase 1 (ALK1) heterozygosity in transforming growth factor-β1 (TGF-β1) receptor expression. Western blot analysis of ALK1, ALK5, and endoglin protein expression from sham-operated (SO), nonobstructed (NO), and obstructed (O) kidneys from ALK1+/+ and ALK1+/- mice. Data represent the average±s.e.m. of the optical density of five different sample groups analyzed in the same conditions. *P<0.05 vs. SO kidneys from ALK1+/+ mice. #P<0.01 vs. SO kidneys from ALK1+/- mice. +P<0.05 vs. O kidneys from ALK1+/+ mice.
Kidney International  , DOI: ( /ki )
Copyright © 2014 International Society of Nephrology Terms and Conditions

8. Figure 7
Activin receptor–like kinase 1 (ALK1) expression in obstructed kidneys from ALK1+/+ and ALK1+/- mice. (a) ALK1 immunofluorescence staining in nonobstructed (NO), and obstructed (O) kidneys. (b1) Double immunofluorescence of ALK1 (green) and α-smooth muscle actin (α-SMA) (red) in NO and O kidneys from ALK1+/+ mice. (b2) ALK1 (green) and α-SMA (red) double immunofluorescence in NO kidney cortex from ALK1+/+ mice, in O kidney cortex from ALK1+/+ mice, and in O kidney medulla from ALK1+/+ mice. (c) Double immunofluorescence of ALK1 (green) and CD68 (red) in NO and O kidneys from ALK1+/- mice. Arrows mark cortical interstitial myofibroblasts (b1, b2), and arrowheads mark CD68-positive cells (c). Original magnification × 630 (a, c) and × 1000 (b1, b2).
Kidney International  , DOI: ( /ki )
Copyright © 2014 International Society of Nephrology Terms and Conditions

9. Figure 8
Effect of activin receptor–like kinase 1 (ALK1) haploinsufficiency in extracellular matrix (ECM) protein expression and transforming growth factor-β1 (TGF-β1) receptor expression in renal fibroblasts. (a) Western blot analysis of ALK1, α-smooth muscle actin (α-SMA), and Erk1/2 protein expression in ALK1+/+ and ALK1+/- renal fibroblasts. (b) Immunofluorescence of total actin and α-SMA in ALK1+/+ and ALK1+/- renal fibroblasts. (c) Western blot analysis of collagen type I, fibronectin, and ALK5 protein expression in ALK1+/+ and ALK1+/- renal fibroblasts. (d) Western blot analysis of collagen type I and fibronectin protein expression in ALK1+/+ and ALK1+/- renal fibroblasts after treatment with 1ng/ml TGF-β1. Data represent the average±s.e.m. of the optical density of three different sample groups analyzed in the same conditions. *P<0.01 vs. ALK1+/+ renal fibroblasts in basal conditions. #P<0.05 vs. ALK1+/- renal fibroblasts in basal conditions. +P<0.05 vs. ALK1+/+ renal fibroblasts in basal conditions.
Kidney International  , DOI: ( /ki )
Copyright © 2014 International Society of Nephrology Terms and Conditions

10. Figure 9
Effect of activin receptor–like kinase 1 (ALK1) haploinsufficiency in transforming growth factor-β1 (TGF-β)-induced Smad phosphorylation in renal fibroblasts. Western blot analysis of phospho-Smad3, phospho-Smad2, phospho-Smad1, and Smad1 and Smad2/3 protein expression in ALK1+/+ and ALK1+/- renal fibroblasts. Data represent the average±s.e.m. of the optical density of six different sample groups analyzed in the same conditions. *P<0.01 vs. ALK1+/+ renal fibroblasts in basal conditions. #P<0.01 vs. ALK1+/- renal fibroblasts in basal conditions. +P<0.05 vs. ALK1+/+ renal fibroblasts after treatment with 1ng/ml TGF-β1.
Kidney International  , DOI: ( /ki )
Copyright © 2014 International Society of Nephrology Terms and Conditions