1. Volume 87, Issue 1, Pages 128-136 (January 2015)
BMP type I receptor inhibition attenuates endothelial dysfunction in mice with chronic kidney disease 
Hidemi Kajimoto, Hisashi Kai, Hiroki Aoki, Hiroki Uchiwa, Yuji Aoki, Suguru Yasuoka, Takahiro Anegawa, Yuji Mishina, Akira Suzuki, Yoshihiro Fukumoto, Tsutomu Imaizumi 
Kidney International 
Volume 87, Issue 1, Pages (January 2015)
DOI: /ki
Copyright © 2015 International Society of Nephrology Terms and Conditions

2. Figure 1
Chronic kidney disease (CKD) activates bone morphogenetic protein receptor signaling in endothelial cells (ECs) and vascular smooth muscle cells (VSMCs). (a) Representative immunoblots and the pooled data demonstrating the effects of serum obtained from healthy subjects or CKD patients on the expression levels of total Smad5 and phosphorylated Smad1/5/8 (p-Smad1/5/8) in human umbilical vein endothelial cells. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. *P<0.05 versus healthy. (b, c) Immunofluorescence (b) and the pooled data (c) demonstrating the effects of Nx on the expression levels of Smad5 and p-Smad1/5/8 in the aortas of wild-type (WT)+Sham and WT+CKD mice. Green, Smad5 or p-Smad1/5/8; red, nuclei; and yellow, merged. Arrows indicate translocation of p-Smad1/5/8 into the nuclei. **P<0.01 versus WT+Sham. AU, arbitrary unit.
Kidney International  , DOI: ( /ki )
Copyright © 2015 International Society of Nephrology Terms and Conditions

3. Figure 2
Bone morphogenetic protein (BMP) receptor activation inhibits the Akt-endothelial nitric oxide synthase (eNOS) pathway by upregulating phosphatase-and-tensin homolog (PTEN) expression in endothelial cells (ECs). (a) Representative immunoblots showing the effects of BMP2 on the basal phosphorylation levels of eNOS, Smad1/5/8, Akt, and PTEN. (b) Scatter analysis of PTEN-labeled ECs. The pooled data showing the percentages of the mean fluorescence intensity of PTEN over the threshold. **P<0.01 versus vehicle. (c) Immunofluorescence staining demonstrating the effects of BMP2 on the expression levels of p-Smad1/5/8 (green) and PTEN (red). Blue, nuclei. The merged image shows that BMP2 increased p-Smad1/5/8 and PTEN in the same cells with nucleus stained as yellow. (d) Representative immunoblots showing basal and vascular endothelial growth factor (VEGF)-stimulated Akt phosphorylation with or without BMP2 stimulation after transfection with Pten small interfering RNA (siRNA; siPten) or non-targeting siRNA (siControl). Photographs of p-Akt immunoblots were obtained from the same blot with short (upper panel) and long (lower panel) exposures. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; DAPI, 4′-6-diamidino-2-phenylindole.
Kidney International  , DOI: ( /ki )
Copyright © 2015 International Society of Nephrology Terms and Conditions

4. Figure 3
Nx increases phosphatase-and-tensin homolog (PTEN) expression and inactivates endothelial nitric oxide synthase (eNOS) in mice. (a) Representative immunoblots and the pooled data showing the effects of chronic kidney disease (CKD) on eNOS signaling in the mouse aorta. *P<0.05 versus wild-type (WT)+Sham. **P<0.01 versus WT+Sham. (b) Immunofluorescence and the pooled data demonstrating the effects of CKD on the expression levels of PTEN (green) in the aorta. Blue, nuclei. Arrows indicate endothelial cells (ECs) overexpressing PTEN. **P<0.01 versus WT+Sham. (c) Pooled data of the acetylcholine (Ach)-induced relaxation in phenylephrine-pretreated rings of the descending aorta obtained from WT+Sham (n=9), WT+CKD (n=4), EC-Pten+/-+Sham (n=12), and EC-Pten+/-+CKD (n=11) mice. *P<0.05 WT+Sham versus WT+CKD, §P<0.05, and §§P<0.01 WT+CKD versus EC-Pten+/-+CKD; Tukey–Kramer’s post-hoc analysis. #P<0.01 WT+Sham versus WT+CKD, and ¶P<0.01 WT+CKD versus EC-Pten+/-+CKD; two-way analysis of variance. AU, arbitrary unit.
Kidney International  , DOI: ( /ki )
Copyright © 2015 International Society of Nephrology Terms and Conditions

5. Figure 4
Representative immunoblots and the pooled data demonstrating the effects of Nx on the expression levels of endothelial nitric oxide synthase (eNOS) and phosphorylated eNOS (p-eNOS) in the aortas of wild-type (WT)+Sham, WT+chronic kidney disease (CKD), Alk3+/-+Sham, Alk3+/-+CKD, EC-Pten+/-+Sham, and EC-Pten+/-+CKD mice. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. *P<0.05 WT+Sham versus WT+CKD, **P<0.01 WT+CKD versus Alk3+/-+CKD, **P<0.01 WT+CKD versus EC-Pten+/-+CKD.
Kidney International  , DOI: ( /ki )
Copyright © 2015 International Society of Nephrology Terms and Conditions

6. Figure 5
Effects of bone morphogenetic protein (BMP) receptor signaling inhibition on the chronic kidney disease (CKD)-induced endothelial dysfunction. (a) Pooled data of acetylcholine (Ach)-induced relaxation in phenylephrine (PE)-pretreated rings of the descending aorta obtained from wild-type (WT)+Sham (n=9), WT+CKD (n=4), Alk3+/-+Sham (n=4), and Alk3+/-+CKD (n=7) mice. *P<0.05 WT+Sham versus WT+CKD, §P<0.05 WT+CKD versus Alk3+/-+CKD; Tukey–Kramer’s post-hoc analysis. #P<0.01 WT+Sham versus WT+CKD, ¶P<0.01 WT+CKD versus Alk3+/-+CKD; two-way analysis of variance (ANOVA). (b) Pooled data of Ach-induced relaxation in PE-pretreated rings of the descending aorta obtained from Sham+vehicle (n=8), Sham+LDN (n=6), CKD+vehicle (n=5), and CKD+LDN (n=7) mice. *P<0.05 WT+Sham+vehicle versus WT+CKD+vehicle, §P<0.05, and §§P<0.01 WT+CKD+vehicle versus WT+CKD+LDN ; Tukey–Kramer’s post-hoc analysis. #P<0.01 WT+Sham+vehicle versus WT+CKD+vehicle, and ¶P<0.01 WT+CKD+vehicle versus WT+CKD+LDN ; two-way ANOVA.
Kidney International  , DOI: ( /ki )
Copyright © 2015 International Society of Nephrology Terms and Conditions