1. Volume 82, Issue 8, Pages 857-866 (October 2012)
Endothelial cell transforming growth factor-β receptor activation causes tacrolimus- induced renal arteriolar hyalinosis 
Valorie L. Chiasson, Kathleen A. Jones, Shelley E. Kopriva, Ashutosh Mahajan, Kristina J. Young, Brett M. Mitchell 
Kidney International 
Volume 82, Issue 8, Pages (October 2012)
DOI: /ki
Copyright © 2012 International Society of Nephrology Terms and Conditions

2. Figure 1
Tacrolimus (TAC) treatment or genetic deletion of FK506-binding protein 12 (FKBP12) from endothelial cells increases SMAD2/3 activation. (a) Mice treated with TAC (10mg/kg/day, 1 week) had increased aortic expression of transforming growth factor (TGF)-β1 compared with controls; however, FK12EC KO mice did not exhibit any changes in TGF-β1 expression. (b) Aortic mRNA levels of angiotensin-converting enzyme (ACE), angiotensinogen (Agt), and TGF-β1 were increased significantly in TAC-treated mice but were not changed in FK12EC knockout (KO) mice. Dotted line represents *P<0.05 vs. control. (c) SMAD2/3 phosphorylation was increased in aortas from mice treated with TAC (10mg/kg/day) and FK12EC KO mice. Densitometry of TGF-β1 to actin or p-SMAD2/3 to SMAD2/3 as a percentage of control; *P<0.05 vs. control. n=8–11 mice per experimental group. C, controls; F, FK12EC KO.
Kidney International  , DOI: ( /ki )
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3. Figure 2
Tacrolimus (TAC) treatment or genetic deletion of FK506-binding protein 12 (FKBP12) from endothelial cells caused renal arteriolar hyalinosis. Hematoxylin and eosin (H&E) staining (top panels) and Masson's trichrome staining (bottom panels) reveal hyalinosis seen as the pink, glassy area indicated by the arrows in (a) TAC-treated mice (10mg/kg/day; 1 week) and in (b) FK12EC knockout (KO) mice compared with controls. Images at × 400 final magnification. Quantification of arteriolar hyalinosis was determined using a scoring system previously described.7 n=6 mice per experimental group; *P<0.05 vs. control.
Kidney International  , DOI: ( /ki )
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4. Figure 3
Tacrolimus (TAC) treatment or genetic deletion of FK506-binding protein 12 (FKBP12) from endothelial cells increased vascular matrix protein production. (a) Increased protein levels of collagen and fibronectin in aortas from mice treated with TAC (10mg/kg/day) and FK12EC knockout (KO) mice. Densitometry of collagen and fibronectin to actin as a percentage of control; *P<0.05 vs. control. (b) Increased mRNA levels of collagen and fibronectin in aortas from mice treated with TAC (10mg/kg/day) and FK12EC KO mice. n=8–11 mice in each group. Dotted line represents *P<0.05 vs. control. C, controls; F, FK12EC KO.
Kidney International  , DOI: ( /ki )
Copyright © 2012 International Society of Nephrology Terms and Conditions

5. Figure 4
Tacrolimus (TAC) treatment, but not calcineurin inhibition, increased SMAD2/3 activation and vascular matrix protein production in isolated aortas. TAC increased SMAD2/3 phosphorylation and protein levels of collagen and fibronectin in aortas isolated from control mice, whereas the calcineurin autoinhibitory peptide (CAIP) had no effect. n=8–11 mice per experimental group. Densitometry of p-SMAD2/3 to SMAD2/3 or collagen and fibronectin to actin as a percentage of control; *P<0.05 vs. control.
Kidney International  , DOI: ( /ki )
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6. Figure 5
Removal of endothelium prevented the tacrolimus (TAC)-induced increase in SMAD2/3 activation and vascular matrix protein production in isolated aortas. Denuding of endothelial cells prevented the TAC-induced increase in SMAD2/3 phosphorylation and protein levels of collagen and fibronectin in aortas isolated from control mice. n=8–11 mice per experimental group. Densitometry of p-SMAD2/3 to SMAD2/3 or collagen and fibronectin to actin as a percentage of control; *P<0.05 vs. control.
Kidney International  , DOI: ( /ki )
Copyright © 2012 International Society of Nephrology Terms and Conditions

7. Figure 6
Inhibition of transforming growth factor (TGF)-β receptor activation prevented the tacrolimus (TAC)-induced increase in SMAD2/3 activation and vascular matrix protein production in isolated aortas. TAC increased SMAD2/3 phosphorylation and protein levels of collagen and fibronectin in aortas isolated from control mice, but this was prevented by the TGF-β receptor inhibitor SB n=8–11 mice per experimental group. Densitometry of p-SMAD2/3 to SMAD2/3 or collagen and fibronectin to actin as a percentage of control; *P<0.05 vs. control.
Kidney International  , DOI: ( /ki )
Copyright © 2012 International Society of Nephrology Terms and Conditions

8. Figure 7
Diagram of molecular mechanisms involved in the development of renal arteriolar hyalinosis. Increased transforming growth factor (TGF)-β receptor activation leads to SMAD2/3 activation and increased production of collagen and fibronectin, which are secreted to the smooth muscle cell causing injury and hyalinosis. CaN, calcineurin; CsA, ciclosporin; FKBP, FK506-binding protein; TAC, tacrolimus.
Kidney International  , DOI: ( /ki )
Copyright © 2012 International Society of Nephrology Terms and Conditions