1. Volume 77, Issue 4, Pages 319-328 (February 2010)
Smad3-dependent and -independent pathways are involved in peritoneal membrane injury 
Pranali Patel, Yoshimi Sekiguchi, Kook-Hwan Oh, Sarah E. Patterson, Martin R.J. Kolb, Peter J. Margetts 
Kidney International 
Volume 77, Issue 4, Pages (February 2010)
DOI: /ki
Copyright © 2010 International Society of Nephrology Terms and Conditions

2. Figure 1
Histology of the anterior abdominal wall 7 days after intraperitoneal adenovirus administration. (a) Smad3+/+ and (b) Smad3-/- mice treated with control adenovirus (AdDL) show normal peritoneal histology with a mesothelial cell layer (thin arrow), a thin submesothelial layer (thick arrow) overlying the abdominal musculature. (c) Smad3+/+ mice treated with adenovirus expressing transforming growth factor-β1 (AdTGF-β1) show a strong fibroproliferative response (arrow). (d) Smad3-/- mice treated with AdTGF-β1 show a moderate proliferative response without significant fibrosis or angiogenesis. (e) Smad3+/+ and (f) Smad3-/- mice treated with adenovirus expressing vascular endothelial growth factor (AdVEGF) show a similar angiogenic response (thick arrows). Masson's trichrome stain, original magnification × 200.
Kidney International  , DOI: ( /ki )
Copyright © 2010 International Society of Nephrology Terms and Conditions

3. Figure 2
Quantification of submesothelium thickness and vascularization. (a) Adenovirus expressing transforming growth factor-β1 (AdTGF-β1)-induced submesothelial thickening is Smad3 dependent. Smad3+/+ animals treated with AdTGF-β1 showed increased submesothelial thickening compared with control adenovirus (AdDL)-treated animals and with Smad3-/- animals treated with AdTGF-β1. (b) Angiogenesis measured as vessels per millimeter length of peritoneum. Animals treated with adenovirus expressing vascular endothelial growth factor (AdVEGF) are included in this analysis. AdTGF-β1 induced angiogenesis in Smad3+/+ but not in Smad3-/- animals. VEGF induced significant but transient angiogenesis in both Smad3+/+ and Smad3-/- animals. (c) Sections were stained with picrosirius red and evaluated using polarized light microscopy. Total birefringence (red + green area) represents total collagen and (d) green birefringent area represents newly deposited collagen. Smad3+/+ animals treated with AdTGF-β1 showed more total and new collagen deposition compared with control AdDL-treated animals and Smad3-/- animals treated with AdTGF-β1. (c) There was a transient but significant increase in total collagen deposited at day 7 in the Smad3-/- animal treated with AdTGF-β1 compared with control AdDL-treated animals. mRNA was extracted from parietal peritoneal tissues and analyzed using quantitative real-time polymerase chain reaction. There was a strong upregulation of (e) plasminogen activator inhibitor 1 (PAI-1) and (f) VEGF in Smad3+/+ mice exposed to TGF-β1. Smad3-/- mice did not show a significant change in the expression of these genes.
Kidney International  , DOI: ( /ki )
Copyright © 2010 International Society of Nephrology Terms and Conditions

4. Figure 3
Sections of parietal peritoneal tissue were stained for cytokeratin (green) and α-SMA (red) with 4,6-diamidino-2-phenylindole (DAPI) counterstaining. At 4 days after infection with adenovirus expressing transforming growth factor-β1 (AdTGF-β1), Smad3+/+ mice showed mesothelial cells that stained for both cytokeratin and α-smooth muscle actin (α-SMA; a–c, thin arrows) along with mesothelial cells without evidence of epithelial-to-mesenchymal transition (thick arrows). Smad3-/- animals (d–f) showed a similar response at this time point with AdTGF-β1-induced dual-labeled cells (thin arrows) and non-transformed mesothelium (thick arrows). (g–i) Control adenovirus-treated Smad3+/+ animals did not show any cellular transition or migration. Original magnification × 200.
Kidney International  , DOI: ( /ki )
Copyright © 2010 International Society of Nephrology Terms and Conditions

5. Figure 4
Sections of parietal peritoneal tissue taken at day 7 were stained for cytokeratin (green) and α-smooth muscle actin (α-SMA; red) with 4,6-diamidino-2-phenylindole (DAPI) counterstaining. (a–c) In Smad3+/+ animals, transforming growth factor-β1 (TGF-β1) induced cellular transition and submesothelial invasion with both dual-labeled (thin arrows) and cytokeratin-labeled (thick arrows) cells intermingled in the submesothelial zone. (d–f) Smad3-/- mice showed similar but attenuated epithelial-to-mesenchymal transition responses 7 days after treatment with adenovirus expressing TGF-β1 (AdTGF-β1). (g–i) Control adenovirus-treated Smad3-/- animals did not show any cellular transition or migration. Original magnification × 200.
Kidney International  , DOI: ( /ki )
Copyright © 2010 International Society of Nephrology Terms and Conditions

6. Figure 5
Epithelial-to-mesenchymal transition occurs in the absence of Smad3. Quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of (a) α-smooth muscle actin (α-SMA) and (b) SNAIL. Transforming growth factor-β1 (TGF-β1) induced a significant increase in α-SMA gene expression in Smad3+/+ animals compared with control treated animals. (b) TGF-β1 also induced a significant increase in SNAIL gene expression in Smad3+/+ mice. (c, d) E-cadherin protein expression is downregulated in mice treated with adenovirus expressing TGF-β1 (AdTGF-β1). A representative western blot is shown in (c) and quantified in (d), which confirmed a significant downregulation in both Smad3+/+ and Smad3-/- mice treated with AdTGF-β1. (e) Epithelial-to-mesenchymal transition (EMT) was quantified by counting the number of dual-labeled α-SMA/cytokeratin-positive cells in the peritoneal tissues. Both Smad3-/- and Smad3+/+ had a quantifiable increase in dual-labeled cells, and Smad3+/+ mice had significantly more dual-labeled cells than Smad3-/- mice treated with AdTGF-β1. (f) The degree of cellular invasion was also quantified. Smad3+/+ mice treated with AdTGF-β1 had prolonged evidence of submesothelial invasion with dual-labeled cells, whereas Smad3-/- mice treated with AdTGF-β1 showed only transient EMT-associated invasion. No evidence of migration of dual-labeled cells was observed in the control adenovirus-treated animals.
Kidney International  , DOI: ( /ki )
Copyright © 2010 International Society of Nephrology Terms and Conditions

7. Figure 6
Rapamycin inhibits epithelial-to-mesenchymal transition in Smad3-/- mice. Sections of parietal peritoneum in Smad3-/- mice at 7 days after treatment with (a–c) adenovirus expressing transforming growth factor-β1 (AdTGF-β1) and vehicle or (d–f) AdTGF-β1 and rapamycin. Sections were stained for (a, d) cytokeratin and (b, e) α-smooth muscle actin (α-SMA) with (c, f) 4,6-diamidino-2-phenylindole (DAPI) counterstaining. (a–c) Smad3-/- mice treated with intraperitoneal AdTGF-β1 and with vehicle by gavage showed epithelial-to-mesenchymal transition (EMT) with evidence of invasive mesothelial cells staining for both cytokeratin and α-SMA (thin arrows) along with mesothelial cells without evidence of EMT (thick arrows). (d–f) Smad3-/- mice treated with AdTGF-β1 and rapamycin showed a significantly reduced EMT response with only occasional dual-labeled cells observed (thin arrows) with no evidence of invasion. Original magnification × 200. (g) The number of dual-labeled cells per millimeter of peritoneum were quantified. Rapamycin had little effect on EMT in Smad3+/+ mice but significantly decreased EMT in Smad3-/- mice.
Kidney International  , DOI: ( /ki )
Copyright © 2010 International Society of Nephrology Terms and Conditions

8. Figure 7
Protein was extracted from peritoneal tissues and analyzed for p-Akt, glycogen synthase kinase (GSK)-3β, and β-catenin expression. (a) Representative western blots. (b) p-Akt/Akt was upregulated by transforming growth factor-β1 (TGF-β1), which was more evident in Smad3-/- mice. (c) pGSK-3β/GSK-3β was increased in the Smad3-/- mice treated with rapamycin (Rapa). (d) β-catenin total protein concentration was increased non-significantly in Smad3-/- treated with adenovirus expressing TGF-β1 (AdTGF-β1) and this effect was reversed by rapamycin. (e) α-smooth muscle actin (α-SMA) gene expression was found to be significantly inhibited by rapamycin.
Kidney International  , DOI: ( /ki )
Copyright © 2010 International Society of Nephrology Terms and Conditions

9. Figure 8
Anterior abdominal wall sections were stained for β-catenin (green) and counterstained with 4,6-diamidino-2-phenylindole (DAPI; blue). (a, b) Smad3-/- mice treated with adenovirus expressing transforming growth factor-β1 (AdTGF-β1) and vehicle show increased β-catenin expression in the peritoneal tissues with evidence of both cytoplasmic and nuclear staining (arrows). (c, d) Smad3-/- mice treated with AdTGF-β1 and rapamycin (Rapa) show minimal β-catenin staining and no evidence of nuclear localization. (e, f) A similar pattern is observed in Smad+/+ mice treated with control adenovirus (AdDL) and vehicle. (g, h) Smad+/+ mice treated with AdTGF-β1 and vehicle show increased β-catenin expression with evidence of nuclear localization (arrow). (i, j) β-catenin expression is present in Smad3+/+ mice treated with AdTGF-β1 (arrow), but nuclear localization seems to be inhibited by treatment with rapamycin.
Kidney International  , DOI: ( /ki )
Copyright © 2010 International Society of Nephrology Terms and Conditions

10. Figure 9
The observations from these experiments suggest that there are at least two parallel pathways involved in transforming growth factor-β1 (TGF-β1)-mediated epithelial-to-mesenchymal transition (EMT). In the absence of Smad3, the phosphatidyl inositol 3 kinase (PI3K)/Akt pathway dominates. The increased expression of Akt in the absence of Smad3 suggests an inhibitory mechanism (dotted line). Smad3-independent EMT is blocked by rapamycin and the main effect occurs through decreased expression and nuclear localization of β-catenin. This may occur directly or indirectly through glycogen synthase kinase (GSK)-3β or mammalian target of rapamycin (mTOR)/p70 S6 kinase (dotted lines).
Kidney International  , DOI: ( /ki )
Copyright © 2010 International Society of Nephrology Terms and Conditions