1. Smad7 gene transfer inhibits peritoneal fibrosis
J. Nie, X. Dou, W. Hao, X. Wang, W. Peng, Z. Jia, W. Chen, X. Li, N. Luo, H.Y. Lan, X.Q. Yu 
Kidney International 
Volume 72, Issue 11, Pages (December 2007)
DOI: /sj.ki
Copyright © 2007 International Society of Nephrology Terms and Conditions

2. Figure 1
Determination of Smad7 gene transfection rate on the peritoneum by confocal microscope. A Dox-regulated flag-m2-Smad7 pTRE plasmid (or empty pTRE) and a Tet-on plasmid (1:1) were mixed with Sonovue in a ratio of 1:1 (vol/vol) and transfected into peritoneum by an ultrasound-mediated system as described in Materials and methods. Smad7 transgene expression was induced by Dox in the drinking water (200 μg/ml) for 3 days, detected by the anti-flag-m2 antibody and examined by confocal microscopy as described in Materials and methods. (a) Control plasmids; (b) Smad7 at a dose of 25 μg/rat; (c) Smad7 at a dose of 50 μg/rat; (d) Smad7 at a dose of 100 μg/rat; (e) quantitative analysis. Note that a strong flag-m2-Smad7 expression (green) is detected within cytoplasm (arrows) in mesothelial cells on the peritoneal surface (s) and also in cells in submesothelial areas, which is enlarged at the inserted area of (d). It is shown that Smad7 transgene expression is, in a dose-dependent manner, peaking at 100 μg/rat, which results in more than 80% of peritoneal cells being positive for flag-m2 (d and e). Data represent mean±s.e.m. for groups of four rats. **P<0.05 compared with a dose of 25 μg/rat; #P<0.05 compared with a dose of 50 μg/rat. Nuclei were counterstained with DAPI (Red). Original magnification × 400.
Kidney International  , DOI: ( /sj.ki )
Copyright © 2007 International Society of Nephrology Terms and Conditions

3. Figure 2
Peritoneal Smad7 transgene expression during the 28-day period determined by confocal microscope and Western blot analysis. An optimal dose of Smad7 plasmid (100 μg/rat) was transfected into rat peritoneum as described in Materials and methods. (a–h) The expression of flag-m2-Smad7 by peritoneal cells was checked on days 0, 3, 7, 14, 17, 21, 24, and 28. Note that the expression of flag-m2-Smad7 (green) by peritoneal cells (arrows) is peaked at day 3 (b, i, and j) and is reduced by 70% at day 14 (d, i, and j). After the second Smad7 gene transfer at day 14, expression of flag-m2-Smad7 reaches the peak again at day 17 (e, i, and j), but is gradually reduced from day 21 onwards. Data represent mean±s.e.m. for groups of four rats. *P<0.05, **P<0.01 compared with the control (0). S, peritoneal surface. Nuclei are counterstained with DAPI (Red). (j) The expression levels of flag-m2Smad7 transgene in rat peritoneum at various time points are detected by Western blot analysis. Original magnification × 400.
Kidney International  , DOI: ( /sj.ki )
Copyright © 2007 International Society of Nephrology Terms and Conditions

4. Figure 3
Smad7 gene transfer attenuates peritoneal fibrosis in a rat model of PD at day 28. (a–d) Hematoxylin and eosin staining. (e–h) Masson Trichrome staining of day-28 parietal peritoneal tissues after PD. (a) Normal control, (b) PD without treatment, (c) empty plasmid control, (d) Smad7 treatment. As compared with the normal (e), a severe peritoneal fibrosis accompanied by the thickening of the peritoneum with marked deposition of ECM and a loss of mesothelial cells, is demonstrated in both untreated (f) and empty plasmid treated (g) animals. However, all these pathological changes are attenuated after gene therapy with Smad7 (h). Representative images were taken from groups of 10 rats. Original magnification × 400.
Kidney International  , DOI: ( /sj.ki )
Copyright © 2007 International Society of Nephrology Terms and Conditions

5. Figure 4
Smad7 gene transfer prevents a loss of peritoneal functions after PD at day 28. (a) Ultrafiltration rate, (b) mass transfer of glucose (MTG). (i) Normal control, (ii) PD without treatment, (iii) empty plasmid control, (iv) Smad7 treatment. Data are expressed as mean±s.e.m. for groups of 10 rats. *P<0.05, **P<0.01 compared with the normal control (i) #P<0.05 compared with both untreated PD (ii) and vector control treatment (iii).
Kidney International  , DOI: ( /sj.ki )
Copyright © 2007 International Society of Nephrology Terms and Conditions

6. Figure 5
Smad7 gene transfer inhibits mRNA and protein levels of peritoneal α-SMA after PD at day 28. (a–d) Confocal microscopy, (e) RT-PCR amplification, (f) Western blot analysis. (i) Normal control, (ii) untreated PD, (iii) PD treated empty plasmids, (iv) Smad7 treatment. Note that there are few α-SMA-positive cells (green) in submesothelial areas of normal peritoneum (a) but numerous α-SMA-positive cells are found in the mesothelial cell layer, indicating a myofibroblast transition in the thickened submesothelial areas of both untreated PD (b), and empty plasmid-treated (c) animals. However, α-SMA-positive cells within the peritoneum after PD are significantly inhibited after gene transfer of Smad7 (d). Similar results are seen at the mRNA (e) and protein (f) levels. S, peritoneal surface. Data represent mean±s.e.m. for groups of 10 rats. *P<0.05 compared with the normal control (i) #P<0.05 compared with both untreated PD (ii) and empty plasmid treatment (iii). Nuclei are counterstained with DAPI (Red). Original magnification × 400.
Kidney International  , DOI: ( /sj.ki )
Copyright © 2007 International Society of Nephrology Terms and Conditions

7. Figure 6
Smad7 gene transfer inhibits peritoneal collagen I mRNA and protein expression after PD at day 28. (a–d) Confocal microscopy, (e) RT-PCR amplification, (f) Western blot analysis. (i) Normal control, (ii) untreated PD, (iii) PD treated empty plasmids, (iv) Smad7 treatment. Note that there is a small amount of collagen I deposition (green) in submesothelial areas of normal peritoneum (a), but massive collagen I accumulation is found in the thickening submesothelial areas in both untreated PD (b) and empty plasmid treated (c) animals. In contrast, collagen I accumulation within the peritoneum after PD is significantly inhibited after gene transfer of Smad7 (d). Similar results are observed at the mRNA (e) and protein (f) levels. S, peritoneal surface. Data represent mean±s.e.m. for groups of 10 rats. *P<0.05, **P<0.01 compared with the normal control (i) #P<0.05, ##P<0.01 compared with both untreated PD (ii) and empty plasmid treatment (iii). Nuclei are counterstained with DAPI (Red). Original magnification × 400.
Kidney International  , DOI: ( /sj.ki )
Copyright © 2007 International Society of Nephrology Terms and Conditions

8. Figure 7
Smad7 gene transfer inhibits peritoneal collagen IV mRNA and protein expression after PD at day 28. (a–b) Confocal microscopy, (e) RT-PCR amplification, (f) Western blot analysis. (i) Normal control, (ii) untreated PD, (iii) PD treated empty plasmids, (iv) Smad7 treatment. Note that there are a few collagen IV depositions (green) in the submesothelial areas of normal peritoneum (a). However, an increased peritoneal collagen IV accumulation is found in the thickening submesothelial areas in both untreated PD (b) and empty plasmid treated (c) animals. Nevertheless, collagen IV accumulation within the peritoneum after PD is significantly inhibited after gene transfer of Smad7 (d). Similar results are seen at the mRNA (e) and protein (f) levels. S, peritoneal surface. Data represent mean±s.e.m. for groups of 10 rats. **P<0.01 compared to the normal control (i) #P<0.05 compared with both untreated PD (ii) and empty plasmid treatment (iii) Nuclei are counterstained with DAPI (Red). Original magnification × 400.
Kidney International  , DOI: ( /sj.ki )
Copyright © 2007 International Society of Nephrology Terms and Conditions

9. Figure 8
Smad7 gene transfer inhibits activation of Smad2/3 in the peritoneum after PD at day 28. (a–d) Confocal microscopy, (e) semiquantitative analysis, (f) Western blot analysis. (i) Normal control, (ii) untreated PD, (iii) PD-treated empty plasmids, (iv) Smad7 treatment. Activation of Smad2/3 was detected by both p-Smad2/3 nuclear translocation by confocal microscopy (a–d) and phosphorylation of p-Smad2 and p-Smad3 by Western blot analysis (f). Note that few p-Smad2/3 (green) positive cells, as identified by its nuclear pattern (arrows), are detected in normal peritoneum (a) but become massive in the thickening peritoneum in both untreated PD (b) and empty plasmid-treated (c) animals. In contrast, p-Smad2/3-positive cells (arrows) within the peritoneum after PD are significantly inhibited after gene transfer of Smad7 (d). Similar results are observed at the phosphorylated protein levels by Western blotting analysis with the specific antibody to p-Smad2 or p-Smad3 (f). S, peritoneal surface. Data represent mean±s.e.m. for groups of 10 rats. *P<0.05, **P<0.01, ***P<0.001 compared with the normal control (i). #P<0.05, ###P<0.001 compared with both untreated PD (ii) and empty plasmid treatment (iii). Nuclei are counterstained with DAPI (Red). Original magnification × 400.
Kidney International  , DOI: ( /sj.ki )
Copyright © 2007 International Society of Nephrology Terms and Conditions

10. Figure 9
Smad7 gene transfer inhibits upregulation of peritoneal TGF-β mRNA and protein after PD at day 28. (a–d) Confocal microscopy, (e) RT-PCR amplification, (f) Western blot analysis. (i) Normal control, (ii) untreated PD, (iii) PD treated empty plasmids, (iv) Smad7 treatment. Although few cells positive for TGF-β expression (green) are found in normal peritoneum (a), expression of TGF-β is significantly increased (arrows) in association with the thickening of the peritoneum in both untreated PD (b) and empty plasmid treated (c) animals. In contrast, peritoneal TGF-β expression is significantly inhibited after gene transfer of Smad7 (d), which is further confirmed at the mRNA and protein levels by RT-PCR (e) and Western blot analysis (f). S, peritoneal surface. Data represent mean±s.e.m. for groups of 10 rats. *P<0.05, **P<0.01 compared with the normal control (i). #P<0.05 compared with both untreated PD (ii) and empty plasmid treatment (iii). Nuclei are counterstained with DAPI (Red). Original magnification × 400.
Kidney International  , DOI: ( /sj.ki )
Copyright © 2007 International Society of Nephrology Terms and Conditions

11. Figure 10
Peritoneal cells expressing TGF-β1 demonstrated by two-color immunofluorescent confocal microscopy and mesothelial cell culture. (a) Macrophages are identified by the mouse monoclonal anti-ED1 antibody followed by Cy3-conjugated goat anti-mouse antibody (Red). (b) Cells expressing TGF-β1 are detected by a rabbit anti-TGF-β1 antibody followed by fluorescein isothiocyanate-conjugated goat anti-rabbit antibody (green). (c) Merged images from (a) and (b), showing TGF-β1 expression by ED1+ macrophages (arrows) and ED1- elongated mesothelial-like cells on the intact peritoneal surface (arrowheads). (d and e) Rat mesothelial cell culture under high glucose and mannitol that mimic 4.25% Dianeal conditions (214 mM). Results show that high glucose, but not mannitol, is able to stimulate TGF-β1 mRNA (d) and protein expression (e). *P<0.05, **P<0.01 compared with the normal control (i). #P<0.05 compared with both untreated PD (ii) and empty plasmid treatment (iii). Nuclei are counterstained with blue. Original magnification × 400.
Kidney International  , DOI: ( /sj.ki )
Copyright © 2007 International Society of Nephrology Terms and Conditions

12. Figure 11
Smad7 gene transfer inhibits upregulation of PAI-1 in the peritoneum after PD at day 28. (a–d) Confocal microscopy, (e) semiquantitative analysis of immunostaining, (f) RT-PCR amplification. (i) Normal control, (ii) untreated PD, (iii) PD treated with empty plasmids, (iv) Smad7 treatment. A few cells are positive for PAI-1 (green) in normal peritoneum (a). However, expression of PAI-1 is largely increased in the thickening peritoneum in both untreated PD (b) and empty plasmid treated (c) animals, which are significantly inhibited after gene transfer of Smad7 (d). Data represent mean±s.e.m. for groups of 10 rats. *P<0.05, **P<0.01 compared with the normal control (i). #P<0.05 compared with both untreated PD (ii) and empty plasmid treatment (iii). Nuclei are counterstained with DAPI (Red). Original magnification × 400.
Kidney International  , DOI: ( /sj.ki )
Copyright © 2007 International Society of Nephrology Terms and Conditions