1. IN-1130, a novel transforming growth factor-β type I receptor kinase (ALK5) inhibitor, suppresses renal fibrosis in obstructive nephropathy 
J.-A. Moon, H.-T. Kim, I.-S. Cho, Y.Y. Sheen, D.-K. Kim 
Kidney International 
Volume 70, Issue 7, Pages (October 2006)
DOI: /sj.ki
Copyright © 2006 International Society of Nephrology Terms and Conditions

2. Figure 1
Dose–response curve of IN-1130 for its inhibitory effects on the constitutively active TGF-β1 type I receptor ALK5 (T204D)-mediated Smad3 phosphorylation. Various concentrations of IN-1130 were mixed with reaction buffer including purified ALK5 protein (200ng) and γ-32P-ATP. The mixtures were incubated onto the Smad3-coated Flash-Plates for 3h. After incubation, the radioactivities of incorporated γ-32P-ATP into the phosphorylated sites of Smad3 by ALK5 were measured. The in vitro IC50 value of IN-1130 was calculated from a dose–response curves generated from two experiments run in duplicate (n=4) using SigmaPlot software.
Kidney International  , DOI: ( /sj.ki )
Copyright © 2006 International Society of Nephrology Terms and Conditions

3. Figure 2
Representative photomicrographs of hematoxylin–eosin-stained kidney sections from sham-operated rats, UUO control rats, and UUO rats treated with IN (a) In sham kidney at day 7, no changes were observed. (b) Histopathological changes manifested by tubular atrophy, loss and dilation, infiltration of inflammatory cells, and proliferation of fibroblastic cells (arrow heads) were prominent in UUO control kidneys at day 7 after UUO. (c) At day 7 after UUO, UUO rats treated once daily with IN-1130 (10mg/kg) reduced the extent of interstitial nephritis and fibrosis (arrowheads). (d) At day 7 after UUO, histopathological changes shown in UUO control kidneys were significantly reduced or absent by IN-1130 treatment (20mg/kg). (e) Severe morphological changes characterized by tubular atrophy and loss, and interstititial expansion with cellular infiltrates and fibrous components (arrowheads) were observed in kidneys with UUO for 14 days. Note the remaining atrophic tubular cells (asterisks) which are surrounded by expanded interstitium. (f) At day 14 after UUO, UUO rats treated once daily with IN-1130 (20mg/kg) reduced the extent of tubular atrophy and loss, and interstitial nephritis and fibrosis (arrowheads) compared to UUO control kidneys. Bar=50μm.
Kidney International  , DOI: ( /sj.ki )
Copyright © 2006 International Society of Nephrology Terms and Conditions

4. Figure 3
Suppression of TGF-β1 mRNA expression in rat UUO kidneys by IN (a) Representative reverse transcriptase-PCR photographs (upper panel) showing levels of TGF-β1 mRNA of kidneys from sham-operated rats, UUO control rats, and UUO rats treated with IN-1130 for 7 and 14 days. Numbers 1 and 2 indicate two individual animals from each group. HPRT served as an internal control. (b) SYBR Green real-time PCR analysis was used to quantify levels of TGF-β1 mRNA in the kidneys of sham-operated rats, UUO control rats, and UUO rats treated with IN-1130 for 7 and 14 days. Treatment of IN-1130 (10 and 20mg/kg/day) significantly decreased the amount of TGF-β1 mRNA in UUO control kidneys. Values are s.e.m. of 7 (day 7) or 4 (day 14) animals per group. *P<0.05 versus sham control group. **P<0.01 versus sham control group. #P<0.05 versus UUO control group. ##P<0.01 versus UUO control group.
Kidney International  , DOI: ( /sj.ki )
Copyright © 2006 International Society of Nephrology Terms and Conditions

5. Figure 4
Suppression of phosphorylation of Smad2 in rat UUO kidneys by IN (a) Representative Western blotting photograph (upper panel) showing levels of pSmad2 protein in the kidneys of sham-operated rats, UUO control rats, and UUO rats once daily treated with either vehicle or IN-1130 for 14 days. The same blot was stripped and reprobed with β-actin to confirm equal loading (lower panel). Numbers 1 and 2 indicate two individual animals from each group. (b) Quantitative analysis of the relative abundance of pSmad2 protein after normalization with β-actin. Values (fold induction relative to sham-operated control) are s.e.m. of four animals per group. *P<0.05 versus sham control group. **P<0.01 versus sham control group. #P<0.01 versus UUO control group.
Kidney International  , DOI: ( /sj.ki )
Copyright © 2006 International Society of Nephrology Terms and Conditions

6. Figure 5
Representative photomicrographs of pSmad2 immunohistochemistry on kidney sections from sham-operated rats, UUO control rats, and UUO rats treated with IN (a and d) In sham-operated animals, no significant nuclear pSmad2 staining was observed at (a) day 7 and (d) day 14. (b and e) Nuclear localization of pSmad2 in tubular and interstitial cells was increased in UUO control kidneys at (b) day 7 and (e) day 14 (brown, arrowheads) compared to sham-operated kidneys. (c and f) IN-1130 (20mg/kg) treatment reduced nuclear staining intensity (brown, arrowheads) in interstitial and tubular areas of kidneys at (c) day 7 and (f) day 14, which are associated with the decrease in the extent of interstitial nephritis and fibrosis compared to UUO control kidneys. Bar=80μm.
Kidney International  , DOI: ( /sj.ki )
Copyright © 2006 International Society of Nephrology Terms and Conditions

7. Figure 6
Suppression of α-SMA expression in rat UUO kidneys by IN (a) Representative Western blotting photographs (upper panel) showing levels of α-SMA protein in the kidneys of sham-operated rats, UUO control rats, and UUO rats once daily treated with either vehicle or IN-1130 for 7 and 14 days. The same blot was stripped and reprobed with β-actin to confirm equal loading (lower panel). Numbers 1 and 2 indicate two individual animals from each group. (b) Quantitative analysis of the relative abundance of α-SMA protein after normalization with β-actin. Values (fold induction relative to sham-operated control) are s.e.m. of 7 (day 7) or 4 (day 14) animals per group. *P<0.01 versus sham control group. #P<0.05 versus UUO control group. ##P<0.01 versus UUO control group.
Kidney International  , DOI: ( /sj.ki )
Copyright © 2006 International Society of Nephrology Terms and Conditions

8. Figure 7
Representative photomicrographs of α-SMA immunohistochemistry on kidney sections from sham-operated rats, UUO control rats, and UUO rats treated with IN (a, d, and g) In sham-operated animals, α-SMA expression (brown color) is limited to blood vessels (arrows). (b and e) Strong immunoreactivity was observed in interstitial and tubular areas of UUO control kidney at day 7 after UUO. (h) The extent of α-SMA intensity and the number of cells expressing α-SMA in UUO control kidney at day 14 was increased compared to those of UUO control kidney at day 7 (b and e). (c and f) Once-daily treatment with IN-1130 (20mg/kg) for 7 and (i) 14 days reduced staining intensity in interstitial and tubular areas of kidneys, which is associated with the decrease in the extent of interstitial nephritis and fibrosis. Bar=30μm.
Kidney International  , DOI: ( /sj.ki )
Copyright © 2006 International Society of Nephrology Terms and Conditions

9. Figure 8
Suppression of type 1 collagen mRNA expression in UUO control kidneys by IN (a) Representative reverse transcriptase-PCR photograph (upper panel) showing levels of collagen type I mRNA of kidneys from sham-operated rats, UUO control rats, and UUO rats treated with IN-1130 for 7 and 14 days. Numbers 1 and 2 indicate two individual animals from each group. HPRT served as an internal control. (b) SYBR Green real-time PCR analysis was used to quantify levels of the collagen type 1 mRNA in the kidneys of sham-operated rats, UUO control rats, and UUO rats treated with IN-1130 for 7 and 14 days. Treatment of IN-1130 (10 and 20mg/kg/day) significantly decreased the amount of type I collagen mRNA in UUO control kidneys. Values are s.e.m. of 7 (day 7) or 4 (day 14) animals per group. *P<0.01 versus sham control group. #P<0.01 versus UUO control group.
Kidney International  , DOI: ( /sj.ki )
Copyright © 2006 International Society of Nephrology Terms and Conditions

10. Figure 9
Effect of IN-1130 on the hydroxyproline content in UUO control kidneys. IN-1130 treatment significantly decreased levels of hydroxyproline in UUO control kidneys at (a) day 7 and (b) day 14. Values are s.e.m. of 7 (day 7) or 4 (day 14) animals per group. *P<0.05 versus sham control group. **P<0.01 versus sham control group. #P<0.05 versus UUO control group. ##P<0.01 versus UUO control group.
Kidney International  , DOI: ( /sj.ki )
Copyright © 2006 International Society of Nephrology Terms and Conditions

11. Figure 10
Representative photomicrographs of Masson's trichrome staining on kidney sections from sham-operated rats, UUO control rats, and UUO rats treated with IN (a, d, and g) In sham-operated animals, collagen expression is limited to blood vessels (blue, arrows). (blue in b and e) Collagen expression is prominent in interstitial and tubular areas of UUO control kidneys at day 7 after UUO (b and e). (blue in h) Collagen expression is present in a widespread pattern at day 14 after UUO. Once-daily treatment with IN-1130 (20mg/kg) for 7 (c and f) and 14 days (i) reduced staining intensity in interstitial and tubular areas of kidneys, which is associated with the decrease in the extent of interstitial nephritis and fibrosis. Collagen is present only around blood vessels (arrows in c) in UUO kidneys once daily treated with IN-1130 (20mg/kg) for 7 days. Bar=30μm.
Kidney International  , DOI: ( /sj.ki )
Copyright © 2006 International Society of Nephrology Terms and Conditions

12. Figure 11
Suppression of fibronectin expression in UUO control kidneys by IN (a) Representative Western blotting photograph (upper panel) showing levels of fibronectin protein in kidneys of sham-operated rats, UUO control rats, and UUO rats once daily treated with IN-1130 for 7 and 14 days. The same blot was stripped and reprobed with β-actin to confirm equal loading (lower panel). Numbers 1 and 2 indicate two individual animals from each group. (b) Quantitative analysis of the relative abundance of fibronectin protein after normalization with β-actin. Values (fold induction relative to sham control) are s.e.m. of 7 (day 7) or 4 (day 14) animals per group. *P<0.05 versus sham control group. **P<0.01 versus sham control group. #P<0.05 versus UUO control group. ##P<0.01 versus UUO control group.
Kidney International  , DOI: ( /sj.ki )
Copyright © 2006 International Society of Nephrology Terms and Conditions