1. HDAC Dependent Transcriptional Repression of Bmp-7 Potentiates TGF-β Mediated Renal Fibrosis in Obstructive Uropathy 
Scott R. Manson, Joseph B. Song, Keith A. Hruska, Paul F. Austin 
The Journal of Urology 
Volume 191, Issue 1, Pages (January 2014)
DOI: /j.juro
Copyright © 2014 American Urological Association Education and Research, Inc. Terms and Conditions

2. Figure 1
Obstruction induced renal injury led to loss of BMP-7 expression. Five mice underwent sham operation or UUO for 7 days (7D) and kidneys were analyzed. A, type IV collagen immunofluorescence (green areas) with DAPI counterstaining (blue areas). BMP-7 immunofluorescence (red areas) with DAPI counterstaining (blue areas). B, quantification of collagen content. Asterisk indicates p <0.05.
The Journal of Urology  , DOI: ( /j.juro )
Copyright © 2014 American Urological Association Education and Research, Inc. Terms and Conditions

3. Figure 2
Loss of BMP-7 expression was mediated at mRNA level in conjunction with histone deacetylation in proximal Bmp-7 promoter. Three mice underwent sham operation or UUO for 7 days (7D) and kidneys were analyzed. A, BMP-7 ELISA. Asterisk indicate p < B, RT-PCR for Bmp-7 and TGF-β1 with GAPDH as control. C, quantification of Bmp-7 expression (B). Asterisks indicate p <0.01. D, chromatin immunoprecipitation with anti-acetylated histone H3 or rabbit IgG as control, followed by PCR directed against proximal region (–407 to –151 bp) of Bmp-7 promoter. E, quantification of HDAC in Bmp-7 promoter (D). Asterisk indicate p <0.005.
The Journal of Urology  , DOI: ( /j.juro )
Copyright © 2014 American Urological Association Education and Research, Inc. Terms and Conditions

4. Figure 3
HDAC proteins repressed BMP-7 mRNA expression in 3 plates of IMCD-3 primary kidney cells per sample treated with PBS as control or 100 nM TSA for 24 hours. Cells were analyzed. A, RT-PCR for Bmp-7 with GAPDH as control. B, quantification of Bmp-7 expression (A). Asterisks indicate p <0.005.
The Journal of Urology  , DOI: ( /j.juro )
Copyright © 2014 American Urological Association Education and Research, Inc. Terms and Conditions

5. Figure 4
HDAC inhibition suppressed activity of TGF-β dependent profibrotic pathways in 3 plates of IMCD-3 primary kidney cells per sample. Cells were serum starved for 24 hours, reintroduced to serum containing medium and treated with PBS as control, 1 ng/ml TGF-β with PBS as control, 10 ng/ml BMP-7 or 100 nM TSA for 24 hours. Cells were analyzed. A, RT-PCR for Bmp-7, TGF-β1, COLIα1 and α-SMA with GAPDH as control. B to E, quantification of expression (A). ns, p >0.05. Open bars indicate control. Light gray bars indicate BMP-7. Dark gray bars indicate TSA. Single asterisk indicates p <0.05. Double asterisks indicate p <0.01.Triple asterisks indicate p < B, COLIα1. C, α-SMA. D, BMP-7. E, TGF-β1.
The Journal of Urology  , DOI: ( /j.juro )
Copyright © 2014 American Urological Association Education and Research, Inc. Terms and Conditions

6. Figure 5
TGF-β suppressing effects of HDAC inhibition depended on BMP activity stimulation. Three plates of IMCD-3 cells per sample were serum starved for 24 hours, reintroduced to serum containing medium and treated with PBS as control or 1 ng/ml TGF-β with PBS as control, 10 ng/ml BMP-7 or 100 nM TSA with PBS as control, or 10 μM dorsomorphin for 24 hours. Cells were analyzed. A, RT-PCR for Bmp-7, TGF-β1, COLIα1 and α-SMA with GAPDH as control. B to E, quantification of expression (A). DM, dorsomorphin. ns, p >0.05. Open bars indicate control. Light gray bars indicate BMP-7. Dark gray bars indicate TSA. Single asterisk indicates p <0.05. Double asterisks indicate p <0.01. Triple asterisks indicate p < B, Bmp-7. C, COLIα1. D, α-SMA. E, TGF-β1.
The Journal of Urology  , DOI: ( /j.juro )
Copyright © 2014 American Urological Association Education and Research, Inc. Terms and Conditions

7. Figure 6
HDAC inhibition restored BMP-7 expression and suppressed TGF-β dependent profibrotic pathways after UUO induced renal injury in 3 mice with sham operation or UUO for 7 days (7D) treated with PBS as control or 500 μg/kg TSA. Kidneys were analyzed. A, BMP-7 ELISA. Open bars indicate control. Gray bars indicate TSA. Double asterisks indicate p <0.01. Triple asterisks indicate p < B, RT-PCR for Bmp-7, TGF-β1, COLIα1 and α-SMA with GAPDH as control. C to F, quantification of expression (B). ns, p >0.05. Single asterisk indicates p <0.05. C, Bmp-7. D, TGF-β1. E, COLIα1. F, α-SMA.
The Journal of Urology  , DOI: ( /j.juro )
Copyright © 2014 American Urological Association Education and Research, Inc. Terms and Conditions

8. Figure 7
HDAC inhibition ameliorated UUO induced renal injury in 5 mice with sham operation or UUO for 7 days (7D) and treatment with PBS as control or 500 μg/kg TSA. Kidneys were analyzed. A, type IV collagen immunofluorescence (green areas) with DAPI counterstaining (blue areas). BMP-7 immunofluorescence (red areas) with DAPI counterstaining (blue areas). B and C, quantification. Open bars indicate control. Gray bars indicate TSA. Single asterisk indicates p <0.05. Double asterisks indicate p <0.01. B, collagen content. C, tubular volume.
The Journal of Urology  , DOI: ( /j.juro )
Copyright © 2014 American Urological Association Education and Research, Inc. Terms and Conditions

9. Figure 8
HDAC dependent transcriptional repression of Bmp-7 potentiated TGF-β mediated renal fibrosis in obstructive uropathy. BMP-7 protein is secreted cytokine that normally functions to stimulate gene transcription by binding to its receptor and activating Smad1/5/8 transcription factors. BMP-7 pathway activation also suppresses profibrotic TGF-β pathway by sequestering Smad4, protein needed for transcription of many TGF-β dependent genes.6 These counter regulatory mechanisms are important to suppress TGF-β dependent profibrotic pathways during kidney repair.8 However, chronic urinary tract obstruction results in HDAC dependent suppression of Bmp-7 transcription. BMP-7 loss results in dysregulation of kidney repair with persistent, inappropriate activation of TGF-β pathway. Pharmacological HDAC inhibitor treatment restores BMP-7 expression, suppresses TGF-β dependent signaling pathways and prevents renal fibrosis after chronic urinary tract obstruction.
The Journal of Urology  , DOI: ( /j.juro )
Copyright © 2014 American Urological Association Education and Research, Inc. Terms and Conditions