HDAC Dependent Transcriptional Repression of Bmp-7 Potentiates TGF-b Mediated Renal Fibrosis in Obstructive Uropathy

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1 HDAC Dependent Transcriptional Repression of Bmp-7 Potentiates TGF-b Mediated Renal Fibrosis in Obstructive Uropathy Scott R. Manson, Joseph B. Song, Keith A. Hruska and Paul F. Austin* From the Division of Urology, Department of Surgery and Division of Pediatric Nephrology, Departments of Medicine and Pediatrics (KAH), Washington University, St. Louis Children s Hospital, St. Louis, Missouri Abbreviations and Acronyms a-sma ¼ a-smooth muscle actin BMP-7 ¼ bone morphogenic protein 7 COLIa1 ¼ type I collagen, a1 chain ELISA ¼ enzyme-linked immunosorbent assay GAPDH ¼ glyceraldehyde-3- phosphate dehydrogenase HDAC ¼ histone deacetylase IMCD ¼ inner medullary collecting duct PCR ¼ polymerase chain reaction RT-PCR ¼ reverse transcriptase-pcr SMAD ¼ SMA- and MAD-related protein TGF-b ¼ transforming growth factor-b TSA ¼ trichostatin A UUO ¼ unilateral ureteral obstruction Accepted for publication June 27, Study received institutional animal care and use committee approval. Supported by NIH/NIDDK Grant 1R01DK * Correspondence: Washington University, 660 South Euclid Ave., Campus Box 8242, St. Louis, Missouri (telephone: ; FAX: ; austinp@wustl.edu). Purpose: Recombinant BMP-7 inhibits the pathogenesis of renal injury in response to various stimuli. However, little is known about the molecular regulation of endogenous BMP-7 and its renal protective functions. We examined transcriptional regulation of Bmp-7 and its role in the pathogenesis of renal injury resulting from urinary tract dysfunction. Materials and Methods: Obstruction induced renal injury was modeled in vivo in mice by unilateral ureteral obstruction and in vitro in primary kidney cells by treatment with transforming growth factor-b, a profibrotic cytokine that is increased in the obstructed kidney. Results: Unilateral ureteral obstruction resulted in the loss of BMP-7 expression in conjunction with histone deacetylation and transcriptional repression of the Bmp-7 promoter. The histone deacetylase inhibitor trichostatin A stimulated Bmp-7 expression in primary kidney cells. Trichostatin A also inhibited the expression of transforming growth factor-b dependent profibrotic genes in a manner that depended on BMP receptor signaling. These findings extended to the obstructed kidney in vivo, in which trichostatin A treatment restored the expression of Bmp-7 along with BMP-7 mediated suppression of transforming growth factor-b dependent signaling pathways. Finally, trichostatin A stimulated activation of the BMP-7 pathway the ameliorated obstruction induced renal injury by preventing disruption of the renal architecture and the development of renal fibrosis. Conclusions: These findings show that histone deacetylase dependent repression of Bmp-7 transcription is a critical event during the pathogenesis of renal injury in obstructive uropathy. Accordingly, treatment with histone deacetylase inhibitors represents a potentially effective strategy to restore BMP-7 expression and its renal protective functions during treatment of obstructive uropathy. Key Words: kidney, ureteral obstruction, bone morphogenetic protein 7, fibrosis, histone deacetylase inhibitors OBSTRUCTIVE uropathy is a leading cause of pediatric kidney disease, accounting for 16.5% of kidney transplants in children. 1 While the kidney has innate repair mechanisms that can restore renal structure and function after acute injury due to urinary obstruction and other stimuli, 2e4 the clinical manifestation of chronic renal injury stems in part 242 j /14/ /0 THE JOURNAL OF UROLOGY 2014 by AMERICAN UROLOGICAL ASSOCIATION EDUCATION AND RESEARCH,INC. Vol. 191, , January 2014 Printed in U.S.A.

2 REPRESSION OF Bmp-7 POTENTIATES RENAL FIBROSIS IN OBSTRUCTIVE UROPATHY 243 from a dysregulated wound healing response, which results in renal fibrosis. 3,4 At the molecular level activation of the TGF-b pathway is classically described as a critical pathological step in the development of renal fibrosis that promotes apoptosis, cellular dedifferentiation, myofibroblast activation and matrix protein synthesis. 5 Given that these processes are involved in injury repair, 4 it is likely that TGF-b also has a physiological role in the early stages of injury repair but when TGF-b activity is dysregulated, it instead promotes disease progression. Nonetheless, mechanisms that counter regulate TGF-b activity during the response to renal injury are poorly understood. Interestingly, another member of the TGF-b superfamily, BMP-7, inhibits TGF-b dependent signaling pathways. 6 The inhibitory effects of BMP-7 are mediated by the activation of SMAD1/5/8 proteins, which in turn suppress the activity of TGF-b dependent transcription factors and their ability to stimulate profibrotic gene expression. 7,8 Treatment with recombinant BMP-7 inhibits the development of renal injury in response to urinary obstruction and various other stimuli. 6e17 While the renal protective effects of recombinant BMP-7 are well established, at our laboratory it was recently noted that endogenous BMP-7 activity is required for cessation of the TGF-b response along with the restoration of renal architecture and the resolution of fibrotic changes in the kidney that occur during the repair of obstruction induced renal injury. 8 The importance of BMP-7 in the repair of renal injury was further supported by studies at our laboratory and by others showing that treatment with recombinant BMP-7 can reverse the progression of chronic renal injury. 8e10 Despite the biological importance of BMP-7 little is known about the molecular mechanisms that regulate endogenous BMP-7 activity and its renal protective functions. However, at our laboratory it was found that BMP-7 expression is up-regulated during kidney repair after acute obstruction induced renal injury. 7,8 Conversely, chronic renal injury in response to various stimuli, including urinary obstruction, is associated with loss of BMP-7 expression. 8,11e13,18 Along with the previous findings this suggests that loss of BMP-7 expression is a critical event during the pathogenesis of chronic renal injury that may lead to the suppression of BMP-7 dependent repair mechanisms and in part the persistent, inappropriate activation of TGF-b dependent profibrotic pathways. In this study we delineated the molecular mechanisms that lead to loss of BMP-7 expression in the obstructed kidney and examined their potential importance for the treatment of obstructive uropathy and other conditions that lead to chronic renal injury. MATERIALS AND METHODS Unilateral Ureteral Obstruction UUO was created in 8 to 10-week-old C57BL/6J mice by placing a microvascular clamp on the proximal ureter. 2 When indicated, mice were treated with 500 mg/kg TSA (SigmaÒ) daily by intraperitoneal injection. All procedures were approved by institutional review. Histology Trichrome staining was done using the AccustainÔ Masson trichrome staining kit. Immunofluorescence was performed using rabbit anti-bmp-7 and rabbit anti-type IV collagen (AbcamÒ), as previously described. 8 Tubular volume was quantified in samples stained for type IV collagen by digitally overlaying a grid on microscopy images and determining the percent of grid points located in the interstitial/tubular regions. 14 Collagen Quantification Kidney samples were hydrolyzed and hydroxyproline was quantified by comparison to standards in a colorimetric reaction, as previously described. 19 Collagen content was calculated using the approximation that collagen contains approximately 14% (weight per weight) hydroxyproline. Values are expressed as a ratio to the dry tissue weight of the sample. BMP-7 Enzyme-Linked Immunosorbent Assay Kidneys were pulverized in liquid nitrogen. They were homogenized in 100 mm tris-hcl (ph 7.4), 150 mm NaCl, 1% Triton Ò, 0.5% sodium deoxycholate, 1 mm ethylenediaminetetraacetic acid, 1 mm ethylene glycol tetraacetic acid and Complete Protease Inhibitor Cocktail (Roche, Mannheim, Germany). Samples were normalized to total protein content using the PierceÔ BCA Protein Assay Kit. BMP-7 levels were quantified using the BMP-7 ELISA Kit (R&D SystemsÒ) according to product specifications. Reverse Transcription-PCR Kidneys were pulverized in liquid nitrogen and homogenized in TRIzolÒ. RNA was isolated. RT-PCR was done using the SuperScriptÔ RT-PCR system with primers specific for BMP-7 (5 0 -GAAAACAGCAGCAGTGACCA-3 0 and 5 0 -GCTCAGGAGAGGTTGGTCTG-3 0 ), TGF-b1 (5 0 -CA AACGTCGGGGCGACCTGG-3 0 and 5 0 -TGCTCCACCTTG GGCTTGCG-3 0 ), a-sma (5 0 -CCCTGAGACGCTGCTCCAG CTA-3 0 and 5 0 -GGCATAGAGGGACAGCACAGCCT-3 0 ), COLIa1 (5 0 -TGGTCCTGCCGGTCCTCCTG-3 0 and 5 0 -ACA CATTGGGGGTAGGAACA-3 0 ), and GAPDH (5 0 -AACTTTG GCATTGTGGAAGG-3 0 and 5 0 -ACACATTGGGGGTAGGA ACA-3 0 ). The relative intensity of PCR bands was quantified using ImageJ ( and normalized as a ratio to GAPDH levels. Chromatin Immunoprecipitation Chromatin immunoprecipitation was performed using the ImprintÒ Chromatin Immunoprecipitation Kit. DNA was sheared to 2 kb fragments by sonication.

3 244 REPRESSION OF Bmp-7 POTENTIATES RENAL FIBROSIS IN OBSTRUCTIVE UROPATHY Immunoprecipitation was performed using rabbit IgG or rabbit anti-acetyl-histone H3 (Sigma). PCR was done with primers specific to the e407 to e151 bp region of the proximal Bmp-7 promoter, including (5 0 -GTTTGTTGCTGGTG CCCGCG-3 0 and 5 0 -GCTCTACGCGCGATCCGGG-3 0 ). The relative intensity of PCR bands was quantified using ImageJ and normalized as a ratio to PCR products from reactions performed on input DNA. Primary Kidney Cell Culture Murine IMCD-3 cells (ATCCÒ) were cultured in Dulbecco s modified Eagle s medium/f12 growth medium supplemented with 10% fetal bovine serum. As indicated, cells were treated with 100 nm TSA, 1 ng/ml TGF-b, 10 ng/ml BMP-7 (R&D Systems) or 10 mm dorsomorphin (Sigma). Statistical Analysis Statistical significance was determined using the t-test or ANOVA with the Bonferroni correction for multiple comparisons, as appropriate. Data are shown as the mean SD. RESULTS Obstruction Induced Renal Injury Associated with Transcriptional Repression of Bmp-7 Expression To examine the molecular regulation of BMP-7 we studied a murine model of obstruction induced renal injury after UUO (fig. 1). 2 UUO resulted in a dramatic decrease in BMP-7 expression in various cell types throughout the kidney (fig. 1, A). When quantifying the loss of BMP-7 expression, we found that UUO induced renal injury was associated with a mean 88.1% 11.1% decrease in BMP-7 protein levels along with a corresponding 83.1% 12.8% decrease in BMP-7 mrna levels (p <0.005 and <0.01, respectively, fig. 2, A to C ). Together these data suggest the possibility that the loss of BMP-7 expression is mediated at the transcriptional level. Accordingly, we determined whether the loss of BMP-7 mrna expression was associated with chromatin modifications in the regulatory regions of the Bmp-7 gene. UUO resulted in a mean 63.0% 8.5% decrease in the acetylation of histone H3 proteins bound to the proximal Bmp-7 promoter (p <0.005, fig. 2, D and E ). This was a significant finding, given that a number of transcriptional factors function by recruiting HDAC complexes to target genes, a process that subsequently promotes chromatin condensation and transcriptional repression. 20 HDAC Proteins Regulated Bmp-7 Expression and BMP-7 Mediated Suppression of TGF-b Dependent Profibrotic Pathways In Vitro To begin to investigate the role of changes in histone acetylation in the regulation of BMP-7 expression we examined transcriptional regulation of Bmp-7 in IMCD-3 cells, which are a significant source of BMP-7 in the kidney. 6 Treatment with the pan-hdac inhibitor TSA 20 stimulated a mean fold increase in BMP-7 mrna levels in IMCD-3 cells (p <0.005, fig. 3). In addition to other results (fig. 2, D and E ), this finding shows that Bmp-7 expression is repressed by HDAC proteins. We then determined the importance of HDAC dependent regulation of Bmp-7 expression in the renal protective functions of the BMP-7 pathway. To accomplish this we studied an in vitro model of TGF-b mediated renal fibrosis and used the suppression of TGF-b dependent profibrotic gene expression as a functional readout for BMP-7 activity. As previously demonstrated, treatment with TGF-b induced the expression of several profibrotic genes central to the pathogenesis of renal injury, including COLIa1, a gene that encodes a protein that significantly contributes to fibrosis, 21 and a-sma, a gene that encodes a protein that contributes to 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.

4 REPRESSION OF Bmp-7 POTENTIATES RENAL FIBROSIS IN OBSTRUCTIVE UROPATHY 245 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-b1 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 (e407 to e151 bp) of Bmp-7 promoter. E, quantification of HDAC in Bmp-7 promoter (D). Asterisk indicate p < the differentiation of profibrotic myofibroblasts 22 (each p <0.005, fig. 4, A to C ). Importantly, co-treatment with recombinant BMP-7 resulted in a mean 67.3% 25.8% decrease in COLIa1 induction and a 68.7% 19.1% decrease in a-sma induction (each p <0.005, fig. 4, A to C ). This shows that BMP-7 pathway activation is sufficient to suppress TGF-b dependent profibrotic gene expression in this model system. When examining the effects of HDAC inhibition on BMP-7 pathway activity, we found that TSA treatment resulted in a mean fold increase in Bmp-7 expression (p <0.005) and stimulation of Bmp-7 expression was not affected by TGF-b co-treatment (fig. 4, A and D). In a manner similar to BMP-7 treatment co-treatment with TSA resulted in a mean 73.7% 21.9% decrease in TGF-b induced COLIa1 expression and a 60.0% 13.6% decrease in TGF-b induced a-sma expression (each p <0.005, fig. 4, A to C ). Given that TGF-b dependent Smad binding sites are required for promoter activity in the COLIa1 and a-sma genes, 21,22 it is likely that the inhibitory effects of TSA treatment are mediated by the suppression of downstream steps in the TGF-b pathway. In support of this possibility TSA treatment had only

5 246 REPRESSION OF Bmp-7 POTENTIATES RENAL FIBROSIS IN OBSTRUCTIVE UROPATHY 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 < minimal effects on baseline expression of COLIa1, a-sma and endogenous TGF-b1 in the absence of recombinant TGF-b (fig. 4, A to C and E ). We next determined the requirement for BMP-7 activity in the ability of TSA to inhibit TGF-b dependent profibrotic gene expression. To accomplish this we examined the effects of dorsomorphin, a pharmacological inhibitor of BMP receptor activity, 23 on the inhibitory effects of TSA. As noted (fig. 4), TSA treatment stimulated Bmp-7 expression and suppressed TGF-b induced COLIa1 and a-sma expression (each p <0.005, fig. 5, A to D). However, when combined with dorsomorphin treatment, the inhibitory effects of TSA on TGF-b induced COLIa1 expression and on TGF-b induced a-sma expression decreased a mean of 66.8% 5.5% and 77.9% 17.1% (p <0.01 and <0.05, respectively, fig. 5, A, C and D). Importantly, dorsomorphin treatment did not affect TSA stimulated Bmp-7 expression and had only minimal effects on baseline expression of COLIa1, a-sma and endogenous TGF-b1 in the absence of recombinant TGF-b (fig. 5). Together these findings show that the TGF-b suppressing effects of HDAC inhibition largely depend on stimulating BMP-7 pathway activity. HDAC Inhibition Restored BMP-7 Expression, Suppressed TGF-b Activity and Ameliorated Renal Injury in Obstructive Uropathy To extend these findings to the pathogenesis of chronic renal injury in vivo we examined the effects of HDAC inhibition on BMP-7 expression in our murine model of obstructive uropathy. While UUO resulted in a significant decrease in BMP-7 protein levels, TSA treatment restored BMP-7 expression in the obstructed kidney by stimulating a mean fold increase in BMP-7 protein levels (p <0.01, fig. 6, A). Importantly, these changes in BMP-7 expression at the protein level were paralleled by a mean fold increase in BMP-7 mrna expression (p <0.01, fig. 6, B and C ). In addition to other findings (fig. 2, D and E ), these results show that Bmp-7 transcription is regulated by HDAC dependent mechanisms in vivo. We subsequently determined whether the HDAC dependent repression of Bmp-7 expression has a role in the molecular mechanisms that respond to renal injury. As shown in previous studies, UUO resulted in significant up-regulation of TGF-b1 expression along with its downstream profibrotic target genes COLIa1 (each p <0.005) and a-sma (p <0.05) during the development of obstruction induced renal fibrosis (fig. 6, B and D to F ). 5 Importantly, TSA treatment not only restored BMP-7 expression in the obstructed kidney but also decreased COLIa1 expression a mean of 41.6% 7.0% and a-sma expression by 61.6% 37.0% (p <0.01, <0.005 and <0.05, respectively, fig. 6, A to C, E and F ). Furthermore, TSA treatment had only minimal effects on TGF-b1 expression (fig. 6, B and D), revealing that the inhibitory effects of HDAC inhibition were mediated by the suppression of downstream steps in the TGF-b pathway. In addition to our prior results (figs. 4 and 5), these results strongly suggest that HDAC dependent repression of Bmp-7 expression contributes to dysregulation of the TGF-b pathway after chronic renal injury. Since previous findings revealed that HDAC inhibition restored BMP-7 expression and the balance in TGF-b/BMP-7 signaling in the obstructed kidney (fig. 6), we examined the therapeutic effects of treatment with HDAC inhibitors during chronic renal injury progression. While UUO resulted in the loss of BMP-7 expression in various cell types in the obstructed kidney, TSA treatment effectively

6 REPRESSION OF Bmp-7 POTENTIATES RENAL FIBROSIS IN OBSTRUCTIVE UROPATHY 247 Figure 4. HDAC inhibition suppressed activity of TGF-b 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-b 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-b1, COLIa1 and a-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, COLIa1. C, a-sma. D, BMP-7. E, TGF-b1. targeted each of these cell populations to restore BMP-7 expression (fig. 7, A). More importantly, TSA treatment also markedly inhibited the development of UUO induced renal injury. TSA treatment was associated with a mean 31.1% 7.6% decrease in total renal collagen content (p <0.05, fig. 7, A and B). This was a striking finding, given that this corresponded to a 65.3% reduction in the increase in collagen expression above baseline after UUO. TSA treatment also resulted in a mean 43.4% 10.1% decrease in tubular volume loss after UUO (p <0.01, (fig. 7, A and C ). Together these findings show that pharmacological HDAC inhibitors are an effective therapeutic approach not only to prevent the loss of BMP-7 expression in the injured kidney but also to inhibit renal fibrosis and the disruption of renal architecture during the progression of chronic renal injury.

7 248 REPRESSION OF Bmp-7 POTENTIATES RENAL FIBROSIS IN OBSTRUCTIVE UROPATHY Figure 5. TGF-b 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-b with PBS as control, 10 ng/ml BMP-7 or 100 nm TSA with PBS as control, or 10 mm dorsomorphin for 24 hours. Cells were analyzed. A, RT-PCR for Bmp-7, TGF-b1, COLIa1 and a-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, COLIa1. D, a-sma. E, TGF-b1. DISCUSSION In the last decade BMP-7 emerged as a critical renal protective protein that safeguards the kidney against various stimuli that cause renal injury. 6e17 However, loss of BMP-7 expression occurs in obstructive uropathy and other conditions that lead to chronic renal injury. 8,11e13,18 While mechanisms that lead to the loss of BMP-7 expression remain unclear, we noted in a mouse model of obstructive uropathy that loss of BMP-7 expression occurs at the mrna level in conjunction with histone deacetylation and transcriptional repression of the Bmp-7 promoter (fig. 2). Importantly, treatment with the pan-specific HDAC inhibitor TSA

8 REPRESSION OF Bmp-7 POTENTIATES RENAL FIBROSIS IN OBSTRUCTIVE UROPATHY 249 Figure 6. HDAC inhibition restored BMP-7 expression and suppressed TGF-b 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 mg/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-b1, COLIa1 and a-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-b1. E, COLIa1. F, a-sma.

9 250 REPRESSION OF Bmp-7 POTENTIATES RENAL FIBROSIS IN OBSTRUCTIVE UROPATHY restored BMP-7 expression in the obstructed kidney (fig. 6). When examining the role of HDAC dependent repression of Bmp-7 expression in molecular mechanisms that respond to renal injury, we found that HDAC inhibition limited the profibrotic potential of renal epithelial cells in vitro by suppressing the downstream steps in the TGF-b signaling pathway in a BMP-7 dependent manner (figs. 4 and 5). These signaling mechanisms also had considerable implications during the in vivo response to renal injury (fig. 8). This was demonstrated by the finding that TSA treatment inhibited the expression of TGF-b induced profibrotic genes in the obstructed kidney (fig. 6). We previously reported that BMP-7 is required for cessation of the TGF-b response and for 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 mg/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.

10 REPRESSION OF Bmp-7 POTENTIATES RENAL FIBROSIS IN OBSTRUCTIVE UROPATHY 251 Figure 8. HDAC dependent transcriptional repression of Bmp-7 potentiated TGF-b 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-b pathway by sequestering Smad4, protein needed for transcription of many TGF-b dependent genes. 6 These counter regulatory mechanisms are important to suppress TGF-b 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-b pathway. Pharmacological HDAC inhibitor treatment restores BMP-7 expression, suppresses TGF-b dependent signaling pathways and prevents renal fibrosis after chronic urinary tract obstruction. the repair of obstruction induced renal injury. 8 The current findings strongly suggest that HDAC dependent repression of Bmp-7 expression contributes to persistent activation of the TGF-b pathway and to dysregulation of kidney repair after chronic renal injury. Based on these findings, we have begun to examine the therapeutic potential of administering pharmacological HDAC inhibitors to limit the progression of chronic renal injury. Importantly, TSA treatment significantly inhibits the development of renal fibrosis and the loss of renal architecture in response to urinary tract dysfunction (fig. 7). While the suppression of TGF-b signaling likely explains many of the antifibrotic effects of TSA stimulated BMP-7 expression, TGF-b independent mechanisms likely also have an important role in inhibiting the pathogenesis of renal injury. We and others previously noted that recombinant BMP-7 promotes the regeneration of renal architecture after injury 8e10,17 in a manner that recapitulates the requirement for BMP-7 during kidney development. 24 A possible role for these functions in the renal protective effects of HDAC inhibition was recently suggested by a group that identified an HDAC inhibitor as the leading candidate in a high throughput screen designed to identify compounds that stimulate regenerative cell populations in the kidney. 25 To fully realize the therapeutic potential of HDAC inhibitors it is necessary to translate these findings to in vivo models that more closely parallel the development of kidney disease after congenital urinary obstruction. While in our study we examined obstruction induced renal injury in the mature kidney, surgical models of fetal/neonatal obstruction as well as genetic models of congenital obstruction may provide useful tools for examining the importance of these biological pathways in the developing kidney. 26 Also, while our treatment regimen for TSA administration was supported by prior pharmacokinetic studies, 27 these protocols may be improved through further optimization. Finally, isoform specific HDAC inhibitors may provide a means of developing more specific, effective therapies for kidney disease. CONCLUSIONS While working toward the development of novel therapies for chronic kidney disease, it has become increasingly evident that loss of BMP-7 is a critical molecular event that contributes to disease progression across a broad spectrum of etiologies. 6e18 Our study shows that loss of BMP-7 occurred due to HDAC dependent repression of Bmp-7 transcription in a murine model of obstructive uropathy. Furthermore, treatment with HDAC inhibitors was an effective therapeutic approach to restore BMP-7 expression and limit the progression of chronic renal injury in the obstructed kidney. The importance of these findings is accentuated by studies showing the renal protective effects of HDAC inhibition in other settings. 28e30 Although the mechanisms underlying these effects remain unclear, our series reveals that the remarkable therapeutic potential of HDAC inhibitors is due in part to

11 252 REPRESSION OF Bmp-7 POTENTIATES RENAL FIBROSIS IN OBSTRUCTIVE UROPATHY their ability to target the BMP-7 pathway. Given that HDAC inhibitors are already in clinical use, it is imperative to further examine the usefulness of these compounds for treating obstructive uropathy and other conditions that lead to chronic kidney disease. REFERENCES 1. Roth KS, Koo HP, Spottswood SE et al: Obstructive uropathy: an important cause of chronic renal failure in children. Clin Pediatr (Phila) 2002; 41: Cochrane AL, Kett MM, Samuel CS et al: Renal structural and functional repair in a mouse model of reversal of ureteral obstruction. J Am Soc Nephrol 2005; 16: Chawla LS and Kimmel PL: Acute kidney injury and chronic kidney disease: an integrated clinical syndrome. Kidney Int 2012; 82: Yang L, Humphreys BD and Bonventre JV: Pathophysiology of acute kidney injury to chronic kidney disease: maladaptive repair. Contrib Nephrol 2011; 174: Bottinger EP: TGF-beta in renal injury and disease. 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