Sodium valproate ameliorates diabetes-induced fibrosis and renal damage by the inhibition of histone deacetylases in diabetic rat

YEXMP-03686; No of Pages 10 Experimental and Molecular Pathology xxx (2015) xxx–xxx

Contents lists available at ScienceDirect

Experimental and Molecular Pathology journal homepage: www.elsevier.com/locate/yexmp

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Sabbir Khan a, Gopabandhu Jena a,⁎, Kulbhushan Tikoo b a

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Article history: Received 6 August 2014 and in revised form 24 December 2014 Accepted 5 January 2015 Available online xxxx

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Keywords: Diabetic nephropathy HDAC inhibitor Myofibroblast Renal fibrosis Sodium valproate Transforming growth factor-beta 1 Histone acetylation

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Facility for Risk Assessment and Intervention Studies, Department of Pharmacology and Toxicology, National Institute of Pharmaceutical Education and Research, Sector-67, S.A.S. Nagar, Punjab 160062, India b Laboratory of Epigenetics and Diseases, Department of Pharmacology and Toxicology, National Institute of Pharmaceutical Education and Research, Sector-67, S.A.S. Nagar, Punjab 160062, India

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Recent reports emphasize the contribution of histone deacetylases (HDACs) in the pathogenesis of diabetic renal injury and fibrosis. Valproic acid (VPA) is a first-line drug used for the treatment of epilepsy and migraine as well as established as a HDAC inhibitor. The present study was aimed to evaluate the anti-fibrotic and renoprotective effects of VPA in diabetic nephropathy (DN). Diabetes was induced by single injection of STZ (50 mg/kg), whereas VPA at the doses of 150 and 300 mg/kg/day was administered for 8 consecutive weeks by oral route in Sprague Dawley rats. The renal injuries and fibrosis were assessed by histology, fibrosis specific staining and fibroblast activation by a transmission electron microscope, while expression of proteins of interest was evaluated by western blotting and immunohistochemistry. VPA treatment ameliorated the histological alterations as well as fibrosis, and decreased the expression of TGF-β1, CTGF, α-SMA, fibronectin, collagen I, COX-2, ICAM-1 and HDAC4/5/7. Further, VPA treatment significantly increased histone H3 acetylation and MMP-2 expression. The present study clearly established that VPA treatment ameliorates the renal injury and fibrosis in diabetic kidney by preventing the myofibroblast activation and fibrogenesis by HDAC inhibition and associated mechanisms, thereby improving the profibrotic and anti-fibrotic protein balance. © 2015 Published by Elsevier Inc.

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1. Introduction

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Diabetic nephropathy (DN) is characterized by the thickening of glomerular basement membranes and increased extracellular matrix (ECM) in the glomerular and tubulo-interstitial region, which finally leads to end-stage renal disease (Ban and Twigg, 2008; Riser et al., 2010). Several mechanisms have been reported to play significant role in the pathogenesis of DN, but transforming growth factor-β1 (TGFβ1)-induced fibrogenesis takes a central position (Noh et al., 2009; Van Beneden et al., 2013). Moreover, connective tissue growth factor (CTGF) in association with TGF-β1, promotes the development of fibrosis in a variety of experimental models (Kliem et al., 1996; Noh et al., 2009). Generally the myofibroblasts are originated from tissue-specific fibroblasts and/or pericytes during tissue injury and repair (Hinz et al., 2007). In the kidney, the myofibroblasts are originated from the differentiation of resident fibroblasts or transformation of epithelial cells,

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Sodium valproate ameliorates diabetes-induced fibrosis and renal damage by the inhibition of histone deacetylases in diabetic rat

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Abbreviations: α-SMA, Alpha smooth muscle actin; COX-2, Cyclooxygenase 2; CTGF, Connective tissue growth factor; DAB, 3,3′-Diaminobenzidine; ECM, Extracellular matrix; HDACs, Histone deacetylases; ICAM-1, Intercellular adhesion molecule 1; MMP-2, Matrix metalloproteinase-2; STZ, Streptozotocin; TGF-β1, Transforming growth factor-beta 1; VPA, Valproic acid. ⁎ Corresponding author. E-mail addresses: [email protected] (S. Khan), [email protected], [email protected] (G. Jena), [email protected] (K. Tikoo).

term as epithelial–mesenchymal transition (EMT) (Cook, 2010; Hinz et al., 2007). Recent reports highlighted that EMT plays a pivotal role in renal fibrotic disease including DN (Fragiadaki and Mason, 2011). It has also been reported that myofibroblasts play an important role in adriamycin-induced nephropathy as well as in ischemia/reperfusion injury (Broekema et al., 2007; Li et al., 2006). The increased myofibroblast population leads to structural abnormalities and reduces the organ function, thereby further accelerates progression of fibrosis. Activated fibroblasts result a series of changes in the morphology and gene expression profile such as increased proliferation, motility, α-SMA expression as well as ECM production and decreased ECM degrading enzymes like matrix metalloproteinase (MMPs) (Aher et al., 2015; Li et al., 2006). Recently, several studies highlighted the importance of epigenetic mechanisms in the pathogenesis of diabetic complications including DN (Gilbert et al., 2011; Kato and Natarajan, 2014; Reddy et al., in press). Histone deacetylases (HDACs) are involved in several molecular signaling relevant to the pathogenesis of DN (Lee et al., 2007; Villeneuve and Natarajan, 2010). HDAC inhibitors including VPA have been acknowledged as potential anti-fibrotic molecules in various fibrotic disorders in multiple organs (Mannaerts et al., 2010; Van Beneden et al., 2013). Further, knockdown of HDAC1 in renal interstitial fibroblasts and tubular epithelial cells confirmed the contribution of HDACs in myofibroblast activation, proliferation and chemokine production (Liu et al., 2013). HDAC inhibitors can prevent the TGF-β1-mediated

http://dx.doi.org/10.1016/j.yexmp.2015.01.003 0014-4800/© 2015 Published by Elsevier Inc.

Please cite this article as: Khan, S., et al., Sodium valproate ameliorates diabetes-induced fibrosis and renal damage by the inhibition of histone deacetylases in diabetic rat, Exp. Mol. Pathol. (2015), http://dx.doi.org/10.1016/j.yexmp.2015.01.003

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2. Methods

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2.1. Animals

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Animal experiment protocol was approved by the Institutional Animal Ethics Committee (IAEC) and experiment was performed on male Sprague–Dawley rat (250–280 g) procured from the Central Animal Facility of the institute in accordance with the Committee for the Purpose of Control and Supervision of Experimentation on Animals (CPCSEA) guidelines. Animals were kept under controlled environment at room temperature (22 ± 2 °C) with humidity (50 ± 10%) and an automatically controlled 12 h light and dark cycle. Feed and water were provided ad libitum. Animals were acclimatized for one week prior to commencement of actual experiment.

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2.2. Chemicals

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Until mentioned otherwise, all the chemical and reagents including sodium valproate (CAS no. 1069-66-5) were purchased from SigmaAldrich chemicals, Saint Louis, MO, USA, while primary and secondary antibodies were purchased from Santa Cruz Biotechnology, CA, USA.

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2.3. Experimental design and animal treatment

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Animals were randomized into five groups; group-1: control (Con, n = 6), received saline; group-2: VPA control (VPA300, n = 6), received VPA 300 mg/kg/day for 8 weeks; group-3: diabetic control (D, n = 8), received single injection of streptozotocin (STZ, 50 mg/kg); groups-4 and 5: VPA treated diabetic animals (D + VPA150 & D + VPA300, n = 8), received VPA at the doses of 150 and 300 mg/kg/day for 8 consecutive weeks. The doses of VPA were selected on the basis of previous studies (Ahmad et al., 2013; Khan et al., 2011; Khan and Jena, in press, 2013). VPA was dissolved in distilled water and administrated by oral route according to body weight.

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fibroblast activation and subsequently reduced the ECM deposition and fibrosis (Gilbert et al., 2011; Khan and Jena, 2014a; Noh et al., 2009). Further, HDAC inhibitors can also modulate the function of renal endothelial and epithelial cells and regulate the expression of CTGF and collagen I as well as other profibrotic molecules (Komorowsky et al., 2009). Recent study shows the expressions of different HDACs in diabetic kidney of patients and STZ treated rats, which proves that HDAC4 is the major player in the pathogenesis of DN (Wang et al., 2014). Together, above reports highlighted the role of HDACs and their inhibitors in the renal injury and fibrosis in DN and various other pathological conditions. Valproic acid (VPA) is a short-chain fatty acid and first-line drug used for the treatment of epilepsy, migraine and other psychiatric disorders. Presently, VPA has been proven as a HDAC inhibitor and subdued the class I and II HDACs (Chateauvieux et al., 2010; Gottlicher et al., 2001). VPA exerts anti-inflammatory and anti-oxidant activity thereby protects the multiple organ damage in several pathological conditions (Khan and Jena, in press; Shang et al., 2010; Zhang et al., 2008). Further, VPA has been reported to reduce the glomerulosclerosis and proteinuria as well as fibrosis in adriamycin-induced nephropathy in mouse as well as DN (Khan et al., in press; Van Beneden et al., 2011). Additionally, VPA prevents the hepatic fibroblast activation in in vitro and in vivo experiments as well as prevents penile fibrosis and erectile dysfunction in cavernous nerve-injured rat (Aher et al., 2015; Hannan et al., 2014; Watanabe et al., 2011). Therefore, we hypothesized that VPA can exert protective effects on TGF-β1-mediated fibrogenesis, myofibroblast activation and renal fibrosis in the kidney of diabetic rat. The present results clearly demonstrated that VPA treatment significantly ameliorates the fibrosis by preventing the diabetes-associated fibrogenesis and activation of myofibroblast in the kidney through HDAC inhibition.

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Fig. 1. Effect of the diabetes and VPA treatment on the (A) body weight and (B & C) absolute as well as relative kidney weight and (D) plasma glucose. All the values are expressed as mean ± SEM, (n = 6–8), #P b 0.001, ‘a’ vs. control ‘b’ vs. VPA control and ‘c’ vs. diabetic control.

Please cite this article as: Khan, S., et al., Sodium valproate ameliorates diabetes-induced fibrosis and renal damage by the inhibition of histone deacetylases in diabetic rat, Exp. Mol. Pathol. (2015), http://dx.doi.org/10.1016/j.yexmp.2015.01.003

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Fig. 2. Effect of the diabetes and VPA treatment on the histological alterations. Representative photomicrographs of the kidney histology evaluated by the PAS staining of the different groups. Inserts on upper-right-side boxes in PAS staining showed the magnified images of the selected glomeruli. Arrows indicate the tubular cell degeneration and cytotoxicity, while asterisks (*) indicate increased glomerular space. All the values are expressed as mean ± SEM, (n = 5), #P b 0.001 and †P b 0.01 ‘a’ vs. control ‘b’ vs. VPA control and ‘c’ vs. diabetic control.

2.4. Induction of diabetes

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Experimental diabetes was induced by a single injection of STZ (50 mg/kg) dissolved in ice-cold 10 mM citrate buffer (pH 4.4) and administered by i.p. route immediately. Age-matched control rat received an equivalent volume of vehicle. After 48 h of STZ injection, animals were kept for 6–8 h fasting and plasma glucose was measured using commercially available kit (ACCUREX, Mumbai, India). Animals with fasting plasma glucose level ≥ 250 mg/dl were considered to be diabetic and used in the present experiment.

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2.5. Histological evaluation

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Histological slides were prepared according to the standardized protocol in our laboratory and previously described (Khan et al., 2011). The kidneys were fixed in 10% neutral buffer formalin, dehydrated gradually in ethanol and xylene then embedded in paraffin. The sections were

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deparaffinized with xylene and rehydrated with alcohol and water, and stained with periodic acid-Schiff (PAS) using a commercial kit (Sigma, MO, USA). Then mounted with DPX and observed under a microscope (Olympus BX51 microscope, Tokyo, Japan). The histological alterations were quantitatively evaluated as described by Thijs W., et al. with some modification (Tervaert et al., 2010). Randomly 15–20 focuses were selected from each animal and histological scores were assigned depending upon the extent of damage as follows; 0 = no interstitial fibrosis and tubular as well as glomerular lesions, 1 = up to 25% interstitial fibrosis with mild tubular and glomerular lesions, 2 = 26–50% interstitial fibrosis and tubular as well as glomerular lesions, 3 = 51– 75% interstitial fibrosis with moderate tubular and glomerular lesions, 4 N 75 interstitial fibrosis with severe tubular and glomerular lesions.

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2.6. Transmission electron microscopy (TEM)

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For TEM, cortex region of kidneys were cut into 1 × 1 × 1 mm blocks 164 and fixed with 2.5% glutaraldehyde and further proceed as per our 165 Q4

Please cite this article as: Khan, S., et al., Sodium valproate ameliorates diabetes-induced fibrosis and renal damage by the inhibition of histone deacetylases in diabetic rat, Exp. Mol. Pathol. (2015), http://dx.doi.org/10.1016/j.yexmp.2015.01.003

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laboratory protocol as described previously (Aher et al., 2015). The sections were taken using ultramicrotome and stained with uranyl acetate and observed with high resolution TEM (FEI TF-20; FEI, Hillsboro, Oregon, USA).

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Fig. 3. Effect of the diabetes and VPA treatment on ECM deposition and fibrosis. Representative photomicrographs of the sirius red and masson trichrome staining along with the quantification of fibrotic area, respectively. Arrows indicate the collagen deposition (fibrotic) area. All the values are expressed as mean ± SEM, (n = 5), #P b 0.001, ‘a’ vs. control ‘b’ vs. VPA control and ‘c’ vs. diabetic control.

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2.7. Immunohistochemistry (IHC) and immunofluorescence

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IHC was performed as described previously (Yadav et al., 2014) with some modifications. The kidney sections (5 μm) were taken on the polylysine-L-lysine coated slides and IHC was performed by Novolink polymer detection system (Leica, Milton Keynes, UK) according to the manufacturer instruction's using primary antibodies for protein of interest (Santa Cruz Biotechnology, USA). Randomly 15–20 fields/focuses from each animal were observed and immune-reactive area was quantified by ImageJ (version 1.46 m) software (http://rsbweb.nih.gov/ij).

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2.8. Western blot analysis

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Proteins were separated in SDS-PAGE and transferred onto nitrocellulose membrane (Pall Corporation, Florida, USA). Immunoblotting was performed using primary antibodies for protein of interest along with anti-actin for loading control (1:1000) and HRP-conjugated secondary antibody (1:5000, Santa Cruz, USA). Proteins were detected by ECL and quantified using Imagequant TL software (GE Healthcare, Hong Kong, China). For western blotting, 4–5 animal tissues were pooled and quantitative data was expressed as average of 3 independent blots from same sample.

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Please cite this article as: Khan, S., et al., Sodium valproate ameliorates diabetes-induced fibrosis and renal damage by the inhibition of histone deacetylases in diabetic rat, Exp. Mol. Pathol. (2015), http://dx.doi.org/10.1016/j.yexmp.2015.01.003

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Results are shown as mean ± SEM for each group and P b 0.05 considered to be statistically significant. Statistical analysis was performed using Sigma Stat (Version 3.5) software (Systat Software, CA, USA). ANOVA was used to determine the level of significance among the different groups, while Tukey's test was used for post-hoc analysis.

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3. Results

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3.1. Effect on the body and organ weight as well as plasma glucose

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Diabetes significantly decreased the body weight of animals as compared control, while VPA treatment restored the reduction in body weight from the 5th week onward, but it was statistically insignificant (Fig. 1A). VPA treatment decreased the diabetes-associated increased relative kidney weight as compared to respective control, but it was statistically insignificant (Fig. 1B and C). Moreover, VPA treatment failed to reduce the plasma glucose level as compared to respective control (Fig. 1D).

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3.2. Effect on the cellular alterations and collagen deposition

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VPA treatment significantly ameliorated the diabetes-induced histological alterations in the kidney such as tubular-intestinal damage including loss of tubular cytoplasm and pyknotic nuclei as well as increased capsular space and glomerular damage as compared to respective controls (Fig. 2). Further, quantitative evaluation of the histological alterations revealed that VPA treatment significantly decreased the diabetes-induced renal damage/injury as compared to respective controls (Fig. 2). Moreover, VPA treatment significantly reduced the diabetes-associated increased ECM and collagen deposition (% fibrotic area) in a dose-dependent manner as evident by sirius red and masson trichrome staining (Fig. 3). In the present study, collagen I expression was also evaluated by IHC as well as immunofluorescence and the results indicated that VPA treatment significantly decreased diabetesassociated over expression of collagen I in a dose-dependent manner as compared to respective controls (Fig. 4 and Suppl. Fig. 1). These

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results confirmed the anti-fibrotic and renoprotective effect of VPA in 221 diabetic nephropathy. 222

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Fig. 4. Representative photomicrographs of IHC along with quantitative evaluation for the expression of collagen I in the kidney. Arrows indicate the immunopositive signal for collagen I expression, which increased in glomerulus and tubules of diabetic kidney. All the values are expressed as mean ± SEM, (n = 5), #P b 0.001 and *P b 0.05, ‘a’ vs. control ‘b’ vs. VPA control and ‘c’ vs. diabetic control.

3.3. Effect on the expression of TGF-β1, fibronectin, α-SMA and fibroblast 223 activation 224 TGF-β1 signaling plays a pivotal role in the fibrogenesis in DN and several other fibrotic disorders. In the present study, VPA treatment significantly decreased the diabetes-induced over expression of TGF-β1, fibronectin and α-SMA in a dose-dependent manner as compared to respective controls (Figs. 5 and 6). Further, fibroblast activation (myofibroblast) was confirmed by TEM and results showed that VPA treatment prevents the diabetes-associated renal myofibroblast activation in a dose-dependent manner as compared to respective controls (Fig. 6). Thus, TEM analysis and α-SMA expression results confirmed that VPA treatment inhibited the fibroblast activation in diabetic kidney.

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3.4. Effect on the expression of CTGF, MMP-2, COX-2 and ICAM-1

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VPA treatment significantly decreased the diabetes-induced over expression of CTGF in a dose-dependent manner as compared to respective control (Fig. 7A and B). Further, VPA treatment also restored the diabetes-associated decreased expression of MMP-2 in diabetic kidney (Fig. 7A and C). Moreover, VPA treatment significantly decreased the diabetes-induced over expression of COX-2 and ICAM-1 in a dosedependent manner as compared to respective controls (Fig. 7D and E).

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3.5. Effect on the HDAC inhibition and histone acetylation

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To confirm the HDAC inhibition potential of VPA in the present study, the expression of HDAC4/5/7 and acetylation of histone H3 in the kidney were evaluated by IHC. The present results showed that VPA treatment significantly decreased diabetes-induced over expression of HDAC4/5/7 in a dose-dependent manner as compared to respective controls (Fig. 8A). The above results were further confirmed by acetylated histone H3 expression, which indicated that VPA treatment significantly restored diabetes-associated decreased acetylation of histone H3 as compared to respective controls (Fig. 8B).

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Please cite this article as: Khan, S., et al., Sodium valproate ameliorates diabetes-induced fibrosis and renal damage by the inhibition of histone deacetylases in diabetic rat, Exp. Mol. Pathol. (2015), http://dx.doi.org/10.1016/j.yexmp.2015.01.003

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Fig. 5. Representative photomicrographs of immunofluorescence along with quantitative evaluation for the expression of TGF-β1 and fibronectin in the kidney stained with DAB at magnification 400×. Arrows and brown staining indicates the immunopositive area for protein of interest. All the values are expressed as mean ± SEM, (n = 5), #P b 0.001, and *P b 0.05 ‘a’ vs. control ‘b’ vs. VPA control and ‘c’ vs. diabetic control. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

4. Discussion

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The present study reports the anti-fibrotic and renoprotective effect of VPA in diabetic rat kidney through HDAC inhibition and associated mechanisms, thereby improving the balance between pro- and antifibrotic genes. VPA treatment significantly reduced diabetesassociated renal fibroblast activation, fibrosis, ECM deposition as well as histological changes such as tubulo-interstitial damages, cytotoxicity and capsular space in a dose-dependent manner as revealed by histology and fibrosis specific staining. The above results confirmed the renoprotective and anti-fibrotic effect of VPA in diabetes-induced renal damage at the structural level. Further, VPA treatment significantly decreased the expression of COX-2, which showed its antiinflammatory property in diabetic kidney. Reduction in the inflammation by VPA is one of the possible mechanisms for its renoprotective and anti-fibrotic efficacy. HDAC inhibitors including VPA have already

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been reported as anti-inflammatory and can improve the renal damages in diabetes and other pathological conditions (Gilbert et al., 2011; Zhang et al., 2008). Inflammation has been known to play a major role in the TGF-β1-mediated fibrogenesis in diabetic condition (Shen et al., 2008). In the present study, VPA treatment significantly decreased the diabetes-induced over expression of TGF-β1, fibronectin, α-SMA, collagen I. These results confirmed that VPA treatment prevents the fibrogenesis and fibroblast activation as revealed by TEM and α-SMA immune-staining, a marker of activated fibroblast. Thus, modulation of TGFβ-1 signaling and inhibition of fibroblasts activation might be one of the plausible mechanisms responsible for the anti-fibrotic and renoprotective effect of VPA. Further, HADC inhibitors including VPA can prevent the TGFβ-1-induced renal fibrogenesis and exert antifibrotic effect in diabetes as well as other fibrotic conditions (Aher et al., 2015; Hemmatazad et al., 2009; Liu et al., 2013).

Please cite this article as: Khan, S., et al., Sodium valproate ameliorates diabetes-induced fibrosis and renal damage by the inhibition of histone deacetylases in diabetic rat, Exp. Mol. Pathol. (2015), http://dx.doi.org/10.1016/j.yexmp.2015.01.003

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Moreover, TGF-β1 signaling and inflammation play a major role in DN as well as chromatin remolding. It has been reported that p300/ CBP and epigenetic histone acetylation involve in TGF-β1-mediated gene transcription in mesangial cells. Additionally, TGF-β1 enhances the expression of profibrotic genes through p300/CBP and Smads2/3/4 as well as microRNAS in renal mesangial cells (Castro et al., 2014; Yuan et al., 2013). Recently, valproic acid has been reported to prevent penile fibrosis and erectile dysfunction in cavernous nerve injured rats (Hannan et al., 2014). Furthermore, blocking the class I histone deacetylase ameliorates the renal fibrosis and inhibits renal fibroblast activation via modulating TGF-beta and EGFR signaling in the murine model of unilateral ureteral obstruction (UUO) and cultured renal interstitial fibroblasts (Liu et al., 2013). It has been reported that HDAC4 plays a critical role in TGF-β1-induced myofibroblast differentiation in cultured human skin fibroblasts (Glenisson et al., 2007). Our findings also suggest that diabetic kidney showed significant over expression of HDAC4/5/7 along with decreased acetylation of histone H3, whereas VPA treatment restored the same. These results confirmed that VPA

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Fig. 6. Representative photomicrographs of immunofluorescence along with quantitative evaluation for the expression of TGF-β1 and fibronectin in the kidney stained with DAB at magnification 400×. Arrows and brown staining indicates the immunopositive area for protein of interest. Bottom panel: Representative photomicrographs of TEM showing the renal fibroblast of different groups. # indicates the activated fibroblast. All the values are expressed as mean ± SEM, (n = 5), #P b 0.001 and †P b 0.01, ‘a’ vs. control ‘b’ vs. VPA control and ‘c’ vs. diabetic control. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

exerts the anti-fibrotic effect by preventing TGF-β1-induced activation of renal fibroblast and subsequently reduces the fibrosis through HDAC inhibition. TGF-β1 is responsible for the excessive production of ECM during the early phase of fibrosis, which subsequently maintained by CTGF (Huber et al., 2007). Moreover, ICAM-1 is another profibrotic molecule, which plays a major role in various vascular pathologies and fibrosis in diabetic condition (Hemmatazad et al., 2009). HDACs and their inhibitors modulate the CTGF and ICAM-1 expression as well as the extent of fibrosis depending on the tissue/cell types (Ahmed et al., 2013; Hellebrekers et al., 2006; Komorowsky et al., 2009). In the present study, VPA treatment significantly reduced the diabetes-associated over expression of CTGF and ICAM-1. On the other hand, MMPs are responsible for the degradation of ECM including collagen and counteract the excessive ECM production to maintain its physiological level. Recent reports highlighted that MMP-2 expression has been reduced in various fibrotic condition including diabetes, which restored by HDAC inhibitors (Aher et al., 2015; Ahmed et al., 2013). Our results provide that VPA

Please cite this article as: Khan, S., et al., Sodium valproate ameliorates diabetes-induced fibrosis and renal damage by the inhibition of histone deacetylases in diabetic rat, Exp. Mol. Pathol. (2015), http://dx.doi.org/10.1016/j.yexmp.2015.01.003

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significantly restored the diabetes-associated down regulation of MMP2 level. Recently, we also reported that VPA treatment restored the MMP-2 level in thioacetamide-induced hepatic fibrosis in rat (Aher et al., 2015). Together, it can be concluded that VPA exerts its antifibrotic effect by maintaining the fine tuning between the production and degradation of ECM. Further, the selective induction of apoptosis in activated fibroblast is one of the strategies for the development of promising anti-fibrotic drug/molecule (Hinz et al., 2012). HDAC inhibitors modulate the apoptosis particularly in the disease cells by altering the expression of pro- and anti-apoptotic proteins and ultimately perturbed the balance between these proteins (Carew et al., 2008; Khan and Jena, 2014b) Apart from this VPA can also induced the selective apoptosis in the activated fibroblast, thereby reduced the production of profibrotic molecules as well as ECM (Aher et al., 2015). It has been reported that suberoylanilide hydroxamic acid (SAHA) altered the expression of apoptosis-associated gene in lung fibroblasts during the post-inflammatory phase injury through HDAC inhibition and histone acetylation (Sanders et al., 2014). Considering the present findings, it can be concluded that VPA may be one of the promising anti-fibrotic drug for the treatment of diabetic renal fibrosis due to its pleiotropic mechanisms. In general, inhibition of HDAC activity leads to increase histone acetylation, which in turn mostly results in gene activation (Moore et al., 2004). However, HDAC inhibition causes both the induction and repression of a large number of genes, although the proportion of upregulated genes is greater than down-regulated, which are involved in

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Fig. 7. (A–D) Representative immunoblots and quantitative analysis showing the effect of diabetes and VPA treatment on the expression of CTGF, MMP-2 and COX-2. (E) Representative photomicrographs of IHC along with quantitative evaluation for the expression of ICAM-1 in the kidney sections. All the values are expressed as mean ± SEM, (n = 4–5), #P b 0.001, †P b 0.01, *P b 0.05, ‘a’ vs. control ‘b’ vs. VPA control and ‘c’ vs. diabetic control.

several cellular processes and functions (Kato and Natarajan, 2014; Moore et al., 2004; Reddy et al., in press). Notably, it has been reported that histone acetylation by TSA down regulate the TGF-β1 and collagen expression, while increased the expression of inhibitors of DNA binding/ differentiation 2 (Id2), bone morphologic protein 7 (BMP-7) and Ecadherin [3]. Further, HDAC2 knockdown reduces the fibronectin and α-SMA expression, but increases the E-cadherin expression, which suggests that HDAC inhibitors modulate specific gene differently (Wang et al., 2012; Yoshikawa et al., 2007). Similarly, the global changes in histone acetylation may or may not be significant, but its change may be significant at the specific genes. Thus gene specific histone acetylation is more appropriate to explore the exact target for epigenetic alteration in a particular condition. Considering above reports, it can be concluded that gene specific acetylation can down- and up-regulated the target gene in particular experimental setups. Furthermore, generally HDAC inhibitors suppress the pathological genes, which are up-regulated in disease condition. Additionally, HDAC inhibitors exert prominent effects in the pathological cell/tissue, because the micro-environment is very much different as compared to non-disease condition (Chateauvieux et al., 2010; Chen et al., 2011). This is the first study that reports the anti-fibrotic efficacy of VPA in diabetic renal fibrosis in vivo. The present results highlighted the antifibrotic and renoprotective effect of VPA as revealed by histological analysis and fibrosis specific staining as well as expression of various profibrotic and anti-fibrotic proteins at clinically relevant doses. VPA exerts its anti-fibrotic effect by preventing fibrogenesis and inactivation of

Please cite this article as: Khan, S., et al., Sodium valproate ameliorates diabetes-induced fibrosis and renal damage by the inhibition of histone deacetylases in diabetic rat, Exp. Mol. Pathol. (2015), http://dx.doi.org/10.1016/j.yexmp.2015.01.003

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Fig. 8. Representative photomicrographs of IHC showing the effect of diabetes and VPA treatment on expression of HDAC4/5/7 and histone H3 acetylation in the kidney of different groups at 400× magnification, while the histograms (A and B) show the quantitative evaluation of the same. All the values are expressed as mean ± SEM, (n = 4), #P b 0.001 and *P b 0.05, ‘a’ vs. control ‘b’ vs. VPA control and ‘c’ vs. diabetic control.

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fibroblast through HDAC4/5/7 inhibition and increased histone acetylation. Because VPA is a well known clinically used drug, therefore, efforts should be made to translate the present findings into a successful therapeutic intervention for renal fibrotic disorders including DN. However, the future detailed investigations are required to demonstrate the exact molecular mechanisms accountable for its renoprotective and antifibrotic effects. Furthermore, exploring and understanding the tissue/ cell specific physiological roles of different HDAC isoforms in DN and associated fibrosis as well as designing and synthesis of selective HDAC inhibitors would contribute in the management of DN. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.yexmp.2015.01.003.

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Conflict of interest The authors declare no conflict of interest.

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This work has been funded by the National Institute of Pharmaceutical Education and Research (NIPER), Mohali, India. The authors would also like to acknowledge Mr. Vinod Kumar, Icon Analytical Equipment Pvt Ltd, Delhi, India for providing the technical assistance in TEM study.

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