Protective Effect of EX-527 against High-Fat Diet-Induced Diabetic Nephropathy in Zucker Rats

Amit Kundu1, Sachan Richa1, Prasanta Dey1, Kyeong Seok Kim1, Ji Yeon Son1, Hae Ri Kim1, Seok-Yong Lee1, Byung-Hoon Lee2, Kwang Youl Lee3, Sam Kacew4, Byung Mu Lee1, Hyung Sik Kim1,*

1School of Pharmacy, Sungkyunkwan University, 2066, Seobu-ro, Jangan-gu, Suwon 440-746, Republic of Korea
2College of Pharmacy, Seoul National University, Seoul 08826, Republic of Korea
3College of Pharmacy & Research Institute of Drug Development, Chonnam National University, Gwangju, Republic of Korea
4McLaughlin Centre for Population Health Risk Assessment, University of Ottawa, Ottawa, ON, Canada

Corresponding author Prof. Hyung Sik Kim
School of Pharmacy, Sungkyunkwan University, Suwon, 440-746, Republic of Korea Tel.: +82-31-290-7789; Fax: +82-31-290-7748; E-mail: [email protected]

ABSTRACT

High-fat diet (HFD)-induced obesity is implicated in diabetic nephropathy (DN). EX-527, a selective Sirtuin 1 (SIRT1) inhibitor, has multiple biological functions ; however, its protective effect against DN is yet to be properly understood. This study was aimed to explore the protective effect of EX-527 against DN in HFD-induced diabetic Zucker (ZDF) rats. After 21 weeks of continually feeding HFD to the rats, the apparent characteristics of progressive DN were observed, which included an increase in kidney weight (~160%), hyperglycemia, oxidative stress, and inflammatory cytokines , and subsequent renal cell damage. However, the administration of EX-527 for 10 weeks significantly reduced the blood glucose concentration and kidney weight (~ 59%). Furthermore, EX-527 significantly reduced the serum concentration of transforming growth factor-β1 (49%), interleukin (IL)-1β (52%), and IL-6 in the HFD-fed rats. Overall, the antioxidant activities significantly increased, and oxidative damage to lipids or DNA was suppressed. Particularly, EX-527 significantly reduced blood urea nitrogen (81%), serum creatinine (71%), microalbumin (43%), and urinary excretion of protein-based biomarkers . Histopathological examination revealed expansion of the extracellular mesangial matrix and suppression of glomerulosclerosis following EX-527 administration. EX-527 downregulated the expression of α-SMA (~ 64%), TGF-β (25%), vimentin, α-tubulin, fibronectin, and collagen-1 in the kidneys of the HFD-fed rats. Additionally, EX-527 substantially reduced claudin -1 and SIRT1 expression, but increased the expression of SIRT3 in the kidneys of the HFD-fed rats. EX-527 also inhibited the growth factor receptors, including EGFR, PDGFR-β, and STAT3, which are responsible for the anti-fibrotic effect of SIRT-1. Therefore, the administration of EX-527 protects against HFD-induced DN.

Keywords: High-fat diet, Diabetic nephropathy, EX-527, Inflammation, Oxidative stress, SIRT.

1.Introduction

Diabetes mellitus is an emerging global health burden owing to its increasing prevalence, and is expected to affect approximately 439 million people by 2030 (Chen et al., 2011). Prolonged diabetes results in vascular alteration, renal dysfunction, and other associated complications, resulting in high morbidity and mortality rates in patients with diabetes. Diabetic nephropathy (DN) should not be solely considered as a general and severe microvascular dysfunction of diabetes mellitus , but also plays a leading role in renal failure. Hyperglycemia is the main risk factor for the development of DN (Giacco and Brownlee, 2010; Kitada et al., 2010). A severe complication of diabetes is chronic kidney disease (CKD), which affects 20-40% of all patients with type 2 diabetes mellitus (Wolf and Ritz, 2003; Yacoub et al., 2007; Mestry et al., 2016).
The progression of the pathogenesis and complications of DN depends on various physiological factors such as oxidative stress, chronic inflammation, and apoptosis (Brownlee, 2005). In DN, reactive oxygen species (ROS) cause oxidative stress -associated renal impairment, leading to noticeable structural and functional alterations in the cells of the glomerulus and renal tubule (Soetikno et al., 2013). Generally, renal cells prevent ROS-induced damage at the cellular level via multiple antioxidative parameters, including superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx). Nuclear factor erythroid 2-related factor 2 (Nrf2) is associated with the antioxidant potential of SOD and CAT (Nakai et al., 2014). The major pathological features of DN include renal fibrosis , which is caused by the upregulated biosynthesis of matrix proteins, collagen IV, and fibronectin in mesangial cells (Valcourt et al., 2005). Collectively, renal interstitial fibrosis plays a crucial role in mediating epithelial-mesenchymal transition (EMT), characterized by the acquisition of α-smooth muscle actin (α -SMA), fibronectin, and vimentin (Badid et al., 2002). The vital clinical features of the kidney have been observed in patients with DN, which include mesangial expansion, thickening of the basement membrane, loss of podocytes within the glomeruli, excretion of urinary albumin, and loss of glomerular function capacity (Ziyadeh, 1993; Horie et al., 1997; Brosius et al., 2010). In particular, various cell surface receptors and transforming growth factor-β (TGF- β) are highly expressed in diabetes-induced renal fibrosis (Zhao et al., 2014).
Several clinical management strategies are recommended for the prevention of DN, which include dietary regulation and controlling the blood sugar and blood pressure (Brownlee, 2005). End-stage renal disease (ESRD) develops rapidly in patients with DN even after adequate multifactorial management. Therefore, novel therapeutic agents are necessary for the treatment of DN. A study demonstrated that the activation of Sirtuin 1 (SIRT1) protects

against kidney dysfunction under hyperglycemic conditions (Li et al., 2010). It was demonstrated by unilateral ureteral obstruction-induced kidney fibrosis in mice, that the SIRT1 activator, resveratrol, suppresses the transforming growth factor-β (TGF-β1)/Smad3-induced upregulation of collagen IV, α-SMA, and fibronectin (Li
et al., 2010). Therefore, these data suggest that SIRT1 plays a significant role in controlling the TGF-β1-induced fibrotic response through Smad3 deacetylation. Contrastingly, SIRT1 inhibitors also show a protective effect against diabetes-induced renal fibrosis (Ponnusamy et al., 2014). It has been reported in a murine model of diabetes that t he SIRT 1 inhibitor, sirtinol, attenuates the expression of collagen I, fibronectin, and α-SMA in the kidney tissues. SIRT1 inhibitors, therefore, have therapeutic efficacy against DN (Peck et al., 2010; Ponnusamy et al., 2014). Furthermore, a new SIRT1 inhibitor, EX-527, has been reported to suppress the stimulation of interstitial fibroblasts and reduce fibrosis in the NRK-49F renal interstitial fibroblast cell line (Ponnusamy et al., 2014). Studies have revealed that EX-527 considerably inhibits the expression of markers of fibroblast activation, including α-SMA, collagen I, and fibronectin, in a dose-dependent manner (Ponnusamy et al., 2014; 2015). The antifibrotic potential of SIRT1 inhibitors are associated with multiple cytokines/growth factor receptors, including epidermal growth factor receptor (EGFR) and platelet-derived growth factor receptor-β (PDGFR β), as well as multiple intracellular signaling pathways. It has been documented that the phosphorylation levels of EGFR and PDGFR increase during the progression of renal fibrosis , and the inhibition of these receptors suppresses the activation of renal fibrosis in animal models (Ludewig et al., 2000; Pang et al., 2009, 2010; Liu et al., 2011, 2012).
The activation of these receptors leads to the stimulation of multiple intracellular signaling pathways, including the signal transducer and activator of transcription 3 (STAT3) pathway. STAT3 has also been associated with the activation of renal interstitial fibroblasts and progress ion of renal fibrosis (Pang et al., 2010). However, the therapeutic efficacy of EX-527 against high-fat diet (HFD)-induced DN and renal fibrosis is not clearly understood.
Therefore, the present study aimed to explore the therapeutic efficacy of EX-527 in HFD-induced DN. In order to elucidate the mechanism underlying the efficacy of EX-527, we assumed that it might protect against renal fibrosis in HFD-induced ZDF rats by modulating blood glucose, oxidative stress , and inflammatory cytokines .

2.Materials and Methods

2.1.Chemicals and materials

EX-527 (6-chloro-2,3,4,9-tetrahydro-1H-carbazole-1 carboxamide) and 3-indoxyl sulfate (3-IS) were purchased from Sigma -Aldrich Biotechnology (St. Louis, MO, USA). The primary antibodies specific to kidney injury molecule-1 (Kim-1), neutrophil gelatinase-associated lipocalin (NGAL), selenium binding protein-1 (SBP-1), collagen-1, E-cadherin, α -tubulin, TGF-β 1, fibronectin, vimentin, α -SMA, SIRT1, SIRT3, SIRT4, claudin-1, and β-actin were procured from Abcam (Cambridge, MA, USA). EGFR, p-EGFR, PDGFRβ, p-PDGFRβ, STAT3, and p-STAT3 were purchased from Cell Signaling Technology. Horseradish peroxidase (HRP)-conjugated secondary antibodies were procured from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

2.2.Experimental design

Male ZDF rats (body weights 300 ± 25 g) were purchased from Central Lab Animal Inc. (Seoul, Korea). The rats were maintained at a temperature of 23 ± 0.5 °C, at 53-57% relative humidity, and a 12-h light and dark cycle, under specific pathogen-free conditions. The rats were allowed to acclimatize for 10 days prior to commencing the experiments. The methodology of this study was approved by the Animal Ethics Committee of Sungkyunkwan University (SKKU-2018-10-32-1). The HFD, comprising protein, fat, carbohydrate, fiber, minerals, and vitamin with 60% fat, was purchased from Research Diets, Inc. (New Brunswick, NJ 08901, USA), and was fed to the rats for 11 weeks for inducing diabetes.
EX-527 was intraperitoneally (i.p.) injected at a dose of 5 μg/kg, twice a week for 10 weeks , to the HFD-fed rats (Velásquez et al., 2011). The normal diet rats received the same diet but was devoid of fats. Blood was collected from the tail vein in the morning, and the glucose concentration was determined using a glucometer (ACCU-CHEK; Daeil Pharm. Co Ltd, Seoul, Korea). The rats with glucose concentration higher than 300 mg/dL were regarded as diabetic, and were selected for further experimentation. The rats with HFD-induced diabetes were randomly divided into three groups , with 6 rats per group. After 21 weeks of feeding, all the rats were anesthetized with CO2 gas, and blood was subsequently collected from the abdominal vein. The serum was collected after centrifuging the blood samples at 2000 × g for 10 min and stored at -80°C until further use. All the rats were placed in metabolic cages for 24-h urine sample collection, and the samples were immediately stored at -20 °C until use. After sacrific ing the rats, their major organs, namely, the kidneys, pancreas, and liver, were perfused with saline for removing the residual

blood, and were stored at -80 °C until use (Fig. 1).

2.3.Analysis of serum biochemical parameters

The serum samples thus collected were transferred to sterile tubes and stored at -80°C within 2 h of collection until analysis. The levels of serum creatinine (SCr) and concentrations of blood urea nitrogen (BUN) were measured using a Vet Scan analyzer (Aba xis, Inc. Union City, CA, USA). The advanced glycation end-products (AGEs ) in the serum samples were analyzed using OxiSelect™ AGEs Competitive ELISA Kit from Cell Biolabs for rapid detection and quantification of the circulating AGEs. The AGEs were quantified using an AGE-BSA standard curve. The AGE-BSA standards and test protein samples were transferred to an AGE conjugate preabsorbed plate. An anti-AGE polyclonal antibody was added after 30 min of incubation, followed by incubation with an HRP-conjugated secondary antibody. The formation of AGEs was analyzed on the basis of the fluorescent intensity or the absorbance of the sample in each well using a fluorescence microplate reader (Molecular Devices, San Jose, CA, USA) at 450 nm. The results were expressed as the relative fluorescence intensity.

2.4.Analysis of urinary parameters

The rats were individually maintained in metabolic cages for 24 h, during which they were made to fast, but were allowed ad libitum access to water. After measuring the total volume of the urine sample over 24 h, the container was gently shaken for 10 s for mixing the urine, following which the samples were sent for urinalysis . The concentrations of microalbumin and creatinine (Cr) in the urine were determined using the Hitachi 7180 auto analyzer (Hitachi, Tokyo, Japan).

2.5.Analysis of oxidative stress biomarkers

Approximately 20 mg of the frozen kidney tissues were homogenized in ice -cold phosphate buffer (pH 7.4) and centrifuged at 1500 × g for 10 min. The supernatant was collected and used in the assay. The concentration of malondialdehyde (MDA) was analyzed by reacting the sample with thiobarbituric acid (TBA) reactive substance using a kit (TBARS assay kit, Cayman, USA). The activity of SOD was quantified using the colorimetric SOD assay kit (Cayman Chemical, Ann Arbor, MI, USA). The concentrations of reduced glutathione (GSH) and oxidized GSH (GSSG) in the renal tissue were analyzed using Ellman’s reagent-based assay with commercially available kits

(Cayman Chemicals Company, Michigan, USA). The intercellular concentration of ROS was measured using an assay kit (Cell Biolabs, San Diego, USA). In order to estimate the concentration of 8-hydroxy-2′-deoxyguanosine (8-OHd G, a product of oxidative DNA damage) in the urine samples, an ELISA kit (Cell Bio labs, USA) was used. The detectable range was 0.07-20 ng/ml, and the total amount of 8-OHdG excreted in the urine over a period of 24 h was determined.

2.6.Analysis of pro-inflammatory cytokines

The concentrations of inflammatory cytokines were measured using ELISA assay kits (Abcam, Cambridge, MA, USA) according to the manufacturer’s instructions. The concentrations of TGF-β, interleukin 1 beta (IL-1β), interleukin 6 (IL-6), and interleukin 10 (IL-10) were determined from the standard plots.

2.7.Western blot analysis

The expression of various proteins in the kidney tissue, including SIRT1 (1:1000), SIRT3 (1:1000), SIRT4 (1:1000), and claudin-1 (1:1000) was measured. The protein markers of fibrosis , including E-cadherin (1:1000), α-SMA (1:500), co llagen-1 (1:500), vimentin (1:1000), α -tubulin (1:1000), TGF-β (1:1000), fibronectin (1:500), EGFR, p-EGFR (1:1000), PDGFRβ (1:1000), p-PDGFRβ (1:1000), STAT3 (1:1000), and p-STAT3 (1:1000) were analyzed by western blotting. The expression of various urinary protein markers , including KIM-1 (1:1000), NGAL (1:1000), SBP-1 (1:1000), and vimentin (1:500) was analyzed by western blotting. Briefly, 50 mg of the renal cortex was homogenized with radioimmunoprecipitation assay (RIPA) lysis buffer. The homogenate was placed on ice for 30 min and centrifuged at 4 °C for 15 min at 12000 × g. The concentration of protein in the supernatant was measured using the bicinchoninic acid (BCA) protein assay kit (Thermo Scientific Pierce, MA, USA). The protein samples (50 µg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis , and were subsequently transferred onto polyvinylidene fluoride or polyvinylidene difluoride (PVDF) membranes that were blocked with 5% milk buffer at 22 °C. The membrane was incubated overnight with the primary antibody at 4 °C. The membrane was subsequently washed with TBST and incubated with the secondary antibody (1:10000) at room temperature for 1 h. Protein expression and band intensity were measured using a chemiluminescent reagent enhanced chemiluminescence (ECL).

2.8.Determination of the concentration of 4-hydroxyproline

The kidney tissue (100 mg) was hydrolyzed by incubation at 120 °C for 12 h and was subsequently homogenized with 10 mL of 5 N HCl. The hydrolyzed sample was passed through a PVDF syringe filter (0.45 µm) and dried by evaporation under vacuum for 45 min at 70 °C. The total amount of 4-hydroxyproline was estimated using a colorimetric assay kit (Hydroxyproline Assay Kit, Cell Biolabs, USA), according to the manufacturer’s instructions. All the samples were assayed in duplicate in a blinded fashion .

2.9.Analysis of the concentration of 3-IS

The concentration of 3-IS in the urine, serum, and kidney tissue was analyzed by high-performance liquid chromatography (HPLC). The samples were extracted using an organic solvent (70% acetonitrile ) by a multistep process. Thereafter, the samples were transferred to a sample tube, which was thoroughly mixed with the internal standard (2-napthalenesulfonic acid) and analyzed by HPLC (Gilson, LC-321 322 350) at 280 nm. The retention time for 3-IS was 6.5 min. The extraction recovery was 87%. The HPLC system used herein comprised a Gilson pump (LC-321 322 350 pump), an auto sampler (Gilson -234), and a UV (UV/VIS -151) detector (Gilson, France). Detection and quantification were performed on a C18 column (250 mm × 4.6 mm, 5 µm) with a pre -column from Agilent (Torrance, USA); the flow rate was 1 ml/min at room temperature. The isocratic mobile phase, comprising a mixture of 0.1% TFA in Milli Q water and acetonitrile, was used at a ratio of 80:20 v/v.

2.10.Histopathological examination

The tissues of the pancreas and kidney were preserved immediately after collection in 10% buffer formaldehyde. The paraffin sections of these tissues were prepared for hematoxylin and eosin (H&E) staining for determining the morphological abnormalities of the tissues, and for Masson’s trichrome (MT) staining for determining the degree of collagen deposition (Trichome, Gomori One -Step, Aniline Blue Stain kit, Newcomer Supply, Middleton, WI).

2.11.Immunohistochemical examination

Immunohistochemical analysis was conducted for investigating the expression of α-SMA, TGF-β, SIRT1,

SIRT3, and claudin-1. The slides were transferred to a xylene chamber and hydrated in pure graded alcohol series

and water prior to transfer to 3% H2O2 for quenching the peroxidase activity. The samples were incubated with 4% BSA containing 5% horse serum for blocking nonspecific binding, at 37 °C for 1 h, followed by washing thrice with Tris-buffered saline (TBS). The samples were incubated overnight with the primary antibodies against α-SMA (1:200), TGF-β (1:500), SIRT1 (1:1000), SIRT3 (1:500), and claudin-1 (1:1000) at 4 °C. After incubation, the samples were washed thrice with TBS, following which the HRP-conjugated secondary antibody was added and incubated at room temperature for 45 min. Diaminobenzidine tetrahydrochloride (DAB) was used as a visualizing agent for immunostaining. The slides were then counterstained with hematoxylin , and histological examination was performed by microscopy for identifying histological changes, without the identity of the various groups.

2.12.Statistical analyses

The data were expressed as the mean ± S.D. and were analyzed by one-way analysis of variance (ANOVA), followed by Tukey’s HSD post hoc test for multiple comparisons. All the statistical analyses were performed using Graph Pad Prism v5.0 (Graph Pad Software, San Diego, CA, USA). A value of p < 0.05 indicated statistical significance.

3.Results

3.1.Effect of EX-527 on the body weight and blood glucose concentration of HFD-induced ZDF rats

The body weights of the rats with HFD-induced diabetes were significantly reduced in comparison to that of the rats that were fed on a normal diet. However, the change in the body weight s of the rats fed on HFD gradually recovered following the administration of EX-527 (Fig. 2A). No mortality or morbidity was observed in the experimental groups. The fasting serum glucose concentration of the HFD-fed rats was significantly increased in comparison to that of the rats fed on a normal diet. The blood glucose concentration after two weeks of HFD feeding was 4-fold higher than that of the rats fed on a normal diet. However, the fasting blood glucose concentration was considerably reduced in the HFD-fed rats following the administration of EX-527 (Fig. 2B).

3.2.Effect of EX-527 on organ weight and histological findings in HFD-induced ZDF rats

We first established a model of differential susceptibility to HFD-induced organ weight gain. The weights of the kidneys and pancreas were measured and represented as relative organ weight with respect to that of the organs of

the rats that were fed on a normal diet (Fig. 2C). The HFD-fed rats showed a significant increase in the relative kidney weight in comparison to that of the rats fed on a normal diet. However, the administration of EX-527 significantly reduced the weight of the kidneys in the HFD-fed rats. Contrastingly, the average weight of the pancreas was significantly reduced in the HFD-fed rats in comparison to that of the rats fed on a normal diet. However, the administration of EX-527 significantly improved the change in the weight of the pancreas (Fig. 2C).
The rats that were fed on a normal diet did not exhibit any abnormal morphological alteration s in the kidneys and pancreas (Fig. 2D). H&E staining of the kidney tissues of the HFD-fed rats displayed an enlargement of the glomerular tissue in comparison to that of the rats fed on a normal diet. The distal tubule was also enlarged and vacuolated in the HFD-fed rats in comparison to that of the rats fed on a normal diet. Tubular dilatation, swelling of tubular cell, brush border loss, and nuclear loss were observed in the HFD-fed rats. Apparently, the architecture of the Bowman capsules in the kidneys was found to be affected in the HFD-fed rats, and interstitial inflammation was also observed. In particular, moderate to severe vacuolar degeneration was observed in the tubules and glomerular space of the kidneys of the HFD-fed rats. The associated abnormal architecture in the HFD-fed rats, including thickening of the glomerular basement membrane ( GBM), podocyte fusion, and lipid droplet accumulation in the tubules, was significantly restored following the administration of EX-527. Moreover, the damage induced by HFD to the glomeruli and tubules was recovered by EX-527. The histopathological outcomes clearly indicated that the structural damage to the kidneys of the HFD-fed rats was protected by EX-527 (Figs. 2D and E). While the pancreatic islets showed round and clear boundaries in the rats that were fed on a normal diet, the islets were rarely visible in the HFD-fed rats, and possessed numerous vacuous areas. However, the administration of EX-527 reduced the number of erratically-shaped islets in the pancreatic tissues of the HFD-fed rats (Fig. 2D).

3.3.Effects of EX-527 on AGEs and serum biochemical parameters in HFD-induced ZDF rats

The formation of AGEs in the serum was found to be significantly higher in the HFD-fed rats than in the rats fed on a normal diet (Fig. 3A). Interestingly, the concentration of AGEs significantly reduced in the HFD-fed rats following the administration of EX-527. The concentrations of BUN and SCr significantly increased in the HFD-fed rats in comparison to those in the rats fed on a normal diet. The concentration of BUN and SCr considerably decreased in the HFD-fed rats following the administration of EX-527 (Fig. 3A).

3.4.Effect of EX-527 on the changes in urinary biomarker expression in HFD-induced ZDF rats

The urinary excretion of microalbumin and Cr was significantly elevated in the HFD-fed rats in comparison to that of the rats fed on a normal diet (Fig. 3B). The urinary excretion of microalbumin and Cr significantly reduced in the HFD-fed rats following the administration of EX-527, and was similar to that of the rats that were fed on a normal diet (Fig. 3B). The urinary excretion of the protein-based biomarkers , namely, KIM-1, NGAL, SBP-1, and vimentin, was measured for exploring the influence of diabetes -induced nephrotoxicity. The urinary excretion of the protein-based biomarkers markedly increased in the HFD-fed rats, and the administration of EX-527 reduced the urinary excretion of these biomarkers of nephrotoxicity (Fig. 3C and D). 3-IS is a novel biomarker of nephrotoxicity and a metabolite of dietary tryptophan. In comparison to that of the rats fed on a normal diet, t he concentration of 3-IS in the kidneys and serum of the HFD-fed rats was considerably increased, however, the urinary excretion of 3-IS was significantly reduced. The concentration of 3-IS was significantly decreased in the kidneys and serum of the HFD-fed rats following the administration of EX-527 (Fig. 3E).

3.5.Effect of EX-527 on oxidative stress in the kidneys of HFD-induced ZDF rats

The concentration of the antioxidant GSH was significantly reduced and that of GSSG was increased in the kidneys of the HFD-fed rats in comparison to that of the rats that were fed on a normal diet. (Fig. 4A). However, the administration of EX-527 significantly increased the concentration of GSH and abruptly decreased the concentration of GSSG (Fig. 4A). The GSG/GSSG ratio was also markedly reduced in the HFD-fed rats in comparison to that in the rats that were fed on a normal diet (Fig. 4A). The activity of SOD was significantly reduced in the HFD-fed rats, whereas the administration of EX-527 restored the activity of SOD in the HFD-fed rats to the normal range (Fig. 4A). The concentration of MDA was significantly increased in the kidneys of the HFD-fed rats in comparison to that of the rats that were fed on a normal diet (Fig. 4B). Additionally, the intercellular concentration of ROS in the renal cortex of the HFD-fed rats was significantly higher than that of the rats that were fed on a normal diet (Fig. 4B). However, the concentration of MDA and the intercellular concentration of ROS were significantly reduced in the renal cortex of the HFD-fed rats following EX-527 administration. The level of the oxidative DNA marker, 8-OHdG, was significantly increased in the rats with HFD-induced diabetes. However, treatment with EX-527 significantly reduced the concentration of 8-OHDG in the HFD-fed rats (Fig. 4B).

3.6.Effect of EX-527 on inflammatory cytokines in HFD-induced ZDF rats

The concentration of the pro-inflammatory cytokines , TGF-β, IL-1β, and IL-6, was increased by 3-5-fold, however, the anti-inflammatory response by IL-10 was significantly reduced in the HFD-fed rats (Fig. 5). However, the administration of EX-527 produced a significant inhibitory effect on the secretion of the pro-inflammatory cytokines, TGF-β, IL-1β, and IL-6, whereas a reverse trend was observed for IL-10 (Fig. 5) as EX-527 induced a significant increase in the concentration of IL-10 in the HFD-fed rats.

3.7.Effect of EX-527 on renal fibrosis in HFD-induced ZDF rats

Clinical and experimental studies have suggested that inflammation and fibrosis of the kidney s are always associated with HFD (Gassler et al., 2001; Mora and Navarro, 2006). Fibronectin and collagen have a significant role in sclerosis (scar formation) following inflammatory injury in tissues as a part of tissue repair (Ponnusamy et al., 2014). We, therefore, focused on studying the molecular markers of renal fibrosis. The results showed that α-SMA, collagen-1, TGF-β, vimentin, α -tubulin, and fibronectin were significantly upregulated in the rats with HFD-induced diabetes. However, the abnormal expression of extracellular matrix (ECM) proteins was restored following EX-527 administration. The administration of normal diet also slightly increased the concentration of fibronectin and α -SMA, but treatment with EX-527 alleviated renal fibrosis (Fig. 6A and B). Similarly, immunohistochemical staining indicated a reduction in the number of αSMA-positive cells in the fibrous septa of the kidneys following EX-527 administration to the HFD-fed rats (Fig. 6C).
TGF-β boosts hypertrophy in the renal cortex and stimulates the accumulation of ECM, which decreases the Glomerular Filtration Rate (GFR), leading to CKD (Cheng et al., 2013; Hasegawa et al., 2013; Lenoir et al., 2015; Saravanan and Pari, 2016). The expression of TGF-β was significantly higher in the kidneys of the HFD-fed rats than in the kidneys of the rats fed on a normal diet. However, the expression of TGF-β decreased following the administration of EX-527 to the HFD-fed rats (Fig. 6C). Thus, EX-527 markedly inhibited the HFD-mediated production of ECM in the kidneys, which inhibited the progression of renal fibrosis. Masson’s staining of the kidney sections revealed an excessive accumulation of collagen in the kidneys of the rats with HFD-induced diabetes, which was accompanied by the formation of nodules. However, these were significantly restored following the administration of EX-527 to the rats with HFD-induced diabetes (Fig. 6D).
As 4-hydroxyproline is an important marker of fibrosis in disorders of renal fibrosis, the concentration of

4-hydroxyproline was determined for estimating fibrosis (Fig. 6E). The abnormality in the ECM proteins was restored following EX-527 administration. A 4-hydroxyproline assay was subsequently performed to provide supportive evidence. The concentration of 4-hydroxyproline in the kidneys of the rats with HFD-induced diabetes was significantly higher by 188% than that in the kidney s of the rats fed on a normal diet. The concentration of 4-hydroxyproline increased significantly (p < 0.01) in the rats with HFD-induced diabetes, whereas treatment with EX-527 significantly restored the collagen concentration in the kidney tissues (Fig. 6E).

3.8.Effect of EX-527 on the expression of SIRT and claudin-1 in the kidneys of HFD-induced ZDF rats

Based on the histological evidence of renal tissue fibrosis in the HFD-fed rats and the known role of the SIRT protein in the development of renal fibrosis, we first examined the expression of known SIRTs in the kidneys of the HFD-fed rats. In order to elucidate whether the increase in SIRT expression correlates with renal pathology, the expression of SIRT1, SIRT3, and SIRT4 were evaluated. The expression of SIRT protein was decreased, whereas the expression of claudin-1 was upregulated in the HFD-fed rats in comparison to those in the normal rats. However, treatment with EX-527 significantly downregulated the expression of SIRT1 and c laudin-1, but significantly upregulated the expression of SIRT3 and SIRT4 (Fig. 7A and 7B). The intensities of the bands were measured and depicted by bar graphs (Fig. 7B).
In order to confirm the protective effects of EX-527 on renal fibrosis in the rats with HFD-induced diabetes, immunohistochemistry analyses were performed for localizing SIRT1, SIRT3, and claudin-1 in the renal tissues. It was observed that SIRT1, SIRT3, and claudin-1 were expressed in the glomeruli and interstitial renal areas in the kidneys of the rats that were fed on a normal diet. Among these, only the expression of claudin-1 was high, whereas those of SIRT1 and SIRT3 were downregulated in the renal tissues of the HFD-fed rats in comparison to those of the rats that were fed on a normal diet. However, the expression was restored to normal following the administration of EX-527, thus corroborating the immunoblot data (Fig. 7C). However, the expression of SIRT1 and c laudin-1 was reduced and that of SIRT3 was increased following EX-527 treatment, corroborating the data obtained by western blotting (Fig. 7C).

3.9.Effect of EX-527 on the Phosphorylation levels of EGFR, PDGFRβ, and STAT3 in the kidneys of HFD-induced ZDF rats

In order to evaluate the effect of EX-527 on EGFR and PDGFRβ activation in the kidney s of the diabetic rats , the phosphorylation levels of EGFR and PDGFRβ were examined by western blot analysis. The results demonstrated that the phosphorylation of EGFR and PDGFRβ was upregulated in the kidneys of the diabetic rats; however, treatment with EX-527, significantly inhibited the phosphorylation without affecting their expression. The total
expression of EGFR and PDGFRβ in the HFD-fed rats was increased. Additionally, the expression of EGFR and

PDGFRβ was upregulated in the kidney s of the HFD-fed rats (Fig. 8A and B). These data correlated with the results of published reports, where it was demonstrated that the inhibition of SIRT1 is a controlling factor in the expression of these tyrosine kinase receptors. Additionally, the effect of EX-527 on STAT3 and its phosphorylation level in the kidneys of the HFD-fed rats was analyzed. Our results demonstrated that the expression of p-STAT3 and STAT3 was dramatically increased in the kidneys of the rats with HFD-induced diabetes. However, the phosphorylation level of STAT3 surprisingly decreased following treatment with EX-527 (Fig. 8A and B).

4.Discussion

This study demonstrated that the SIRT1 inhibitor, EX-527, has a protective effect against DN in the HFD-induced ZDF rat model. ZDFRs, an animal model of type 2 diabetes, revealed significant a significant higher level of HbA1c, urinary albumin and L-FABP excretion, tubular cell damage, histological evidence of renal tissue fibrosis and urinary 8-OHdG excretion, which is directly associated to high glucose levels (Peterson et al., 1990; Janssen et al., 1999; Ogura et al., 2002; Yokoi et al., 2013). The male fa/fa rats in the ZFDM strain are fertile, exhibit obesity, hyperglycemia, and hyperlipidemia and develop diabetes as early as 10 weeks of age, reaching 100% incidence at around 20 weeks of age. The progressive nephropathy of ZDF rat by 12 wk of age, earlier than in most other models of diabetes with increasing proteinuria resulting in chronic renal insufficiency (Kasiske et al., 1992; Magil., 1995; Coimbra et al., 2000; Gassler et al., 2001; Hoshi et al., 2002). To the best of our knowledge, this study is the first to demonstrate that EX-527 protects against the progression of HFD-induced renal dysfunction resulting from hyperglycemia.

It is known that SIRT1 expression changes under different physiological and morbid conditions. In general, SIRT1 is decreased in conditions of chronic metabolic stress, oxidative stress, or hypoxia that drives the pathophysiologies of age-related diseases including diabetes, cardiovascular, and renal diseases. Furthermore, a number of studies showed that resveratrol and SRT1720 are not directly targeting SIRT1 but show multiple off -target effects (Pacholec

et al., 2010).

In this respect, Ponnusamy et al. (2015) revealed that the activation of SIRT1 with SRT1720 developed renal fibrosis. Additionally, exposure of cultured renal interstitial fibroblasts to YK-3-237 (another SIRT1 activator), also boosted their activation as described by increased expression of fibronectin and α-SMA. These results confirmed that SIRT1 plays a vital role in interfering renal fibrogenesis after CKD. Hence, identifying the functional role of SIRT1 is very important in tissue fibrosis by employing more specific inhibitors. The mechanistic approach of SIRT1 activators is not clearly understood on tissue fibrosis. It may be associated with their nonspecific properties. However, the function of SIRTs on tissue-specific fibrosis regulation remains controversial. In this study, we also cannot clearly exclude the possibility that a SIRT1 inhibitor, EX527, might play a role in the inhibition of renal fibrogenesis.

Previous study by Ponnusamy et al. (2014) exhibited the blocking of SIRT1 and SIRT2 with a pan-inhibitor (sirtinol) or isoform-selective inhibitors (EX527), inhibited activation and proliferation of cultured renal interstitial fibroblasts. Sirtinol inhibited the development of renal fibrosis in a murine model. SIRT1 med iate activation and proliferation of renal interstitial fibroblasts and progress of renal fibrogenesis, suggesting that SIRT1 inhibitors might be beneficial for the treatment of CKD. It was found that blocking SIRT1/2 also inhibits STAT3 phosphorylation in both obstructed kidney and cultured renal fibroblasts (Ponnusamy et al., 2014). Additionally, it was noticed that SIRT1/2 inhibitors not only inhibited UUO -induced STAT3 phosphorylation in the kidney, but also decreased its phosphorylation in the sham-operated kidney.

Besides, activation of multiple signaling pathways such as EGFR and PDGFRβ as well as TGF-β signaling plays a significant role in the development of renal fibrosis (Terzi et al., 2000; Bonner, 2004; Lautrette et al., 2005; Liu, 2006). These reports revealed that EGFR and PDGFRβ are also associated with fibrogenesis and fibroblast activation after kidney injury. EGFR not only is regarded as a convergent pathway but also transduces the fibrotic signal from its ligands, which directly/indirectly effects many other fibrogenic factors. Moreover, the EGFR signaling plays a significant role to regulate the renal epithelial cell proliferation and dedifferentiation (Liu et al, 2013; Zhuang et al, 2004). Activation of multiple growth factor receptors induced proliferation of renal tubular cells. Among them, EGFR was recognized to be critically associated with renal regeneration and kidney development (Chen et al, 2012; He et al, 2013; Wang et al, 2003). Therefore, the EGFR and PDGFRβ are also potentia l targets for the prevention

and treatment of renal fibrosis.

The beneficial effect of EX527 may be mediated by its capacity to block activation of growth factor receptors and subsequent inactivation of the STAT3 signaling pathway. STAT3 is highly expressed and activated in renal interstitial fibroblasts after kidney injury (Kuratsune et al., 2007). The phosphorylated STAT3 was expressed in interstitial fibroblasts of fibrotic kidney (Kuratsune et al., 2007; Wynn., 2008). Our recent observation also supported this hypothesis that the pharmacologic blockade of STAT3 signaling inhibited expression of all of those inflammatory mediators. Our experimental results revealed that the EX527 has a potential to abolish the phosphorylation of EGFR, PDGFR, and STAT3. In hibition of phosphorylation of EGFR, PDGFR and STAT3, also downregulated the expression of fibroblast markers, such as (α -SMA (~64%), α -Tubulin (~69%), TGF-β (25%)). These fibroblast markers provide predominant pathological characteristic, which is conside red as a common final pathway in all forms of CKD (Pang et al., 2009). In addition, STAT3 is the key factor of inflammation due its major contribution to renal fibrosis. It is believed that EX527 inhibit inflammation by STAT3 and attenuate renal fibrosis in DN. Therefore, the downregulation of these receptors may explain, at least in part, the anti-fibrotic potential of EX-527 in HFD-induced diabetic rats. Following ligand binding, EGFR and PDGFR undergo dimerization, which is followed by the phosphorylation of tyrosine residues. Multiple intracellular signaling pathways, including the STAT3 pathway, is activated following the activation of these receptors (Vignais et al., 1996; Lo et al., 2007). STAT3 is also associated with the activation of renal interstitial fibroblasts and the progression of renal fibrosis (Pang et al., 2010).

Here, the administration of HFD to rats for 16 weeks induced insulin resistance, characterized by elevated blood glucose concentration. The size of the pancreatic islets and the number of acinar cells were markedly reduced in the HFD-fed rats, similar to that reported in a previous study (Monday and Uzoma , 2013). The islets were damaged and shrunken, and lymphocytic infiltration was detected in the pancreas of the HFD-fed rats. However, the damages to the structure of the pancreas were alleviated following the administration of EX-527 to the HFD-fed rats. It was therefore assumed that EX-527 can restore insulin sensitivity and protect against the pancreatic damages induced by HFD.

In the rat model of HFD-induced diabetes, the concentrations of BUN and SCr were elevated, indicating the onset of renal dysfunction due to HFD, consistent with the results of previous studies (Zheng et al., 2015; Chen et al.,

2019). A significantly high concentration of glucose in the blood plasma and an increase in body weight, cholesterol concentration, and triglyceride concentration were observed following the administration of HFD for 13 weeks (from the 8th to the 21st week). The high rate of body weight gain in the HFD-fed rats was accentuated 8 week onwards. These results indicated that hyperglycemia was maximum after 13 weeks of HFD administration. Therefore, the HFD-fed ZDF rats are generally used as a model for human type II diabetes due to their necrotic effects on the pancreatic beta cells, which reduce insulin secretion (Junod et al., 1969; Soler et al., 2012; Hsu et al., 2016).
Here, treatment with EX-527 effectively improved the blood glucose concentration and renal dysfunction. The concentrations of SCr, BUN, antioxidants, and pro-inflammatory cytokines were measured for establishing the correlation with renal dysfunction. The concentrations of BUN, Cr, and urinary proteins are commonly higher in HFD-induced DN (Gassler et al., 2001; Ponnusamy et al., 2014; Zhang et al., 2014; Lenoir et al., 2015). The urinary excretion of microalbumin is a key factor of diabetes -induced renal dysfunction (Viswanathan et al., 2004; Xue et al., 2011; Saravanan and Pari, 2016). The quantity of microalbumin in the urine of the HFD-fed rats was significantly higher in comparison to that in the rats that received a normal diet. EX-527 significantly reduced the concentration of microalbumin in the urine of the HFD-fed rats. In order to confirm the onset of diabetes-induced CKDs, the urinary excretion of protein-based biomarkers of nephrotoxicity, including KIM-1, SBP-1, NGAL, and vimentin were measured. Previous studies have demonstrated that the expression of these protein-based biomarkers are significantly increased in the urine of rats and patients with nephrotoxicity ( Vaidya et al., 2008; Fiseha, 2015; Kim et al., 2017). Here, EX-527 markedly reduced the urinary excretion of KIM-1, SBP-1, NGAL, and vimentin in the HFD-fed rats. Therefore, EX-527 plays a vital role in controlling blood glucose concentration and has protective effects on renal dysfunction in DN. Furthermore, it has been reported that 3-IS accumulates in the serum and kidneys of patients with CKD or acute kidney injury (AKI) (Wu et al., 2013; Yabuuchi et al., 2016). Our previous study reported that the urinary excretion of 3-IS is dramatically reduced in AKI (Won et al., 2016). Generally, 3-IS is excreted by active secretion from the proximal tubules , and therefore a reduction in the urinary concentration of 3-IS indicates tubular damage. Furthermore, 3-IS generates ROS in the renal tubular region and stimulate s nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), which in turn augments the expression of plasminogen activator inhibitor (PAI)-1. Our data revealed that the concentrations of 3-IS in the serum and kidney tissues of the HFD-fed rats were high, but treatment with EX-527 significantly reduced the concentrations of 3-IS in the serum and kidneys and restored them nearly up to normal levels . The e xcessive formation of AGEs in hyperglycemia plays a

vital role in the development of DN (Forbes et al., 2003). AGEs damage blood vessels and the renal cortex (Niedowicz and Daleke , 2005; Sohn et al., 2013). The structural abnormalities in the kidneys are directly correlated with the accumulation of AGEs in DN (Forbes et al., 2003; Kishore et al., 2017). However, AGE reduces SIRT1 expression, resulting increased acetylation STAT3. In diabetic kidneys, the expression of proinflammatory genes was induced by acetylated STAT3 (Chuang et al., 2011; Liu et al., 2014). Both phosphorylation and acetylation of STAT3 are induced by AGE. Therefore, the inhibition of AGE formation is considered to be a therapeutic milestone against DN. Our results demonstrated that the concentration of AGEs in the HFD-fed rats was high, but EX-527 significantly inhibited the formation of AGEs.
ROS have a leading role in the development of diabetes -associated complications, especially DN (King and Loeken, 2004; Cheng et al., 2013; Gujjala et al., 2016). Antioxidant management offers a new therapeutic window for patients with diabetes. The defensive mechanistic approach of antioxidant s at the cellular level is hindered by ROS. Here, the high level of intracellular ROS formation in the HFD-fed rats was significantly reduced by EX-527. In accordance with ROS formation, EX-527 significantly reduced the formation of MDA and 8-OHd G in the kidneys and their excretion in the urine of the HFD-fed rats. The urinary excretion of 8-OHd G is a popular biomarker for oxidative DNA damage and systemic oxidative stress (Shigenaga et al., 1989; Halliwell, 1996). A significant increase in the quantity of 8-OHdG in the urine of diabetic rats is associated with the progression of diabetic nephritis (Hinokio et al., 2002). A previous study reported that the concentration of microalbumin in patients with DN is directly correlated with the high excretion of 8-OHdG (Xu et al., 2004). We therefore suggest that EX-527 has protective effects against DN by inhibiting the formation of A GEs, and by preventing oxidative damage to lipids and DNA in the renal cortex by increasing the concentrations of SOD, GSH, and catalase, suggesting the strong antioxidant potential of EX-527. The high urinary concentration of microalbumin, SCr, and BUN, accompanied by other morphological alterations in the HFD-fed rats were the characteristic features of renal injury associated with oxidative stress. The alteration of renal pathophysiology associated with glomerular hypertrophy, proliferation of mesangial matrix, impairment of the glomerular filtration membrane, infusion of p odocytes, dilation of tubules, swelling of tubular cells, and loss of brush border cells and nuclei were clearly observed in the HFD-fed rats. Treatment with EX-527 considerably improved the renal microarchitecture and restored the tubular cells and glomerular spaces. Therefore, our data indicated that EX-527 exerted renoprotective effects in the HFD-fed rats by reducing oxidative stress mediated by the downregulation of pro-inflammatory cytokines, leading to an improvement

in proteinuria and the concentrations of BUN and SCr.

TGF-β1 is primarily a profibrotic cytokine involved in the proliferation and growth of the cells of the basement membrane. It has been reported that expression of TGF-β1 receptor is higher in chronic renal fibrotic diseases in humans and animal models (Bottinger and Bitzer, 2002; Liu, 2006). Our study demonstrated that the expression of TGF-β1 reduced after the administration of EX-527. Glomerular or interstitial inflammation plays a vital role in renal impairment (Gassler et al., 2001; Ponnusamy et al., 2014; Lenoir et al., 2015). The concentration of IL-6 has been reported to be abnormally elevated in rats with HFD-induced diabetes (Dalla Vestra et al., 2005). Similar to the results of a previous study, our study demonstrated that the concentration of IL-6 in the HFD-fed rats was high. EX-527 significantly decreased the concentration of IL-6 in the HFD-fed rats. A previous study demonstrated that the concentration of IL-6 was markedly increased in patients with diabetes. An increase in the concentration of IL-6 is responsible for mesangial cell proliferation, changes in endothelial permeability, and thickening of the glomerular membrane in DN (Mora and Navarro, 2006). It has been reported that IL-1β significantly increases in the kidneys of db/db mice in an age-dependent manner (Shahzad et al., 2015). IL-1β increases vascular endothelial permeability and is responsible for the proliferation of mesangial cells , matrix synthesis , and progression of intraglomerular hemodynamic changes associated with the synthesis of prostaglandin by the mesangial cells (Mora and Navarro, 2006). Here, the concentration of IL-1β was significantly increased in the HFD-fed rats in comparison to that in the rats that were fed on a normal diet. The serum concentration of IL-1β significantly declined following treatment with EX-527. The concentration of IL-10 is one of the important parameters in the development of DN, and a high concentration of IL-10 improves renal function and suppresses inflammation in CKD (Mu et al., 2005). Here, the concentration of IL-10 was abruptly reduced in the HFD-fed rats in comparison to that in the rats that were fed on a normal diet, but treatment with EX-527 considerably increased the concentration of IL-10 in the HFD-fed rats.
Pang et al. (2010) demonstrated that in the renal injury, the accumulation of inflammatory cells, expression of TGF-β, and the increase in pro-inflammatory cytokines (IL-β and TNF -α) were mediated by STAT3 activation. The mechanistic approach of inflammation in initiation and maintenance of renal damage, and reduced inflammatory response are associated with attenuation of renal fibrosis (Wynn, 2008; Misseri et al., 2004).
The development of renal fibrosis depends on the rate of accumulation of ECM proteins, the deposition of mesangial cell matrix, and the thickening of the glomerular basement membrane (GBM ) (Lu et al., 2015). In particular, the pathophysiology of interstitial fibrosis is associated with the proliferation of renal fibroblasts, which

leads to the myofibroblast phenotype and the deposition of ECM (Ponnusamy et al., 2014). One of the principle factors behind the overproduction of ECM is the presence of α-SMA-positive myofibroblasts in the kidneys (Wynn, 2008). The production and alteration of TGF-β in the tubules is also responsible for renal fibrosis (Cheng et al., 2013; Hasegawa et al., 2013; Lenoir et al., 2015; Saravanan and Pari 2016). Here, our results demonstrated that the accumulation of collagen in the kidney tissues of the HFD-fed rats was considerably reduced following treatment with EX-527. Masson's trichrome staining also showed that the expression of collagen was significantly higher in the HFD-fed rats in comparison to that in the rats that were fed on a normal diet, and the administration of EX-527 abruptly reduced collagen deposition in the kidney s. Furthermore, the abnormal architecture associated with renal injury in the HFD-fed rats was restored following treatment with EX-527. This was confirmed by analyzing the expression of biomarkers of renal fibrosis, including α-SMA, α-tubulin, TGF-β, fibronectin, and vimentin in the HFD-fed rats. EX-527 administration significantly reduced the expression of the indicators of HFD-induced fibrosis in the HFD-fed rats. TGF-β is one of the most important factors for the generation of the extracellular matrix, and therefore has a significant role in DN. A previous study suggested that DN can activate the TGF-β pathway, which is responsible for renal fibrosis (Zhao et al., 2014). Contrastingly, the expression of E-cadherin was reduced in the kidneys of the HFD-fed rats, but its expression was restored following treatment with EX-527. Another vital parameter of kidney fibrosis is 4-hydroxyproline (Wang et al., 2017; Li et al., 2017), and the concentration of 4-hydroxyproline was significantly reduced in the HFD-fed rats following treatment with EX-527. Thus, EX-527 has an anti-fibrotic effect in HFD-induced nephrotoxicity.
To date, it is unclear how SIRT1 regulates STAT3 function and expression, but inactivation of SIRT1 or other HDACs can induce or enhance protein acetylation, and that STAT3 acetylation was also observed in renal fibroblasts exposed to the class I/II HDAC inhibitor. Therefore, it is possible that acetylation of STAT3 can function as positive or negative factors to regulate various aspects of STAT signaling (Zhuang, 2013).
Previous studies have demonstrated that SIRT1 exhibits its anti-inflammatory activity via deacetylation or inactivation of the p65 subunit by decreasing ROS production ( Yeung et al., 2004; Lee et al., 2008). The expression of SIRT1 in the renal tissues is low in DN (Kitada et al., 2011; Xu et al., 2012). SIRT3 regulates mitochondrial function by scavenging the ROS for protecting against apoptosis and cell senescence (Lombard et al., 2007; Salminen et al., 2013; Zhao et al 2016). It has been reported that that SIRT3 prevents inflammation associated with oxidative stress in diabetes -induced kidney disorders. The protective function of SIRT3 against inflammation is

closely associated with the reduction in ROS production, attenuation of inflammasome formation, and reduction in the concentrations of IL-18 and IL-1β in diabetes -induced kidney disorder (Zhao et al., 2016). Moreover, SIRT3 controls the activity of SOD by deacetylating several lysine residues (Qiu et al., 2010; Tao et al., 2014; Zhang et al., 2017). Qiu and coworkers (2010) suggested that calorie restriction by SIRT3 reduces oxidative stress by activating SOD. Therefore, the upregulation of SIRT3 has a beneficial effect on kidney injury by alleviating mitochondrial dysfunction. Current study demonstrated that SIRT1 and claudin-1 had opposite expression in the kidneys of the HFD-fed rats. It was observed that the expression of SIRT1 was significantly reduced, whereas the expression of claudin-1 was increased in the HFD-fed rats. The expression of SIRT1 and claudin-1 proteins was significantly decreased in the HFD-fed rats following treatment with EX-527.
Functions of SIRT1 in kidney fibrosis remain controversial. In this respect, Wang et al. (2008) demonstrated that SIRT1 levels were upregulated in patient’s with primary myelofibrosis. Generally, SIRT1 has the protective effect in kidney disorders (He et al., 2010; Villalba and Alcain, 2012; Fan et al., 2013). However, function of different isoforms of SIRT1 (ant-)agonist in renal interstitial fibroblast activation and development of renal fibrosis is not well understood. Recent literatures revealed that the currently available SIRT1 agonists may not be specific for SIRT1 receptor (Beher et al., 2009; Pacholec et al., 2010). Additionally, SIRT1 agonists exhibits both protective as well as harmful effects (Accili et al., 2011). Hasegawa et al. (2013) explained that albuminuria was developed in proximal tubular (PT)-specific SIRT1 knockout mice. However, the upregulatory effect of SIRT1 in PT reveled the protective function of mice in diabetic kidney injury. The downregulation of SIRT1 expression in PT triggered the upregulation of claudin-1 in the podocytes, which results disruption of glomerular filtration, podocyte dysfunction and albuminuria in mice. However, Liu et al. (2014) revealed that albuminuria was not developing in the basal condition for podocyte-specific SIRT1 knockout mice. In case of diabetic mice, more severe kidney injury was manifested. These discrepancies between the two models proposes that SIRT1 may assist different functions in PT and podocytes. However, further studies are necessary to describe the cellular mechanism, which underlies the phenotypic variations between these SIRT1 loss -of-function models .
As therapeutic function of SIRT1 against HFD -induced renal fibrosis are not clearly understood, we evaluated the effect of SIRT1 inhibitor on the therapeutic efficacy of renal fibroblast in this study. SIRT1 play an important role to inhibit renal fibrosis, other subtype SIRTs might be act to blocking or therapy progression of renal fibrosis induced by hyperglycemia or HFD.

The expression of claudin-1 in the HFD-fed rats was observed in the podocytes owing to their dedifferentiation in diabetes -induced kidney disorder. The expression of SIRT3 and SIRT4 was low in the HFD-fed rats in comparison to that in the rats that were fed on a normal diet; however, the expression of SIRT3 and SIRT4 was increased in the HFD-fed rats following EX-527 administration. Immunohistochemistry data revealed a significantly low expression of SIRT3 in the HFD-fed rats, and the administration of EX-527 was observed to increase SIRT3 expression. Therefore, a high expression of SIRT3 might cause adverse outcomes by suppressing mitochondrial oxidative stress and the production of inflammatory cytokines. The uprgulation of SIRT4 may produce beneficial role in HFD induced renal fibrosis. However, the higher expression of claudin-1 in the HFD-fed rats was significantly reduced following treatment with EX-527.
It appears from our experimental data that the downregulation of SIRT1 and the upregulation of SIRT3 by EX-527 might prevent renal fibrosis. The downregulation of SIRT1 expression in the kidneys of the HFD-fed rats triggered the upregulation of claudin-1 in the glomeruli. These changes are highly correlated with the primary alteration in diabetes -induced albuminuria. Additionally, our study demonstrated that the inhibition of SIRT1 by EX-527 could attenuate HFD-induced fibrosis. The anti-fibrotic potential of SIRT1 inhibitor could be related with the downregulation of EGFR, PDGFR, and other intracellular signaling pathways. However, further research is necessary for understanding the mechanistic approach underlying the therapeutic effect of EX-527 against DN.

5.Conclusions

We successfully developed an in house rat model of HFD-induced glucose metabolic disorder and renal injury. To the best of our knowledge, this study is the first to demonstrate the protective effect of EX-527 against hyperglycemia -induced renal fibrosis, mediated by the oxidative stress pathway, pro-inflammatory cytokines, and the cellular signaling pathways that are involved in renal fibrosis. In agreement with all recent findings of SIRT1 Inhibitors, our finding also highlight that the inhibition of SIRT1 with EX527 might block the phosphorylation level of EGFR and PDGFR and attenuates the activation of renal fibrosis in well -defined animal model. Additionally, the beneficial effect of EX527 might be associated by blocking activation of TGF -β and/or other growth factor receptors,
which might results consequent inactivation of the STAT3 signaling pathway. This pharmacologic blockade of STAT3 signaling might inhibit expression of downstream inflammatory mediators. Therefore, anti -fibrosis and anti-inflammation might provide protective effect in nephropathy. Essentially, our finding provide a first-hand clue

that SIRT1 inhibitor might play critical role on via fibrous growth factors. Besides, we also observed that downstream regulator of SIRT1 (e.g. fibrous growth factor, oxidative stress and inflammation) was inhibited due to SIRT1 suppression. In conclusion, we believe that our finding is not entirely contradictory for SIRT1 pathway in diabetic nephropathy disorders, rather it provides a deep insight in (patho -) physiological role of SIRT1 in nephropathy. These findings can be used to develop a novel treatment strategy for DN. Our findings therefore support the possible clinical application of EX-527 as a potential renoprotective agent in HFD-induced diabetic patients. Additional studies are required to explore the underlying mechanism by which SIRT1 inhibition results in inhibition of growth factor receptors and signaling related with activation and proliferation of renal fibroblasts and renal fibrogenesis.

Ethical approval : All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. All procedures involving animals were in accordance with the ethical standards of the institution at which the studies were conducted.

Author contributions : A.K, R.S., P.D., K.S.K., and J.S.L. performed the in vivo experiments, conducted data analysis, and prepared the manuscript. J.S.L. and K.B.K. performed a part of the in vivo experiments, discussed the data, and provided conceptual advice. S.K. and B.M.L. assisted in experimental design, discuss ion and analysis of data, and provided conceptual advice. H.S.K. generated the hypotheses, developed the experimental design, conceptualized the study, supervised the experimental work, obtained funding, and prepared the manuscript. All the authors discussed the results and commented on the manuscript.

Funds: This study was supported by grants from National Research Foundation of Korea (NRF) , funded by the Korean Government (grant numbers : NRF-2016R1A2B2011071 and NRF-2017R1D1A1B03036571).

Conflict of interest: The authors declare that there are no conflicts of interest.

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Figure Legends

Fig. 1. Experimental design. After one-week of adaption, the ZDF rats were divided randomly into two groups: the normal diet group, that was fed a standard chow diet (ND, n = 6) and the experimental group was fed a high -fat diet (HFD, n = 12). After 10 weeks of feeding the HFD, the rats were divided into two groups (n = 6 per group), of which one group was fed HFD (n= 6) and the other group was fed HFD followed by EX-527 administration (HFD+EX-527) for 21 weeks. ND, normal diet; HFD, high-fat diet.

Fig. 2. Effect of EX-527 on the gain in body weight, organ weight, blood glucose concentration, and histopathological structures in rats with HFD-induced diabetes. (A) HFD-induced body weight gain in ZDF rats. A significant increase in body weight was observed in the HFD-fed rats, which was normalized after EX-527 treatment.
(B)Effect of HFD on the fasting blood glucose concentration (mg/dL). Treatment with EX-527 normalize the blood glucose level in HFD group. (C) Changes in the weights of the kidney and pancreas. EX-527 significantly restored both kidney and pancreas weights. (D) Histological changes in the pancreatic tissue stained with H& E. The control group showed normal pancreatic architecture. The exocrine component of the pancreas was closely packed with acinar cells and was arranged into small lobules. Pancreatic lobules separated by intact intralobular and interlobular connective tis sue septa. The islet cells are seen interspersed between the acinar cells. The islets appeared to be lightly stained in comparison to the surrounding acinar cells. The rats with HFD-induced diabetes showed pathological changes in both the exocrine and endocrine components. The acinar cells were swollen and small vacuoles were observed in almost all the acinar cells. The interlobular ducts were lined with flattened epithelium [indicated by black arrow]. The β -cells of the islets were almost entirely lost in the HFD-fed rats. However, the HFD-fed rats that were treated with EX-527 showed distortion of the general architecture. The atrophic changes in the acinar cells were less severe and the border between the exocrine and endocrine portions were more distinct. The images represent the results obtained from three animals per experimental group. (magnification 200X; bar = 50 μm. ND, normal diet; HFD, high-fat diet). Representative histology of the H&E-stained kidney sections from the experimental group. The rats with HFD-induced diabetes showed an enlarged cortex with glomerular sclerosis (arrowheads), expansion (asterisk), and dilation at 21 weeks. The medulla exhibited tubular dilatation, interstitial nodular sclerosis , and fibroplasia (asterisk). The cortex of the rats that were administered EX-527 exhibited normal sized renal cortex (RC), a lower incidence of tubular and medullar injury, and displayed a normal histological structure comprising thin

tubules (arrows) and collecting ducts (arrowheads) (magnification 200X; bar = 50 μm. ND, normal diet; HFD, high-fat diet). E) Quantitative analysis of the Bowman capsules, glomerular space, and interstitial regions of the H&E-stained kidney sections from the experimental groups. The values are represented as the mean ± S.D. of six rats per group. Statistical analysis was performed one-way analysis of variance (ANOVA) followed by Tukey’s HSD post hoc test for multiple comparis ons. *p < 0.05, **p < 0.01, and ***p < 0.001 as compared with the group that received a normal diet (ND); #p < 0.05, ##p < 0.01, and ###p < 0.001 as compared with the HFD -fed group. ND, normal diet; HFD, high fat diet.

Fig. 3. Effect of EX-527 on the biochemical parameters of the serum and urine of the rats with HFD-induced diabetes. (A) Serum levels of advanced glycation end products (AGEs), blood urea nitrogen (BUN), and creatinine (sCr) in the rats with HFD-induced diabetes . EX-527 significantly reduced the serum-level of AGEs, BUN, and sCr in the HFD-induced diabetic rats. (B) Changes in the urinary excretion of microalbumin and creatinine. EX-527 significantly reduced the level of urinary biomarkers (microalbumin and creatinine) in the HFD-induced diabetic rats.
(C)Representative western blots of NGAL, KIM-1, SBP-1, and vimentin expression in the urine, using an experimental model of rats with HFD-induced diabetes. Changes in the urinary excretion of biomarkers of kidney damage in the rats with HFD-induced diabetes following treatment with EX-527. (D) The intensity of the bands was analyzed densitometrically by Image J software. (E) Changes in the concentration of 3-indoxyl sulfate (3-IS) in the HFD-fed rats following treatment with EX-527 (5 μg/kg). The concentration of 3-IS in the serum, urine, and kidney tissues was measured by high-performance liquid chromatography (HPLC). The IS concentration in serum, urine and kidney was restored significantly in the EX-527 treatment group. The values are represented as the mean ± S.D. of six rats per group. Statistical analysis was performed one-way analysis of variance (ANOVA) followed by Tukey’s HSD post hoc test for multiple comparisons . *p < 0.05, **p < 0.01, and ***p < 0.001 as compared with the group that received a normal diet (ND); #p < 0.05, ##p < 0.01, and ###p < 0.001 as compared with the HFD -fed group. ND, normal diet; HFD, high fat diet.

Fig. 4. Effect of EX-527 on the activity of antioxidant enzymes in the rats with HFD-induced diabetes . (A) Changes in the oxidative biomarkers (reduced glutathione (GSH), oxidized GSH (GSSG), GSH/GSSG, and superoxide dismutase (SOD)) were measured in the kidneys of the HFD-induced rats. These oxidative biomarkers recovered

after EX527 treatment. (B) Lipid peroxidation (malondialdehyde (MDA)), intracellular reactive oxygen species (ROS), and the marker of oxidative DNA damage, 8-hydroxy-2'-deoxyguanosine (8-OHDG), were measured in the HFD-induced ZDF rats. All these oxidative biomarkers were significantly reduced in the EX-527 treatment group. The values are represented as the mean ± S.D. of duplicate experiments (6 animals/group). Statistical analysis was performed one-way analysis of variance (ANOVA) followed by Tukey’s HSD post hoc test for multiple comparisons . *p < 0.05, **p < 0.01, and ***p < 0.001 as compared with the group that received a normal diet (ND); #p < 0.05, ##p
< 0.01, and ###p < 0.001 as compared with the HFD-fed group. ND, normal diet; HFD, high fat diet.

Fig. 5. Effect of EX-527 on the serum concentrations of pro-inflammatory cytokines and anti-inflammatory cytokine in the rats with HFD-induced diabetes . The pro-inflammatory cytokines (IL-1β , IL-6, and TGF-β1) were significantly reduced and anti-inflammatory cytokine (IL-10) was significantly increased after EX-527 treated HFD-rats. The values are represented as the mean ± S.D. of duplicate experiments (6 animals/group). Statistical analysis was performed by one way ANOVA followed by Tukey’s HSD post hoc test for multiple comparisons . *p < 0.05, **p < 0.01, and ***p < 0.001 as compared with the group that received a normal diet (ND); #p < 0.05, ##p < 0.01, and ###p < 0.001 as compared with the HFD-fed group. ND, normal diet; HFD, high fat diet.

Fig. 6. Effect of EX-527 on the biomarkers of renal fibrosis in the rats with HFD-induced diabetes . (A) The expression of E-cadherin, collagen-1, α-SMA, α -tubulin, TGF-β, vimentin, and fibronectin was measured by western blotting, using an experimental rat model of HFD-induced diabetes. The results of western blot represent the results obtained from three separate experiments. The expression of β-actin was used as the loading control. (B) The intensity of the bands was analyzed densitometrically by Image J software. (C) Representative immunohistochemical
staining of α-SMA and TGF-β1 in the kidneys of the rats with HFD-induced diabetes . The black arrows represent

α-SMA and TGF-β1 expression. Original magnification: 200X, scale bar: 100 μm. (D) Representative Masson’s

trichrome-stained kidney sections. The black arrows represent collagen accumulation (blue color). Representative

photomicrographs of the kidney sections stained with Masson’s Trichrome showing renal collagen deposition. Quantification of tubular interstitial collagen (Sirius Red staining quantified by Image analysis). Original magnification: 200X, scale bar: 100 μm. (E) The concentration of 4-hydroxyproline in the kidney of the rats with HFD-induced diabetes . 4-hydroxyproline was reduced significantly after EX -527 treatment. The values are

represented as the mean ± S.D of duplicate experiments (6 animals/group). Statistical analysis was performed by one way ANOVA followed by Tukey’s HSD post ho c test for multiple comparisons . *p < 0.05, **p < 0.01, and ***p < 0.001 as compared with the group that received a normal diet (ND); #p < 0.05, ##p < 0.01, and ###p < 0.001 as compared with the HFD-fed group. ND, normal diet; HFD, high fat diet.

Fig. 7. Effect of EX-527 on SIRT and c laudin-1 expression in the kidneys of the rats with HFD-induced diabetes by western blotting. (A) The expression of SIRT1, SIRT3, SIRT4, and claudin-1 was measured by western blotting, using an experimental rat model of HFD-induced diabetes. The expression of β-actin was used as the loading control. The results of western blot represent the results obtained from three separate experiments. (B) The intensity of the bands was analyzed densitometrically. Statistical analysis was performed by one-way ANOVA followed by Tukey’s HSD post hoc test for multiple comparisons. *p < 0.05, **p < 0.01, and ***p < 0.001 as compared with the group that received a normal diet (ND); #p < 0.05, ##p < 0.01, and ###p < 0.001 as compared with the HFD -fed group. ND, normal diet; HFD, high fat diet. ND, normal diet; HFD, high fat diet. ( C) Representative Immunohistochemical staining of SIRT1, claudin-1 and SIRT3 in the kidneys of the rats with HFD-induced diabetes . Original magnification: 200X, scale bar: 50 μm. ND, normal diet; HFD, high-fat diet.

Fig. 8. Effect of EX-527 on the phosphorylation of EGFR, PDGFRβ, and STAT3 in the rats with HFD-induced diabetes. Kidney tissue lysates were prepared and subjected to western blot analysis by incubating with antibodies for p-EGFR, p-PDGFRβ, p -STAT3, EGFR, PDGFRβ, and STAT3. (A) The phosphorylated and total levels of EGFR and PDGFRβ, and STAT3 expression were measured by western blotting using an experimental rat model of HFD-induced diabetes. The expression of β-actin was used as the loading control. The results of western blot represent the results obtained from three separate experiments. (B) The intensity of the bands was analyzed densitometrically by Image J software. Statistical analysis was performed by one-way ANOVA followed by Tukey’s HSD post hoc test for multiple comparisons . *p < 0.05, **p < 0.01, and ***p < 0.001 as compared with the group that received a normal diet (ND); #p < 0.05, ##p < 0.01, and ###p < 0.001 as compared with the HFD -fed group. ND, normal diet; HFD, high fat diet.

Highlights

 Protective effect of EX-527 against DN in HFD-induced diabetic Zucker (ZDF) rats. EX-527 significantly reduced the blood glucose level, restored kidney morphology. Oxidative stress, cytokines markers significantly restored with EX-527 in HFD rats. Urinary biomarkers expression in HFD rats were significantly reduced with EX-527. It down-regulated the expression of fibrous biomarkers in the kidneys of HFD rats.

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Declaration of interests

☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:
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EX 527

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