Experimental comparison of protective characteristics of enalapril and trimetazidine in diabetic nephropathy

http://informahealthcare.com/rnf ISSN: 0886-022X (print), 1525-6049 (electronic) Ren Fail, Early Online: 1–8 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/0886022X.2014.930331

LABORATORY STUDY

Experimental comparison of protective characteristics of enalapril and trimetazidine in diabetic nephropathy Tugba Karadeniz1, Turker Cavusog˘lu2, Engin Turkmen3, Yigit Uyanıkgil2, Muammer Karadeniz4, Ovunc Akdemir5, M. Ibrahim Tuglu6, Utku Ates2, and Oytun Erbas7

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Department of Pathology, Izmir Tepecik Training and Research Hospital, Izmir, Turkey, 2Faculty of Medicine, Department of Histology and Embryology, Ege University, Bornova, Izmir, Turkey, 3Department of Internal Medicine, Bahcelievler Medikal Park Hospital, Istanbul, Turkey, 4Faculty of Medicine, Department of Endocrinology, Sifa University, Bornova, Izmir, Turkey, 5Department of Plastic, Reconstructive and Aesthetic Surgery, Bahcelievler Medikal Park Hospital, Istanbul, Turkey, 6Faculty of Medicine, Department of Histology and Embryology, Celal Bayar University, Manisa, Turkey, and 7Medicine Faculty, Department of Physiology, University of Tokat Gaziosmanpasa, Tokat, Turkey Abstract

Keywords

Hyperglycemia, hypertension, dyslipidemia, and inflammation have been proposed to account for the development of nephropathy in diabetic subjects. The beneficial effects of enalapril on diabetic nephropathy are known. However, the effects of trimetazidine (TMZ) are still unknown. We aimed at comparing the effects of the enalapril and TMZ treatment on fibronectin expression, inducible nitric oxide synthase expression, urine proteinuria, blood glucose and glomerular, and mesangial structures of kidney in rats that take streptozotocin (STZ). In this study, 32 male Sprague–Dawley albino mature rats of 8 weeks, weighing 200–220 g were used. Diabetes was induced by intraperitoneal injection of STZ (60 mg/kg) for 24 rats. We made four groups (Group 1: control, non-diabetic rats (n ¼ 8), Group 2: diabetes, without treatment (n ¼ 8), Group 3: diabetes treatment with enalapril (n ¼ 8), Group 4: diabetes treatment with TMZ (n ¼ 8). The positive effects of renal tissue and tubules in the mesangium immunohistochemical were shown in TMZ receiving rat groups. These positive effects were in parallel with the reduction in fibronectin and I-NOS expression and reduction in the proteinuria. TMZ and enalapril treatment of diabetic rats and renal parenchyma in this study are shown to have positive effects on the different levels.

Enalapril, diabetic, nephropathy, rats, trimetazidine

Introduction Diabetes mellitus is characterized by hyperglycemia, which induces oxidative stress and perturbs a number of pathways, leading to renal injury. Hyperglycemia, hypertension, dyslipidemia, and inflammation have been proposed to account for the development of nephropathy in diabetic subjects.1–3 Hyperglycemia, angiotensin, angiotensin 1 receptor activation and oxidative stress increase, and intracellular ATP reduction play an important role in the formation of diabetic nephropathy (DN). Enalapril is a non-thiol angiotensin-converting enzyme inhibitor, which is commonly used in the treatment of diabetes-associated hypertension. Inducible nitric oxide synthase (iNOS) can be expressed by different cells, for example macro-phages, vascular smooth muscle cells, and mesangial glomerular cells. Stimulation of iNOS via hyperglycemia may lead to an amplified production of NO, which in turn is attributed to hyperfiltration and glomerular pathology in diabetic patients.4

History Received 18 December 2013 Revised 19 April 2014 Accepted 17 May 2014 Published online 10 July 2014

In diabetes, increased amounts of fibronectin are showed in thickened glomerular basement membranes and mesangium of the kidney.5,6 Also, another molecule name of trimetazidine (TMZ; 1-[2,3,4-trimethoxybenzyl] piperazine) is a cellular anti-ischemic agent that selectively inhibits the activity of the final enzyme of fatty acid oxidation pathway, 3-ketoacylcoenzyme A thiolase. Administration of this drug leads to a switch in preference of the energy substrate, resulting in partial inhibition of fatty acid oxidation and increased glucose oxidation. Clinical studies showed that TMZ has cardioprotective effects in the setting of myocardial ischemia including acute myocardial infarction.7–10 However, the data related to DN of the TMZ are limited. Since the beneficial effects of ACEi on DN are known, the objective of our study was mainly focused on the effects of TMZ to the development of DN. Our aim in this study is to examine and compare the effect of enalapril and trimetozine on DN in rats developed diabetes by streptozocin.

Materials and methods Address correspondence to Ovunc Akdemir, MD, Department of Plastic, Reconstructive and Aesthetic Surgery, Bahcelievler Medikal Park Hospital, Istanbul 34160, Turkey. Tel: 00 90 532 2043139; E-mail: [email protected]

Animals In this study, 32 male Sprague–Dawley albino mature rats of 8 weeks, weighing 200–220 g were used. Animals were fed

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ad libitum and housed impairs in steel cages having a temperature-controlled environment (22 ± 2  C) with 12-h light/dark cycles. Experimental procedures were approved by the Committee for Animal Research of Celal Bayar University. All animal studies strictly conformed to the animal experiment guidelines of the Committee for Human Care.

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Experimental protocol Diabetes was induced by intraperitoneal (i.p.) injection of STZ (Sigma-Aldrich, Inc.; Saint Louis, MO) (60 mg/kg in 0.9% NaCl, adjusted to a pH 4.0 with 0.2 M sodium citrate) for 24 rats. No drug was administered to the remainder of rats whose blood glucose levels were lower than 120 mg/dL (n ¼ 8) (Group 1, control group). Diabetes was verified after 24 h by evaluating blood glucose levels with the use of glucose oxidase reagent strips (Boehringer-Mannheim, IN). The rats with blood glucose levels 250 mg/dL and higher were included in this study as diabetic rat group (n ¼ 24). Then, 24 diabetic rats were randomly divided into three groups; no medication was given to Group 2 (diabetes without treatment group, eight rats), but 4 mL per day of tap water was given by oral gavage; 3 mg/kg/d of oral enalapril was given to Group 3 (diabetes + enalapril group, eight rats) for 4 weeks’ 5 mg/kg/d of oral TMZ was given to Group 4 (diabetes + TMZ group, eight rats) for 4 weeks. Tablets containing 35 mg TMZ (Vasterel MR, Sandoz) and 5 mg enalapril (Enalapril, Servier) were crushed and suspended in tap water to yield a concentration of 10 mg/mL. According to the weight of each rat, suspended drug solution was completed to 4 mL with tap water. The medications were given by means of orogastric tubes. Then, the animals were euthanized and blood samples were collected by cardiac puncture for blood glucose and nephrectomy were performed for histopathological examination and urine samples were taken on stick for proteinuria. Histopathological examination of renal tissue For histological and immunohistochemical studies, all animals were anesthetized by an i.p. of ketamine (40 mg/kg, AlfamineÕ , Ege Vet, Alfasan International B.V., Holland)/ xylazine (4 mg/kg, AlfazyneÕ ) and perfused with 200 mL of 4% formaldehyde in 0.1-M phosphate-buffer saline (PBS). Formalin-fixed renal sections (5 mm) were stained with hematoxylin and eosin (H&E). All sections were photographed with Olympus C-5050 digital camera mounted on Olympus BX51 microscope. Morphological analysis was assessed by computerized image analysis system. The degree of glomerulosclerosis and expansion of the mesangial matrix in the glomeruli in the sections were examined by light microscopy. Glomerular hypertrophy was calculated from the cross-sectional area of the glomerular tuft. The cross section yielding the maximum diameter of the glomerulus was photographed and converted into a digital image by an examiner blinded to the source of the tissue. The glomerular tuft and mesangial areas were measured with the image analysis software Image-Pro Express 1.4.5, Media Cybernetics, Inc., Bethesda, MD. Fifty glomeruli from each animal were examined, and the average was used for analysis. A semi-quantitative score (SI) was used

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to evaluate the degree of glomerulosclerosis. Sclerosis was defined as collapse and/or obliteration of glomerular capillary tuft accompanied by hyaline material and/or increase of matrix. Severity of sclerosis for each glomerulus was graded from 0 to 4+ as follows: 0, no lesion; 1+, sclerosis of525% of the glomerulus; 2+, 3+, and 4+, sclerosis of 25 to 50%, 450 to 75%, and 475% of the glomerulus, respectively. A wholekidney average SI was obtained from biopsy and autopsy specimens by averaging scores from all glomeruli on one section.11 Fibronectin, i-NOS (NOS-2) immunoexpression For immunohistochemistry, sections were incubated with H2O2 (10%) for 30 min to eliminate endogenous peroxidase activity and blocked with 10% normal goat serum (Invitrogen, Carlsbad, CA) for 1 h at room temperature. Subsequently, sections were incubated in primary antibodies (Fibronectin, NOS-2, Bioss, Inc.; 1/100) for 24 h at 4  C. Antibody detection was performed with the Histostain-Plus Bulk kit (Bioss, Inc., Woburn, MA) against rabbit IgG, and 3,30 diaminobenzidine (DAB) was used to visualize the final product. All sections were washed in PBS and photographed with an Olympus C-5050 digital camera mounted on Olympus BX51 microscope. Brown cytoplasmic staining was scored positive for immunoexpression. The number of immunoexpression positive cell was assessed by systematically scoring at least 50 glomeruli and tubular cell per field in 10 fields of tissue sections at a magnification of 100. Fibronectin and iNOS expression was evaluated by a semi-quantitative score. Scores of 0 to 4 represent negative, trace, 510%, 10–25%, and 425% staining, respectively, in each glomerulus.11 Urine dipstick analysis for proteinuria Urine of rats were tested with a dipstick (Urinalysis Dipstick, Bayer, Elkhart, IN). This test is based on the color change of the indicator tetrabromophenol blue. A positive reaction is indicated by a color change from yellow through green and then to greenish-blue. The minimum sensitivity of this test is 10 mg/dL of protein in urine. This assay is also semiquantitative; results of 1+ correlate to a quantitative amount of 30 mg/dL, 2+ to 100 mg/dL, 3+ to 300 mg/dL, and 4+ to 1000 mg/dL.12–15 Statistical analysis All quantitative data were analyzed by using non-parametric (Mann–Whitney U) test. Student’s t-test was used to evaluate the differences between the groups. Data are presented as mean values ± standard error of the mean (SEM). p Values of 0.05 or less were regarded as statistically significant.

Results In diabetic rats (Group 2), volume of glomerular area, fibronectin and i-NOS expression were increased significantly when compared to control group (p50.05) (Group 1). Administration of enalapril or TMZ in diabetic rats causes a significant reduction both in glomerular area, fibronectin and i-NOS expression (p50.05; Figure 1). According to these

The influences of trimetazidine on diabetic nephropathy

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Figure 1. Comparison between the groups in terms of immunoexpression percentage.

Table 1. Comparison between the groups according to the level of blood sugar, glomerular area (% of control), immunoexpression fibronectin percentage, immunoexpression i-NOS percentage, proteinuria (mg/dL), blood glucose (mg/dL). Control Glomerular area (% of control) Immunoexpression fibronectin percent (%) Immunoexpression i-NOS percent (%) Proteinuria (mg/dL) Blood glucose (mg/dL)

a

100 10.2 ± 2.3a 8.6 ± 1.2a 0.18 ± 0.05a 95.6 ± 8.5a

Diabetes 165 ± 10.24 78.6 ± 8.2 65.2 ± 10.3 2.56 ± 0.2 423.1 ± 15.6

Diabetes and enalapril 123 ± 6.32 35.1 ± 6.8a 32.5 ± 4.5a 0.56 ± 0.2a 418.3 ± 12.3

a

Diabetes and trimetazidine 135 ± 3.56a 23.8 ± 3.6a 12.4 ± 4.8a 0.45 ± 0.1a 435.2 ± 10.2

Note: aStatistically significantly lower (p50.05).

results, the fibronectin and i-NOS expression of TMZ group was statistically significantly lower than enalapril group (p50.05). However, there were no statistically significant differences between enalapril and TMZ in terms of the volume of glomerular area (p40.05). Protein in urine levels were increased dramatically in diabetic rats according to control group. Administration of enalapril or TMZ decreased urine protein levels significantly in diabetic rats. The proteinuria levels of TMZ group were statistically significantly lower than enalapril group and diabetic group (p50.05). Blood glucose levels were significantly increased in diabetic rats expectedly, but there was no important difference between diabetic rats and enalapril or TMZ-administrated diabetic rats in terms of blood glucose levels (p40.05; Table 1). Histopathologic evaluation Glomerulus and tubular structures were observed as normal in renal sections belonging to renal tissues in the control group. Constriction in Bowman’s space, tubular dilatation, separation and impairments in brush borders of tubule epithelium in glomerular structures in diabetic structures were observed. Besides, an increase was determined in glomerular mesangial matrix and reticular fibrils. Bowman’s space constriction seen in diabetic rats was not observed in the glomerulus in DM + Enalapril group. In contrast, mesangial matrix increase finding was determined. However, tubular dilatations were observed less than the

diabetic group and close to the control group. The appearance of brush borders of tubular epithelium was better when compared to that of the diabetic group, and separations and impairments in brush borders of tubular epitheliums were observed less clearly. Besides, a thickening was observed in tubular basal membranes. An increase was determined in Bowman’s space in the glomerulus in DM + TMZ group. It was observed that the structure of mesangial matrix is close to that of the control group. Tubular dilatations were observed less and close to the control group. It was seen that deletion in brush borders of tubular epitheliums, separations and impairments in brush borders of tubular epitheliums were less clear. Besides, it was seen that the thickening in tubular basal membranes were the least among diabetes-applied groups (Figure 2). When fibronectin immune staining is examined, it is seen that there is a low degree of staining in basal lamina and stromal structures in conformity with normal tissue in the control group. However, in the group, a very high immunoreactivity was determined when compared to the control group. A high immunoreactivity was determined in treatment groups than control groups while a lower level of immunoreaction was determined when compared to diabetic group (Figure 3). When Inos immune staining is evaluated, minimal staining was determined in tubular structures rather than glomerulus structure in the control group. There is a very high level of immunoreactivity in glomerulus and tubular structure in diabetic group. There is a very level of staining in glomerulus structure in Enalapril group while an immunoreaction was partly determined in tubular structures. It was seen that the

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Figure 2. H & E staining (100 magnification): (a) control group (non-diabetes group), (b) diabetes group, (c) diabetes and enalapril, (d) diabetes and trimetazidine.

Figure 3. Fibronectin immunostaining (100 magnification); (a) control group (non-diabetes group), (b) diabetes group, (c) diabetes and enalapril, (d) diabetes and trimetazidine.

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Figure 4. NOS-2 (i-NOS) immunostaining (100 magnification): (a) control group (non-diabetes group), (b) diabetes group, (c) diabetes and enalapril, (d) diabetes and trimetazidine.

glomerulus structure reacted in a manner very close to the control in TMZ group but gave an immune reaction only in some distal tubules in terms of tubular structures (Figures 4 and 5). The mean glomerulosclerosis scores of group 1, 2, 3, and 4 were 0.12 ± 0.35, 3.75 ± 0.46, 2.12 ± 0.83, and 2.25 ± 0.46, respectively. There was statistically significantly different between control and diabetic group about glomerulosclerosis scores (p50.05). The mean glomerulosclerosis scores of group 3 and 4 were statistically significantly lower than diabetic group (p50.05). The mean fibronectin scores of group 1, 2, 3, and 4 were 0.25 ± 0.46, 3.5 ± 0.75, 1.87 ± 0.35, 1.25 ± 0.46, respectively. There was statistically significantly difference between control and diabetic group according to fibronectin scores (p50.05). The mean fibronectin scores of TMZ group was statistically significantly lower than enalapril and diabetic group (p50.05). The mean iNOS scores of group 1, 2, 3, and 4 were 0.12 ± 0.35, 3.62 ± 0.51, 1.87 ± 0.35, and 1 ± 0.53, respectively. There was statistically significantly difference between control and diabetic group according to iNOS scores (p50.05). The mean iNOS scores of TMZ group was statistically significantly lower than enalapril and diabetic group (p50.05; Table 2).

Discussion DN is a major cause of end-stage renal disease (ESRD) worldwide. Despite the recent progress in our understanding of disease processes, the silent slow progression of DN has limited our success in identifying specific causative factors or factors that may predict the development of kidney disease in

diabetic patients.3,5 In the last four decades, the most commonly used model was streptozotocin (STZ)-induced type 1 diabetes in rats. Although use of this model allowed several potentially important pathogenic pathways of DN to be identified, it does not share primary features of metabolic syndrome: insulin resistance/hyperinsulinemia, obesity, and hypertension. Furthermore, STZ rats develop mild hyperlipidemia and are resistant to development of nephropathy: animals very slowly develop mild glomerular and tubulointerstitial lesions.16 Importantly, in patients with metabolic syndrome and type 2 diabetes, only one-third develop renal lesions typical of type 1 diabetes renal pathology, with diffuse and nodular mesangial expansion, glomerular basement membrane thickening, and with an approximately balanced severity of glomerular, tubulointerstitial, and arteriolar changes.16 Significant numbers of type 2 diabetic patients (535%) develop an atypical pattern of renal injury with significant tubulointerstitial changes and global glomerulosclerosis,16 features not seen in STZ-induced kidney injury in rodents. In literature, there are many studies related to the effects of Enalapril on DN. However, there are few study about TMZ.3,4,5,9,10 TMZ is a cellular anti-ischemic agent.17,18 First studies showed that TMZ inhibits damaging effects of ischemia–reperfusion at both cellular and mitochondrial levels, and shows an antioxidant effect.19,20 TMZ reduces an increased release of oxygen-free radicals, amplifies glucose metabolism, restricts intracellular acidosis, and inhibits membrane lipid peroxidation and inflammation.21,22 An increase was determined in Bowman’s space in the glomerulus immunohistochemically in diabetic rat group

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Table 2. Comparison between the groups according to the mean glomerulosclerosis, immunoexpression fibronectin, immunoexpression i-NOS score. Control Glomerulosclerosis score Immunoexpression fibronectin score Immunoexpression i-NOS score

Diabetes a

0.12 ± 0.35 0.25 ± 0.46a 0.12 ± 0.35a

3.75 ± 0.46 3.5 ± 0.75 3.62 ± 0.51

Diabetes and enalapril a

2.12 ± 0.83 1.87 ± 0.35a 1.87 ± 0.35a

Diabetes and trimetazidine 2.25 ± 0.46a 1.25 ± 0.46a 1 ± 0.53a

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Note: Standard deviation; ±aStatistically significantly lower (p50.05).

Figure 5. H & E staining (40 magnification); (a) control group (non-diabetes group), (b) diabetes group, (c) diabetes and enalapril, (d) diabetes and trimetazidine.

receiving TMZ in our study. It was shown that the structure of mesangial matrix is close to the control group. Also, it was observed that the deletion of brush borders of tubular epithelium, separations and impairments in brush borders of tubular epithelium are less clear, and the thickening in tubular basal membranes is the lowest among diabetes applied groups. It is possible that these positive results occurred as a result of antioxidant and anti-ischemic events in TMZ receiving diabetic rats. TMZ is a cellular anti-ischemic agent.17,18 First studies showed that TMZ inhibits damaging effects of ischemia–reperfusion at both cellular and mitochondrial levels, and shows an antioxidant effect.19,20 TMZ reduces an increased release of oxygen-free radicals, amplifies glucose metabolism, restricts intracellular acidosis, and inhibits membrane lipid peroxidation and inflammation.20,21 An increase was determined in Bowman’s space in the glomerulus immunohistochemically in diabetic rat group receiving TMZ in our study. It was shown that the structure of mesangial matrix is close to the control group. Also, it was observed that the deletion of brush borders of tubular epithelium, separations and impairments in brush borders of tubular epithelium are less clear, and the thickening in tubular

basal membranes is the lowest among diabetes applied groups. It is possible that these positive results occurred as a result of antioxidant and anti-ischemic events in TMZ receiving diabetic rats. First studies have reported the favorable effects of ACEs on the progression of renal failure.22–24 Oxidative stress and inflammation are implicated in both renal and cardiovascular diseases.25,26 In hypertension, renal iNOS expression can be augmented in response to an increment in ROS.27,28 Broadbent et al.29 showed that increasing glomerular pressure in cultured proximal tubular epithelial cells can also lead to early iNOS induction. In both cases, iNOS inhibition was linked with an important advance in glomerular filtration rate. Captopril treatment prevented increase in intrarenal ANG II, and reversed expression of nephrin, TGFbeta1, collagen and fibronectin.30 Locally increased synthesis of angiotensin II (ANG II) in the kidney has been associated with glomerular hypertrophy and tubulo-interstitial fibrosis observed in chronic kidney failure. This action of ANG II is way to be mediated chiefly with transforming growth factor-beta, which stimulates the synthesis and decreases the degradation of extracellular

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DOI: 10.3109/0886022X.2014.930331

matrix components, as well as collagen types and fibronectin.31 In our study, in diabetic rats (without treatment), volume of glomerular area, glomerulosclerosis, fibronectin and i-NOS expression were increased significantly when compared to control group (Group 1). However, a statistically meaningful reduction was determined in glomerulus area in diabetic rat group receiving enalapril and TMZ when compared to control rat group in our study. Enalapril and TMZ were also reduced the glomerulosclerosis. Again in our study, a statistically meaningful reduction was determined in terms of fibronectin immune expression percentage and score when compared to control and diabetic rat group in our study. These results show us, TMZ and enalapril are potent protector of glomerular area. Additionally, TMZ prevents the harmful effects of fibronectin and i-NOS expression, it maintains tubular and interstitial area stabilization. Bowman’s space constriction seen in unhealed diabetic rats was not observed in glomerulus in diabetic rat group receiving enalapril in our study by immunohistochemical studies. In contrast, a mesangial matrix increase finding was determined. However, tubular dilatations were observed to be lower than the diabetic group and close to the control group. The appearance of brush borders of tubular epitheliums are better when compared to the diabetic group, separations and impairments in brush borders of tubular epitheliums were observed to be less clear. Besides, a thickening was observed in tubular basal membranes. As a biochemical finding, no statistically meaningful difference was determined in terms of rat blood glucose levels between the control group and the groups receiving enalapril and TMZ. Angiotensin-converting enzyme (ACE) inhibition reduces proteinuria and protects against progressive renal damage.32–34 The increase in Bowman’s space was not determined in enalapril receiving group when compared to the groups receiving TMZ and enalapril. However, it was determined that mesangial matrix structure in TMZ receiving rat group was similar to that of the control group, the tubular dilatation were lesser, and the deletion in brush borders of tubular epitheliums separations and impairments in brush borders of tubular epitheliums were less clear. These effects may be associated with the reduction in free oxygen radicals of TMZ molecule, the increase in glucose metabolism, and the reduction in the inflammation. On the other hand, no statistically meaningful reduction was determined when compared to the control and diabetic rat group in both groups in rat urine protein excretion levels between the control group and the groups receiving enalapril and TMZ. This common effect can be explained by the reduction in enalaprilic intraglomerular hypertension and hyper-infiltration. However, the TMZ can be affected by a reduction in irreversible advanced glycation end products (AGEs) and cytokines, and increased serum levels of inflammatory markers. As a result, TMZ was more protective on tubular and interstitial region, enalapril was more protective in the glomerular region, but TMZ also affected the glomerular region in our DN model. It was shown that both molecules produced positive results with constriction in glomerulus in renal tissue and also different effects on the tubule in the

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mesangium immunohistochemically. It was shown that these positive effects might be associated with the reduction in fibronectin and I-NOS expression and the reduction in the proteinuria in experimental rat models. This study is a preliminary study, the number of rats in our study are small, for that reason further studies are necessitated.

Declaration of interest There is no conflict of interest and no funding or grants in this study.

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