The Superiority of Rosmarinic Acid over Vitamin E in Preventing Development and Inhibiting Progression of Diabetic Nephropathy in Rats
Nur Samsu1, Setyawati Soeharto2, Muhaimin Rifai3, Achmad Rudijanto4
1Department of Internal Medicine, Division of Nephrology and Hypertension,
Faculty of Medicine, Universitas Brawijaya, Malang, Indonesia.
2Department of Pharmacology, Faculty of Medicine, Universitas Brawijaya, Malang, Indonesia.
3Department of Biology, Faculty of Mathematics and Sciences, Universitas Brawijaya, Malang, Indonesia.
4Department of Internal Medicine, Division of Endocrinology and Metabolic, Faculty of Medicine,
Universitas Brawijaya, Malang, Indonesia.
*Corresponding Author E-mail: drnursamsu@gmail.com, watikaryono@gmail.com, rifai123@ub.ac.id, achmadrudijanto@yahoo.co.id
ABSTRACT:
Background: Oxidative stress plays an important role in podocyte injury and hence in the development and progression of diabetic nephropathy (DN). The present study was to compare the effect of antioxidant rosmarinic acid (RA) and vitamin E (VtE) in preventing development and inhibiting progression of DN in rats. Methods: Sixteen male Wistar rats were divided into 4 groups each of 4. The 1st group acts as normal rats. The 2nd group acts as untreated-diabetic rats. The 3rd group acts as treated diabetic rats with RA at doses 75 mg/kg/day, and the 4th group acts as treated diabetic rats with VtE at dose 400 mg/kg/day and treatment continued for 8 weeks. Urinary level of podocin, nephrin, albumin, and serum cystatin C levels were measured at the end of the study by ELISA. Immunohistochemistry were used to examined the expression of p65 NF-kB whereas the expression of podocin and nephrin glomerulus were examined by immunofluorescence. Results: The excretion of nephrin, podocin and albumin urine, expression of p65 NF-kB, and level of cystatin C serum were significantly lower in the RA group compared with the VtE group, whereas expression of glomerular nephrin and podocin in the RA group were higher compared with the VtE group. Compared to the untreated-diabetic rats, the expression of glomerular nephrin and podocin, and cystatin C serum level in the VtE group did not differed significantly. Conclusions: In streptozotocin-induced diabetic rats, RA superior than VtE in preventing development and inhibiting progression of diabetic nephropathy.
KEYWORDS: Antioxidant, Diabetic nephropathy, Podocyte, Rosmarinic acid, Vitamin E.
1. INTRODUCTION:
Diabetic nephropathy (DN) is the leading cause of chronic kidney disease (CKD) and end-stage renal disease (ESRD). Oxidative stress and inflammatory process are important factors contributing to kidney damage by increasing production of oxidants.1 In the glomerulus there are podocytes which are highly specialized epithelial cells that have complex functions. Proper podocyte function demands a high and continuous energy supply that is mainly derived from the respiratory chain of the inner mitochondrial membrane. Dysregulations in the metabolic homeostasis of podocytes may result in podocyte damage and glomerular disease. Podocyte damage is considered a key factor in the development and progression of DN. Excessive production of reactive oxygen species (ROS) is found in injured podocytes as in DN.2 Furthermore, existing oxidants directly cause damage to the podocytes3, damage to the glomerular basement membrane (GBM), and interfere the 3-integrins4, all of which can contribute to the detachment of podocytes, either alone or in synergy.
Detachment and apoptosis cause podocytopenia and will later lead to the formation of synechia and glomerular sclerosis.5 Given the important role of oxidative stress in pathological changes in the kidneys, therapy with a radical approach, which can protect podocytes by reducing oxidative stress, and subsequent podocyte detachments, is important to prevent glomerulosclerosis, CKD and ESRD. However, in order to achieve this goal, it requires multi-property antioxidants, which have strong antioxidant capacity, have anti-inflammatory effects, long half-lives, have high permeability to mitochondria and can improve the body's antioxidant enzyme activity.6
Vitamin E is proposed for the prevention of kidney injuries associated with ROS.7 Vitamin E is incorporate into the plasma membrane of cells, thereby scavenging free radicals, mainly the peroxyl radical, and halting lipid peroxidation chain reactions.8 It has been proven the renoprotective effect of vitamin E in several experimental diabetic animals by reducing oxidative stress.9-11 Whereas rosmarinic acid (RA) is a potent polyphenol that has the function as an antioxidant and anti-inflammatory [6], decreases nuclear factor-kB (NF-kB), increases glutathione transferase (GSTs), anti-activity of Bcl-2, peroxynitrite scavengers12,13, able to prevent decreased activity of superoxide dismutase (SOD), Catalase (CAT), and Glutathione (GSH)14 and inhibits CTGF.15 The high levels of GSH directly detoxifies ROS and protects cellular proteins against oxidative stress through the glutathione redox cycle.16 Based on the information above, we designed to investigate the comparative effect of RA and vitamin E in preventing development and inhibiting progression of DN in streptozotocin (STZ)-induced diabetic in rats.
2 MATERIALS AND METHODS:
2.1. Materials:
This study is part of a previously published study.17 Streptozotozin (STZ) purchased from Bio World with catalog number 41910012-3 (714990). Rosmarinic acid (RA, molecular formula: C18H16O8, molecular weight: 360.31) 96% from Sigma-Aldrich Co (St Louis, MO, USA) with Product Number: 536954. D-Alpha-Tocopherol 97% (VtE) 25 gram from TCI with catalog number T2309. Podocin urine, nephrin urine and albumin was measured by rat NPHS2 Elisa kit 96T (cat. no. E1595Ra), Rat NPHN Elisa kit 96T (cat. no. E1366Ra), and Rat Albumin Elisa Kit 96T (cat. no. E1276Ra), respectively, whereas cystatin C was measured by Rat Cys-C Elisa kit 96T (cat. no. E0145Ra). All of Elisa kit were purchased from Bioassay Technology Laboratory (Shanghai Korain Biotech Co Ltd, China). Expression of p65 NF-kB was determined by antibody NF-kB p65 (A-12) (cat. no. sc-514451) from Santa Cruz Biotechnology, California, USA. Nephrin expression was determined by Nephrin (G-8) HRP mouse monoclonal IgG2a (cat. no. sc-376522), from Santa Cruz Biotechnology, California, USA, whereas podocin expression by Rabbit anti-NPHS2/Podocin polyclonal antibody (cat. no. bs-6597R) from BIOSS Inc. (Woburn, MA, USA).
2.2. Animals:
Adult male Wistar rats (7–9 weeks; 150–170g) obtained from the Central Animal House of the Brawijaya University, Malang, were used in the experiment. Before starting the experiment, the rats went through an adjustment period of 7 days. The rats were caged individually and maintained at a constant temperature and fed with a composition of 60% calories from fat to get diabetic rat with insulin resistance and free access to water. All animal procedures were approved by the Animal Ethics Committee of Brawijaya University, Malang, Indonesia. The ethics committee review the research protocol based on IACUC (institutional animal care and use committee) guidelines.
Diabetes induction:
After 1 week of adjustment, rats were given a high-fat diet for 3 weeks. On the 22nd day, the rats were fasted for 6 to 8 hours (starting at 7 a.m) before giving STZ. The study group received STZ to induce diabetes mellitus, whereas the control group received citrate buffer without STZ. Diabetes was induced by a single intraperitoneal injection of 40mg/kg STZ, diluted in 0.1 M sodium-citrate buffer (pH 4.5).18 Age-matched control rats received an equivalent amount of sodium-citrate buffer. After STZ injection, rats get a free diet and water. Blood samples were collected from the tail vein 5 days after STZ or vehicle injection. Glucose levels were measured with a portable glucometer (Nesco). Only rats with fasting blood glucose over 280mg/dL were considered diabetic and used for the study.
2.3. Experimental design:
The rats were randomly divided into four groups (4 rats per group). Group 1 was negative control (normal, non-diabetic rats), group 2 was positive control (diabetic rats without therapy) and 2 groups of diabetic rats who received therapy consisting of group 3 treated with RA 75mg/kg/day, and group 4 treated with Vitamin E 400
mg/kg/day. At the first day of the blood sugar target is reached, the rats get therapy according to the treatment group, while the rats in the control groups received saline solution. RA and D-Alpha-Tocopherol was prepared freshly in 25% ethanol and administered via gavage, between 07 and 08 a.m. once a day for 8 weeks, at a volume not exceeding 0.1mL/100g rat weight.
In order to correct for the interference of ethanol, a group of control rats received a solution of 25% ethanol. Eight weeks after the treatment, urine specimens were collected and then the rats were anesthetized for blood collection by cardiac puncture, and the kidney were carefully removed for subsequent biochemical analysis. The biological material that was not used was disposed of following biosecurity standards.
Measurement of albumin urine, urinary nephrin and podocin, and serum cystatin C level by ELISA. 24-hour urine collection was performed using metabolic cage at the end of the study for measurement of albumin urine, urinary nephrin and podocin excretion, whereas serum cystatin C level was measured from a blood sample. The measurement method refers to the protocol in the ELISA kit for each examination and reading using an ELISA reader.
2.4. Renal histological analysis:
The kidneys were fixed in 10% buffered formalin, embedded in paraffin, cut into 3 μm sections, and stained with hematoxylin and eosin. The sections were viewed with a light microscope (Olympus) under 400 × magnification. Forty glomeruli per sample sections were photographed randomly. Glomerular area (GA) and mesangial area (MA) were assessed with ImageJ 1.48 software. Glomerular volume (GV) was calculated using formula, GV = 1.2545 (GA)1.5. Mesangial expansion was calculated in percent by measuring MA proportion per GA or (MA/GA) × 100%.19
2.5. Immunohistochemical assay:
Paraffin embedded 3µm tissue sections were deparaffinized with xylene and gradually dehydrated in ethanol. The sections were incubated with 4% hydrogen peroxidase (15 minutes at RT) to block endogenous peroxidase activity. Nonspecific binding of antibodies was blocked by incubating slides with 3% fetal bovine serum (FBS) + 0.25% Triton X-100 (1 hour at RT).
The sections were stained using rabbit anti-p65 NF-kB antibody (90 minutes at RT), which were dissolved in PBS + 3% FBS + 0.25% Triton X-100 for expression of p65 NF-kB. The slides then were incubated with biotinylated secondary antibody (1 hour at RT), followed by streptavidin horseradish peroxidase (40 minutes at RT). Slides were visualized using diaminobenzidine (DAB) and counterstained with hematoxylin. Slides were mounted and observed under light microscope (Olympus) with 400× magnification. Ten glomeruli per sample section were observed randomly. Then we calculated the total number of DAB-staining area, then divided by 10 to get the average of each glomerulus per sample observed.
2.6. Immunofluorescence staining:
In brief, tissue sections were fixed with 4% paraformaldehyde for 15 minutes, washed with PBS three times, permeabilized with 2% Triton X-100 for 5 minutes, blocked with blocking solution (2% BSA and 2% FBS in PBS) for 30 minutes. Primary and secondary antibodies were applied at the appropriate dilutions according to standard techniques. Double-staining was achieved by incubating with primary antibody and FITC-conjugated secondary antibody and then incubated with rhodamine-conjugated secondary antibody for 1 hour. The negative control was performed using 2% BSA in PBS instead of the primary antibody. Images were obtained by confocal laser-scanning microscopy (Fluoview FV1000, Olympus, Tokyo, Japan) and recorded on a computer using Fluoview Ver.4.2a viewer and processed with ImageJ 1.50 software (NIH, MD, USA).
2.7. Statistical analysis:
All data are presented as mean ± SD of two independent experiments. Differences between groups were determined by one-way ANOVA test, followed by post hoc test using Least Significant Difference. All technical data processing results were analyzed by computerization using Statistical Product and Service Solution software, IBM SPSS Statistics 20 with a significance level of 0.05 (p = 0.05) and a confidence level of 95% (α = 0.05).
3 RESULTS:
3.1. Diabetic induction:
The average blood glucose level after 5 days of STZ injection in the diabetes group (PC, RA, and VtE groups) was 352±61.57mg/dL, 388±37.17mg/dL, and 317± 83.09mg/dL, respectively, which were not statistically different (p> 0.05). In the negative control group, the average blood glucose level was 95 ± 9.29 mg / dL which was significantly different compared to the diabetes group (Fig. 1).
Fig. 1: Weekly blood glucose levels were measured by portable glucometer.
Notes: I, blood glucose pre-STZ injection; II, blood glucose post-STZ injection; III-VII, blood glucose levels at week 3 to week 7. Blood glucose levels at week 7 in the PC group, RA group and VtE group were 244 ± 42 mg/dL; 157 ± 29 mg/dL, and 198 ± 36 mg/dL, respectively, which were not significant different statistically (p>0.05).
Abbreviations: NC, negative control; PC, positive control; RA, rosmarinic acid; VtE, vitamin E; SD, standard deviation.
3.2. Effect of RA and Vitamin E on Development of DN:
Evaluation of DN was assessed from the presence of albuminuria, glomerular hypertrophy and mesangial expansion in the histological picture of the kidneys (Fig. 2). Significantly elevated albuminuria was shown in the PC group and VtE group compared with the NC group (P<0.05). The PC group and VtE group revealed significant increase of GV and ME compared with the NC group and RA group (Fig. 2B and 2C). For RA group, there were no significant differences for albuminuria, GV and ME compared with the NC group (P<0.05). Conversely, for VtE group, there were no significant differences for albuminuria, GV and ME are compared with the PC group (P<0.05). This result indicated that RA group, but not VtE group, slowed down functional and pathological deterioration of DN in diabetic rats.
Fig. 2: Treatment Effect on Development of Diabetic nephropathy.
Notes: Effect of treatment on (A) albuminuria measured by ELISA, (B) glomerular volume, and (C) mesangial expansion measured by renal histology analysis. (D) Representative photomicrographs of groups I–IV (original magnification, ×400) of the he stained kidney section among groups. I. NC group; II. PC group; III. RA group; IV. VtE group. Results are expressed as the mean ± SD. a and b denote the difference annotations that imply significant differences (p<0.05). a vs b (P < 0.05).
Abbreviations: NC, negative control; PC, positive control; RA, rosmarinic acid; VtE, vitamin E; SD, standard deviation
3.3. Effect of RA and Vitamin E on urinary nephrin and Podocin:
The levels of urinary nephrin dan podocin in the treated diabetic rat groups, differed significantly compared to the positive control group. RA group differed significantly compared to the VtE group (Fig. 3). These results indicate that RA treatment prevent podocyte detachment better than VtE treatment.
3.4. Effect of RA and Vitamin E on expression of Glomerular Nephrin and Podocin:
The expression of glomerular nephrin and podocin in the RA group were significantly higher compared with the control group (both negative and positive control groups) and VtE group. There was no different effect on expression of glomerular nephrin and podocin between VtE treatment and positive control (Fig. 4B).
Fig. 3: Treatment effect on nephrin and podocin urine excretion. The concentration of urinary nephrin and podocin by ELISA.
Notes: Results are expressed as the mean ± SD. a, b, and c denote the difference annotations that imply significant differences (p<0.05). a vs b (P < 0.05); a vs c (P<0.05); b vs c (P<0.05).
Abbreviations: NC, negative control; PC, positive control; RA, rosmarinic acid; VtE, vitamin E; SD, standard deviation
Fig. 4: Treatment effect on glomerular podocin and nephrin.
Notes: A. Immunofluorescence from podocin is characterized by FITC staining (green) and nephrin characterized by rodamin staining (red). The image was taken using a confocal laser scanning microscope (magnification 400 X, line scale 50 = µm); B. Histogram expression of glomerular podocin and nephrin. Results are expressed as the mean ± SD. a and b denote the difference annotations that imply significant differences (p<0.05). a vs b (P < 0.05).
Abbreviations: NC, negative control; PC, positive control; RA, rosmarinic acid; VtE, vitamin E; SD, standard deviation.
3.5. Effect of RA and Vitamin E on expression of p65 NF-kB:
3.6. Effect of RA and Vitamin E on Renal Function:
Serum Cystatin C levels were significantly increased in the positive control group in comparison with the normal control group. Compared with the positive control group, the RA group significantly inhibited the increase in serum cystatin C level, although it was significantly higher than the negative control group. Conversely, VtE group cannot inhibit the increase of serum cystatin C level (Fig. 6). These results indicate that RA is better than VtE in preventing kidney function decline although not achieve to normal level.
Fig. 5: Treatment effect on p65 NF-kB expression.
Notes: A. Immunohistochemical images of glomerular rats. The arrow indicates the expression of p65 NF-kB in the glomerulus. The image was taken using a microscope with the master scan dot slide application with a magnification of 400 X. I. negative control group; II. positive control group; III. RA group; IV. VtE group. B. Histogram expression of glomerular p65 NF-kB. Results are expressed as the mean ± SD. a, b, and c denote the difference annotations that imply significant differences (p<0.05). a vs b (P < 0.05); a vs c (P<0.05); b vs c (P<0.05).
Abbreviations: NC, negative control; PC, positive control; RA, rosmarinic acid; VtE, vitamin E; SD, standard deviation.
Fig. 6: Treatment effect on serum cystatin C level.
Notes: The concentration of serum cystatin C by ELISA. Results are expressed as the mean ± SD. a, b, and c denote the difference annotations that imply significant differences (p<0.05). a vs b (P < 0.05); a vs c (P<0.05); b vs c (P<0.05).
Abbreviations: NC, negative control; PC, positive control; RA, rosmarinic acid; VtE, Vitamin E; SD, standard deviation.
4 DISCUSSION:
Diabetic nephropathy (DN) is a progressive and irreversible kidney disease that is characterized by initial hyperfiltration, albuminuria, expansion of mesangial matrix, interstitial fibrosis, thickening of basement membranes, and renal cell damage.1 In the present study, urinary albumin, glomerular hyperthropy and mesangial expansion (ME) were noted to be increased in diabetic rats which suggest the development of DN. Furthermore, we demonstrated that antioxidants may have therapeutic potential for the treatment of DN. Our results showed that GV, ME, albuminuria and cystatin C serum level in the diabetic rats differed significantly compared to the normal rats (Fig. 2). There was no significant difference in albuminuria, GV and ME in the RA group compared to the normal rats, whereas VtE therapy showed the opposite results, which was not significantly different than the non-treated diabetic rats group. Our results differ from other researchers that in animal models. Zhao et al, 2019, high dose of VtE can significantly decrease serum creatinine, urinary albumin and urinary protein.20 Hayashi et al, 2017, showed that VtE at a dose of 40 mg/kg intraperitoneally (ip) were ameliorate urine albumin and creatinine.11 Haidara et al, 2009, showed that at dose of VtE 300mg/kg or 600mg/kg intramuscularly (im) 3 times a week for 4 weeks significantly reduced both urea and creatinine levels to almost control levels Haidara et al, 2009. In human study,in 19 diabetic patients, showed that the teatment with VtE decreased microalbuminuria, and that VtE nephroprotective effect VtE was mediated by the antioxidant action.21. In contrary, the results from Hirnerová et al., 2004, showed that VtE did not show a baneficial effect on diabetic complication including albuminuria.22 Farvid et al., 2005, has been reported that there is no significant changes in proteinuria after combined treatment with zinc + magnesium or Vitamin C + Vitamin E.23 Gaede et al., 2001, also showed no significant difference was observed in end of treatment serum creatinine values between the intervention and the control group.24 These different results might be related to differences in the VtE dose used, differences in the therapeutic method of administration, i.e. orally, intaperitoneally or intramuscularly.
Study have shown that a decrease in the number of podocytes occurs in the early stages of DN and is caused by apoptosis or podocyte detachment.25 Our data show that RA therapy at a dose of 75mg/kg for 8 weeks showed urinary nephrin and podocin levels were significantly lower compared to the diabetic rats group and not significantly different compared to the normal rats group. Therapy with VtE in our study actually can also prevent podocyte detachment but cannot achieve such normal rats as therapy with RA (Fig. 3). Our results are in accordance with results from Zhao et al., that VtE ameliorates podocyte pathology and prevents podocyte loss.20 Based on these results, we conclude that therapy with RA or VtE in diabetic rat, can prevent podocyte detachment from GBM, in which treatment with RA gives better results than treatment with VtE. In the glomerular, RA therapy showed significantly higher expression of nephrin and podocin compared to the VtE therapy, even in VtE therapy the expression of nephrin and podocin was lower than that of PC but not significant statistically (Fig. 4). This evidence clearly shows that RA therapy, but not VtE therapy, increases the expression of nephrin and podocin in STZ-induced diabetic rats.
RA has the main effect as an antioxidant and anti-inflammatory6 as well as VtE as an antioxidant and has an anti-inflammatory effect.7 The better preventive effect on detachment of podocytes and increased expression of glomerular podocin and nephrin with RA therapy compared to the VtE therapy is probably due to the better antioxidant and anti-inflammatory effects of RA compared to VtE. We proved this by decreasing the expression of p65 NF-kB with RA therapy which was significantly different compared to VtE therapy (Fig. 5). RA was found to reduce NFkB.12,13 In oxidative stress-induced kidney disease, NF-kB is activated by ROS and initiates signaling pathways involved in renal fibrosis.26 Increased activation of NF-kB was observed in DN in animal and human studies.27,28 Unfortunately, in present study we not assessing the phosphorylation of the IkBα inhibitor molecule to assess NF-kB activity. Increased expression of the p65 NF-kB subunit can reduce nephrin expression.29 Increase of nephrin expression is due to a decrease in the expression of p65 NF-kB, which has a negative feedback response to nephrin expression.29 Activation of NF-kB in podocytes can also induce epithelial transition to the mesenchymal, thereby reducing nephrin expression.29,30
Several study have been proved the hypoglicemic effect or insulin sensitivity of RA and VtE.31,33 VtE reduced HbA1C level in type 2 diabetes.34 Our results also show that RA and VtE have the effect of lowering blood glucose levels (Fig. 1). Nevertheless, the beneficial effects of VtE was due to a reduction in oxidatve stress, not secondary to a decrease in plasma glucose.31 Similarly, ameliorating renal function by RA therapy not by reducing glycemia levels but by a direct nephroprotective effect.15
Two major goals for controlling development of CKD are early detection and slowing progression to ESRD. Assessment of GFR by cystatin C, urine or serum is considered a sensitive biomarker as it detects minor glomerular injury, and this method not influenced by body mass, being comparable and even better than methods using serum creatinine.35-37 Our result showed that treatment with RA is better in preventing elevated serum cystatin C levels than VtE therapy (Fig. 6). This result was in line with the effects of RA therapy on albuminuria (Fig. 2A). The results of this study have proven that RA therapy can inhibit the progression of DN in diabetic rats. The renoprotective effect of RA is dose dependent, as evidenced by Jiang et al., who have shown that RA therapy with doses of 7.5mg/kg and 15 mg/kgfor 8 weeks significantly decreases 24-hour urine albumin and serum cystatin C levels, where at a dose of 15mg/kg better than 7.5mg/kg.37
CONCLUSION:
Our results have been proved that oxidative stress plays an important role in the development and progression of diabetic nephropathy by detachment of podocyte cell. Treatment with RA or VtE can prevent the detachment of podocytes. But on overall therapy with RA gives better results than vitamin E in the prevention of podocyte detachment and several parameters of DN progressivity.
CONFLICT OF INTEREST:
The authors report no conflicts of interest in this work.
ACKNOWLEDGEMENTS:
The authors thank the University of Brawijaya Postgraduate Program for their financial support and also the Central Laboratory of Life Sciences and the Laboratory of Bioscience, Brawijaya University, Malang, for providing the laboratory facilities.
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Received on 07.01.2020 Modified on 24.02.2020
Accepted on 19.03.2020 ©Asian Pharma Press All Right Reserved
Asian J. Pharm. Res. 2020; 10(3):131-139.
DOI: 10.5958/2231-5691.2020.00025.8