|Year : 2019 | Volume
| Issue : 1 | Page : 20-24
Mitochondrial nephrotoxicity induced by tacrolimus (FK-506) and modulatory effects of Bacopa monnieri (Farafakh) of Tabuk Region
Atif Abdulwahab A. Oyouni1, Shalini Saggu1, Ehab Tousson2, Anand Mohan3, Abdullah Farasani4
1 Department of Biology, Faculty of Sciences, University of Tabuk, Tabuk, Saudi Arabia
2 Department of Zoology, Faculty of Sciences, Tanta University, Tanta, Egypt
3 Department of Botany, School of Bioengineering and Biosciences, Lovely Professional University, Phagwara, Punjab, India
4 Department of Medical Laboratory, Faculty of Applied Medical Sciences, Jazan University, Jazan, Saudi Arabia
|Date of Web Publication||20-Feb-2019|
Dr. Atif Abdulwahab A. Oyouni
Department of Biology, Faculty of Sciences, University of Tabuk, Tabuk 71491
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Background: Tacrolimus is a known immunosuppressive drug used widely for organ transplantation, but its nephrotoxicity mechanism is still unclear. Objectives: The present study investigates the protective efficacy of Bacopa monnieri (BM), against tacrolimus-induced nephrotoxicity in rats. Materials and Methods: Group 1 (control group); administered orally with normal saline for 30 days; Group 2 (BM extract treated group); Group 3 (tacrolimus-treated group); and Group 4; (tacrolimus plus BM extract treated group). Tacrolimus-treated rats received 1 mg/kg body weight of tacrolimus intraperitoneally for 30 days, and BM-pretreated rats were administered with the dose of 200 mg/kg orally by gavage once a day for 30 days. Results: Tacrolimus-induced nephrotoxicity was assessed biochemically and histopathologically. Pretreatment with BM has shown to possess a significant protective effect against tacrolimus-induced kidney functions regarding urea, creatinine, and albumin levels, respectively. The creatinine, mitochondrial lipid peroxidation (thiobarbituric acid reactive substances), and protein carbonyl levels were significantly increased dramatically, and however, the total proteins, albumin, glutathione, superoxide dismutase, and glutathione peroxidase were decreased when pretreated with tacrolimus. The nephroprotective efficacy of the BM extract was further evident by histopathological analysis and DNA fragmentation. Conclusion: The outcome of this study indicates that BM extracts exerted protection against tacrolimus-induced kidney toxicity.
Abbreviations Used: ANOVA: Analysis of variance, BM: Bacopa monnieri, BUN: Blood urea nitrogen, DNPH: Dinitrophenylhydrazine, DPPH: 2,2-diphenyl-1-picrylhydrazyl, DSR: Deanship of Scientific Research, EOBPV: Egyptian Organization for Biological Products and Vaccines, GPx: Glutathione peroxidase, GSH: Glutathione, H and E: Hematoxylin and eosin, H2O2: Hydrogenperoxide, IAEC: Institutional Animals Ethics Committee, IC: Inhibitory concentration, Ip: Intraperitoneal, mLPO: Mitochondrial lipid peroxidation, Mn-SOD: Mn-superoxide dismutase, PC: Protein carbonyl, ROS: Reactive oxygen species.
Keywords: Antioxidant activity, Bacopa monnieri, DNA fragmentation, nephrotoxicity, tacrolimus
|How to cite this article:|
Oyouni AA, Saggu S, Tousson E, Mohan A, Farasani A. Mitochondrial nephrotoxicity induced by tacrolimus (FK-506) and modulatory effects of Bacopa monnieri (Farafakh) of Tabuk Region. Phcog Res 2019;11:20-4
|How to cite this URL:|
Oyouni AA, Saggu S, Tousson E, Mohan A, Farasani A. Mitochondrial nephrotoxicity induced by tacrolimus (FK-506) and modulatory effects of Bacopa monnieri (Farafakh) of Tabuk Region. Phcog Res [serial online] 2019 [cited 2019 Mar 26];11:20-4. Available from: http://www.phcogres.com/text.asp?2019/11/1/20/252556
- Nephroprotective properties of the ethanol extract of Bacopa monnieri and its antioxidant activity was evaluated. It showed anti-oxidative and protective efficacy against tacrolimus by significant attenuation of oxidative stress parameters in mitochondria isolated from the kidney, augmentation in renal function biochemistry as well as the restoration of renal structures.
| Introduction|| |
Humans are incessantly exposed to exogenous chemicals, drugs, and environmental pollutants deliberately and inadvertently that harm the kidney. Kidneys are extremely susceptible to damage caused by reactive oxygen species (ROS) due to oxidative stress. Tacrolimus is a known immunosuppressive drug used in organ transplantation and has both short-term and long-term advantages over conventional drugs. After transplantation, toxicity of tacrolimus is an important determinant of morbidity and mortality. This drug has higher nephrotoxicity, neurotoxicity, and diabetogenicity. Tacrolimus is absorbed in the duodenum and jejunum after oral administration and have a great erraticism of pharmacokinetics.
Herbal remedies have been used for a long time for the management and detoxification from drugs of addiction. Because of the efficiency, fewer side effects and comparatively low cost, medicinal plants, and herbal drugs are prescribed widely. Bacopa monnieri (Linn.) Pennell, from family Scrophulariaceae, has been studied widely for its hepatoprotective and nephroprotective properties., Numerous clinical studies inveterate the valuable actions of BM. These pharmacological actions are mainly attributed to the saponin compounds present in the alcoholic extract of the plant. The primary chemical constituents isolated from BM are triterpenoids, saponins with jujubogenin and pseudojujubogenin as the aglycones including bacosides A1–A3, bacopasaponins A-G, and bacopasides I-V., It has also been shown to possess potent antioxidant activity.
However, the efficacy of the BM against an immunosuppressive drug is scarce. Therefore, the present study was aimed to reconnoiter the protective efficacy of BM against tacrolimus-induced nephrotoxicity in rats.
| Materials and Methods|| |
Plant collection and extract preparation
BM was collected from the local market, authenticated by the departmental botanist and was shade dried, blended to form the coarse powder. Briefly, 70 g of dry powder was taken, and to it, 70% ethanolic (v/v) was added with intermittent shaking and kept overnight. The process was repeated three times, pooled, filtered and extracted in soxhlet apparatus at 60°C.
A total of 40 male adult Sprague Dawley rats weighing 150–180 g were used for the experiments and purchased from the breeding unit of Egyptian Organization for Biological Products and Vaccines, Abbassia, Cairo. They were housed in steel mesh cages and maintained for 1 week acclimatization period on commercial standard and pellet diet and drinking water ad libitum. The housing cycle was 12:12 h light-dark cycle under controlled temperature (20°C–22°C). The animal use protocol had been approved by the Institutional Animals Ethics Committee of Tanta University.
Phytochemical screening of the plant
BM extracts were subjected to phytochemical testing for the identification of chemical constituents, including, tannins, saponins, phenols, total antioxidant capacity, and flavonoids as reported by Saggu et al.,
Randomly animals were divided into four groups of ten rats each. Group I animals served as control. Group II animals were given BM extract at a dose of 200 mg/kg body weight, Group III animals were injected 1 mg/kg body weight of tacrolimus intraperitoneally, daily for 30 days, and Group IV received tacrolimus + BM extract, respectively, for 30 days. Oral administration of the BM extract was given orally by gavage before the administration of tacrolimus. After 24 h, the animals were sacrificed by cervical dislocation, and their kidneys were removed immediately. One kidney of each animal was processed for the preparation of mitochondria, and the other kidney was fixed in 10% buffered formalin (chilled) for histopathological studies.
Preparation of homogenate and determination of antioxidant markers
Samples of blood were taken from the orbital sinus, and centrifugation separated the serum at 3000 g for 10 min using capillary tubes, and the Auto-analyzer estimated renal function parameters.
The kidneys were removed and dissected free from the surrounding fat and connective tissue. Subsequently, renal tissue was homogenized, and mitochondria from tissues were isolated from the fasted animal by the differential centrifugation method as illustrated., The protein concentration in the stock suspension was determined by using the Lowry method.
Quantitative determination of glutathione, superoxide dismutase, and mitochondrial glutathione peroxidase activity
The reduced glutathione (GSH) content was determined by the method Beutler et al. The activity of Mn-superoxide dismutase (MnSOD) was established by the way as described by Flohe and Otting  The change in absorbance was measured at 438 nm and expressed as U/mg of mitochondrial protein.
The activity of GSH peroxidase was assayed by the method described by Flohe. Results were expressed as U/mg of mitochondrial protein.
Estimation of mitochondrial lipid peroxidation and protein carbonyl
The mitochondrial lipid peroxidation was quantitated by monitoring the formation of thiobarbituric acid reactive substances (TBARS) according to the method by Waseem and Parvez. The rate was determined as n moles of TBARS formed/mg of protein using a molar extinction coefficient of 1.56 × 105 M −1 cm −1. The protein carbonyl (PC) content was measured using dinitrophenylhydrazine as described by Sohal et al.
The histopathology was carried out according to Scheuer and Chalk. Briefly, after excision of kidneys, instantly they were washed using chilled saline solution and treated for further bioassays. A small section was straightaway fixed in 10% formalin and later embedded in paraffin, 5 μm sectioned were cut and stained with hematoxylin and eosin, and examined under a light microscope.
Estimation of fragmentation of DNA.
Samples from the kidney of the ten animals/group were mechanically dissociated in hypotonic lysis buffer and centrifuged at 13,800 g for 15 min. Colorimetric determination by Diphenylamine assay was performed, and small DNA fragments from the supernatant were immediately separated, and the pellet containing large DNA pieces were used for the estimation. The developed blue color was colorimetrically quantified spectrophotometrically at 578 nm. The formula expressed percentage of DNA fragmentation in each sample: % DNA fragmentation = (O. D supernatant/O. D supernatant + O. D pellet) × 100.(O. D. optical density).
All the data are presented as means ± standard deviation for at least three replications for each prepared sample. One-way analysis of variance followed by Student–Newman–Keul's test was done to assess the significant differences among the groups, using SigmaPlot, Systat Software program version 11.
| Results|| |
Initial phytochemical screening of Bacopa monnieri extract
Phytochemical analysis of the BM extract showed the presence of tannins, saponins, phenols, and flavonoids. Total phenol content present in BM extract was found to be 40.8 mg/g GAE and the total flavonoid is 16.2 mg/g of CE equivalent, respectively [Table 1].
|Table 1: Total flavonoids; total phenolics contents in Bacopa monnieri extract|
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Effect of tacrolimus and Bacopa monnieri on kidney function test
In the present study, treatment with tacrolimus induced a significant elevation in serum blood urea nitrogen (BUN) and creatinine. However, the levels of total protein and albumin were significantly decreased (P < 0.001) when compared to healthy animals (Group 1). These alterations were attenuated considerably in rats treated with the BM extract (P < 0.05) [Table 2].
|Table 2: Effect of Bacopa monnieri leaves extract administration on kidney function test: Creatinine, blood urea nitrogen, total protein and albumin levels in tacrolimus-induced nephrotoxicity in rats|
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Effect of Bacopa monnieri extract on tacrolimus induced perturbations on isolated mitochondrial enzymatic and nonenzymatic antioxidants
Tacrolimus induces oxidative stress and produces a generation of free radicals and peroxidative damage to the tissues. The activities of antioxidant enzymes, namely, GSH, Mn-superoxide dismutase, GPx were significantly reduced in tacrolimus-induced rats when compared to control animals. [Figure 1] reflects the reduced mitochondrial GSH level was significantly decreased in the Group III as compared to the normal control (P < 0.001) (Group I) but increased dramatically in the Group IV as compared to the tacrolimus treated group (P < 0.05). A similar trend of the decrease was also observed in the activities of GPx and MnSOD.
|Figure 1: Effect of Bacopa monnieri extracts administration (200.0 mg/kg) on GSH activity in tacrolimus induced nephrotoxicity in experimental rats. Values are mean ± standard deviation, N = 10 animals in each group. ***Statistically significance at P < 0.001, in comparison to the normal control group; **Statistically significance at P < 0.01, compared with tacrolimus treated group|
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The TBARS values increased significantly in the tacrolimus-treated group when compared with the control, and BM-treated animals (P < 0.001) [Table 3]. The PC values were significantly reduced in comparison with controls. However, BM oral extract treatment in rats resulted in significant increases in values (P < 0.05) [Table 3]. The significant elevation of plasma PCs indicates the presence of oxidative stress and upregulation of NAPQI production, which stimulates protein oxidations.
|Table 3: Effect of Bacopa monnieri extracts on Mn-superoxide dismutase and glutathione peroxidase activities in total antioxidant capacity-treated rats|
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The histopathological changes in the kidney sections in the different groups are shown in [Figure 2]a, [Figure 2]b, [Figure 2]c, [Figure 2]d, [Figure 2]e, [Figure 2]f. Kidney sections in control group and BM revealed normal structure of glomeruli, proximal convoluted tubules, and distal convoluted tubules in both cortex and medulla [Figure 2]a and [Figure 2]b, on the other hand, kidney sections in groups treated with tacrolimus showed some histopathological lesions in glomeruli and some parts of the urinary tubules such as marked atrophied glomeruli, leukocytic infiltrations, congestion in blood vessels and marked degeneration in proximal and distal convoluted tubules and vacuolization in tubular cells focal necrosis [Figure 2]c and [Figure 2]d at the concentration 1 mg/kg body weight for 30 days. In contrast; kidney sections in rats treated with TAC + BM revealed a better improvement in glomerular damage with minimal vacuolization and degeneration in tubular cells and the Malpighian corpuscles appeared as normal structure [Figure 2]e and [Figure 2]f.
|Figure 2: (a-f) Photomicrographs of kidney sections in the different groups under study stained with hematoxylin and Eosin. (a and b) Kidney sections in control and Bacopa monnieri showed normal structure of renal cortex which comprised renal corpuscles (G), proximal and distal convoluted tubules. (c and d) Kidney sections in treated group with TAC showed atrophied glomeruli (G), leukocytic infiltrations (Black arrows), and congestion in blood vessels and marked degeneration in proximal and distal convoluted tubules (White arrows). (e and f) Kidney sections in groups treated with TAC + Bacopa monnieri revealed minimal vacuolization and degeneration in tubular cells (White arrows) and the malpighian corpuscles appeared as normal structure|
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As compared with the normal control group, the mean percentage of DNA fragmentation when treated with 1 mg/kg of Tacrolimus for 30 days is significantly increased (P < 0.001). Treatment with 200 mg/kg of BM extracts prevents the cellular damage substantially [Figure 3].
|Figure 3: DNA fragmentation of rats treated with tacrolimus, Bacopa monnieri extract, and combination of both. Statistical significances are indicated by ***P < 0.001 when compared with normal control groups|
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| Discussion|| |
Tacrolimus is a known immunosuppressive drug which binds to FKBP12 and works by inhibiting calcineurin, a calcium-calmodulin-dependent phosphatase which ultimately inhibits T-cell activation.
The current study aimed to analyze the potential impact of ethanolic extract of BM on tacrolimus-induced nephrotoxicity. We have examined the antioxidant profile, renal markers, and renal morphology in tacrolimus-treated rats together with the phytochemical screening of the plant. We have found that the extract possessed tannins, saponins, phenols, flavonoids and showed radical scavenging activity [Table 1].
Mitochondria play an essential role in the cytoxicities of several drugs and chemicals., In the present work, the observed nephrotoxicity due to immunosuppressive tacrolimus treatment was manifested by marked increases in serum creatinine, BUN clearance, and a decrease in total protein and albumin.
During stressful situations increase in energy demand necessitates a multifold upsurge in oxygen supply to active tissues to maximize energy yield. The augmented metabolic rate results in the generation of ROS free radicals in massive amounts, causing an imbalance between ROS production and antioxidant defense. PC is a universally accepted biomarker of PC accumulation and oxidation of protein. Tacrolimus pretreatment aggravated the increase in protein oxidation, and BM pretreatment restored the levels in insolated kidney mitochondria. It has been found that BM contains saponins, alkaloids, phenols, and antioxidants which might be the reason for the protective efficacy of the extract against the ROS-induced oxidative stress.
In this study, it has been found that the antioxidant and nonantioxidant enzymes were substantially suppressed in tacrolimus-treated rats. However, treatment with BM substantially up-regulated the levels of antioxidant and non-antioxidants when compared with the tacrolimus-treated animals. Commonly, increased levels of MDA are used as an index of oxidative injury due to oxygen free radicals. Some of the authors reported the protective efficacy of juniper oil against tacrolimus induced nephrotoxicity in rats and also showed the anti-inflammatory and antinociceptive influences of some juniper species in Swiss male albino mice., In this study in tacrolimus-treated rats, there was a significant increase of MDA levels along with decreased levels of GPx. It is a well-known fact that ROS generation plays a vital role in peroxidation of lipid membranes of the tissue, resulting in subcellular damage as evident in the histopathological examination [Figure 2]. In our study, the kidney of tacrolimus-treated rats has shown characteristic morphological findings such as glomerular atrophy, degeneration in renal tubules, necrosis, and vacuolation. Similarly, Oyouni et al. reported the nephroprotective efficacy of Ocimum basilicum in mice treated with tacrolimus at the dose of 3 mg/kg b. wt. Nephroprotective effects of BM extracts were further confirmed by measuring the DNA fragmentation in rat kidney tissue. It prevents the cellular DNA damage and safe to use.
| Conclusion|| |
Taken together, BM pretreatment in rats protected against tacrolimus-induced nephrotoxicity may be through free radical scavenging activity due to the presence of antioxidants and flavonoids in the extract. Protective efficacy was accompaniment by a significant attenuation of oxidative stress parameters in mitochondria isolated from kidney, augmentation in renal function biochemistry as well as the restoration of renal structures evident by histopathological, DNA fragmentation findings.
The authors would like to acknowledge monetary support from the Deanship of Scientific Research, University of Tabuk, Tabuk, Saudi Arabia, under the grant no. S-1438-128. The group would also like to broaden the thanks especially to Dr. Ehab Tousson, Department of Zoology, Faculty of Sciences, Tanta University, for continuous support, guidance and carrying out the histopathology.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
U.S. Multicenter FK506 Liver Study Group. A comparison of tacrolimus (FK 506) and cyclosporine for immunosuppression in liver transplantation. N Engl J Med 1994;331:1110-5.
Hebert MF. Contributions of hepatic and intestinal metabolism and P-glycoprotein to cyclosporine and tacrolimus oral drug delivery. Adv Drug Deliv Rev 1997;27:201-14.
Sumathy T, Subramanian S, Govindasamy S, Balakrishna K, Veluchamy G. Protective role of Bacopa monniera
on morphine induced hepatotoxicity in rats. Phytother Res 2001;15:643-5.
Sumathi T, Niranjali Devaraj S. Effect of Bacopa monniera
on liver and kidney toxicity in chronic use of opioids. Phytomedicine 2009;16:897-903.
Russo A, Borrelli F. Bacopa monniera
, a reputed nootropic plant: An overview. Phytomedicine 2005;12:305-17.
Deepak M, Sangli GK, Arun PC, Amit A. Quantitative determination of the major saponin mixture bacoside A in Bacopa monnieri
by HPLC. Phytochem Anal 2005;16:24-9.
Murthy PB, Raju VR, Ramakrisana T, Chakravarthy MS, Kumar KV, Kannababu S, et al.
Estimation of twelve Bacopa saponins
in Bacopa monnieri
extracts and formulations by high-performance liquid chromatography. Chem Pharm Bull (Tokyo) 2006;54:907-11.
Tripathi YB, Chaurasia S, Tripathi E, Upadhyay A, Dubey GP. BACOPA monniera
linn. as an antioxidant: Mechanism of action. Indian J Exp Biol 1996;34:523-6.
Abbas ZK, Saggu S, Sakeran MI, Zidan N, Rehman H, Ansari AA. Phytochemical, antioxidant and mineral composition of hydroalcoholic extract of chicory (Cichorium intybus
L.) leaves. Saudi J Biol Sci 2015;22:322-6.
Saggu S, Sakeran MI, Zidan N, Tousson E, Mohan A, Rehman H, et al.
Ameliorating effect of chicory (Chichorium intybus
L.) fruit extract against 4-tert-octylphenol induced liver injury and oxidative stress in male rats. Food Chem Toxicol 2014;72:138-46.
Parvez S, Winkler-Stuck K, Hertel S, Schönfeld P, Siemen D. The dopamine-D2-receptor agonist ropinirole dose-dependently blocks the Ca2+-triggered permeability transition of mitochondria. Biochim Biophys Acta 2010;1797:1245-50.
Waseem M, Parvez S. Mitochondrial dysfunction mediated cisplatin induced toxicity: Modulatory role of curcumin. Food Chem Toxicol 2013;53:334-42.
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951;193:265-75.
Beutler E, Duron O, Kelly BM. Improved method for the determination of blood glutathione. J Lab Clin Med 1963;61:882-8.
Flohe L, Otting F. Superoxide dismutase assays. In: Methods in Enzymology. Vol. 105. 1984. p. 93-104.
Flohe L. Glutathione peroxidase. In: Oxygen Radicals in Biology and Medicine. Boston, MA: Springer; 1988. p. 663-8.
Sohal RS, Kamzalov S, Sumien N, Ferguson M, Rebrin I, Heinrich KR, et al.
Effect of coenzyme Q10 intake on endogenous coenzyme Q content, mitochondrial electron transport chain, antioxidative defenses, and life span of mice. Free Radic Biol Med 2006;40:480-7.
Scheuer PJ, Chalk BT. Clinical Tests: Histopathology. London: Wolfe Medical Publication; 1986.
Perandones CE, Illera VA, Peckham D, Stunz LL, Ashman RF. Regulation of apoptosis in vitro
in mature murine spleen T cells. J Immunol 1993;151:3521-9.
Naesens M, Kuypers DR, Sarwal M. Calcineurin inhibitor nephrotoxicity. Clin J Am Soc Nephrol 2009;4:481-508.
Begriche K, Igoudjil A, Pessayre D, Fromenty B. Mitochondrial dysfunction in NASH: Causes, consequences and possible means to prevent it. Mitochondrion 2006;6:1-28.
Cardoso CM, Custódio JB, Almeida LM, Moreno AJ. Mechanisms of the deleterious effects of tamoxifen on mitochondrial respiration rate and phosphorylation efficiency. Toxicol Appl Pharmacol 2001;176:145-52.
Butani L, Afshinnik A, Johnson J, Javaheri D, Peck S, German JB, et al.
Amelioration of tacrolimus-induced nephrotoxicity in rats using juniper oil. Transplantation 2003;76:306-11.
Orhan N, Akkol E, Ergun F. Evaluation of antiinflammatory and antinociceptive effects of some Juniperus
species growing in Turkey. Turk J Biol 2012;36:719-26.
Oyouni AA, Saggu S, Tousson E, Rehman H. Immunosuppressant drug tacrolimus induced mitochondrial nephrotoxicity, modified PCNA and bcl-2 expression attenuated by Ocimum basilicum
L. in CD1 mice. Toxicol Rep 2018;5:687-94.
[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2], [Table 3]