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Nephro-protective action of P. santalinus against alcohol-induced biochemical alterations and oxidative damage in rats
Biomedicine & Pharmacotherapy 84 (2016) 740–746
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Nephro-protective action of P. santalinus against alcohol-induced biochemical alterations and oxidative damage in rats Saradamma Bullea , Vaddi Damodara Reddya , Ananda Vardhan Hebbania,b , Pannuru Padmavathic , Chandrasekhar Challad , Pavan Kumar Puvvadae , Elisha Repallee, Devanna Nayakantid, Chandrakala Aluganti Narasimhuluf , Varadacharyulu Nallanchakravarthulaa,* a
Department of Biochemistry, Sri Krishnadevaraya University, Anantapur, 515 003, Andhra Pradesh, India Department of Biotechnology, New Horizon College of Engineering, Bangalore, 560103, India c Oil Technological Research Institute, Jawaharlal Nehru Technological University, Anantapur, 515 001, Andhra Pradesh, India d Department of Chemistry, Jawaharlal Nehru Technological University, Anantapur, 515 002, Andhra Pradesh, India e DR Biosciences, Research and Development Institute, Jayanagar, Bangalore, Karnataka, 560 011, India f Department of Medicine, University of Central Florida, Orlando, FL, 32816, USA b
A R T I C L E I N F O
Article history: Received 21 August 2016 Received in revised form 23 September 2016 Accepted 27 September 2016 Keywords: Alcohol P. santalinus Nephro-toxicity Oxidative stress/Nitrosative stress
A B S T R A C T
The present study investigated the antioxidant potential of P. santalinus heartwood methanolic extract (PSE) against alcohol-induced nephro-toxicity. The results indicated an increase in the concentration of kidney damage plasma markers, urea and creatinine with a concomitant decrease in the concentration of uric acid in alcohol-administered rats. A significant decrease in plasma electrolytes and mineral levels with increased kidney thiobarbituric acid reactive substances (TBARS) and nitric oxide (NOx) levels was also observed. PSE treatment to alcohol-administered rats effectively prevented the elevation in TBARS and NOx levels. Decreased activity of Na+/K+-ATPase in alcohol administered rats was brought to near normal levels with treatment of PSE. Chronic alcohol consumption affects antioxidant enzymatic activity and reabsorption function of the kidney which is evident from the decreased level of GSH as well as the activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR) and glutathione s-transferase (GST). However, treatment with PSE to alcoholadministered rats significantly enhanced these enzymatic activities and reduced glutathione (GSH) content close to normal level. Alcohol-induced organ damage was evident from morphological changes in the kidney. Nevertheless, administration of PSE effectively restored these morphological changes to normal. The flavonoid and tannoid compounds might have protective activity against alcohol-induced oxidative/nitrosative stress mediated kidney damage. ã 2016 Elsevier Masson SAS. All rights reserved.
1. Introduction Chronic alcohol consumption induces dramatic changes in the biochemical and physiological processes of every organ system including kidney [1,2]. Chronic ethanol consumption alters cellular functions by acting on DNA, RNA, protein and metabolites and leading to kidney dysfunction [3]. Emerging evidence indicates alcohol-induced toxicity is due to some common mechanisms like oxidative stress, inflammation and apoptotic or other signaling pathways [4]. In kidney, reactive oxygen species (ROS) mediated
* Corresponding author. E-mail address: [email protected] (V. Nallanchakravarthula). http://dx.doi.org/10.1016/j.biopha.2016.09.103 0753-3322/ã 2016 Elsevier Masson SAS. All rights reserved.
toxicity and oxidative stress has been considered as the primary pathways to alcohol-induced injury [5]. The kidney is an important organ actively involved in maintaining body fluid homeostasis by reabsorbing important material and excreting waste products. Electrolytes and minerals are the chief components of body fluids, which act as catalysts in most cellular enzyme catalyzed reactions. Alcohol consumption produces functional abnormalities in the kidney depending on the quantity of alcohol ingested [6]. Abnormal concentrations of these catabolites and electrolytes in the plasma or serum can serve as a clear indication of renal function impairment [7]. Studies have revealed that the renal tissue membrane is highly vulnerable to damage caused by ROS due to the abundance of long chain polyunsaturated fatty acids in renal lipids [8]. Na+/K+-ATPase
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present on the renal tubular epithelial cells may promote the renal tubular reabsorption of sodium. But previous studies have shown that alcohol interferes with the carrier functions of proximal tubular cells by decreasing the Na+/K+-ATPase activity in rats [9]. Chronic alcohol consumption induces the expression of inducible nitric oxide synthase (iNOS) in the kidney [10]. Increased NOx and its active metabolite peroxynitrite (ONOO ) cross cell membranes through anion channels and inactivate many biologically important proteins and enzymes through nitration of tyrosine [11]. Increased ROS/RNS may damage the renal membrane at the glomerular region or Henle's loop, altering the reabsorption of electrolytes and minerals. Knowledge gained by a better understanding of various mechanisms of tissue injury will provide new avenues for developing effective treatment of alcohol-induced organ damage [12]. Now the world is turned towards plant medicine with less or no side effects, in particular, treatments using locally available phyto- extracts which are found to be good resources for the prevention of diseases. Pterocarpus santalinus, a Fabaceae member, grows in the tropical regions of the world, especially in India, Sri Lanka, Taiwan and China [13]. Phytochemical analysis of plant extract showed the presence of several specific components in heartwood powder in particular santalin A, B and Y, pterocarpol, pterocarptriol, isopterocarpalone, pterocarpodiolone, cryptomeridol and several non specific compounds such as isoflavones, isoflavonoid glucosides, triterpenes, sesquiterpenes, b-sitosterol, lupeol, epicatechin, lignans and pterostilbeans [14,15]. P. santalinus heartwood extract showed antioxidant [16], anti-cancer [17], hepato-protective [18], gastro-protective [19], and anti-diabetic properties [20], along with angiogenic and wound-healing properties [21]. The risk of cell injury resulting from oxidative stress may be prevented by natural phytocompounds. It has been established that phytocompounds can restore the balance between the endogenous antioxidants and free radicals directly by scavenging ROS and the induction of endogenous defences like glutathione and superoxide dismutase [22]. Beneficial effects of several phyto-extracts might result from the combination of phytochemicals acting via additive or synergistic mechanisms. Despite P. santalinus diverse medicinal properties, no study has been carried out using the heart wood extract against alcohol-induced biochemical alterations and neuronal damage. Hence, the present study was carried out to study the therapeutic potential of P. santalinus against alcoholinduced nephro-toxicity.
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light-dark cycle in the University animal house. After acclimatization for a week, animals were divided into four groups (n = 8) viz., group-I, served as controls, group-II, alcohol, group-III, PSE alone and group-IV, alcohol and PSE. Alcohol (20%) was administered at a dose of 5 g/kg b.wt/day, PSE was administered at a dose of 250 mg/ kg b.wt/day. Control rats received iso-caloric glucose instead of alcohol. All treatments were given for a period of 60 days. Experimentation and animal maintenance were done with prior approval of the institutional animal ethics committee. At the end of the experimental period the animals were fasted over night and sacrificed by cervical dislocation. Immediately blood was collected into heparinised tubes by cardiac puncture and plasma was separated from cells by centrifugation at 3000 rpm for 10 min. Tissues were collected and stored at 80 C until assays were carried out. A Small section of the kidney was fixed in 10% neutral formalin solution for histo-pathological analysis. 2.3. HPLC analysis of PSE extract PSE (5 mg) was dissolved in 1 ml of methanol and 20 ml of this was injected in to an HPLC system equipped with UV-vis detector (Shimadzu SPD10A UV–vis, Japan) set at 280 nm. Polyphenols were chromatographically separated on a reverse phase Luna 5 mm C18 (100 Å, LC Column 250 mm 4.6 mm). A solvent mixture of methanol/phosphate buffer (pH 3.0) taken in the ratio of 70:30 was used as a mobile phase and with a flow rate of 1 ml/min for the isocratic elution of hydrophobic polyphenols was achieved. Presence and quantification of polyphenols in PSE was achieved by comparing the chromatogram with that of standards (100 mg/ ml) such as gallic acid, rutin and quercetin. 2.4. Quantification of total phenolics, flavonoids and tannins Quantification of total phenolics and flavonoids present in the PSE was analyzed as described by Cetkovic et al. [23] and the tannin content was determined as described previously [24]. 2.5. Measurement of nitrogenous compounds, electrolytes and minerals
2. Materials and methods
Plasma nitrogenous compounds were measured by using commercially available kits (Span Diagnostics, Surat, India). Plasma sodium, potassium, chloride, calcium, urea, creatinine, uric acid and magnesium were determined as described previously [25,26].
2.1. Preparation of extract
2.6. Measurement of TBARS and nitric oxide levels
P. santalinus heartwood pieces were procured from locally available plant and the voucher specimens were deposited at Sri Krishnadevaraya University Herbarium, Anantapur. The pieces were used to prepare P. santalinus heartwood extract (PSE). Heartwood powder was placed in a mixture of methanol and sterile distilled water (80:20, v/v) for 48 h and the mixture was thoroughly stirred until the extract had been dissolved. The mixture was then centrifuged at 2500 rpm for 10 min and the supernatant was then filtered and dried at 45 C using rotary evaporator. The extract was dissolved in water for experimental use.
Kidney tissue was homogenized (10% w/v) in Tris buffer (0.1 M, pH 7.4), centrifuged (10,000g for 20 min at 4 C) and the supernatant was used for all the biochemical parameters. Kidney thiobarbituric acid reactive species (TBARS) were measured by the formation of malondialdehyde as described previously [27]. Total NOx in the form of nitrite and nitrate levels in the kidney was measured as described by Sastry et al. [28].
2.2. Animals Two month old male albino Wistar rats weighing 120–140 g were procured from Sri Venkateswara Agencies, Bangalore, India. Animals were maintained on a standard pellet diet (M/s. Hindustan Lever Ltd., Mumbai, India) and water ad libitum with 24 h
2.7. Assays of non-enzymic, enzymic antioxidants and Na+/K+-ATPase in kidney Total reduced glutathione content was measured in the kidney by following the method of Ellman’s [29]. Activities of glutathione reductase [30], glutathione peroxidase [31], glutathione-s-transferase [32], catalase [33] and superoxide dismutase [34] were measured. Protein concentration was estimated by the method of Lowry et al. [35]. Na+/K+-ATPase activity was measured by following the method of Kartz and Epstein [36].
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2.8. Histopathological studies A portion of the kidney was dissected and fixed in 10% neutral buffered formalin solution for 24 h [37]. The fixed tissue was processed routinely, and then embedded in paraffin, sectioned to 3–5 mm thickness, deparaffinised and rehydrated using standard techniques. Morphological changes in kidney sections were observed by staining with hematoxylin and eosin, original magnification 100. 2.9. Statistical analysis Data were subjected to statistical analyses, values are mean SD of 8 rats in each group. Student t-test followed by Duncan’s Multiple Range (DMR) test was performed to determine significance between groups. 3. Results HPLC analysis of heartwood extract of P. santalinus reveals the presence of multiple polyphenolic compounds (Fig. 1a). The individual phytocompounds present in extract were compared with standards, gallic acid, rutin and quercetin. Total phytocompounds, total phenolics, flavonoids and tannins present in PSE
were quantified (Fig. 1b). The chemical structures of significant phytocompounds pterolinus B (Fig. 1c) and pterostilbene (Fig. 1d) were also presented. Plasma nitrogenous compounds, urea and creatinine levels were significantly (p < 0.05) increased in alcohol-administered rats with concomitant decrease in uric acid concentration (Table 1). PSE treatment to alcohol administered rats restored the levels of urea, creatinine and uric acid to near normal compared to alcohol alone administered rats. In addition, chronic alcohol administration also altered the serum electrolytes and minerals (Table 2). Sodium, potassium and chloride levels were decreased in alcohol-administered rats as compared to controls. Moreover, calcium and Table 1 Effect of PSE on plasma urea, uric acid and creatinine levels in alcohol-administered rats. Parameter
Control
Alcohol
PSE
Alcohol + PSE
Urea Uric acid Creatinine
25.2 1.7 2.3 0.05 0.42 0.06
34.8 2.7* 1.52 0.07* 0.95 0.05*
25.7 2.0 2.3 0.05 0.42 0.08
27.5 2.5ns 2.2 0.08ns 0.48 0.07ns
Values are represented as mean SD. A p < 0.05 is considered as significantly different between groups. Asterisk “*” indicates significant from controls, “ns” indicates not significant from controls and PSE alone administered rats. Urea, uric acid and creatinine values are expressed as mg/dL.
Fig. 1. (a) High performance liquid chromatography analysis (HPLC) of PSE, (b) quantification data of total phenolics, flavonoids and tannins. The chemical structures of pterolinus B (c) and pterostilbene (d). Total Phenolics and flavonoids were expressed in terms of gram equivalent of gallic acid/gram extract, tannins were expressed in terms of gram equivalent of tannic acid/gram extract.
S. Bulle et al. / Biomedicine & Pharmacotherapy 84 (2016) 740–746 Table 2 Effect of PSE on plasma electrolytes and minerals in alcohol-administered rats. Control
Alcohol
PSE
Alcohol + PSE
Sodium Potassium Chloride Calcium Magnesium
110 2.4 5.2 0.18 92 2.2 9.4 0.16 1.8 0.08
96 2.8* 3.4 0.16* 64 3.2* 5.4 0.18* 0.85 0.09*
111 2.8 5.3 0.15 92 3.2 9.4 0.18 1.8 0.06
107 3.6ns 5.2 0.14ns 90 2.7ns 9.3 0.21ns 1.7 0.05ns
Values are represented as mean SD. A p < 0.05 is considered as significantly different between groups. Asterisk “*” indicates significant from controls, “ns” indicates not significant from controls and PSE alone administered rats. Values of electrolytes are expressed as Mm/L and minerals values are expressed as mg/dL.
magnesium levels were also decreased in alcohol administered rats. PSE-administration to alcohol-administered rats significantly restored these alterations to normal levels compared to alcohol alone administered rats. Lipid peroxidation measured in terms of TBARS (Fig. 2a). Kidney TBARS levels were significantly elevated in alcohol-administered rats. However, administration of PSE to alcohol receiving rats (p < 0.05) prevented this elevation when compared with alcohol alone administered rats. Total nitrites and nitrate levels, an index of nitric oxide production were measured in kidney (Fig. 2b). Alcoholadministered rats showed a significant increase in NOx levels. However, administration of PSE to alcohol-administered rats significantly prevented the elevation of NOx levels in the kidney. Furthermore, the effect of alcohol on kidney Na+/K+-ATPase enzyme activity were determined (Fig. 3). Administration of PSE to alcohol-administered rats indicates the restoration of Na+/K+ATPase activity to near normal levels. The effect of PSE on kidney enzymatic and non-enzymic antioxidants in alcohol- administered rats were determined (Table 3). The activities of antioxidant enzymes viz., GPx, GST, GR, SOD, catalase and the concentration of GSH was markedly decreased in alcohol- administered rats when compared with other experimental groups. Administering PSE to alcohol treated rats significantly (p < 0.05) increased these antioxidant enzyme activities close to normal levels. PSE alone administered rats did not show any significant difference compared with controls. Histological changes brought about by alcohol administration in kidney were noticed (Fig. 4). Microscopic examination of kidney sections of alcohol-administered rats showed degeneration and necrotic changes of tissue with distortion of normal architecture
0
40
*
35
ns
Nitrite/nitrate levels (nmol/mg protein)
Kidney TBARS (nmol MDA/mg protein)
0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1
12 10 8
*
6 4 2 0
Fig. 3. Effect of PSE on kidney Na+/K+-ATPase activity in alcohol-administered rats. Values are represented as the mean SD (n = 8). A p < 0.05 is considered as significantly different between groups. Asterisk“*” indicates significant from controls, “ns” indicates not significant from controls and PSE alone administered rats.
Table 3 Effect of PSE on kidney antioxidants and GSH content in alcohol-administered rats. Parameter
Control
Alcohol
PSE
Alcohol + PSE
GSH GPx GR GST SOD CAT
2.4 0.2 7.9 0.15 31.4 1.5 67.5 2.4 23.4 0.8 5.9 0.06
1.2 0.1* 4.5 0.15* 22.6 1.6* 50 1.4* 16.3 0.5* 2.6 0.12*
2.4 0.3 7.8 0.16 30.8 1.5 67.9 2.6 23.5 0.7 5.9 0.08
2.3 0.12ns 7.4 0.18ns 29.4 1.5ns 66.2 2.2ns 22.2 0.8ns 5.8 0.08ns
Values are represented as the mean SD of eight rats in each group. GSH is expressed as mg/mg protein and remaining values are expressed as mmole/min/mg protein. A p < 0.05 is considered as significantly different between groups. Asterisk “*” indicates significant from controls, “ns” indicates not significant from controls and PSE alone administered rats.
and tissue congestion in comparison with other experimental groups. PSE-administration to alcohol rats restored these histopathological changes in the kidney to near normal.
b
a
ns
14 Na+/K+-ATPase activity (µmole/min/mg ptn)
Parameter
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4. Discussion
*
30 25
ns
20 15 10 5 0
Fig. 2. Effect of PSE on kidney (a) TBARS and (b) NOx levels in alcohol-administered rats. Values are represented as the mean SD (n = 8). A p < 0.05 is considered as significantly different between groups. Asterisk“*” indicates significant from controls, “ns” indicates not significant from controls and PSE alone administered rats.
Kidneys along with liver, which are the primary organs for the oxidation of ingested alcohol result in the production of harmful metabolites such as acetaldehyde and acetate. Several reports indicated renal tubular necrosis and dysfunction in cases with prolonged alcohol consumption [9,38]. In the present study, plasma urea and creatinine levels were increased with a decrease of uric acid concentration in alcohol-administered rats compared to controls. Urea, creatinine and uric acid are the catabolites of ammonia, creatine and purine nucleotides respectively released into blood and are eliminated by the kidney. Due to a higher sensitivity of the glomerular region to oxidative damage induced by alcohol would have decreased the filtration rate and clearance of substances in alcohol-administered rats [5]. Previous studies also reported that increased plasma urea and creatinine levels cause impairment of kidney function such as acute glomerulonephritis, nephrosclerosis and tubular necrosis [39]. Administration of PSE to alcoholic rats significantly lowered the elevated levels of plasma
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Fig. 4. Histological micrograph of kidney sections of rats stained with haematoxylin and eosin, original magnification at 100. Control rats kidney showed normal renal parenchyma, tubules and glomeruli. Alcohol-administered rats showed congestion of blood vessels, necrosis of renal cells and degenerative changes in tubules. PSE alone administered rats showed normal kidney architecture similar to controls. Alcohol and PSE co-administered rats showed regeneration of blood vessels, renal cells and tubules.
urea and creatinine levels with increase in uric acid concentration, further confirming the protective effect of the PSE against alcoholinduced glomerular damage from the rectified histopathological observations. Chronic alcohol consumption disrupts mineral homeostasis and significantly increases the risk of kidney disease [26]. In this study, biochemically and physiologically important electrolytes and minerals concentrations were measured. Plasma sodium, potassium, chloride, calcium and magnesium were decreased in alcoholadministered rats compared to the controls. Plasma electrolytes maintain intravascular homeostasis with interstitial and intracellular space. Alcohol consumption induces hyperurecemia and severe loss of electrolytes and minerals, which would result in plasma hyponatremia, hypokalemia, hypomagnesia, hypocalcemia and hypochloremia. Adewale and Ifudu studies reported that alcohol generated ROS/RNS cause severe membrane damage, which causes a decrease in the reabsorption of minerals at the level of the distal tubules and ascending limb of Henles loop [7]. Membrane stability is vital for the normal functioning of membrane lipids and proteins, which include receptors and enzymes. Na+/K+-ATPase is a membrane bound enzyme which actively transports Na+ and K+ ions across cell membranes to establish and maintain the characteristic transmembrane gradients of Na+ and K+ ions [40]. In this study, Na+/K+-ATPase activity in the kidney was decreased in alcohol-administered rats, however, the administration of PSE restored the activity of the enzyme. The effect of alcohol on the activity of Na+/K+-ATPase might have decreased the tubular reabsorption of calcium along with sodium and potassium. PSE-administration to alcoholic rats restored the plasma electrolytes and minerals to normal levels. The therapeutic effect of phytocompounds present in PSE would have modified membrane fatty acid composition, fluidity, permeability and electrolyte homeostasis by decreasing alcohol-induced
oxidative/nitrosative stress through free radical scavenging activity. To further understand the alcohol-induced damage of the kidney, TBARS, NOx levels and antioxidant enzyme activities in alcohol-administered rats were studied. Lipid peroxidation has been reported as a marker of oxidative stress. Free radicals induce lipid peroxidation, resulting in reactive molecules such as malondialdehyde MDA and 4-hydroxy-3-nonenal (HNE) [41]. In this study, kidney TBARS levels were significantly decreased in PSE-administration to alcoholic rats compared with alcohol alone administered rats. The radical scavenging potential of lignans and malanoxoin compounds present in PSE might have inhibited the propagation of lipid radicals, thereby preventing the formation of lipid peroxidation. Alcohol consumption causes over-production of NOx by various mechanisms. Much toxicity of NOx is due to formation of potent oxidants like peroxynitrite, which participates in ethanol induced oxidative stress [42]. In this study, kidney NOx levels were increased in alcohol-administered rats compared to the controls, but PSE-administration to alcoholic rats prevented this elevation. The strong nitric oxide radical scavenging property of PSE would have modulated the overproduction of NOx in alcoholic rats. Thus, in one way the augmentation of the oxidative stress is prevented by the PSE-administration. Renal metabolism of alcohol takes place through renal alcohol dehydrogenase, CYP2E1 and CYP24A1 which causes the production of ROS/RNS, eliciting the oxidative stress in the kidney [43,44]. ROS provoked damage in organisms is counteracted by enzymatic mechanisms of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR) and non-enzymatic mechanisms, including reduced glutathione (GSH), uric acid, specific chelating proteins and essential mineral and nutritional antioxidants [45]. In the present study, antioxidant enzymes, GPx, GST, GR, CAT, SOD activities and the content of GSH
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significantly decreased in alcohol-administered rats compared to controls. Free radical dependent or oxidative enzyme inactivation might be the reason for the decreased activities of antioxidant enzymes in alcohol fed rats. PSE- administration to alcohol rats restored the SOD, CAT, GPx, GR, GST activities and the content of GSH. SOD catalizes the conversion of superoxide radical to hydrogen peroxide and CAT catalizes the reduction of hydrogen peroxide, and thereby protects the tissues against reactive hydroxyl radicals. GSH is an endogenous antioxidant and plays a significant role in the detoxification of xenobiotics and maintenance the redox status of the cells. To reverse the oxidative stress induced by alcohol in the kidney, many authors have recently focused their research towards antioxidant phytoextracts [46,47]. Phenolic compounds act as potent scavengers of superoxide, hydroxyl and nitric oxide radicals. Pterolinus and pterostilbenes present in PSE might have scavenged O , OH and N radicals [18,22] or reversed the oxidative inactivation of antioxidant enzymes or further would have triggered antioxidant enzyme synthesis in PSE-administered rats. PSE might have maintained the cellular status of antioxidant enzymes, making it available to detoxify the toxic metabolites produced in the course of ethanol metabolism and decrease oxidative stress. Alcohol-induced nephro-toxicity was further evidenced from histopathological studies. Photomicrographs represent the glomerular tubules, interstitium and blood vessels in controls and different experimental groups. Alcohol-administered rats showed degenerative changes, constriction of blood vessels and inflammatory infiltration compared to control rats and these results are in agreement with previous reports [48]. Alcohol and PSE-administered rats showed a marked reduction in the degenerative changes of glomerular as evidenced by the normal cellularity of the interstitium and reduction in the constriction of blood vessels. PSE alone administered rats showed a normal physiology of glomerular and blood vessels. The inhibition of the deleterious effects produced by free radicals, enhanced supply of antioxidants and regeneration of glomerular region in the kidney might result from the potential therapeutic phytocompounds like lignans and pterostilbenes present in PSE. In conclusion, multiple phytocompounds present in PSE might act at different levels of altered biochemical, pathological and morphological changes in the kidney and finally alleviate the alcohol-induced nephro-toxicity. Conflict of interest The authors declare that there are no conflicts of interest. Acknowledgement Authors thank University Grants Commission (UGC), New Delhi, India for providing UGC-BSR-MF to Saradamma Bulle. References [1] M. Epstein, Alcohol's impact on kidney function, Alcohol Health. Res. World 21 (1997) 84–92. [2] D. Dinu, M.T. Nechifor, L. Movileanu, Ethanol-induced alterations of the antioxidant defense system in rat kidney, J. Biochem. Mol. Toxicol. 19 (2005) 386–395. [3] C. Latchoumycandane, L.E. Nagy, T.M. Mclntyre, Chronic ethanol ingestion induces oxidative kidney injury through taurine-inhibitable inflammation, Free Radic. Biol. Med. 69 (2014) 403–416. [4] P.S. Harris, S.R. Roy, C. Coughlan, D.J. Orlicky, Y. Liang, C.T. Shearn, J.R. Roede, K. S. Fritz, Chronic ethanol consumption induces mitochondrial protein acetylation and oxidative stress in the kidney, Redox. Biol. 6 (2015) 33–40. [5] M.L. Ojeda, M.J. Barrero, M.L. Noales, M.L. Murillo, O. Carreras, Oxidative effects of chronic ethanol consumption on the functions of heart and kidney: folic acid supplementation, Alcohol Alcohol. 47 (2012) 404–412. [6] S.D. Kumar, D.M. Vasudevan, Alcohol induced effects on kidney, Indian J. Clin. Biochem. 23 (2008) 4–9.
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Nephro-protective action of P. santalinus against alcohol-induced biochemical alterations and oxidative damage in rats Saradamma Bullea , Vaddi Damodara Reddya , Ananda Vardhan Hebbania,b , Pannuru Padmavathic , Chandrasekhar Challad , Pavan Kumar Puvvadae , Elisha Repallee, Devanna Nayakantid, Chandrakala Aluganti Narasimhuluf , Varadacharyulu Nallanchakravarthulaa,* a
Department of Biochemistry, Sri Krishnadevaraya University, Anantapur, 515 003, Andhra Pradesh, India Department of Biotechnology, New Horizon College of Engineering, Bangalore, 560103, India c Oil Technological Research Institute, Jawaharlal Nehru Technological University, Anantapur, 515 001, Andhra Pradesh, India d Department of Chemistry, Jawaharlal Nehru Technological University, Anantapur, 515 002, Andhra Pradesh, India e DR Biosciences, Research and Development Institute, Jayanagar, Bangalore, Karnataka, 560 011, India f Department of Medicine, University of Central Florida, Orlando, FL, 32816, USA b
A R T I C L E I N F O
Article history: Received 21 August 2016 Received in revised form 23 September 2016 Accepted 27 September 2016 Keywords: Alcohol P. santalinus Nephro-toxicity Oxidative stress/Nitrosative stress
A B S T R A C T
The present study investigated the antioxidant potential of P. santalinus heartwood methanolic extract (PSE) against alcohol-induced nephro-toxicity. The results indicated an increase in the concentration of kidney damage plasma markers, urea and creatinine with a concomitant decrease in the concentration of uric acid in alcohol-administered rats. A significant decrease in plasma electrolytes and mineral levels with increased kidney thiobarbituric acid reactive substances (TBARS) and nitric oxide (NOx) levels was also observed. PSE treatment to alcohol-administered rats effectively prevented the elevation in TBARS and NOx levels. Decreased activity of Na+/K+-ATPase in alcohol administered rats was brought to near normal levels with treatment of PSE. Chronic alcohol consumption affects antioxidant enzymatic activity and reabsorption function of the kidney which is evident from the decreased level of GSH as well as the activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR) and glutathione s-transferase (GST). However, treatment with PSE to alcoholadministered rats significantly enhanced these enzymatic activities and reduced glutathione (GSH) content close to normal level. Alcohol-induced organ damage was evident from morphological changes in the kidney. Nevertheless, administration of PSE effectively restored these morphological changes to normal. The flavonoid and tannoid compounds might have protective activity against alcohol-induced oxidative/nitrosative stress mediated kidney damage. ã 2016 Elsevier Masson SAS. All rights reserved.
1. Introduction Chronic alcohol consumption induces dramatic changes in the biochemical and physiological processes of every organ system including kidney [1,2]. Chronic ethanol consumption alters cellular functions by acting on DNA, RNA, protein and metabolites and leading to kidney dysfunction [3]. Emerging evidence indicates alcohol-induced toxicity is due to some common mechanisms like oxidative stress, inflammation and apoptotic or other signaling pathways [4]. In kidney, reactive oxygen species (ROS) mediated
* Corresponding author. E-mail address: [email protected] (V. Nallanchakravarthula). http://dx.doi.org/10.1016/j.biopha.2016.09.103 0753-3322/ã 2016 Elsevier Masson SAS. All rights reserved.
toxicity and oxidative stress has been considered as the primary pathways to alcohol-induced injury [5]. The kidney is an important organ actively involved in maintaining body fluid homeostasis by reabsorbing important material and excreting waste products. Electrolytes and minerals are the chief components of body fluids, which act as catalysts in most cellular enzyme catalyzed reactions. Alcohol consumption produces functional abnormalities in the kidney depending on the quantity of alcohol ingested [6]. Abnormal concentrations of these catabolites and electrolytes in the plasma or serum can serve as a clear indication of renal function impairment [7]. Studies have revealed that the renal tissue membrane is highly vulnerable to damage caused by ROS due to the abundance of long chain polyunsaturated fatty acids in renal lipids [8]. Na+/K+-ATPase
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present on the renal tubular epithelial cells may promote the renal tubular reabsorption of sodium. But previous studies have shown that alcohol interferes with the carrier functions of proximal tubular cells by decreasing the Na+/K+-ATPase activity in rats [9]. Chronic alcohol consumption induces the expression of inducible nitric oxide synthase (iNOS) in the kidney [10]. Increased NOx and its active metabolite peroxynitrite (ONOO ) cross cell membranes through anion channels and inactivate many biologically important proteins and enzymes through nitration of tyrosine [11]. Increased ROS/RNS may damage the renal membrane at the glomerular region or Henle's loop, altering the reabsorption of electrolytes and minerals. Knowledge gained by a better understanding of various mechanisms of tissue injury will provide new avenues for developing effective treatment of alcohol-induced organ damage [12]. Now the world is turned towards plant medicine with less or no side effects, in particular, treatments using locally available phyto- extracts which are found to be good resources for the prevention of diseases. Pterocarpus santalinus, a Fabaceae member, grows in the tropical regions of the world, especially in India, Sri Lanka, Taiwan and China [13]. Phytochemical analysis of plant extract showed the presence of several specific components in heartwood powder in particular santalin A, B and Y, pterocarpol, pterocarptriol, isopterocarpalone, pterocarpodiolone, cryptomeridol and several non specific compounds such as isoflavones, isoflavonoid glucosides, triterpenes, sesquiterpenes, b-sitosterol, lupeol, epicatechin, lignans and pterostilbeans [14,15]. P. santalinus heartwood extract showed antioxidant [16], anti-cancer [17], hepato-protective [18], gastro-protective [19], and anti-diabetic properties [20], along with angiogenic and wound-healing properties [21]. The risk of cell injury resulting from oxidative stress may be prevented by natural phytocompounds. It has been established that phytocompounds can restore the balance between the endogenous antioxidants and free radicals directly by scavenging ROS and the induction of endogenous defences like glutathione and superoxide dismutase [22]. Beneficial effects of several phyto-extracts might result from the combination of phytochemicals acting via additive or synergistic mechanisms. Despite P. santalinus diverse medicinal properties, no study has been carried out using the heart wood extract against alcohol-induced biochemical alterations and neuronal damage. Hence, the present study was carried out to study the therapeutic potential of P. santalinus against alcoholinduced nephro-toxicity.
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light-dark cycle in the University animal house. After acclimatization for a week, animals were divided into four groups (n = 8) viz., group-I, served as controls, group-II, alcohol, group-III, PSE alone and group-IV, alcohol and PSE. Alcohol (20%) was administered at a dose of 5 g/kg b.wt/day, PSE was administered at a dose of 250 mg/ kg b.wt/day. Control rats received iso-caloric glucose instead of alcohol. All treatments were given for a period of 60 days. Experimentation and animal maintenance were done with prior approval of the institutional animal ethics committee. At the end of the experimental period the animals were fasted over night and sacrificed by cervical dislocation. Immediately blood was collected into heparinised tubes by cardiac puncture and plasma was separated from cells by centrifugation at 3000 rpm for 10 min. Tissues were collected and stored at 80 C until assays were carried out. A Small section of the kidney was fixed in 10% neutral formalin solution for histo-pathological analysis. 2.3. HPLC analysis of PSE extract PSE (5 mg) was dissolved in 1 ml of methanol and 20 ml of this was injected in to an HPLC system equipped with UV-vis detector (Shimadzu SPD10A UV–vis, Japan) set at 280 nm. Polyphenols were chromatographically separated on a reverse phase Luna 5 mm C18 (100 Å, LC Column 250 mm 4.6 mm). A solvent mixture of methanol/phosphate buffer (pH 3.0) taken in the ratio of 70:30 was used as a mobile phase and with a flow rate of 1 ml/min for the isocratic elution of hydrophobic polyphenols was achieved. Presence and quantification of polyphenols in PSE was achieved by comparing the chromatogram with that of standards (100 mg/ ml) such as gallic acid, rutin and quercetin. 2.4. Quantification of total phenolics, flavonoids and tannins Quantification of total phenolics and flavonoids present in the PSE was analyzed as described by Cetkovic et al. [23] and the tannin content was determined as described previously [24]. 2.5. Measurement of nitrogenous compounds, electrolytes and minerals
2. Materials and methods
Plasma nitrogenous compounds were measured by using commercially available kits (Span Diagnostics, Surat, India). Plasma sodium, potassium, chloride, calcium, urea, creatinine, uric acid and magnesium were determined as described previously [25,26].
2.1. Preparation of extract
2.6. Measurement of TBARS and nitric oxide levels
P. santalinus heartwood pieces were procured from locally available plant and the voucher specimens were deposited at Sri Krishnadevaraya University Herbarium, Anantapur. The pieces were used to prepare P. santalinus heartwood extract (PSE). Heartwood powder was placed in a mixture of methanol and sterile distilled water (80:20, v/v) for 48 h and the mixture was thoroughly stirred until the extract had been dissolved. The mixture was then centrifuged at 2500 rpm for 10 min and the supernatant was then filtered and dried at 45 C using rotary evaporator. The extract was dissolved in water for experimental use.
Kidney tissue was homogenized (10% w/v) in Tris buffer (0.1 M, pH 7.4), centrifuged (10,000g for 20 min at 4 C) and the supernatant was used for all the biochemical parameters. Kidney thiobarbituric acid reactive species (TBARS) were measured by the formation of malondialdehyde as described previously [27]. Total NOx in the form of nitrite and nitrate levels in the kidney was measured as described by Sastry et al. [28].
2.2. Animals Two month old male albino Wistar rats weighing 120–140 g were procured from Sri Venkateswara Agencies, Bangalore, India. Animals were maintained on a standard pellet diet (M/s. Hindustan Lever Ltd., Mumbai, India) and water ad libitum with 24 h
2.7. Assays of non-enzymic, enzymic antioxidants and Na+/K+-ATPase in kidney Total reduced glutathione content was measured in the kidney by following the method of Ellman’s [29]. Activities of glutathione reductase [30], glutathione peroxidase [31], glutathione-s-transferase [32], catalase [33] and superoxide dismutase [34] were measured. Protein concentration was estimated by the method of Lowry et al. [35]. Na+/K+-ATPase activity was measured by following the method of Kartz and Epstein [36].
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2.8. Histopathological studies A portion of the kidney was dissected and fixed in 10% neutral buffered formalin solution for 24 h [37]. The fixed tissue was processed routinely, and then embedded in paraffin, sectioned to 3–5 mm thickness, deparaffinised and rehydrated using standard techniques. Morphological changes in kidney sections were observed by staining with hematoxylin and eosin, original magnification 100. 2.9. Statistical analysis Data were subjected to statistical analyses, values are mean SD of 8 rats in each group. Student t-test followed by Duncan’s Multiple Range (DMR) test was performed to determine significance between groups. 3. Results HPLC analysis of heartwood extract of P. santalinus reveals the presence of multiple polyphenolic compounds (Fig. 1a). The individual phytocompounds present in extract were compared with standards, gallic acid, rutin and quercetin. Total phytocompounds, total phenolics, flavonoids and tannins present in PSE
were quantified (Fig. 1b). The chemical structures of significant phytocompounds pterolinus B (Fig. 1c) and pterostilbene (Fig. 1d) were also presented. Plasma nitrogenous compounds, urea and creatinine levels were significantly (p < 0.05) increased in alcohol-administered rats with concomitant decrease in uric acid concentration (Table 1). PSE treatment to alcohol administered rats restored the levels of urea, creatinine and uric acid to near normal compared to alcohol alone administered rats. In addition, chronic alcohol administration also altered the serum electrolytes and minerals (Table 2). Sodium, potassium and chloride levels were decreased in alcohol-administered rats as compared to controls. Moreover, calcium and Table 1 Effect of PSE on plasma urea, uric acid and creatinine levels in alcohol-administered rats. Parameter
Control
Alcohol
PSE
Alcohol + PSE
Urea Uric acid Creatinine
25.2 1.7 2.3 0.05 0.42 0.06
34.8 2.7* 1.52 0.07* 0.95 0.05*
25.7 2.0 2.3 0.05 0.42 0.08
27.5 2.5ns 2.2 0.08ns 0.48 0.07ns
Values are represented as mean SD. A p < 0.05 is considered as significantly different between groups. Asterisk “*” indicates significant from controls, “ns” indicates not significant from controls and PSE alone administered rats. Urea, uric acid and creatinine values are expressed as mg/dL.
Fig. 1. (a) High performance liquid chromatography analysis (HPLC) of PSE, (b) quantification data of total phenolics, flavonoids and tannins. The chemical structures of pterolinus B (c) and pterostilbene (d). Total Phenolics and flavonoids were expressed in terms of gram equivalent of gallic acid/gram extract, tannins were expressed in terms of gram equivalent of tannic acid/gram extract.
S. Bulle et al. / Biomedicine & Pharmacotherapy 84 (2016) 740–746 Table 2 Effect of PSE on plasma electrolytes and minerals in alcohol-administered rats. Control
Alcohol
PSE
Alcohol + PSE
Sodium Potassium Chloride Calcium Magnesium
110 2.4 5.2 0.18 92 2.2 9.4 0.16 1.8 0.08
96 2.8* 3.4 0.16* 64 3.2* 5.4 0.18* 0.85 0.09*
111 2.8 5.3 0.15 92 3.2 9.4 0.18 1.8 0.06
107 3.6ns 5.2 0.14ns 90 2.7ns 9.3 0.21ns 1.7 0.05ns
Values are represented as mean SD. A p < 0.05 is considered as significantly different between groups. Asterisk “*” indicates significant from controls, “ns” indicates not significant from controls and PSE alone administered rats. Values of electrolytes are expressed as Mm/L and minerals values are expressed as mg/dL.
magnesium levels were also decreased in alcohol administered rats. PSE-administration to alcohol-administered rats significantly restored these alterations to normal levels compared to alcohol alone administered rats. Lipid peroxidation measured in terms of TBARS (Fig. 2a). Kidney TBARS levels were significantly elevated in alcohol-administered rats. However, administration of PSE to alcohol receiving rats (p < 0.05) prevented this elevation when compared with alcohol alone administered rats. Total nitrites and nitrate levels, an index of nitric oxide production were measured in kidney (Fig. 2b). Alcoholadministered rats showed a significant increase in NOx levels. However, administration of PSE to alcohol-administered rats significantly prevented the elevation of NOx levels in the kidney. Furthermore, the effect of alcohol on kidney Na+/K+-ATPase enzyme activity were determined (Fig. 3). Administration of PSE to alcohol-administered rats indicates the restoration of Na+/K+ATPase activity to near normal levels. The effect of PSE on kidney enzymatic and non-enzymic antioxidants in alcohol- administered rats were determined (Table 3). The activities of antioxidant enzymes viz., GPx, GST, GR, SOD, catalase and the concentration of GSH was markedly decreased in alcohol- administered rats when compared with other experimental groups. Administering PSE to alcohol treated rats significantly (p < 0.05) increased these antioxidant enzyme activities close to normal levels. PSE alone administered rats did not show any significant difference compared with controls. Histological changes brought about by alcohol administration in kidney were noticed (Fig. 4). Microscopic examination of kidney sections of alcohol-administered rats showed degeneration and necrotic changes of tissue with distortion of normal architecture
0
40
*
35
ns
Nitrite/nitrate levels (nmol/mg protein)
Kidney TBARS (nmol MDA/mg protein)
0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1
12 10 8
*
6 4 2 0
Fig. 3. Effect of PSE on kidney Na+/K+-ATPase activity in alcohol-administered rats. Values are represented as the mean SD (n = 8). A p < 0.05 is considered as significantly different between groups. Asterisk“*” indicates significant from controls, “ns” indicates not significant from controls and PSE alone administered rats.
Table 3 Effect of PSE on kidney antioxidants and GSH content in alcohol-administered rats. Parameter
Control
Alcohol
PSE
Alcohol + PSE
GSH GPx GR GST SOD CAT
2.4 0.2 7.9 0.15 31.4 1.5 67.5 2.4 23.4 0.8 5.9 0.06
1.2 0.1* 4.5 0.15* 22.6 1.6* 50 1.4* 16.3 0.5* 2.6 0.12*
2.4 0.3 7.8 0.16 30.8 1.5 67.9 2.6 23.5 0.7 5.9 0.08
2.3 0.12ns 7.4 0.18ns 29.4 1.5ns 66.2 2.2ns 22.2 0.8ns 5.8 0.08ns
Values are represented as the mean SD of eight rats in each group. GSH is expressed as mg/mg protein and remaining values are expressed as mmole/min/mg protein. A p < 0.05 is considered as significantly different between groups. Asterisk “*” indicates significant from controls, “ns” indicates not significant from controls and PSE alone administered rats.
and tissue congestion in comparison with other experimental groups. PSE-administration to alcohol rats restored these histopathological changes in the kidney to near normal.
b
a
ns
14 Na+/K+-ATPase activity (µmole/min/mg ptn)
Parameter
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4. Discussion
*
30 25
ns
20 15 10 5 0
Fig. 2. Effect of PSE on kidney (a) TBARS and (b) NOx levels in alcohol-administered rats. Values are represented as the mean SD (n = 8). A p < 0.05 is considered as significantly different between groups. Asterisk“*” indicates significant from controls, “ns” indicates not significant from controls and PSE alone administered rats.
Kidneys along with liver, which are the primary organs for the oxidation of ingested alcohol result in the production of harmful metabolites such as acetaldehyde and acetate. Several reports indicated renal tubular necrosis and dysfunction in cases with prolonged alcohol consumption [9,38]. In the present study, plasma urea and creatinine levels were increased with a decrease of uric acid concentration in alcohol-administered rats compared to controls. Urea, creatinine and uric acid are the catabolites of ammonia, creatine and purine nucleotides respectively released into blood and are eliminated by the kidney. Due to a higher sensitivity of the glomerular region to oxidative damage induced by alcohol would have decreased the filtration rate and clearance of substances in alcohol-administered rats [5]. Previous studies also reported that increased plasma urea and creatinine levels cause impairment of kidney function such as acute glomerulonephritis, nephrosclerosis and tubular necrosis [39]. Administration of PSE to alcoholic rats significantly lowered the elevated levels of plasma
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Fig. 4. Histological micrograph of kidney sections of rats stained with haematoxylin and eosin, original magnification at 100. Control rats kidney showed normal renal parenchyma, tubules and glomeruli. Alcohol-administered rats showed congestion of blood vessels, necrosis of renal cells and degenerative changes in tubules. PSE alone administered rats showed normal kidney architecture similar to controls. Alcohol and PSE co-administered rats showed regeneration of blood vessels, renal cells and tubules.
urea and creatinine levels with increase in uric acid concentration, further confirming the protective effect of the PSE against alcoholinduced glomerular damage from the rectified histopathological observations. Chronic alcohol consumption disrupts mineral homeostasis and significantly increases the risk of kidney disease [26]. In this study, biochemically and physiologically important electrolytes and minerals concentrations were measured. Plasma sodium, potassium, chloride, calcium and magnesium were decreased in alcoholadministered rats compared to the controls. Plasma electrolytes maintain intravascular homeostasis with interstitial and intracellular space. Alcohol consumption induces hyperurecemia and severe loss of electrolytes and minerals, which would result in plasma hyponatremia, hypokalemia, hypomagnesia, hypocalcemia and hypochloremia. Adewale and Ifudu studies reported that alcohol generated ROS/RNS cause severe membrane damage, which causes a decrease in the reabsorption of minerals at the level of the distal tubules and ascending limb of Henles loop [7]. Membrane stability is vital for the normal functioning of membrane lipids and proteins, which include receptors and enzymes. Na+/K+-ATPase is a membrane bound enzyme which actively transports Na+ and K+ ions across cell membranes to establish and maintain the characteristic transmembrane gradients of Na+ and K+ ions [40]. In this study, Na+/K+-ATPase activity in the kidney was decreased in alcohol-administered rats, however, the administration of PSE restored the activity of the enzyme. The effect of alcohol on the activity of Na+/K+-ATPase might have decreased the tubular reabsorption of calcium along with sodium and potassium. PSE-administration to alcoholic rats restored the plasma electrolytes and minerals to normal levels. The therapeutic effect of phytocompounds present in PSE would have modified membrane fatty acid composition, fluidity, permeability and electrolyte homeostasis by decreasing alcohol-induced
oxidative/nitrosative stress through free radical scavenging activity. To further understand the alcohol-induced damage of the kidney, TBARS, NOx levels and antioxidant enzyme activities in alcohol-administered rats were studied. Lipid peroxidation has been reported as a marker of oxidative stress. Free radicals induce lipid peroxidation, resulting in reactive molecules such as malondialdehyde MDA and 4-hydroxy-3-nonenal (HNE) [41]. In this study, kidney TBARS levels were significantly decreased in PSE-administration to alcoholic rats compared with alcohol alone administered rats. The radical scavenging potential of lignans and malanoxoin compounds present in PSE might have inhibited the propagation of lipid radicals, thereby preventing the formation of lipid peroxidation. Alcohol consumption causes over-production of NOx by various mechanisms. Much toxicity of NOx is due to formation of potent oxidants like peroxynitrite, which participates in ethanol induced oxidative stress [42]. In this study, kidney NOx levels were increased in alcohol-administered rats compared to the controls, but PSE-administration to alcoholic rats prevented this elevation. The strong nitric oxide radical scavenging property of PSE would have modulated the overproduction of NOx in alcoholic rats. Thus, in one way the augmentation of the oxidative stress is prevented by the PSE-administration. Renal metabolism of alcohol takes place through renal alcohol dehydrogenase, CYP2E1 and CYP24A1 which causes the production of ROS/RNS, eliciting the oxidative stress in the kidney [43,44]. ROS provoked damage in organisms is counteracted by enzymatic mechanisms of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR) and non-enzymatic mechanisms, including reduced glutathione (GSH), uric acid, specific chelating proteins and essential mineral and nutritional antioxidants [45]. In the present study, antioxidant enzymes, GPx, GST, GR, CAT, SOD activities and the content of GSH
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significantly decreased in alcohol-administered rats compared to controls. Free radical dependent or oxidative enzyme inactivation might be the reason for the decreased activities of antioxidant enzymes in alcohol fed rats. PSE- administration to alcohol rats restored the SOD, CAT, GPx, GR, GST activities and the content of GSH. SOD catalizes the conversion of superoxide radical to hydrogen peroxide and CAT catalizes the reduction of hydrogen peroxide, and thereby protects the tissues against reactive hydroxyl radicals. GSH is an endogenous antioxidant and plays a significant role in the detoxification of xenobiotics and maintenance the redox status of the cells. To reverse the oxidative stress induced by alcohol in the kidney, many authors have recently focused their research towards antioxidant phytoextracts [46,47]. Phenolic compounds act as potent scavengers of superoxide, hydroxyl and nitric oxide radicals. Pterolinus and pterostilbenes present in PSE might have scavenged O , OH and N radicals [18,22] or reversed the oxidative inactivation of antioxidant enzymes or further would have triggered antioxidant enzyme synthesis in PSE-administered rats. PSE might have maintained the cellular status of antioxidant enzymes, making it available to detoxify the toxic metabolites produced in the course of ethanol metabolism and decrease oxidative stress. Alcohol-induced nephro-toxicity was further evidenced from histopathological studies. Photomicrographs represent the glomerular tubules, interstitium and blood vessels in controls and different experimental groups. Alcohol-administered rats showed degenerative changes, constriction of blood vessels and inflammatory infiltration compared to control rats and these results are in agreement with previous reports [48]. Alcohol and PSE-administered rats showed a marked reduction in the degenerative changes of glomerular as evidenced by the normal cellularity of the interstitium and reduction in the constriction of blood vessels. PSE alone administered rats showed a normal physiology of glomerular and blood vessels. The inhibition of the deleterious effects produced by free radicals, enhanced supply of antioxidants and regeneration of glomerular region in the kidney might result from the potential therapeutic phytocompounds like lignans and pterostilbenes present in PSE. In conclusion, multiple phytocompounds present in PSE might act at different levels of altered biochemical, pathological and morphological changes in the kidney and finally alleviate the alcohol-induced nephro-toxicity. Conflict of interest The authors declare that there are no conflicts of interest. Acknowledgement Authors thank University Grants Commission (UGC), New Delhi, India for providing UGC-BSR-MF to Saradamma Bulle. References [1] M. Epstein, Alcohol's impact on kidney function, Alcohol Health. Res. World 21 (1997) 84–92. [2] D. Dinu, M.T. Nechifor, L. Movileanu, Ethanol-induced alterations of the antioxidant defense system in rat kidney, J. Biochem. Mol. Toxicol. 19 (2005) 386–395. [3] C. Latchoumycandane, L.E. Nagy, T.M. Mclntyre, Chronic ethanol ingestion induces oxidative kidney injury through taurine-inhibitable inflammation, Free Radic. Biol. Med. 69 (2014) 403–416. [4] P.S. Harris, S.R. Roy, C. Coughlan, D.J. Orlicky, Y. Liang, C.T. Shearn, J.R. Roede, K. S. Fritz, Chronic ethanol consumption induces mitochondrial protein acetylation and oxidative stress in the kidney, Redox. Biol. 6 (2015) 33–40. [5] M.L. Ojeda, M.J. Barrero, M.L. Noales, M.L. Murillo, O. Carreras, Oxidative effects of chronic ethanol consumption on the functions of heart and kidney: folic acid supplementation, Alcohol Alcohol. 47 (2012) 404–412. [6] S.D. Kumar, D.M. Vasudevan, Alcohol induced effects on kidney, Indian J. Clin. Biochem. 23 (2008) 4–9.
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