Renoprotective Effect of Grape Seed Extract against Oxidative Stress Induced by Gentamicin and Hypercholesterolemia in Rats

Abstract

Rationale: Kidneys are dynamic organs and represent one of the major systems maintaining the body homeostasis; they are affected by many chemicals and drugs. Grape seed extract (GSE) has been targeted to prevent drug-induced renal toxicity. Objectives: This study investigates the possible renoprotective effect of GSE against oxidative stress, renal impairment, and hypercholesterolemia (HC) induced by gentamicin (GM) and cholesterol-enriched diet. Seventy adult male Wistar rats (160 ± 10 g) were divided into seven groups: (1) served as control, (2) GSE, (3) GM, (4) GSE + GM, (5) hypercholesterolemic (HC) group, (6) GM + HC group, and (7) GM + HC + GSE. Kidney functions, inflammatory mediators, cytokines, lipid profile, nitric oxide (NO), cyclic guanosine monophosphate (cGMP), and oxidative and antioxidative stress parameters were assessed in all groups. Main findings: GM induced renal dysfunction, which was exacerbated by the presence of HC as confirmed by laboratory determinations. Administration of GSE attenuated the renal toxicity evidenced in significant reduction in elevated kidney function, inflammatory cytokines as well as lipid profile, NO, cGMP, enzymatic, and nonenzymatic antioxidants. Conclusion: Administration of GSE simultaneously with GM attenuated oxidative stress, diminished renal toxicity, and improved lipid profile induced by GM and HC.

• nephrotoxicity
• hypercholesterolemia
• gentamicin
• oxidative stress
• rats

INTRODUCTION

A number of environmental contaminants, chemicals, and drugs including antibiotics and high cholesterol diet dramatically alter the structure and function of kidney tissues and produce multiple deleterious effects.^1,2

Aminoglycoside antibiotics, particularly gentamicin (GM), are frequently used in the treatment of severe gram-negative infection.³ However, their clinical exploitation is limited due to the chance of nephrotoxicity.⁴ GM nephrotoxicity has a complex phenomenon characterized by an increase in plasma creatinine and urea levels as well as severe proximal renal tubular necrosis, followed by deterioration and renal failure.¹ It is apparent that the toxicity of GM involves renal free radical generation, reduction in antioxidant defense mechanism, acute tubular necrosis and glomerular congestion,³ renal inflammatory cascades, high renal oxidative stress, and associating pathological signaling mechanisms.⁵

Hypercholesterolemia (HC) plays an important role in the development and progression of atherosclerotic process, accelerates the development of fibrosis in the stenotic kidney, and increases the propensity for end stage of renal disease.⁶ Cholesterol-rich diet significantly elevated urinary excretion of urea, creatinine, lipid peroxidation (LPO) products in the kidney and erythrocytes, while significantly attenuated reduced glutathione (GSH) and antioxidative enzymes activities.⁷ It exacerbated the development of nephropathy with elevations of renal hypertrophy and mesangial matrix,⁸ affected the level of hemoglobin and its derivatives which cause anemia, and may produce reactive oxygen species (ROS) and other free radicals increasing thiobarbituric acid reactive substances (TBARS) and decreasing superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) enzyme activities.² HC aggravates contrast media-induced nephrotoxicity through the reduced production of nitric oxide (NO).⁹ The concurrent feeding of cholesterol-enriched diet with the GM treatment resulted in significantly greater biochemical and morphological changes in renal tissue than those following GM alone.¹⁰

Grape seed extract (GSE) contains important vitamins, minerals, and polyphenols including flavonoids and tannins.^11,12 Tannins are secondary plant metabolites subdivided into condensed and hydrolyzable compounds. Condensed tannins are known as proanthocyanidins, which are the most abundant flavonoids present in GSE.¹³ GSE demonstrated the strongest antioxidant effects and free radical scavenging ability¹⁴ as well as being anti-inflammatory,¹⁵ anticancer,¹⁶ antibacterial¹⁷; it has platelet aggregation inhibiting and metal chelating properties¹⁸ and vasodilatory, immunostimulating, antiallergic, antiviral activates¹⁹; it reduced plasma cholesterol in animals fed a high cholesterol diet and that may reduce risk of atherosclerosis.²⁰ Recently, GSE has been marketed as dietary supplement and advocated for its multiple beneficial properties.¹⁴

Based on the reported antioxidant and anti-inflammatory properties and vasodilatory action of polyphenolic compounds that exist in GSE, the hypothesis was made that GSE could attenuate oxidative stress, renal damage, and atherosclerosis induced by GM and HC. Therefore, the aim of this study was to investigate the renoprotective effect of GSE against oxidative stress, renal damage, and atherosclerosis induced by GM and HC in rats through the assessment of kidney biochemical parameters and lipid profile.

MATERIAL AND METHODS

Seventy adult male Wistar rats of similar age and weight (160 ± 10 g) were obtained from the Egyptian Organization of Biological Products and Vaccines (Helwan, Egypt). Animals were housed in stainless steel wire mesh suspended rodent cages under standard laboratory conditions of humidity 62%, temperature 25 ± 2°C, and 12 h light/dark cycle. Rodent chow diet and water were allowed ad libitum. All animals received human care in compliance with guidelines of the Ethical Committee of Medical Research of National Research Centre, Egypt.

Chemicals

GSE is a dried powder in capsules (150 mg) commercially known as Gervital® and was obtained from Mepco Pharma (Cardiff, UK) produced by Arab Company for Pharmaceuticals and Medicinal Plants, Cairo, Egypt. GM was purchased from Sigma Chemicals (St. Louis, MO, USA). All other chemicals and reagents used in the experiment were of analytical grade.

Experimental Design

Two weeks after acclimatization, rats were divided into 7 groups of 10 animals each. The first group was kept on saline and served as control. The second group was orally (16 G ball-tipped feeding needle with syringe) administered 150 mg/kg/day GSE for 30 days²¹ and served as GSE group. The third group was injected subcutaneously with 50 mg/kg GM twice daily for 15 days^1,22 and served as GM group. The fourth group was injected subcutaneously with 50 mg/kg GM twice daily for 15 days and orally administered GSE (150 mg/kg day) concomitantly with GM and 15 days after and served as GSE + GM group. The fifth group was kept on cholesterol-rich diet (4% cholesterol, 1% cholic acid, and 0.5% thiouracil) first for 6 weeks and continued throughout the drugs administration for another 30 days²³ and served as hypercholesterolemic (HC) group. The sixth group was kept on cholesterol-rich diet and injected subcutaneously with 50 mg/kg GM twice daily for 15 days and served as HC + GM group. The seventh group was kept on cholesterol-rich diet and injected with 50 mg/kg GM twice daily for 15 days and was orally administered 150 mg/kg/day GSE concomitantly with GM and 15 days after and served as HC + GSE + GM group.

Blood Sampling

At the end of treatment period (30 days), animals were fasted overnight and anesthetized using diethyl ether. Blood samples were collected from retro-orbital venous plexus using a glass capillary collector. One portion of each blood sampling was collected in dry centrifuge tubes for serum separation, centrifuged at 3000 × g for 15 min (4°C), then stored at −20°C for further determination of creatinine, urea, uric acid, N-acetyl-β-glucosaminidase (NAG), interleukin 1β (IL-1β), tumor necrosis factor α (TNF-α), monocyte chemoattractant protein 1 (MCP-1), total cholesterol (TC), triglyceride (TG), high-density lipoprotein (HDL), and low-density lipoprotein (LDL). The other portion of each blood sample was collected in heparinized tubes for plasma separation, centrifuged at 3000 × g for 20 min (4°C) and stored at −20°C for subsequent determinations of NO and cyclic guanosine monophosphate (cGMP).

Tissue Sampling

After blood collection, all animals were rapidly killed and the right kidneys were removed and washed with 0.9% NaCl and quickly frozen in liquid nitrogen (–170°C ), then stored at −20°C for further determinations of LPO, SOD, GSH, GSH-Px, and catalase (CAT).

Biochemical Analysis

Serum creatinine concentration was measured kinetically as described by Henry²⁴ using Diamond Diagnostic Kits (Cairo, Egypt) following the manufacturer and expressed as mg/dL. Serum urea was quantitatively determined using the QCA kit (urea Quimica Clinica Aplicada, S.A. Tarragona, Spain) with the modified Searcy method²⁵ and expressed as mg/dL. Uric acid concentration was determined colorimetrically as described by Haisman and Muller,²⁶ using Stanbio uric acid Liquicolor kit (Boerne, TX, USA) and expressed as mg/dL. Serum NAG was determined colorimetrically as described by Luqmani et al.,²⁷ using Diagnostic FAR kit (Verona, Italy) and expressed as microunit/mL. Serum cytokines IL-1β and TNF-α as well as MCP-1 were determined according to the methods of solid phase enzyme-linked immunosorbent assay using rat kits (Biosource International, Tucson, AZ, USA) and microtiter plate reader, Fisher Biotech (Ebersberg, Germany) and expressed as pg/mL. Serum TC, TG, and HDL were determined colorimetrically according to Allian et al.,²⁸ Wahlefeld,²⁹ and Lopes-Virella et al.,³⁰ respectively. LDL was calculated mathematically according to Friedwald et al.,³¹ using the following equation:

Plasma NO measured as nitrite was determined using Griess reagent according to the method of Moshage et al.³² and expressed as μmol/L. The quantitative determination of cGMP in plasma was carried out according to Kojda et al.,³³ using cGMP radioimmunoassay kit (IPL Com., Radolfzell, Germany) and expressed as pmol/mL.

The stored kidneys were homogenized in Teflon-glass homogenizer with buffer containing 1.5% potassium chloride to obtain 1:10 (w/v) of whole homogenate to determine LPO and antioxidant enzyme activates in renal tissue. The homogenate was prepared for analysis by centrifugation at 18,000 × g (4°C) for 30 min.

The amount of produced malondialdehyde (MDA) was used as an index of LPO. MDA concentration was assayed according to a modified method of Ohkawa et al.,³⁴ based on reaction with thiobarbituric acid and expressed as nmol MDA/g tissue. Tissue superoxide anion was determined according to the modified method of Hassoun and Stohs,³⁵ following the principle of Babior et al.,³⁶ on the basis of cytochrome c reduction and was expressed as nmol cytochrome c reduced/min/g tissue. Measurement of tissue SOD activity was performed according to the method described by Sun et al.,³⁷ which consists of the inhibition of nitroblue tetrazolium reduction with xanthine–xanthine oxidase used as superoxide generator and expressed as U/mg protein. Tissue GSH concentration was measured by kinetic assay using a dithionitrobenzoic acid recycling method³⁸ and expressed as μmol/mg tissue. The GSH-Px activity in renal tissue was determined according to Beutler,³⁹ based on oxidation of reduced GSH coupled to the appearance of NADPH by glutathione reductase measured at 37°C and 340 nm and expressed as U/g protein. CAT activity was determined by measuring the decomposition of hydrogen peroxide (H₂O₂) at 240 nm according to the method of Aebi⁴⁰ and was expressed as U/g. Protein concentration was measured according to the method described by Lowry et al.⁴¹

Statistical Analysis

The obtained data were statistically analyzed. The differences between groups were assessed by one-way analysis of variance (ANOVA), using the SPSS software (IBM, New York, USA). The values are expressed as mean ± SD. Intergroup comparisons were performed by Duncan’s multiple rank test using MSTAT-C computer program. Mean values followed by the same alphabetical letter are not significantly different. Differences were considered to be significant at p < 0.05.

Table 1. Effect of GSE on blood fractions of hypercholesterolemic rats treated with gentamicin.

Parameters	Control	GSE	GM	GSE + GM	HC	HC + GM	HC + GSE – GM
Creatinine (mg/dL)	0.8 ± 0.07c	0.7 ± 0.06c	3.1 ± 0.22a	1.2 ± 0.1b	1.1 ± 0.1bc	3.6 ± 0.25a	1.6 ± 0.13b
Urea (mg/dL)	20.8 ± 1.6d	21.1 ± 1.7d	43.7 ± 3.1a	28.3 ± 2.6bc	26.1 ± 2.4c	46.8 ± 3.6a	32.0 ± 2.5b
Uric acid (mg/dL)	4.2 ± 0.5e	4.0 ± 0.3e	10.1 ± 0.8b	6.1 ± 0.7cd	5.0 ± 0.5de	12.2 ± 0.9a	7.8 ± 0.8c
NAG (microunit/mL)	26.3 ± 2.2e	25.8 ± 2.1d	69.8 ± 4.3a	34.2 ± 2.6c	30.1 ± 2.4cd	72.5 ± 5.8a	40.4 ± 3.7b
IL-1β (pg/mL)	50.5 ± 4.1d	51.2 ± 3.9d	84.3 ± 6.1b	60.4 ± 5.2c	62.3 ± 5.1c	92.6 ± 8.3a	63.7 ± 5.1c
TNF-α (pg/mL)	30.4 ± 2.6d	28.1 ± 2.3d	78.6 ± 6.3a	36.5 ± 2.9c	39.1 ± 3.1bc	82.4 ± 6.8a	40.5 ± 3.4b
MCP-1 (pg/mL)	72.8 ± 5.6e	70.2 ± 6.1e	156.3 ± 9.4b	85.2 ± 7.2d	94.1 ± 8.5c	168.6 ± 11.6a	89.2 ± 7.3cd
NO (μmol/L)	24.3 ± 1.8a	25.1 ± 2.1a	14.4 ± 0.9de	21.4 ± 1.2bc	22.5 ± 1.4b	12.5 ± 0.8e	19.8 ± 1.1c
cGMP (pmol/mL)	4.8 ± 0.3ab	5.2 ± 0.4a	2.4 ± 0.2d	4.1 ± 0.2bc	3.8 ± 0.2c	2.1 ± 0.1d	3.9 ± 0.2bc

Notes: NAG, N-acetyl-β-glucosaminidase; IL-1β, interleukin 1β ; TNF-α, tumor necrosis factor α; MCP-1, monocyte chemoattractant protein 1; NO, nitric oxide; cGMP, cyclic guanosine monophosphate. Means followed by the same alphabetical letter for each parameter are not significantly different at p < 0.05.

RESULTS

Feeding healthy rats with 150 mg/kg/day GSE for 30 consecutive days had insignificant effects on blood fractions level as compared to the control group. However, injecting rats with 50 mg/kg GM twice daily for 15 consecutive days resulted in significant increases in serum levels of creatinine (288%), urea (110%), uric acid (141%), NAG (165%), IL-1β (67%), TNF-α (159%), and MCP-1 (115%) (Table 1). Also, significant increases were detected in serum levels of TC (13%), TG (64%), and LDL (84%) (Figure 1); while significant attenuation was recorded in serum levels of HDL (25%) (Figure 1) and plasma NO (40%) as well as cGMP (50%) (Table 1) as compared with the control group.

Feeding the GM group concomitantly with GSE and 15 days after produced significant protection against renal toxicity. The amelioration of renal toxicity was evidenced in significant reductions in the elevated serum levels of creatinine (61%), urea (35%), uric acid (40%), and NAG (51%) (Table 1) in comparison with the GM group. In addition, GSE supplementation significantly reduced the elevated serum levels of IL-1β (28%), TNF-α (54%), MCP-1 (45%) (Table 1) as well as serum TG (28%) and LDL (21%) (Figure 1) while significantly increased the attenuated serum level of HDL (24%) (Figure 1) and plasma NO (49%) as well as cGMP (71%) (Table 1) as compared with the GM group.

The current results demonstrated that keeping rats on cholesterol-rich diet significantly elevated the serum levels of urea (25%), NAG (14%), IL-1β (23%), TNF-α (29%), MCP-1 (29%), TC (21%), TG (116%), and LDL (135%), while significantly attenuated serum HDL (26%) and plasma cGMP (21%) as compared with the control group. The fluctuations of serum creatinine, uric acid, and plasma NO in HC rats did not reach the level of significance when compared with the normal group.

Injecting HC rats with 50 mg/kg GM twice daily for 15 days caused highly significant elevations in the serum levels of creatinine (350%), urea (125%), uric acid (190%), NAG (187%), IL-1β (83%), TNF-α (171%), MCP-1 (132%) (Table 1), TC (28%), TG (143%), LDL (173%) (Figure 1), while significantly attenuated serum HDL (33%) (Figure 1) and the plasma levels of NO (49%) and cGMP (56%) (Table 1) as compared with the normal diet control group. On the other hand, feeding HC GM-treated rats with GSE produced significant amelioration against renal toxicity, which was manifested in significant reductions in serum creatinine (56%), urea (32%), uric acid (36%), and NAG (44%) as compared with the HC GM group. Moreover, GSE supplementation significantly attenuated the elevated serum levels of IL-1β (31%), TNF-α (51%), MCP-1 (47%), TC (10%), TG (29%), and LDL (31%), while significantly elevated the attenuated serum level of HDL (29%) and plasma NO (58%) as well as plasma cGMP (86%) as compared with the HC rats injected with GM.

Healthy rats that received GSE for 30 days had insignificant effects on both oxidative and antioxidative stress parameters of renal tissue as compared with the normal control. However, injecting rats with GM twice daily for 15 days caused marked increases in LPO (90%) and superoxide anion (198%), while significantly reduced the levels of SOD (55%), GSH (53%), GSH-Px (54%), and CAT (56%) (Figure 2) in renal tissue of rat compared with the normal control. On the other hand, supplementing GM group with 150 mg/kg/day GSE concomitantly with GM administration and 15 days after significantly attenuated the elevated levels of LPO (38%) and superoxide anion (54%) while significantly elevated the attenuated activity of antioxidant stress parameters, that is, SOD (71%), GSH (83%), GSH-Px (98%), and CAT (209%) in renal tissue of rats as compared with GM-treated rats.

The results illustrated in Figure 2 revealed that HC significantly raised oxidative stress levels, that is, LPO (12%) and superoxide anion (61%), while significantly attenuated antioxidative stress activities, that is, SOD (17%), GSH (30%), and CAT (8%) in the renal tissue of rats as compared with the normal diet control rats. Injecting HC rats with GM significantly elevated the levels of oxidative stress and markedly reduced the activities of antioxidant parameters in renal tissue of rats as compared with the normal control and other studied groups. Otherwise, feeding HC-injected GM rats 150 mg/kg/day GSE concomitantly with GM administration and 15 days after produced significant protection against renal toxicity which was evidenced by significant reductions in the elevated levels of oxidative stress parameters LPO (37%) and superoxide anion (49%), while significantly increased the attenuated antioxidant parameters levels, that is, SOD (74%), GSH (108%) GSH-Px (97%), and CAT (101%) in renal tissue of rats in comparison with the HC rats injected with GM group.

Figure 1. Effect of GSE on lipid profile of hypercholesterolemic rats treated with gentamicin. Columns followed by the same alphabetical letter for each parameter are not significantly different at p < 0.05. GSE, grape seed extract; GM, gentamicin group; HC, hypercholesterolemic group; TC, total cholesterol; TG, triglyceride; HDL, high-density lipoprotein; LDL, low-density lipoprotein.

Figure 2. Effect of GSE on renal tissue fractions of hypercholesterolemic rats treated with gentamicin. Columns followed by the same alphabetical letter for each parameter are not significantly different at p < 0.05. GSE, grape seed extract; GM, gentamicin group; HC, hypercholesterolemic group; LPO, lipid peroxidation; SOD, superoxide dismutase; GSH, reduced glutathione; GSH-Px, glutathione peroxidase; CAT, catalase.

DISCUSSION

Recent interest in GSE has greatly increased due to its antioxidant capacity and free radical scavenging ability¹⁴ as well as being anti-inflammatory,¹⁵ having platelet aggregation inhibiting and metal chelating properties,¹⁸ and having possible beneficial implications in human health. This study investigates the possible renoprotective effects of GSE against oxidative stress, renal damage, and atherosclerosis induced by GM and HC in rats through the assessment of kidney biochemical parameters and lipid profile.

In this study, in the normal control and GSE groups, rats demonstrated normal renal functions (normal serum creatinine, urea, uric acid, and NAG). However, the toxic renal effects of GM were manifested in the GM group in significant increases in serum levels of creatinine, urea, uric acid, and NAG. Similar degrees of GM toxicity have been reported by different investigators.^1,42 The increases in urea and creatinine levels may indicate a reduction in the glomerular filtration rate (GFR) as a result of GM intoxication, since the serum concentration of these two parameters depends largely on the glomerular filtration.⁴³ NAG is a lysosomal enzyme widely distributed in body tissue; it is released into serum from cells by exocytosis or from the breakdown of cells.⁴⁴ The elevated serum NAG activity may be related to atherosclerosis, elevated serum cholesterol, increased uric acid,⁴⁵ and increased oxygen free radicals, which lead to destruction of lysosomal membrane with subsequent release of NAG enzyme.⁴⁶ Serum NAG activity has gained increased importance as an aid in the diagnosis of atherosclerosis.⁴⁵ The toxic renal effects of GM may be due to the interaction between molecular and subcellular components (lysosomes and mitochondria), where GM binds to the anionic phospholipids of cell membranes and gains access to the cell interior before binding to the subcellular organelles or being taken up by lysosomes. Accumulation of GM within the renal cortex is intimately related to the pathogenesis of renal failure.⁴⁷

This study demonstrated that GM exposure significantly elevated serum levels of IL-1β, TNF-α, and MCP-1. The inflammatory cytokine TNF-α itself causes injury to renal epithelial cells because it stimulates the production of ROS and produces oxidative stress by sensitizing infiltrating leukocytes.⁴⁸ TNF-α and IL-1β are proinflammatory cytokines and often elevated in parallel. They stimulate the production of one another and act synergistically to stimulate the production of other cytokines and chemokines.⁴⁹ A major chemotactic factor of monocytes is also induced by TNF-α and IL-1β in response to vascular injury and it is critical for the progression of atherosclerosis.⁵⁰ TNF-α appears to be involved in many types of inflammatory processes and has been negatively correlated with SOD, CAT, and renal tissue GSH.⁵¹The obtained results showed that the administration of GM significantly elevated serum TC, TG, and LDL, while markedly decreased HDL as compared with the control group. Confirming these results, Rashid et al.⁵² found that serum cholesterol was increased significantly in rats following GM exposure and also caused prominent damage in the kidney tissue. High serum TG levels have been reported to be an important risk factor as it influences lipid deposition and clotting mechanism.⁵³ The increase in serum LDL has also been pointed out as one of the risk factors for the development of atherosclerosis.⁵⁴ Elevation of serum cholesterol concentration played a major role in the progress of the GM-induced nephrotoxicity.¹⁰ Therefore, the high lipid levels detected in GM-treated rats may be associated with the accumulation of filtered LDL fractions in the glomeruli, leading to a reduction in the glomerular filtration surface area.⁵⁵

When compared with normal control rats, GM injection induced significant decrease in both plasma NO and cGMP. This result may be due to the inhibition of endothelium-dependent vasodilatation.⁵⁶ NO with the heme moiety of soluble guanylyl cyclase leads to the formation of cGMP from GTP.⁵⁷

The toxic renal effects of GM observed in this study are manifested in significant elevation in MDA and superoxide anion production and significant reduction in GSH level and SOD, GSH-Px, and CAT activities. It is believed that reduced activity of one or more antioxidant systems because of the direct toxic effect of GM causes an increase of LPO and oxidative stress and consequently renal toxicity.^3,5

Feeding cholesterol-rich diet to rats induced significant increases in serum levels of urea, NAG, IL-1β, TNF-α, MCP-1, TC, TG, and LDL as well as MDA and superoxide anion levels in renal tissue, while significantly reduced the serum level HDL and plasma cGMP as well as enzymatic and nonenzymatic antioxidants (SOD, GSH, GSH-Px, and CAT) activities in renal tissue. In this connection, feeding rats with cholesterol-rich diet resulted in increased LDL level and LPO, and reduced CAT and SOD activities.⁵⁸ In addition, HC elevated TC, TGs, LPO coupled with decreased enzymatic and nonenzymatic antioxidants in renal tissue, which indicate the onset of oxidative changes in the renal tissue.⁵⁹ Moreover, Abdelhalim² concluded that HC produces ROS and other free radicals increasing TBARS, serum level and decreasing plasma enzymes SOD and GSH-Px activity which confirmed the results of this study. However, there was no significant difference (p < 0.05) in the levels of serum creatinine, uric acid, NAG, and plasma NO of both animals fed on the control diet and those fed on the HC diet.

The concurrent feeding of the cholesterol-enriched diet with the GM treatment resulted in significantly greater biochemical changes and functional exacerbation of glomerular disorder than those following GM alone. These results were manifested in significant increases in the serum levels of creatinine (350%), urea (125%), uric acid (190%), NAG (176%), IL-1β (83%), TNF-α (171%), and MCP-1 (132%) as compared with the control group. This abnormal concentration of these catabolites in serum is a clear indication of renal function impairment.^60,61 There were also significant elevations in the serum levels of TC (28% and 13%), TG (143% and 48%), and LDL (173% and 48%) as compared with the normal control and GM groups, respectively. The elevation in serum cholesterol concentration probably played a major role in the progression of the GM-induced renotoxicity.¹⁰ HC rats injected with GM showed significant reductions in the plasma levels of NO (48% and 20%) and cGMP (56% and 12%) as compared with the normal control and GM groups, respectively. This result is in the same line with Pear et al.,⁶² who reported that the reduced production of NO may be implicated in the hemodynamic changes of HC animals. The accelerated generation of ROS induced by HC was accompanied by the depletion of their specific scavengers (enzymatic and nonenzymatic antioxidants),⁵⁹ which are in harmony with the results of this study.

The results of this study indicate the ability of GSE to ameliorate GM-induced renotoxicity in normal diet and HC rats, as serum levels of creatinine, urea, uric acid, and NAG were decreased in both GM and HC + GM groups to be approximately near their normal levels. Similarly, different studies that tested the protective effect of GSE against chemical assaults in various organs using animal models showed that pre-exposure GSE rescued cells from toxic assaults, significantly protected genomic integrity, and minimized apoptotic, necrotic, and apocrotic cell deaths.⁶³ The amelioration of serum creatinine and urea levels due to supplementation of GSE indicated the increase in GFRs. Moreover, the reduction of serum NAG and uric acid in GM and GM HC groups due to administration of GSE indicated the protective effect of GSE against the toxicity of GM and HC.

The hypothesis that GSE exhibits anti-inflammatory properties has been supported by our findings where the intake of GSE significantly reduced the serum level of TNF-α, IL-1β, and MCP-1 of the rats exposed to GM and HC. TNF-α and IL-1β as proinflammatory cytokines are often changed in parallel and act synergistically in production of other cytokines and chemokines.⁴⁹ Through this connection, Terra et al.¹⁵ suggested that GSE procyanidin reduced the expression of proinflammatory cytokines TNF-α and IL-6 and enhanced production of the anti-inflammatory cytokines adiponectin.

Administration of GSE markedly suppressed the elevation of serum TC, TG, and LDL, while increased the attenuated serum level of HDL in GM and HC + GM groups. These results are in agreement with the findings of Adisakwattana et al.⁶⁴ HDL may play a protective role through reversing cholesterol transport, enhancing the oxidation of LDL, and neutralizing the atherogenesis effect of oxidized LDL.² The anti-hyperlipidemic activities of GSE may act through the inhibition of lipid digestion and absorption. Supplementation of GSE reduces plasma lipid profiles by improving adipokine secretion and oxidative stress⁶⁵ and inhibiting pancreatic lipase, cholesterol esterase, cholesterol micellization, and bile acid binding.⁶⁴

This study showed that supplementation of GSE to GM or GM HC rats significantly elevated the suppressed plasma levels of NO and cGMP. These results are confirmed by the findings of Fitzpatrick et al.⁶⁶ GSE reduced platelet aggregation, releasing NO from endothelial cells, activating the guanylate cyclase, increasing the cGMP production, and finally inducing vascular relaxation.⁶⁷

Providing both GM and GM HC rats with GSE significantly lowered the elevated superoxide anion and LPO in renal tissue, while it markedly raised the attenuated level of GSH and the activities of SOD, GSH-Px, and CAT. Elevating GSH in renal tissue is essential for the regulation of cell functions, maintaining cell integrity, cell protection against variety of toxins such as free radicals and their accompanied oxidative injury due to its reducing properties, and participation in the cell metabolism.⁶⁸ Many studies have reported that the monomers catechin, epicatechin (monomeric flavonols), and procyanidins are the major phenolic compounds in GSE that show antioxidant activity.^13,14

The renoprotective effects of GSE intake may be due to its multiple effects on LPO, inflammatory mediators such as MCP-1, and cytokines such as TNF-α and IL-1β, enhanced NO release and cGMP, and improved lipid profile. From this point of view, the intake of GSE may be a feasible therapeutic strategy for prevention and treatment of patients with GM and HC-induced renal impairment. Further studies in both animals and humans are necessary to demonstrate the antioxidant properties and other effects of GSE on renal diseases. This investigation is helpful for understanding the mechanism of action of GSE and its active ingredients and also reveals the potential of GSE for use as a natural oral agent with both renotoxicity and HC effects.

Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.