Pomegranate Extract Attenuates Gentamicin-Induced Nephrotoxicity in Rats by Reducing Oxidative Stress

Abstract

Nephrotoxicity is a major complication of gentamicin (GEN), which is widely used in the treatment of severe Gram-negative infections. Reactive oxygen species are important mediators of GEN-induced nephrotoxicity. Because of the strong antioxidant properties of pomegranate extract (PE), we evaluated the protective effect of PE against GEN-induced nephrotoxicity. Thirty-two adult male rats were randomly divided into four equal groups: (1) controls; (2) treated with GEN for 14 consecutive days (100 mg/kg/day); (3) treated with GEN plus distilled water; and (4) treated with GEN plus PE (100 μL). After 15 days, the rats were killed and their kidneys were taken, and blood analysis was performed. Tubular necrosis and interstitial fibrosis scores were determined histopathologically; and biochemically, nitric oxide (NO), malondialdehyde (MDA), and reduced glutathione (GSH) levels in kidneys were determined. Urea, creatinine, Na⁺, and K⁺ levels were investigated in the blood analysis. Statistical analyses were made by the chi-square test and analysis of variance. Serum urea and creatinine levels were significantly higher in rats treated with GEN alone than rats in the control and the GEN + PE-treated groups. The GSH level in renal tissue of only GEN-treated rats was significantly lower than those in the control group, and administration of PE to GEN-treated rats significantly increased the level of GSH. The group that was given GEN and PE had significantly lower MDA levels in kidney cortex tissue than those given GEN alone. There was no significant difference of NO levels between the groups. In rats treated with GEN + PE, despite the presence of mild tubular degeneration and tubular necrosis is less severe, and glomeruli maintained a better morphology when compared with the GEN-treated group. We think that PE prevents kidney damage by decreasing oxidative stress in kidney.

• gentamicin
• pomegranate extract
• nephrotoxicity
• nitric oxide
• reactive oxygen species

INTRODUCTION

Gentamicin (GEN) is an antibiotic of aminoglycoside group and is widely used in the treatment of Gram-negative infections. A major complication of this drug is nephrotoxicity, and it has been estimated that approximately 10–20% of cases are treated with aminoglycoside therapy.¹ Despite the introduction of less nephrotoxic antibiotics against Gram-negative microorganism, it is still used because of its low cost and efficacy against resistant beta-lactam positive microorganisms.² GEN is not metabolized in the body but is essentially eliminated by glomerular filtration and partially reabsorbed by proximal tubular cells.³ No conclusive evidence exists of tubular secretion of GEN, and consequently, most aminoglycosides are excreted in the urine correspond to filtrates.⁴ The specificity of GEN for renal toxicity is apparently related to its preferential accumulation in the renal proximal convoluted tubules, reaching a concentration of 5–50 times higher than plasma in the tubular renal cell.⁵

It has been demonstrated that nephrotoxicity induced by GEN is characterized by direct tubular necrosis, which is localized mainly in the proximal tubules. The exact mechanisms of GEN-induced nephrotoxicity still remain unclear. However, several studies demonstrated that reactive oxygen species (ROS) may be important in GEN-induced nephrotoxicity.⁶ Moreover, GEN has also been shown to enhance the generation of ROS. Lipid peroxidation (LPO) mediated by ROS has been suggested as a causative agent of cell death in different pathological states including various models of renal diseases.^7,8 Besides their direct damaging effects on tissues, ROS seem to trigger the accumulation of leukocytes in the tissue involved, and thus cause tissue injury indirectly through activated neutrophils. It has been shown that activated neutrophils secrete enzymes such as myeloperoxidase, elastase, and proteases and liberate oxygen radicals.⁹ On the other hand, in vivo and in vitro studies have shown that the scavengers of reactive oxygen metabolites are protective in GEN-induced renal failure.^10,11

Pomegranate extract (PE) and its derivatives have been used for centuries to confer health benefits in a number of inflammatory diseases. Edible parts of pomegranate fruit represent 52% of total fruit weight, comprising 78% juice and 22% seeds.¹² Fresh juice is rich in vitamin C, and polyphenolic compounds such as anthocyanins, punicalagin, and ellagic and gallic acids.^13,14 PE is a rich source of potent polyphenolic, flavonoid antioxidants (anthocyanins). The soluble polyphenol content in PE varies within the limits of 0.2%–1.0% depending on the variety and includes mainly anthocyanins that have been shown to possess antiatherogenic properties. Anthocyanins were shown to be effective inhibitors of LPO, production of nitric oxide (NO), and inducible nitric oxide synthase (iNOS) activity in different model systems.^15,16

Pomegranate has become more popular because of the attribution of important physiological properties, such as anticancer,^17,18 cholesterol-lowering, cardioprotective,¹⁹ and so on. Many investigators have reported that pomegranate and its derivatives have free radical scavenger and potent antioxidant activity.^20–22 It has also been shown that pomegranate can suppress nuclear factor kappa B (NF-κB) activation through a novel mechanism in vascular endothelial cells.²³

As a result of this information, in this study we investigated the possible inhibitory effects of pomegranate against the GEN-induced oxidative stress and renal injury in rat models. The nephroprotective effect of PE is previously established in many studies, however, to our knowledge this is the first study in literature concerning the protective role of pomegranate against GEN nephrotoxicity.

MATERIALS AND METDODS

Drugs

GEN was purchased from Bilim Pharmaceuticals (Istanbul, Turkey), and PE (100% pure, pasteurized pomegranate juice, 250 mL; Elite Natural Beverage Co., Ankara, Turkey) was purchased from a local store. GEN was dissolved in saline and injected intraperitoneally. PE was dissolved in distilled water and administered via nasogastric gavage. The average of 2.5 mL diluted PE contains 100-μL PE, which is equivalent to 2.8 μmol of total polyphenol per day.

Animals

Adult male Wistar albino rats (200–250 g) were housed in clean plastic cages in a temperature and humidity-controlled facility with a constant 12 h light/dark cycle with free access to food and water. The use of animals and the experimental protocol were approved by the Institutional Animal Care and Use Committee, and animals were treated in accordance with the Guide for the Care and Use of Laboratory Animals of Research Council.

Treatment and Experimental Design

After a quarantine period of 7 days, 32 rats were randomly divided into four equal groups, each consisting of eight animals as follows: (1) controls; (2) intraperitoneally injected with GEN for 14 consecutive days (100 mg/kg/day); (3) treated with GEN plus distilled water via nasogastric gavage for 14 days; (4) treated with GEN plus PE (100 μ/L) for 14 days. Rats were treated for 14 days. After 15 days, rats were killed and their kidneys were taken, and blood analysis was performed. Tubular necrosis and interstitial fibrosis scores were determined histopathologically in a part of kidneys; NO, malondialdehyde (MDA), and reduced glutathione (GSH) levels were determined in the other part of kidneys. Urea, creatinine, Na⁺, and K⁺ levels were investigated in a blood analysis.

Sample Collection and Biochemical Assays

Twenty-four hours after the administration of last doses of GEN and PG, on the 15th day, the rats were anesthetized by intraperitoneal injection of ketamine and were sacrificed. Twenty-four-hour urine collections were obtained in standard metabolic cages a day before the rats were killed. Renal cortical tissues were separated into two parts for biochemical analysis and light microscopic examination. Blood samples were also obtained by cardiac puncture to assess the serum levels of urea, creatinine, Na⁺, and K⁺ concentrations. The tissues were shock-frozen in liquid nitrogen and were kept in −80°C. In the frozen tissues, MDA, end product of LPO, reduced GSH, nonenzymatic antioxidant, and total nitrite (NOx), a stable product of NO, were evaluated biochemically as a means of oxidative stress.

Renal impairment was assessed by serum urea and creatinine levels, as well as by the kidney histology. Serum urea and creatinine levels were determined using an autoanalyzer (SYNCHRON LX20, Beckman Coulter Inc., Ireland) by using commercial Becman Coulter diagnostic kits. Kidney tissue (300 mg) was homogenized in ice-cold tamponade containing 150 mM KCl for determination of MDA. MDA levels were assayed for products of LPO. MDA referred to as thiobarbituric acid reactive substance was measured with thiobarbituric acid at 532 nm using a spectrofluorometer, as described previously.²⁴ GSH was determined by the spectrophotometric method, which was based on the use of Ellman’s reagent.²⁵

NOx was quantified by the Griess reaction²⁶ after incubating the supernatant with nitrate reductase from Escherichia coli to convert nitrate (NO₃) to nitrite (NO₂). Griess reagent (1 mL—1% sulfanilamide, 0.1% naphthyl-ethylenediamine hydrochloride, and 2.5% phosphoric acid; Sigma Chemical Co., St. Louis, MO, USA) was then added to 1 mL of supernatant. The absorbance was read at 545 nm after 30-min incubation. The absorbance was compared with the standard graph of NaNO₂, obtained from the reduction of NaNO₃ (1–100 lmol/L). The accuracy of the assay was checked in two ways; the interassay and intraassay coefficients of variation were 7.52% and 4.61%, respectively. To check conversion of nitrate to nitrite (recovery rate), known amounts of nitrate were added to control plasma samples; these samples were deproteinized and reduced as above.

Histopathological Examinations

Histopathological evaluation of the kidney tissues was done. Paraffin-embedded specimens were cut into 6-μm thickness and stained with Hematoxylin–Eosin stain for light microscopic examination using a conventional protocol²⁷ (Olympus BH-2, Olympus, Tokyo, Japan). A semiquantitative evaluation of renal tissues was accomplished by scoring the degree of severity according to previously published criteria.²⁸ All sections of the kidney samples were examined for parietal cell hyperplasia, tubular vacuolization, and tubular necrosis. Briefly, minimum of 50 proximal tubules associated with 50 glomeruli were examined for each slide, and an average score was obtained. Severity of lesion was graded from 0 to 3 according to the percentage of tubular involvement. Slides were examined and assigned for severity of changes using scores on scale of none (0), mild (1), moderate (2), and severe (3) damages, in which (0) denotes no change; grade (1) changes affecting <25% tubular damage (mild); grade (2) changes affecting 25%–50% of tubules (moderate); grade (3) changes affecting >50% of tubules (severe).

To evaluate kidney fibrosis, the specimens obtained from kidney were embedded in paraffin, sectioned at 6-μm sections, and stained with Masson’s trichrome. Specimens were scored after painting with, in brief, (−) no fibrosis, (+) fibrosis in <25% of total kidney tissue (mild), (++) fibrosis in 25%–50% of total kidney tissue (moderate), (+++) fibrosis in >50% of total kidney tissue (serious).²⁹

Statistical Analyses

Results of all groups were shown as mean values ± standard deviation (SD). Statistical analyses of the histopathological evaluation of the groups were carried out by the chi-square test, and biochemical data were analyzed by the one-way analysis of variance (ANOVA). The significance between two groups was determined by the Dunnett’s multiple comparison test, and p < 0.05 was accepted as statistically significant value.

RESULTS

No deaths or remarkable signs of external toxicity were observed in the groups of rats given GEN either alone or combination with PE. The biochemical and histopathological results were similar for the control and PE groups, and we decided to consider them without distinction and report only the control group.

Urinary Volume

The 24-h urine volume in the GEN-treated group was significantly higher than in the group control (p < 0.01), indicating the presence of GEN-induced polyuria, whereas in the group treated with GEN + PE, it was not different from that of the group control, pointing out the protective role of PE against acute tubular necrosis (Table 1).

Table 1.  Effects of GEN alone and its combination with PE on plasma urea, creatinine, Na⁺, K⁺, and 24-h urine volume levels in rats.

Parameters	Control	GEN	GEN + vehicle	GEN + PE
Urea (mg/dL)	34 ± 8.1	109 ± 14.9^a	105 ± 14.4	45 ± 10.8^b
Creatinine (mg/dL)	0.44 ± 0.1	2.1 ± 0.5^a	1.99 ± 0.5	0.8 ± 0.3^b
Na^+ (mmol/L)	138.5 ± 1.4	140.3 ± 2.7	139.1 ± 2.4	138.8 ± 1.6
K^+ (mmol/L)	3.9 ± 0.2	4.2 ± 0.5	4.1 ± 0.4	4.1 ± 0.3
24-h Urine volume (mL)	8.8 ± 1.1	21.8 ± 4.5^a	20.3 ± 3.4	9.6 ± 1.7^b

Notes: Values are expressed as mean ± SD for eight rats in each group.

Groups: control, GEN-treated, GEN + vehicle-treated, and GEN + PE-treated.

^aSignificantly different from the control group.

^bSignificantly different from the GEN-treated group (p < 0.001).

Abbreviations: NO, nitric oxide; MDA, malondialdehyde; GSH, reduced glutathione; GEN, gentamicin.

Biochemical Variables in Plasma and Tissue

Na⁺ and K⁺ concentrations among four groups were similar. Serum urea and creatinine levels were significantly higher in rats treated with GEN alone than rats in the control and GEN + PE-treated groups (p < 0.01). Administration of PE to the GEN-treated rats caused decrease in serum urea and creatinine levels (Table 1).

The GSH level in renal tissue of only GEN-treated rats was significantly lower than those in the control group (p < 0.05), and administration of PE to GEN-treated rats significantly increased the level of GSH (p < 0.05; Table 2).

Table 2.  Effects of PE on NO, MDA, and GSH levels of rat kidney.

Parameters	Control	GEN	GEN + vehicle	GEN + PE
NO (nmol/g wet tissue)	30.3 ± 9.3	47.2 ± 7.3^a	44.5 ± 5.8	43.7 ± 11.9
MDA (nmol/g wet tissue)	2.6 ± 0.7	4.1 ± 1.1^a	3.9 ± 1.1	2.2 ± 0.9^b
GSH (umol/g wet tissue)	2.3 ± 0.8	1.4 ± 0.7^a	1.3 ± 0.6	2.4 ± 0.4^b

Notes: Values are expressed as mean ± SD for eight rats in each group.

^aSignificantly different from the control group.

^bSignificantly different from the GEN-treated group (p < 0.05).

Abbreviations: NO, nitric oxide; MDA, malondialdehyde; GSH, reduced glutathione; GEN, gentamicin.

The group that was given GEN and PE had significantly lower MDA levels in kidney cortex tissue than those given GEN alone. There was no significant difference for any levels between the groups (Table 2).

Results of Histopathological Examinations

The histopathological examination of kidney showed no pathologic findings in the control group (Figure 1A). In rats treated with GEN and GEN + vehicle, there were mild and severe tubular necrosis, tubular degeneration, and epithelial vacuolization in the proximal tubules, and parietal cell hyperplasia compared with the control group (Figure 1B and C). In rats treated with GEN + PE, despite the presence of mild tubular degeneration and epithelial vacuolization in the proximal tubules, parietal cell hyperplasia and tubular necrosis are less severe, and glomeruli maintained a better morphology when compared with the GEN group (Figure 1D). These changes are summarized in Table 3.

Figure 1.  (A) Normal tubules and glomeruli in kidney cortex, staining with H&E ×100 (control group). (B) Severe tubular necrosis, tubular degeneration, and epithelial vacuolization in the proximal tubules, staining with H&E ×100 (GEN-treated group). (C) Moderate tubular necrosis, tubular degeneration, and epithelial vacuolization in the proximal tubules, staining with H&E ×100 (GEN + vehicle-treated group). (D) Mild epithelial vacuolization in the proximal tubules and normal glomeruli, staining with H&E ×100 (GEN + PE-treated group).

Figure 2.  (A) No fibrosis in the control group, staining with Masson’s trichrome ×100. (B) Mild fibrosis in interstitium, staining with Masson’s trichrome ×400 (GEN-treated group). (C) Mild fibrosis in interstitium, staining with Masson’s trichrome ×200 (GEN + vehicle-treated group). (D) No fibrosis in GEN + PE-treated group, staining with Masson’s trichrome ×100.

Table 3.  Semiquantitative analysis of tubular necrosis, tubular vacuolization, parietal cell hyperplasia in the control, GEN-, GEN + vehicle-, and GEN + PE-treated rats.

	Tubular necrosis				Tubular vacuolization				Parietal cell
Hyperplasia	n	0	1	2	3	0	1	2	3	0	1	2	3
Control	8	8	0	0	0	7	1	0	0	8	0	0	0
GEN^a	8	0	2	5	1	0	2	4	2	0	2	5	1
GEN + vehicle	8	1	2	4	1	1	2	3	2	0	3	4	1
GEN + PE^b	8	4	3	1	0	1	6	1	0	3	4	1	0

Notes: Score 0, no degeneration; 1, mild degeneration; 2, moderate degeneration; 3, severe degeneration.

^aStatistical significant difference from the control group.

^bStatistical significant difference from the GEN-treated group (p < 0.05).

Table 4.  Analysis of kidney fibrosis in the control, GEN-, GEN + vehicle-, and GEN + PE-treated rats.

	n	(−)	(+)	(++)	(+++)
Control	8	0	0	0	0
GEN	8	3	4	1	0
GEN + vehicle	8	4	4	0	0
GEN + PE	8	6	2	0	0

Notes: Score (−), no fibrosis; (+), mild fibrosis; (++), moderate fibrosis; (+++), serious fibrosis.

No statistical difference between the groups (p > 0.05).

After staining with Masson’s trichrome, no statistical difference was found between the groups in kidney fibrosis scores. However, in GEN-treated rats, there was more mild and moderate fibrosis in kidney specimen (Table 4; Figure 2A and D).

DISCUSSION

The kidneys are easily susceptible to damage from drugs because of larger perfusion and accumulation of excreted compounds that occur in renal tubular cells during absorption and secretion. Aminoglycoside is an antibiotic whose clinical use is limited by its nephrotoxicity. GEN is a widely used aminoglycoside antibiotic and has been shown to cause marked histological damage in particular to renal proximal convoluted tubules,^30,31 resulting in swelling, vacuolization, and necrosis of epithelial cells and accumulation of myelin-like bodies.³²

Furthermore, the results of many studies have shown that altered concentrations of various biochemical indicators of oxidative stress in kidney tissue are due to GEN.³³ Because of the obvious responsibility of ROS in GEN-induced renal damage, several antioxidant agents have been used to prevent GEN nephrotoxicity.³⁴ PE is rich in antioxidants of the polyphenolic class, which includes tannins and anthocyanins. These antioxidants are more potent, on a molar basis, than many other antioxidants, including vitamins C and E, coenzyme Q-10, and lipoic acid. The antioxidant levels in PE were found to be higher than that in other natural juices, such as blueberry, cranberry, and orange, as well as in red wine.¹⁵

Pomegranate has become more popular because of the attribution of important physiological properties, such as anticancer, cholesterol-lowering, and cardioprotective. This study demonstrated ameliorative effects of PE, a phenolic antioxidant, on GEN-induced nephrotoxicity, in line with the consideration that oxygen-free radicals are important mediators of GEN-induced acute renal failure. The understanding of aminoglycoside nephrotoxicity is clinically important; such nephrotoxicity is typically associated with anoliguric acute renal failure, that is, azotemia in the presence of 1–2 L/day urine output. In this study, the 24-h urine volume in the GEN group was significantly higher than in the control group, indicating the presence of GEN-induced polyuria, whereas in the GEN + PE-treated group, it was not different from that of the control group, suggesting the protective role of PE against acute tubular necrosis. In addition, increased serum urea and creatinine levels in GEN-treated rats reflect the renal damage. In contrast to the previous studies, serum K⁺ levels were similar between the GEN-induced nephrotoxicity and the control groups. The final studies show that K⁺ levels were not increased in rats with GEN-induced nephrotoxicity.^35–37 Administration of PE protects the kidney function from GEN as indicated by preventing an increase in serum urea and creatinine levels. PE restores the renal function by preserving the structural integrity of renal cells against GEN challenge, evidenced by significantly preventing an increase in the levels of serum creatinine and urea.

Since brush border membrane (BBM) and other intracellular organelles such as mitochondria and lysosomes are known GEN target,^38,39 the structural/functional integrity was assessed by the status of their respective biomarker enzymes. So, here, we measured the MDA, GSH, and NO, as a means of oxidative stress. Our findings corroborate those of the earlier studies demonstrating that an enhanced endogenous oxidative stress has a major role in the severity of GEN-induced acute renal failure.^40,41 MDA, a stable lipid hydroperoxide, provides an index of the LPO in biological tissues.⁴² In this study, we found increased MDA levels in the GEN-treated group, and as a protective effect of PE, lower MDA levels were found in the group determined by GEN + PE. The GSH antioxidant system is considered as the most notable cellular protective mechanism. GSH has a very important role in protecting against oxygen-free radical damage by providing reducing equivalents for several mechanism, as well as scavenging hydroxyl radicals and singlet oxygen. Its depletion is a common consequence of increased formation of ROS⁵ like GEN-induced nephrotoxicity. In the group treated with GEN + PE, we found increased GSH levels. However, our study has shown that PE does not affect NO levels protectively in contrast to some previous studies with different antioxidant agents.^3,43 So, it may enhance LPO by a different way. These findings strongly indicate that PE is important in protecting the kidney from GEN-induced injury through improvement in oxidant status.

GEN treatment provokes acute tubular necrosis and acute renal failure in about 30% of high-risk patients.⁶ Animal models of aminoglycoside nephrotoxicity also present residual areas of interstitial fibrosis in the renal cortex and progressive tubular injury.^44,45 After staining with Masson’s trichrome, no statistical difference was found between the groups in kidney fibrosis scores. However, in GEN-treated rats, there was more mild and moderate fibrosis in kidney specimen as previous studies. In this study, the histopathological examination of kidneys showed severe and extensive damage in GEN-treated rats which have tubular necrosis and edema. This could be due to the formation of highly reactive radicals as a consequence of oxidative stress caused by GEN. The kidneys of the control group showed normal histological features, but the GEN-treated group revealed more extensive and marked tubular necrosis. On the other hand, the tubules from rats of the GEN + PE-treated group were nearly normal in histological appearance except for a slight desquamation and atrophy of the tubular epithelial cells. Similar changes were also reported by some studies, which demonstrated structural changes in renal tissue of GEN-treated animals and its reversal by various agents.^3,46,47

In conclusion, the data indicated that GEN-induced nephrotoxicity might be related to oxidative damage. The results obtained in this study suggested that PE has an overall protective effect against GEN-induced nephrotoxicity in rat model. The observed protective effects can be attributed to the antioxidant properties of PE. So, PE is a highly free-radical scavenger agent and offers protection against GEN-induced acute renal failure.

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