Sage Journals: Discover world-class research

Abstract

Gentamicin (GNT) is an aminoglycoside antibiotic used for treatment of serious infections, and the nephrotoxic adverse effect is one of the main therapeutic limitations. This study aimed to investigate the possible protective effect of apocynin (APO) on nephrotoxicity induced by GNT in rats. Twenty-four rats were allocated into three groups: control, GNT (100 mg/kg, intraperitoneally (i.p.)), and GNT plus APO (10 mg/kg, i.p.). All rats were killed at the end of the experiment, and then the blood, urine, and kidneys samples were taken. GNT-induced nephrotoxicity was manifested by a significant (p < 0.05) increase in the weight of kidney, 24-h urine volume, renal somatic index (RSI), protein in urine, serum lactate dehydrogenase (LDH), creatinine (Cr), blood urea nitrogen (BUN), renal Fas ligand (CD95), nitric oxide (NO), and malondialdehyde (MDA). Furthermore, a significant reduction in body weight, creatinine clearance (CCr), serum albumin, renal superoxide dismutase (SOD), and glutathione activities were detected when compared with the control rats. APO ameliorated the nephrotoxic effect and oxidative damage caused by GNT by improving tissue morphology and significantly decreasing 24-h urine volume, RSI, serum Cr, LDH and BUN, protein in urine, and renal content of MDA, CD95, and NO. Additionally, APO caused a significant elevation in renal SOD activity and CCr when compared with the GNT group. These results confirm that APO by its anti-inflammatory, antiapoptotic, and antioxidant effects can ameliorate GNT-induced nephrotoxicity.

Keywords

Gentamicin apocynin nephrotoxicity nitric oxide apoptosis antioxidant

Introduction

Gentamicin (GNT) is an aminoglycoside antibiotic used for the treatment of serious gram-positive and gram-negative bacterial infections.¹ Although it is a powerful antibacterial agent, the nephrotoxic adverse effect is one of the main therapeutic limitations.²

The nephrotoxicity induced by GNT is a complex situation involving different pathways, such as the reduction of renal blood flow, oxidative stress, inflammation, nitric oxide (NO) generation,³ lipid peroxidation, the nuclear factor κB pathway,⁴ apoptosis,⁵ and the reduction of efficiency of kidney antioxidant enzymes such as superoxide dismutase (SOD), catalase, glutathione peroxidase, and reduced glutathione (GSH).⁶ The administration of compounds with anti-inflammatory, antiapoptotic, or antioxidant activity has been successfully used to block GNT nephrotoxicity.^7,8

Apocynin (APO) is a prodrug that is converted by peroxidase-mediated oxidation to a dimer (diapocynin), which has been shown to be more efficient than APO itself.⁹ Its mechanism of inhibition of NADPH oxidases (NOXs) is not totally known.¹⁰ APO showed potential antioxidant activities, and it suppresses the generation of reactive oxygen species (ROS) that is implicated in lipopolysaccharide (LPS)-induced acute lung injury (ALI).¹¹ Additionally, it showed inhibitory effects on pro-inflammatory cytokines¹² and apoptosis pathway.^13,14

APO has shown potential therapeutic effects in many diseases by its NOX inhibition, such as diabetic nephropathy,¹⁵ nephrotoxicity induced by cyclosporine,¹⁶ ALI,¹⁷ arteriosclerosis,¹⁸ and arthritis. ¹⁹ APO has been used as one of the most promising drugs in experimental models of inflammatory and neurodegenerative diseases but its effect on nephrotoxicity induced by GNT has not been reported.

In the light of the aforementioned information, the hypothesis was made that APO, by its NOX inhibition, could attenuate GNT-induced renal damage and this was investigated by measuring kidney biochemical parameters, oxidative stress biomarkers, and histological examination of the renal tissues.

Materials and methods

Drugs and chemicals

APO was purchased from Sigma-Aldrich Chemical Co. (St. Louis, Missouri, USA), which was kindly provided by Dr Mohammed S El-Awady. GNT as pharmaceutical ampoules (80 mg) preparation was purchased from Alexandria Chemical Co., Egypt.

Experimental animals

Twenty-four adult male Wistar rats, weighing 200 ± 20 g with average age of 6–8 weeks, were purchased from “Egyptian Organization for Biological Products and Vaccines,” Giza, Egypt. All the procedures and care administered to the animals have been approved by the “Research Ethics Committee” of the Faculty of Pharmacy, Mansoura University, Egypt, which is in accordance with the “Principles of Laboratory Animal Care” (NIH publication no. 85-23, revised 1985).

Experimental protocol

The animals were allocated into the following three groups each consisting of eight rats: (1) in control group, rats did not receive any drug or solvent; (2) in GNT group, for 7 successive days, rats were injected by GNT (100 mg/kg, intraperitoneally (i.p.))²⁰; (3) in APO/GNT group, APO (10 mg/kg, i.p.)¹¹ was administrated to rats starting 7 consecutive days before GNT (100 mg/kg, i.p.) injection and another 7 days together with GNT injection.

For a collection of 24-h urine samples, after the last dose (without prior adaptation), animals were immediately kept in individual metabolic cages. These samples were centrifuged for 15 min at 1000 × g/4°C and kept at −80°C until analyzed.

After collection of the urine samples, all the rats were anesthetized with diethyl ether. Blood was collected in tubes from the retro-orbital venous plexus; blood was allowed to clot for 60 min at 25°C. The serum samples were obtained by centrifuging blood at 1000 × g for 15 min using a cooling centrifuge and stored frozen for biochemical assays.

After collection of the blood samples, the animals were sacrificed by cervical dislocation. The kidneys were immediately removed, rinsed with ice-cold normal saline, blotted with a piece of filter paper, and weighed for calculation of the renal somatic index (RSI). RSI = (the weight of kidney (g)) ÷ (the final weight of the body (g)) × 100. The left kidneys (50 mg) were used for detection of CD95 (apoptosis) by the flow cytometric method. The remaining part of left kidneys was homogenized in phosphate buffer (0.1 M, pH 7.4) as 10% (w/v). The homogenates were centrifuged for 15 min at 2000 × g/4°C, and the supernatant was used for biochemical assays. The right kidneys were cleaned and fixed in 10% buffered formalin for histopathological examination.

Biochemical measurements

Biochemical measurements in the serum

Serum creatinine (Cr) and blood urea nitrogen (BUN) level were determined according to Bartels et al.²¹ and Fawcett and Scott,²² respectively. Serum Cr and BUN were spectrophotometrically measured at 550 nm and expressed as milligrams per deciliter (mg/dl). In addition, lactate dehydrogenase (LDH) activity and albumin level were assessed according to the method of Henry et al.²³ and Doumas et al.,²⁴ respectively, and expressed as a unit per liter and grams per deciliter, respectively.

Biochemical measurements in urine

Urine (Cr) and protein levels were measured according to Bartels et al.²¹ and Daughaday et al.,²⁵ respectively, and expressed as milligrams per deciliter and milligrams per day, respectively.

Creatinine clearance (CCr) was used to determine the glomerular filtration, calculated using the formula “CCr = Cr in urine (mg/dl) × urine flow (ml/min)/Cr in serum (mg/dl).” Urine flow was calculated by dividing urine volume of 24 h by 1440 (the number of minutes in day) and expressed in milliliters per minute.

Biochemical measurements in kidney tissue homogenate

Biomarkers of oxidative stress including thiobarbituric acid reactive substances (TBARS) levels, GSH, and the activities of SOD were determined in kidney homogenate according to the method of Ohkawa et al.,²⁶ Ellman,²⁷ and Marklund and Marklund,²⁸ respectively. The absorbance was determined spectrophotometrically at 532, 412, and 420 nm, respectively, and expressed as nanomoles per gram tissue (nmol/g.tissue), millimoles per gram tissue (mmol/g.tissue), and units per gram tissue (U/g.tissue), respectively.

Additionally, an indicator of NO synthesis, total nitrate/nitrite (NOx), was measured by following the manufacturers instruction of NO assay kit (R&D Systems, Minneapolis, Minnesota, USA).

Flow cytometry detection of apoptosis

CD95 as a marker for apoptosis was detected according to Cifone et al.²⁹ by following the manufacturers instruction of fluorescein isothiocyanate antihuman Fas (CD95) assay kit (BD Biosciences, California, USA), catalog no/size (555673/100 test). Flow cytometry data was analyzed by flow cytometry (FACS [fluorescence-activated cell sorting] caliber flow cytometer; Becton Dickinson, Sunnyvale, California, USA) with a compact air cooked low power 15-m watt argon ion laser beam (488 nm) at the Mansoura Children Hospital. The average number of evaluated nuclei per specimen 20,000 and the number of nuclei scanned were 120 per second using propidium iodide.³⁰

Histological examinations

At the end of the experiment, the right kidney was rapidly dissected out, fixed in 10% buffered formalin, then embedded in paraffin, and prepared as (5-μm thick) sections stained with hematoxylin and eosin (H&E). The pathologist doing histopathological assessment was blinded to the protocol of work.

Statistical analysis

Data are expressed as mean ± standard error of the mean, where n = number of rats. Statistical analysis was carried out using one-way analysis of variance followed by Tukey–Kramer multiple comparisons post hoc test. The level of significance was set at p < 0.05. GraphPad Prism V 5.02 (GraphPad Software Inc., San Diego, California, USA) was used for statistical analysis and graphing. BD Accuri V 1.0.264.21 (BD Accuri C6 Software Inc., Ann Arbor, USA) was used for flow cytometry graphing.

Results

General animal and laboratory data

Effect of APO on GNT-induced decrease in body weight of rats

The animals were weighed before and after the experiments. A severe body weight loss caused by GNT was observed when compared with the control group. Pretreatment with APO significantly (p < 0.05, n = 8) attenuated the GNT-induced decrease in body weight, but the reduction in body weight still significantly from the control group (Table 1).

Table 1.

General animal and laboratory data.^a

Group	Change in body weight (g)	Kidney weight (g)	Renal somatic index	24-h urine volume (ml/100 g body weight)
Control	16.25 ± 0.88	0.74 ± 0.056	0.36 ± 0.028	1.99 ± 0.093
GNT	−14.48 ± 0.97^b	1.04 ± 0.045^b	0.48 ± 0.02^b	4.18 ± 0.26^b
APO/GNT	−6.5 ± 0.44^b, ^c	0.82 ± 0.036^c	0.397 ± 0.02^c	1.63 ± 0.11^c

GNT: gentamicin (100 mg/kg, i.p.); APO: apocynin (10 mg/kg, i.p.); SEM: standard error of the mean; ANOVA: analysis of variance.

^aData are expressed as mean ± SEM, n = 8.

^b p < 0.05, significantly different from control using one-way ANOVA followed by Tukey -Kramer multiple comparisons post hoc test.

^c p < 0.05, significantly different from GNT group using one-way ANOVA followed by Tukey -Kramer multiple comparisons post hoc test.

Effect of APO on GNT-induced increase in kidney weight and RSI of rats

Table 1 shows a significant (p < 0.05, n = 8) increase in the weight of kidney and RSI compared to the control group caused by GNT. Preinjection with APO significantly lowered the kidney weight and RSI when compared to GNT-treated group (Table 1).

Effect of APO on GNT-induced increase in urine volume of rats

GNT caused a significant (p < 0.05, n = 8) elevation in the 24-h urine volume indicating the presence of polyuria compared to the control group. The 24-h urine volume was significantly (p < 0.05, n = 8) decreased in APO/GNT group when compared to GNT-treated rats (Table 1).

Biochemical parameters

Effect of APO on GNT-induced change in kidney function in rats

A significant (p < 0.05, n = 8) elevation in urine protein, serum BUN, and Cr compared to the control group was observed after 7 days of treatment with GNT. Moreover, GNT caused significant decrease in CCr and albumin. Preinjection with APO significantly (p < 0.05, n = 8) ameliorated changes induced by GNT in CCr, serum Cr, BUN, and proteinuria. Conversely, treatment with APO for 14 days did not significantly reverse the reduction in serum albumin induced by GNT (Table 2).

Table 2.

Effect of APO on GNT-induced change in kidney function in rats.^a

Group	Serum creatinine (mg/dl)	CCr (ml/min)	BUN (mg/dl)	Serum albumin (g/dl)	Proteinuria (mg/day)
Control	0.54 ± 0.023	0.33 ± 0.015	31.75 ± 1.18	3.75 ± 0.019	90.13 ± 8.77
GNT	1.18 ± 0.12^b	0.18 ± 0.022^b	59 ± 8.32^b	3.29 ± 0.035^b	172 ± 8.89^b
APO/GNT	0.71 ± 0.036^c	0.32 ± 0.044^c	38.75 ± 1.79^c	3.39 ± 0.035	106.6 ± 18.72^c

GNT: gentamicin (100 mg/kg, i.p.); APO: apocynin (10 mg/kg, i.p.). CCr: creatinine clearance; BUN: blood urea nitrogen; SEM: standard error of the mean; ANOVA: analysis of variance.

^aData are expressed as mean ± SEM, n = 8.

^b p < 0.05, significantly different from control using one-way ANOVA followed by Tukey -Kramer multiple comparisons post hoc test.

^c p < 0.05, significantly different from GNT group using one-way ANOVA followed by Tukey -Kramer multiple comparisons post hoc test.

Effect of APO on GNT-induced increase in serum LDH activity of rats

Figure 1 shows that GNT significantly (p < 0.05, n = 8) increased LDH activity compared to control rats. Preinjection with APO in rats produced a significant decrease in LDH activity induced by GNT.

Figure 1.

Effect of APO on GNT-induced increase in serum LDH activity of rats. GNT (100 mg/kg, i.p.); APO (10 mg/kg, i.p.). Data are expressed as mean ± SEM, n = 8. *p < 0.05, significantly different from control using one-way ANOVA followed by Tukey–Kramer multiple comparisons post hoc test. APO: apocynin; GNT: gentamicin; LDH: lactate dehydrogenase; SEM: standard error of the mean; ANOVA: analysis of variance. $ p < 0.05, significantly different from GNT group.

Effect of APO on GNT-induced change in kidney oxidative biomarkers in rats

The results in Figure 2 show that GNT significantly (p < 0.05, n = 8) increased the malondialdehyde (MDA) levels (Figure 2(a)) but decreased both GSH (Figure 2(b)) and SOD (Figure 2(c)) activities in rat kidney homogenate. Pretreatment with APO significantly (p < 0.05, n = 8) reduced the MDA levels and significantly (p < 0.05, n = 8) increased SOD activities, without significantly affecting the GSH activities compared to GNT-treated rats.

Figure 2.

Effect of APO on GNT-induced change in kidney oxidative biomarkers in rats. GNT (100 mg/kg, i.p.); APO (10 mg/kg, i.p.). Data are expressed as mean ± SEM, n = 8. (a) MDA. (b) Reduced GSH. (c) SOD. *^, ^$ p < 0.05, significantly different from control or GNT group, respectively, using one-way ANOVA followed by Tukey–Kramer multiple comparisons post hoc test. APO: apocynin; GNT: gentamicin; SEM: standard error of the mean; MDA: malondialdehyde; GSH: glutathione; SOD: superoxide dismutase; ANOVA: analysis of variance.

Effect of APO on GNT-induced increase in renal nitric oxide (NOx) content

GNT caused significant (p < 0.05, n = 8) elevation in the renal NOx level when compared to control rats. Treatment of rats with APO significantly (p < 0.05, n = 8) decreased the renal NOx level when compared to the GNT group (Figure 3).

Figure 3.

Effect of APO on GNT-induced increase in renal NOx content. GNT (100 mg/kg, i.p.); APO (10 mg/kg, i.p.). Data are expressed as mean ± SEM, n = 8. *^, ^$ p < 0.05, significantly different from control or GNT group, respectively, using one-way ANOVA followed by Tukey–Kramer multiple comparisons post hoc test. APO: apocynin; GNT: gentamicin; NOx: nitric oxide; SEM: standard error of the mean; ANOVA: analysis of variance.

Flow cytometry results

The data in Figure 4 show that GNT significantly (p < 0.05, n = 4) increased the CD95 amount in kidney tissues compared to control rats. Conversely, there was a significant decrease (p < 0.05, n = 4) in the amount of CD95 in rats that were pretreated with APO compared to the GNT group, but still significantly different from the control group.

Figure 4.

Effect of APO on GNT-induced increase in renal Fas ligand (CD95). GNT (100 mg/kg, i.p.); APO (10 mg/kg, i.p.). Data are expressed as mean ± SEM, n = 4. *^, ^$ p < 0.05, significantly different from control or GNT group, respectively, using one-way ANOVA followed by Tukey–Kramer multiple comparisons post hoc test. APO: apocynin; GNT: gentamicin; SEM: standard error of the mean; ANOVA: analysis of variance.

Histopathological results

Effect of APO on GNT-induced changes in histopathological examination of kidney in rats

Histopathological investigation of the kidney using H&E (×200) stain showed normal glomeruli and normal renal tubules with normal lining renal tubular epithelium (Figure 5(a)). Kidney of GNT-induced rats showed hypercellularity in mesangial cells and adhesion between parietal and visceral layers of Bowman’s capsule (proliferative glomerulonephritis; arrow), with degeneration of renal tubular epithelium (Figure 5(b)). Animals treated with APO (Figure 5(c)) showed normal renal tubular epithelium lining renal tubules.

Figure 5.

Effect of APO on GNT-induced histopathological changes in kidney of rats. GNT (100 mg/kg, i.p.); APO (10 mg/kg, i.p.). Photomicrograph of the kidney using H&E (×200) stain, n = 4. (a) Kidney of control rat showed normal glomeruli and normal renal tubules with normal lining renal tubular epithelium. (b) Kidney of GNT-induced rats showed hypercellularity in mesangial cells and adhesion between parietal and visceral layers of Bowman’s capsule (proliferative glomerulonephritis), with degeneration of renal tubular epithelium. (c) Pretreatment of rats with APO for 14 days showed normal renal tubular epithelium lining renal tubules. APO: apocynin; GNT: gentamicin; H&E: hematoxylin and eosin.

Discussion

This study is the first to show that APO could attenuate GNT-induced kidney dysfunction. In this study, APO has an ameliorative effect against nephrotoxicity induced by GNT as illustrated by decreasing GNT-induced elevation in kidney weight, RSI, serum Cr, BUN, proteinuria, LDH level, kidney MDA, NO level, and CD95 amount, in addition to normalizing CCr and improving kidney histopathological changes in rats.

GNT is a member of aminoglycoside antibiotics; nephrotoxicity as a side effect of all aminoglycosides, especially GNT, limits its therapeutic use.² In the present study, GNT injection for 1 week caused a significant reduction in body weight of rats and a significant increase in the RSI and in the weight of kidney compared to control rats. This loss in weight may be due to either direct injury in renal tubules resulting in inability of the tubular cells to reabsorb water, leading to dehydration and loss of body weight³¹ or increased catabolism resulting in acidosis, anorexia, and decreased food intake.³² The increase in kidney weight after GNT injection is a result of inflammation and edema.³³ The weight loss and the increase in kidney weight were also observed in previous studies.^34,35

APO administration significantly decreased the kidney weight and RSI when compared to the GNT-treated rats; this result may be due to APO’s anti-inflammatory effect by reducing the inflammatory cytokines, such as TNF-α, IL-1β, and IL-6.¹²

GNT induced a typical pattern of nephrotoxicity that was associated with significant increase in serum Cr, BUN levels, and a higher volume of urine and protein. During renal dysfunction and as a result of diminished glomerular filtration rate, there is a reduction of the kidney’s ability to filter Cr and the nonprotein waste product is produced. Moreover, during renal dysfunction, the levels of urea and uric acid are elevated,³⁶ and protein level in urine is increased which might be a sensitive indicator of tubular damage, impaired reabsorptive capability of tubular protein, or impaired protein filtration of glomerular barrier.³⁷

The results of this study show that intraperitoneal injections of GNT to Wistar rats induced nephrotoxicity which was manifested by elevation in serum urea and Cr levels indicating glomerular damage. Additionally, a reduction in GFR is produced by GNT, clarified by the reduction in CCr and elevated levels of urine protein indicating tubular damage. These results came in line with other studies.^34,38,39

APO has a very good safety profile in animal studies as well, and several studies used long-term treatment without any signs of ill-health effects.⁴⁰ Previous studies reported that the renal function and the morphology of normal kidney were not altered by the administration of APO in normal rats, APO was given in the drinking water (2 g/L)⁴¹ or injected in dose of (20 mg/kg, i.p.) in rats.⁴²

APO produced a significant reduction in serum Cr and BUN level, and normalizing CCr. The ameliorative effect of APO on the kidney markers may be attributed to the protection against oxidative injury. These findings are in line with Chirino et al. who showed that administration of APO in the drinking water (2 g/L) 7 days before and 3 days after cisplatin injection ameliorates the increased serum Cr, BUN, and urinary excretion of total protein induced by cisplatin.⁴¹ In addition, Altintas et al. stated that APO (20 mg/kg, i.p., before ischemia and during ischemia) has a protective effect against renal ischemia/reperfusion (I/R)-induced kidney damage by significantly decreasing the elevated BUN and Cr levels.⁴²

Treatment with GNT has been shown to involve renal inflammatory responses in experimental animals.^43
–45 In the present study, GNT caused a significant elevation in serum LDH, with a significant decrease in the level of albumin in serum; this result agrees with El-Kashef et al.⁷ Treatment of rats with APO attenuated the elevation of serum LDH induced by GNT; this may be due to the anti-inflammatory properties of APO.

The components of NOX in the kidney are expressed in the glomerular mesangial, renal vessels, macula densa, distal tubule, the thick ascending limb, and collecting ducts.⁴⁶ NOX are the major source of ROS in many tissues that play both physiological and pathophysiological roles.⁴⁷ Due to the sensitivity of urinary system toward oxidative stress, ROS play an important role in the pathogenesis of drug-induced kidney diseases.^48
–50 Oxidative stress has participated in nephrotoxicity; ROS is the central key in the mechanisms that lead to a decrease in glomerular filtration rate and tubular necrosis.⁵¹

Another mechanism of GNT-induced nephrotoxicity is oxidative stress. GNT exerts its adverse renal toxicity by the generation of ROS in the kidney such as superoxide anions,⁵² hydrogen peroxide, hydroxyl radicals, and reactive nitrogen species (RNS).⁵³ Interaction of excessively produced ROS with cellular components, such as lipids (peroxidation of unsaturated fatty acids in the cell membrane), proteins (denaturation), carbohydrates, and nucleic acids resulted in cell and tissue damage.⁵⁴ GNT-induced tissue damage may be due to the depletion of renal GSH which permits lipid peroxidation.⁵⁵ Therefore, ROS scavengers and antioxidant molecules have the capacity to partially reduce or eliminate the deleterious effects induced by GNT.

In the current study, one of the causes of GNT-induced renal damage is oxidative stress. This view is supported by a significant elevation in the renal tissue levels of MDA as reflected by an increase in TBARS which is an end product of lipid peroxidation, while kidney antioxidant enzymes like SOD and GSH levels were reduced in the kidney tissue. The same observations were also showed in other studies.^7,56

APO by its NOX inhibition activity showed a significant reduction in the level of MDA and prevented the reduction in SOD activity compared to rats treated with GNT. The mechanism of inhibition of NOX is not totally known but involves inhibition of NOX component expressions such as p67-phox, p47-phox, and gp91-phox.¹⁸

These results are in line with other studies that demonstrated that APO prevents the generation of oxidative stress in the kidney after cisplatin-induced nephrotoxicity,⁴¹ cyclosporine-induced nephrotoxicity,¹⁶ renal I/R injury,⁵⁷ and after streptozotocin-induced diabetic nephropathy.¹⁵ Additionally, APO prevents the elevation of oxidative stress in the lung after LPS¹¹ and after bleomycin.⁵⁸ The protective effect of APO might be due to the reduction of the free radicals induced by GNT.

NO plays an important role in pathological and physiological pathways in the kidney. NO is important in the renal tubular function regulation and renal hemodynamics regulation.⁵⁹ Narita et al.⁶⁰ reported that decreased glomerulosclerosis and glomerular injury was produced by limiting NO production. In the kidney, GNT induced increase in the level of NO; the free radical nature of NO has a role in the acute renal failure caused by GNT leading to tubular damage.^61,62 In addition, NO reacts with superoxide radical and generates a cytotoxic peroxynitrite, resulting in renal failure and damage of the tubular cells.³

In this study, GNT caused a significant increase in NO level; this result agrees with El-Kashef et al.⁷ APO, by its NOX inhibition, significantly decreases the high level of NO induced by GNT injection. This result is in agreement with the other study reported that APO significantly reduced the activity of inducible nitric oxide synthase (iNOS) in renal tissues compared to the renal (I/R) injury group.⁵⁷

In addition to oxidative stress, one of the main side effects of GNT is in vivo and in vitro apoptosis in mesangial and proximal tubule cells. The ROS pathway is known to be important mediators in apoptosis induced by GNT.⁴⁵ GNT-induced apoptosis is associated with an increase in the proapoptotic protein Bax, in the survival promoting protein Bcl-2,⁶³ and in CD95.⁶⁴ CD95 (APO-1/Fas) is a member of the death receptor family, a subfamily of the tumor necrosis factor receptor (TNF-R) superfamily. Its main and best-known function in signaling is the induction of apoptosis. Within seconds after CD95 stimulation, the death-inducing signaling complex is formed.⁶⁵

In this study, Fas and Fas ligand were detected as a marker of apoptosis, which was detected by CD95. GNT caused a significant increase in CD95 amount which may be due to increased production of ROS; this result is in line with Asmaa et al.⁶⁴ APO significantly reduced the high amount of CD95 induced by GNT injection. This result may be explained by the antioxidant and antiapoptotic properties of APO. This result is in agreement with the other study which reported that APO, by its anti-apoptotic effect, protects against apoptosis in renal (I/R) injury⁴² and in diabetic nephropathy.⁶⁶

The histological investigation of kidneys from rats injected with GNT showed hypercellularity in mesangial cells and adhesion between parietal and visceral layers of Bowman’s capsule (proliferative glomerulonephritis), with degeneration of renal tubular epithelium. Animals treated with APO for 14 days showed normal renal tubular epithelium lining renal tubules, establishing its protective effect against tissue damage induced by GNT.

In conclusion, this is the first study reported that APO protects rats from nephrotoxicity induced by GNT by exerting antioxidant, antiapoptotic, and anti-inflammatory properties. This was supported by biochemical measurements and histopathological examination. Therefore, APO represents a new therapy for GNT-induced nephrotoxicity; however, more investigation is needed to determine its precise mechanism of action.

Footnotes

Acknowledgments

The author thanks Dr Mohammed S El-Awady, Department of Pharmacology and Toxicology, Faculty of Pharmacy, Mansoura University, Mansoura, Egypt, for providing APO. The author acknowledges Dr Mohamed F Hamed, Department of Pathology, Faculty of Veterinary Medicine, Mansoura University, Mansoura, Egypt, for his assistance in the histopathological examination. The author is grateful to Dr Mona S Gouida, Assistant Consultant of Molecular Immunology, Genetics Unit, Head of Flow Cytometry Unit, Mansoura Children Hospital, for providing assistance in flow cytometry technique.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

References

Choi

Moffett

McDade

. Altered gentamicin serum concentrations in obese pediatric patients. Pediatr Infect Dis J 2011; 30: 347–349.

Ali

Blunden

. Experimental gentamicin nephrotoxicity and agents that modify it: a mini-review of recent research. Basic Clin Pharmacol Toxicol 2011; 109: 225–232.

Christo

Rodrigues

Mouro

. Nitric oxide (NO) is associated with gentamicin (GENTA) nephrotoxicity and the renal function recovery after suspension of GENTA treatment in rats. Nitric Oxide 2011; 24: 77–83.

Rangan

Wang

Harris

. NF-kappaB signalling in chronic kidney disease. Front Biosci (Landmark Ed) 2009; 14: 3496–3522.

Laurent

Mingeot-Leclercq

. Gentamicin-induced apoptosis in renal cell lines and embryonic rat fibroblasts. Toxicol Sci 2000; 56: 229–239.

Dam

Scott

Ross

. Inhibition of cystathionine gamma-lyase and the biosynthesis of endogenous hydrogen sulphide ameliorates gentamicin-induced nephrotoxicity. Eur J Pharmacol 2012; 685: 165–173.

El-Kashef

El-Kenawi

Suddek

. Flavocoxid attenuates gentamicin-induced nephrotoxicity in rats. Naunyn Schmiedebergs Arch Pharmacol 2015; 388: 1305–1315.

Otunctemur

Ozbek

Cekmen

. Protective effect of montelukast which is cysteinyl-leukotriene receptor antagonist on gentamicin-induced nephrotoxicity and oxidative damage in rat kidney. Ren Fail 2013; 35: 403–410.

Johnson

Schillinger

Kwait

. Inhibition of NADPH oxidase activation in endothelial cells by ortho-methoxy-substituted catechols. Endothelium 2002; 9: 191–203.

10.

Barbieri

Cavalca

Eligini

. Apocynin prevents cyclooxygenase 2 expression in human monocytes through NADPH oxidase and glutathione redox-dependent mechanisms. Free Radic Biol Med 2004; 37: 156–165.

11.

Abdelmageed

El-Awady

Suddek

. Apocynin ameliorates endotoxin-induced acute lung injury in rats. Int Immunopharmacol 2016; 30: 163–170.

12.

Kim

Moon

. Anti-inflammatory effects of apocynin, an inhibitor of NADPH oxidase, in airway inflammation. Immunol Cell Biol 2012; 90: 441–448.

13.

Impellizzeri

Esposito

Mazzon

. Effect of apocynin, a NADPH oxidase inhibitor, on acute lung inflammation. Biochem Pharmacol 2011; 81: 636–648.

14.

Paterniti

Galuppo

Mazzon

. Protective effects of apocynin, an inhibitor of NADPH oxidase activity, in splanchnic artery occlusion and reperfusion. J Leukoc Biol 2010; 88: 993–1003.

15.

Asaba

Tojo

Onozato

. Effects of NADPH oxidase inhibitor in diabetic nephropathy. Kidney Int 2005; 67: 1890–1898.

16.

Ciarcia

Damiano

Florio

. The protective effect of apocynin on cyclosporine a-induced hypertension and nephrotoxicity in rats. J Cell Biochem 2015; 116: 1848–1856.

17.

Ghosh

Kanthasamy

Joseph

. Anti-inflammatory and neuroprotective effects of an orally active apocynin derivative in pre-clinical models of Parkinson’s disease. J Neuroinflammation 2012; 9: 241.

18.

Liu

Zhuan

. Effects of apocynin on oxidative stress and expression of apoptosis-related genes in testes of diabetic rats. Mol Med Rep 2013; 7: 47–52.

19.

Nauseef

. Biological roles for the NOX family NADPH oxidases. J Biol Chem 2008; 283: 16961–16965.

20.

Jafarey

Changizi

Najafi

. Calcium dobesilate for prevention of gentamicin-induced nephrotoxicity in rats. Iran J Kidney Dis 2014; 8: 46–52.

21.

Bartels

Bohmer

Heierli

. Serum creatinine determination without protein precipitation. Clin Chim Acta 1972; 37: 193–197.

22.

Fawcett

Scott

. A rapid and precise method for the determination of urea. J Clin Pathol 1960; 13: 156–159.

23.

Henry

Chiamori

Golub

. Revised spectrophotometric methods for the determination of glutamic-oxalacetic transaminase, glutamic-pyruvic transaminase, and lactic acid dehydrogenase. Am J Clin Pathol 1960; 34: 381–398.

24.

Doumas

Watson

Biggs

. Albumin standards and the measurement of serum albumin with bromcresol green. Clin Chim Acta 1971; 31: 87–96.

25.

Daughaday

Lowry

Rosebrough

. Determination of cerebrospinal fluid protein with the Folin phenol reagent. J Lab Clin Med 1952; 39: 663–665.

26.

Ohkawa

Ohishi

Yagi

. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal Biochem 1979; 95: 351–358.

27.

Ellman

. Tissue sulfhydryl groups. Arch Biochem Biophys 1959; 82: 70–77.

28.

Marklund

. Involvement of the superoxide anion radical in the autoxidation of pyrogallol and a convenient assay for superoxide dismutase. Eur J Biochem 1974; 47: 469–474.

29.

Cifone

Roncaioli

. Apoptotic signaling through CD95 (Fas/Apo-1) activates an acidic sphingomyelinase. J Exp Med 1994; 180: 1547–1552.

30.

Yoshino

Ami

Terao

. Upgrading of flow cytometric analysis for absolute counts, cytokines and other antigenic molecules of cynomolgus monkeys (Macaca fascicularis) by using anti-human cross-reactive antibodies. Exp Anim 2000; 49: 97–110.

31.

Ali

Al-Qarawi

Haroun

. The effect of treatment with gum Arabic on gentamicin nephrotoxicity in rats: a preliminary study. Ren Fail 2003; 25: 15–20.

32.

Ali

Abdel Gayoum

Bashir

. Gentamicin nephrotoxicity in rat: some biochemical correlates. Pharmacol Toxicol 1992; 70: 419–423.

33.

Erdem

Gundogan

Usubutun

. The protective effect of taurine against gentamicin-induced acute tubular necrosis in rats. Nephrol Dial Transplant 2000; 15: 1175–1182.

34.

El-Kashef

El-Kenawi

Rahim

. Agmatine improves renal function in gentamicin-induced nephrotoxicity in rats. Can J Physiol Pharmacol 2016; 94: 278–286.

35.

Nasri

Nematbakhsh

Ghobadi

. Preventive and curative effects of ginger extract against histopathologic changes of gentamicin-induced tubular toxicity in rats. Int J Prev Med 2013; 4: 316–321.

36.

Perrone

Madias

Levey

. Serum creatinine as an index of renal function: new insights into old concepts. Clin Chem 1992; 38: 1933–1953.

37.

Mueller

Lash

Price

. Urinary biomarkers to detect significant effects of environmental and occupational exposure to nephrotoxins. I. Categories of tests for detecting effects of nephrotoxins. Ren Fail 1997; 19: 505–521.

38.

Hosaka

Santos

Seguro

. Effect of cyclooxygenase inhibitors on gentamicin-induced nephrotoxicity in rats. Braz J Med Biol Res 2004; 37: 979–985.

39.

Soliman

Abdul-Hamid

Othman

. Effect of carnosine on gentamicin-induced nephrotoxicity. Med Sci Monit 2007; 13: BR73–BR83.

40.

Weiwer

Linhardt

. The role of the methoxyphenol apocynin, a vascular NADPH oxidase inhibitor, as a chemopreventative agent in the potential treatment of cardiovascular diseases. Curr Vasc Pharmacol 2008; 6: 204–217.

41.

Chirino

Sanchez-Gonzalez

Martinez-Martinez

. Protective effects of apocynin against cisplatin-induced oxidative stress and nephrotoxicity. Toxicology 2008; 245: 18–23.

42.

Altintas

Polat

Vardi

. The protective effects of apocynin on kidney damage caused by renal ischemia/reperfusion. J Endourol 2013; 27: 617–624.

43.

Bledsoe

Crickman

Mao

. Kallikrein/kinin protects against gentamicin-induced nephrotoxicity by inhibition of inflammation and apoptosis. Nephrol Dial Transplant 2006; 21: 624–633.

44.

Geleilete

Melo

Costa

. Role of myofibroblasts, macrophages, transforming growth factor-beta endothelin, angiotensin-II, and fibronectin in the progression of tubulointerstitial nephritis induced by gentamicin. J Nephrol 2002; 15: 633–642.

45.

Quiros

Vicente-Vicente

Morales

. An integrative overview on the mechanisms underlying the renal tubular cytotoxicity of gentamicin. Toxicol Sci 2011; 119: 245–256.

46.

Chabrashvili

Tojo

Onozato

. Expression and cellular localization of classic NADPH oxidase subunits in the spontaneously hypertensive rat kidney. Hypertension 2002; 39: 269–274.

47.

Carlstrom

Lai

. Role of NOX2 in the regulation of afferent arteriole responsiveness. Am J Physiol Regul Integr Comp Physiol 2009; 296: R72–R79.

48.

Ozen

Akyol

Iraz

. Role of caffeic acid phenethyl ester, an active component of propolis, against cisplatin-induced nephrotoxicity in rats. J Appl Toxicol 2004; 24: 27–35.

49.

Yilmaz

Ilhan

Naziroglu

. Ceftriaxone ameliorates cyclosporine A-induced oxidative nephrotoxicity in rat. Cell Biochem Funct 2011; 29: 102–107.

50.

Parlakpinar

Tasdemir

Polat

. Protective role of caffeic acid phenethyl ester (cape) on gentamicin-induced acute renal toxicity in rats. Toxicology 2005; 207: 169–177.

51.

Lopez-Novoa

Quiros

Vicente

. New insights into the mechanism of aminoglycoside nephrotoxicity: an integrative point of view. Kidney Int 2011; 79: 33–45.

52.

Yaman

Balikci

. Protective effects of nigella sativa against gentamicin-induced nephrotoxicity in rats. Exp Toxicol Pathol 2010; 62: 183–190.

53.

Balakumar

Chakkarwar

Kumar

. Experimental models for nephropathy. J Renin Angiotensin Aldosterone Syst 2008; 9: 189–195.

54.

Gutteridge

. Lipid peroxidation and antioxidants as biomarkers of tissue damage. Clin Chem 1995; 41: 1819–1828.

55.

Manikandan

Beulaja

Thiagarajan

. Ameliorative effects of curcumin against renal injuries mediated by inducible nitric oxide synthase and nuclear factor kappa B during gentamicin-induced toxicity in Wistar rats. Eur J Pharmacol 2011; 670: 578–585.

56.

Aygun

Akcam

Kaya

. Caffeic acid phenethyl ester modulates gentamicin-induced oxidative nephrotoxicity in kidney of rats. Biol Trace Elem Res 2012; 145: 211–216.

57.

Wang

. Effect of NADPH oxidase inhibitor-apocynin on the expression of Src homology-2 domain-containing phosphatase-1 (SHP-1) exposed renal ischemia/reperfusion injury in rats. Toxicol Rep 2015; 2: 1111–1116.

58.

Kilic

Parlakpinar

Taslidere

. Protective and therapeutic effect of apocynin on bleomycin-induced lung fibrosis in rats. Inflammation 2015; 38: 1166–1180.

59.

Patel

McAndrew

Sellak

. Biological aspects of reactive nitrogen species. Biochim Biophys Acta 1999; 1411: 385–400.

60.

Narita

Border

Ketteler

. Nitric oxide mediates immunologic injury to kidney mesangium in experimental glomerulonephritis. Lab Invest 1995; 72: 17–24.

61.

Rivas-Cabanero

Montero

Lopez-Novoa

. Increased glomerular nitric oxide synthesis in gentamicin-induced renal failure. Eur J Pharmacol 1994; 270: 119–121.

62.

Nakas-Icindic

Avdagic

Mijanovic

. Nitric oxide in gentamicin-induced acute tubular necrosis in rats. Bosn J Basic Med Sci 2005; 5: 70–74.

63.

Tey

Singh

Piredda

. Influence of bcl-2 on cell death during the cultivation of a Chinese hamster ovary cell line expressing a chimeric antibody. Biotechnol Bioeng 2000; 68: 31–43.

64.

Asmaa

Mahmoud

A-M

Mohamed

. Flow cytometric evaluation of apoptosis on the effect of mirazid on gentamicin-induced renal damage in rats. J Biochem Microbiol Biotechnol 2014; 1: 17–22.

65.

Lavrik

Golks

Krammer

. Death receptor signaling. J Cell Sci 2005; 118: 265–267.

66.

Ahmad

Mondello

. Protective effect of apocynin, an NADPH-oxidase inhibitor, against contrast-induced nephropathy in the diabetic rats: a comparison with n-acetylcysteine. Eur J Pharmacol 2012; 674: 397–406.

Protective effect of apocynin against gentamicin-induced nephrotoxicity in rats