Protective effects of naringenin on carbon tetrachloride-induced acute nephrotoxicity in mouse kidney

Chemico-Biological Interactions 205 (2013) 138–147

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Chemico-Biological Interactions

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Protective effects of naringenin on carbon tetrachloride-induced acutenephrotoxicity in mouse kidney

0009-2797/$ - see front matter � 2013 Elsevier Ireland Ltd. All rights reserved.http://dx.doi.org/10.1016/j.cbi.2013.06.016

⇑ Corresponding author at: Department of Histology, Faculty of Medicine,Pharmacy and Dentistry, Vasile Goldis Western University of Arad, 86 Rebreanustr., 310414 Arad, Romania. Tel.: +40 747347150; fax: +40 257282839.

E-mail address: [email protected] (A. Hermenean).

Anca Hermenean a,b,⇑, Aurel Ardelean b, Miruna Stan c, Hildegard Herman b, Ciprian-Valentin Mihali b,Marieta Costache c, Anca Dinischiotu c

a Department of Histology, Faculty of Medicine, Pharmacy and Dentistry, Vasile Goldis Western University of Arad, 86 Rebreanu str., 310414 Arad, Romania.b Department of Experimental and Applied Biology, Institute of Life Sciences, Vasile Goldis Western University of Arad, 86 Rebreanu, 310414 Arad, Romaniac Department of Biochemistry and Molecular Biology, University of Bucharest, 91-95 Splaiul Independentei, 050095 Bucharest, Romania

a r t i c l e i n f o

Article history:Received 2 March 2013Received in revised form 16 June 2013Accepted 18 June 2013Available online 9 July 2013

Keywords:NaringeninCCl4

Oxidative stressNephroprotectionAntioxidant effects

a b s t r a c t

The ability of naringenin (NGN) to protect the kidney against CCl4-induced renal toxicity in male Swissmice was investigated. The flavonoid was given orally to mice for 7 days and then on the 8th day, thesewere intraperitoneally injected with 10 mmol/kg CCl4. When the toxicant was administrated alone, anincrease of malondialdehyde (MDA) concentration was observed and a significant decrease in superoxidedismutase (SOD), catalase (CAT) glutathione-peroxidase (GPx) specific activities as well as glutathione(GSH) levels was detected after 24 h. These were accompanied by glomerular and tubular degenerations,vascular congestion, necrosis and fatty changes. Marked collagen deposition and strong TGF-b1 expres-sion were observed mainly in the mesangial cells of the glomeruli and tubulointerstitial areas. Ultrastruc-tural investigations showed proximal and distal tubular epithelial cells alterations including numerouslysosomes and dense granular bodies, altered mitochondria, appearance of ‘‘myeloid bodies’’ and basalenfolding dilatation. Pre-treatment with NGN resulted in the return of biochemical markers to controlvalues. Histopathological and electron-microscopic examinations confirmed the biochemical results. Inconclusion, NGN showed antioxidant and renal protective effects against injuries induced by CCl4.

� 2013 Elsevier Ireland Ltd. All rights reserved.

1. Introduction

Carbon tetrachloride (CCl4), an industrial solvent, cleaner, anddegreaser, has been extensively used in animal models to explorechemical toxin-induced hepatic injury [1]. It was demonstratedthat liver is not the only target organ of CCl4 toxicity, and trichlo-romethyl radicals generation (CCl3

�and/or CCl3OO�) also occurred inother tissues, such as the kidney [2–5], lung [6,7], testis [8,9], brain[10,11] and blood [12]. It has also been reported that exposure toCCI4 results in renal injuries [13].

The kidney removes the metabolic waste, the xenobiotics andtheir metabolites, while also regulating the water and ion contentin the blood. Xenobiotics can be excreted unchanged into the urine,bile, faeces, expired air or can be converted through biotransforma-tion processes into more water soluble metabolites. The kidneyspossess the common xenobiotic metabolizing enzymes, mainlylocalized in proximal tubular cells [14]. The initial step in biotrans-formation of CCl4 is reductive dehalogenation [15]. As electrophils,

free radicals initiate the lipid peroxidation process and if this pro-cess overwhelms the antioxidant defense system, oxidative stressleading to kidney damage occurs.

Many studies have reported that antioxidants can preventnephropathy and hepatic damage by counteracting free radicalsand preventing lipid peroxidation. Additionally, previous reportshave shown that hepatic and renal toxicity caused by CCl4 werediminished by natural products such as vitamin C which restoredthe normal level of reduced glutathione [16] and melatonin whichdecreased the elevated TBARS and up-regulated the low activitiesof antioxidant enzymes [5].

Flavonoids are phenolic compounds widely present in the fruitand vegetables characteristic of the human diet. The former havebeen suggested to exhibit a powerful antioxidant activity due totheir ability to reduce free radical formation and scavenge free rad-icals, together with the up-regulation of antioxidant defenses [17].

Naringenin (NGN) is a bioflavonoid highly enriched in citrusfruit, tomatoes and cocoa. This compound has been investigatedfor its pharmacological activities, including anti-tumor [18–22],anti-inflammatory [23–25], anti-viral [26,27] and anti-diabetic[28] effects.

As far as we know, there is no study concerning the effect ofnaringenin against CCl4-induced renal injury. Therefore, our workwas carried out to establish the protection of naringenin against

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CCl4–induced damage in mice kidney. In order to evaluate oxida-tive stress, the specific activities of SOD, CAT and GPX, as well asGSH and MDA concentrations were determined. The changes inmice renal tissue were also evaluated from a structural and ultra-structural point of view.

2. Materials and methods

2.1. Animals and experimental design

Swiss male mice (25 ± 3 g), supplied by the Animal House of theVasile Goldis Western University of Arad, were used. The animalswere acclimatized at a temperature of 20–25 �C under naturallight/dark conditions for two days and were fed with food andwater ad libitum. Prior to the experiment, the animals were keptunder fasting overnight. All experimental procedures wereapproved by the ethics and regulations of animal experiments ofVasile Goldis Western University of Arad.

Thirty-two animals were used for the experiment and thesewere divided into 4 groups, as follows:

Group 1: Control animals; received by gavage only the vehicle(i.e. olive oil and 0.5% carboxymethyl cellulose) every day for7 days and on 8th day were deprived of food for 24 h.

Group 2: CCl4 group; received the vehicle (i.e. olive oil and 0.5%carboxymethyl cellulose) every day for 7 days and were subse-quently i.p. injected with CCl4 at a dose of 10 mmol/kg b.w. in50% olive oil (1:1) on the 8th day.

Group 3: NGN pre-treated group; received naringenin solubilizedin 0.5% carboxymethyl celulose (NGN, Sigma 98%) (50 mg/kg b.w.)orally for 7 days and were subsequently i.p. injected with CCl4

(10 mmol/kg b.w.) on the 8th day.Group 4: NGN group; received naringenin (NGN) solution alone

(50 mg/kg b.w.) orally for 7 days and on 8th day were deprived offood for 24 h.

After 8 days from the start of treatment of groups 1 and 3, and24 h after CCl4 i.p. injection for groups 2 and 3, the mice were sac-rificed by cervical dislocation. Kidney samples were used for histo-pathology, electron microscopy and biochemical analyses.

Fig. 1. Protective effect of the 50 mg/kg dose of NGN on MDA concentrationinduced by CCl4 exposure in mice kidneys. Values are expressed as means ± SD(n = 8). ⁄⁄⁄Statistical significance at p < 0.001 as compared to control. ###Statisticalsignificance at p < 0.001 as compared to CCl -treated group.

2.2. Kidney tissue homogenate preparation

Fresly isolated kidney (1 g of tissue) was added to 10 volumes ofice-cold buffer (0.1 M TRIS–HCl, 5 mM EDTA buffer, pH 7.4) con-taining a protease inhibitor cocktail (Sigma–Aldrich, USA, 1:50dilution) and homogenized for 2 min at 16 Hz using a ball mill(type MM 301, Retsch GmbH & Co, Haan, Germany). The homoge-nate was centrifuged at 8000g for 30 min at 4 �C to remove the celldebris. The supernatant was collected and used for biochemicalassays.

4

Table 1Protective effect of 50 mg/kg b.w. NGN on specific activities of SOD, CAT, GPX andconcentration of GSH in mice kidneys.

Group SODU/mg protein

CATU/mg protein

GPXU/mg protein

GSHnmol/mgprotein

Control 1.85 ± 0.34 0.66 ± 0.12 40.14 ± 4.16 9.53 ± 1.00CCl4 1.02 ± 0.22*** 0.39 ± 0.03*** 25.86 ± 4.14*** 7.02 ± 1.88***

NGN 2.35 ± 0.34*** 0.68 ± 0.03 41.40 ± 5.81 10.22 ± 1.25NGN + CCl4 2.05 ± 0.24### 0.63 ± 0.11### 37.21 ± 1.90### 10.22 ± 1.41###

Note. Values are expressed as means ± SD (n = 8).*** Significance versus control p < 0.001.### Significance versus CCl4-treated group p < 0.001.

2.3. Assessment of antioxidant status

Renal catalase (CAT) activity was assessed according to the Aebimethod [29] that records the decrease in the absorbance at 240 nmcorresponding to H2O2 decomposition. One unit of CAT activity wasrepresented by the decomposition of 1 lmol H2O2/min/mL. Kidneysuperoxide dismutase (SOD) activity was measured by the methoddescribed by Paoletti and Mocali [30]. One unit of SOD activity wasequal to the amount of enzyme that inhibits the oxidation of NADHby 50% at 37 �C. The activity of glutathione peroxidase (GPx) wasassayed by monitoring the oxidation of NADPH by t-butyl-hydro-peroxide at 340 nm (V-530 JASCO spectrophotometer) [31]. Allenzymatic activities were calculated as specific activities (units/mg of protein).

GSH content was determined using a commercial kit (Sigma–Aldrich, MO, USA) according to the manufacturer’s instructions.The absorbance was measured at 405 nm using a microtiter platereader (GENIOS Tecan) and the concentration was expressed innmol GSH/mg protein.

2.4. Assessment of peroxidative stress

The renal malondialdehyde (MDA) content was assayed as ameasure of lipid peroxidation using a fluorimetric method de-scribed by Del Rio et al [32]. The kidney tissue homogenate sample(200 lL) was incubated with 0.1 M HCl (700 lL) for 20 min at roomtemperature. After that, 900 lL of 0.025 M thiobarbituric acid wasadded, and the mixture was incubated for 65 min at 37 �C. Subse-quently, samples were subjected to fluorescence analysis(kex = 520 nm; kem = 549 nm) (Spectrofluorometer FP-6300 JASCO).Relative fluorescence units (RFU) recorded were converted to nmolmalondialdehyde (MDA) using 1,1,3,3-tetramethoxypropane asstandard.

2.5. Protein concentration measurement

The protein content was determined after Lowry’s methodusing bovine serum albumin as standard [33].

2.6. Histopathology

Freshly prelevated fragments of mice kidneys were fixed inBouin solution, dehydrated in ethanol, cleared in toluene andembedded in paraffin. Five micrometer thickness kidney sectionswere deparaffinized and processed routinely for Hematoxylin–

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eosin (H&E) and Mallory trichrome staining according to Bio Opticastaining kit. Frozen sections were cut at 8 lm with the SLEE MNTcryotome, fixed in 10% buffered formaldehyde and stained withOil Red O kit according to the methods of Bio-Optica staining kits.Mounted slides were examined under a light microscope (OlympusBX43 microscope) and photographed using a digital camera Olym-pus XC30.

Fig. 2. Photomicrographs of kidney sections stained with hematoxylin–eosin under the lituff of glomerular capillaries (⁄) surrounded by Bowman’s space (arrowhead). Normal pmice medulla region. (B1) CCl4-treated mice renal cortex showing tubular renal epithevascular congestion (arrow) and necrosis (arrowhead) into medulla area of CCl4-treatedhistological aspect which is comparable to that of the control group. (C2) NGN + CCl4 groucontrol group. (D1) NGN alone group showing a normal aspect of the cortex area whichaspect of the cortex area which is comparable to that of the control group.

2.7. Immunohistochemical determination of TGF-b1

Immunohistochemical studies were performed on paraffinembedded kidney tissues using rabbit polyclonal anti-TGFb1 anti-body diluted 1:50 (Santa Cruz Biotechnology, California, USA).Slides were incubated with a peroxidase block. After washing,monoclonal antibodies diluted in phosphate buffered saline

ght microscope. (A1) Control mice cortex showing normal renal corpuscles formed ofroximal and distal convoluted tubules are also seen. (A2) Normal aspect of controllial vacuolization (arrowhead) and glomerular atrophy (arrow). (B2) Dilatation ormice. (C1) NGN + CCl4 group displaying a marked improvement of the renal cortexp showing the normal aspect of the medulla area which is comparable to that of the

is comparable to that of the control group. (D2) NGN alone group showing a normal

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supplemented with bovine serum albumin were added to tissuesamples and incubated overnight at 4 �C in a humid environment,followed by incubation with peroxidase labeled polymer conju-gated to secondary antibodies. The immunoreaction product wasvisualized by adding the substrate-chromogen diaminobenzidine(DAB) solution, resulting in a brownish coloration at antigen sites.Tissues were counterstained with hematoxylin, dehydrated in agradient of alcohol and mounted.

The specificity of the reaction was confirmed by substitution ofprimary antibodies with irrelevant immunoglobulins of matchedisotype, used in the same conditions and dilutions as primary

Fig. 3. Photomicrographs of kidney sections stained with Mallory tricrome under the lisurrounding the renal corpuscles and tubules. (B) CCl4-treated mice kidney showing magroup displaying marked improvement of the renal histological aspect which is comparathe kidney which is comparable to that of the control group.

antibodies. Stained slides were analyzed by light microscopy(Olympus BX43, Tokyo, Japan).

2.8. Immunohistochemical determination of TIM-1

Immunohistochemical studies were performed on paraffinembedded kidney tissues using rabbit polyclonal anti-TIM1 anti-body diluted 1:50 (Thermo Scientific, USA). Slides were incubatedwith rabbit ABC staining system (sc-2018), according to the meth-ods of Santa Cruz staining kits (Santa Cruz Biotechnology, USA).Tissues were counterstained with hematoxylin, dehydrated in a

ght microscope. (A) Control mice kidney showing few collagen fibers (arrowhead)ny collagen fibers (arrowhead) around renal corpuscles and tubules. (C) NGN + CCl4

ble to that of the control group. (D) NGN alone group showing the normal aspect of

Fig. 4. Photomicrographs of kidney sections stained with Oil Red O under the light microscope. (A) Control mice kidney showing few lipid drops in renal parenchyma. (B)CCl4-treated mice kidney showing proliferation of lipids (red aspect) in renal parenchyma compared with control. (C) NGN + CCl4 group displaying marked improvement ofthe renal histological aspect which is comparable to that of the control group. (D) NGN alone group showing a normal aspect of kidney which is comparable to that of thecontrol group. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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gradient of alcohol and mounted. The specificity of the reactionwas confirmed by substitution of primary antibodies with blockingsolution, used in the same conditions and dilutions as primaryantibodies. Stained slides were analyzed by light microscopy(Olympus BX43, Tokyo, Japan).

2.9. Electron microscopy

Electron microscopic kidney specimens were prefixed in 2.7%glutaraldehyde solution in 0.1 M phosphate buffer for 1.5 h, at4 �C. Following this, they were washed in 0.15 M phosphate buffer(pH 7.2) and post-fixed in 2% osmic acid solution in 0.15 M phos-phate buffer for 1 h at 4 �C. Dehydration was performed in acetone,and inclusion was done in the epoxy embedding resin Epon 812.The blocks were cut with an ultramicrotome type LKB, at 70 nmthickness. The sections were doubly contrasted with solutions ofuranyl acetate and lead citrate and analyzed with a TEM Tecnai12 Biotwin electron microscope.

2.10. Statistical analysis

All results were analyzed and plotted using GraphPad Prismsoftware (Version 5; GraphPad Software, Inc., La Jolla, CA) and ex-pressed as mean values ± SD (n = 8). Comparisons between groupswere evaluated by one-way ANOVA followed by a post hoc Bonfer-roni test. A value of p < 0.05 was considered to be statisticallysignificant.

3. Results and discussion

NGN is generally present in food as its b-glycoside, i.e. naringin,which is deglycosylated prior to intestinal absorption [34]. Thereare important inter-individual differences in the ability of humansto convert naringin to NGN due to the presence or absence

of certain bacterial strains in the gut [35]. Furthermore, humanintestinal bacteria can metabolize naringin to NGN and then to4-hydroxybenzoic acid, phloroglucinol, 2,4,6-trihydroxybenzoicacid and 4-hydroxyphenyl acetic acid [36].

Also, it was noticed that a large percentage of NGN absorbed inhumans is conjugated as glucuronide derivates [35] which can beexcreted in bile and to a lesser extent in urine.

Depending on the diet type, the human intake of NGN couldvary between 2.2 mg/day in Denmark [37], 4.1 mg/day in Japan[38], 8.3 mg/day in Finland [39] and 58.1 mg/day in UK [40].

In our experiments we used a single dose of 50 mg/kg b.w.,which was proved to be protective in other toxicant mediated oxi-dative damage in rodents [41,42].

3.1. Lipid peroxidation and effects on renal antioxidant status

As highlighted in Fig. 1, the exposure to CCl4 increased the levelof MDA, which is generated by the lipid peroxidation cascade, by80%. As one of the end products of this process, MDA reacts withprotein amino, sulfhydryl and imidazole groups [43], as well aswith DNA bases [44]. Due to its bifunctional aldehydic property,MDA has the potential to cross-link proteins, which can reduceor abolish their function [45]. On the other hand, MDA adductswith DNA bases are mutagenic and carcinogenic [46]. The pre-treatment with NGN decreased lipid peroxidation significantlycompared to CCl4 group to a level almost identical with the controllevel. This occurrence might be explained by the capacity of NGN,as other polyphenolic compounds, to scavenge the reactive species[47], by acting as terminators of free radical chains and as chelatorsof redox-active metal ions that are able to catalyze lipid peroxida-tion [48].

SOD, CAT and GPX specific activities in the kidney were signif-icantly decreased in CCl4 treated mice in comparison with thecontrol group (Table 1), which is in accordance with other data

Fig. 5. The effect of NGN on the expression and specific distribution of TGF-b1 in the kidney. (A1–2) In the control kidney, TGF-b1 expression was insignificant. (B1–2) TGF-b1-immunopositivity in CCl4-intoxicated mice in both glomeruli and tubular areas (arrowhead). (C1–2) Pre-treatment with NGN (50 mg/kg) markedly reduced TGF-b1immunopositivity compared to CCl4-intoxicated alone mice. (D1–2) The kidneys of mice receiving NGN (50 mg/kg) alone were similar to control. 1. Kidney cortex; 2. Kidneymedulla.

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which proved that oxidative stress reduced the aforementionedenzymatic activities [49]. The decrease of these enzymatic activi-ties could be explained by the crosslinking of their moleculescaused by the formation of MDA-protein adducts which affectstheir conformation and biological activity, as well as by the celldeath. The NGN + CCl4 treated group had a significant increasein renal antioxidant enzymes activity when compared to CCl4

treated mice. These results indicate that the inhibition of antiox-idant enzymes activity in kidney intoxicated with CCl4 wasprevented by NGN treatment. Previous studies have reported par-

tial NGN-related protection of the kidney against increased lipidperoxidation and the decline of the antioxidant enzymes systemdue to cadmium [50], cisplatin [51], arsenic [52] or lead [53]administration in rats.

In the group treated with NGN only, the specific activities ofCAT and GPX were at the same level with the control one, whereasan up-regulation of SOD activity by 15% was observed. Thismight be a consequence of the increased expression of SOD atprotein level as previously observed in rats exposed to polyphenols[54,55].

Fig. 6. The effect of NGN on the expression and specific distribution of TIM-1 in the kidney. (A1) In the control kidney, TIM-1 expression was absent. (B1) TIM-1-immunopositivity in CCl4-intoxicated mice in cortical area (arrowhead). (C) In the pre-treated NGN group, TIM-1 expression was insignificant. (D) The kidneys of micereceiving NGN (50 mg/kg) alone were similar to control.

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The administration of 50 mg/kg b.w. NGN to mice did not affectthe level of GSH compared to the control, suggesting that thisnatural compound does not affect GSH biosynthesis and/or regen-eration (Table 1). Our findings differ from other studies showingeither a non-protective in vitro effect on oxidation of GSH [56] oran increase in the level of this non-enzymatic antioxidant in the li-ver of young rats upon NGN administration [57].

3.2. Protective effects of naringenin against nephropathy induced byCCl4

The kidney sections of the control group showed normalappearance of tubules, glomeruli and tubulointerstitial cells(Fig. 2A1–2). Collagen deposition (Fig. 3A1–2) and lipids (Fig. 4A)were at normal range in the control group.

The group of mice treated with NGN alone preserved normalkidney morphology and architecture (Figs. 2D1–2, 3D1–2 and4D). Histopathological studies revealed that CCl4 induced glomer-ular and tubular degenerations varying from glomerular basementthickening (Figs. 2B1 and 3B1) and mild dilatation to congestion ofBowman’s space with glomerular atrophy (Fig. 2B1). In addition,some of the renal tubules were dilated and their epithelial cellstended to be vacuolated (Fig. 2B1). As far as the renal cortex ofCCl4-treated group is concerned, the aforementioned lesions werespread to the subcortical and medulla areas as well as where vas-cular congestion and necrosis were present (Fig. 2B2). Marked col-lagen deposition (Fig. 3B1–2) and fat tissue changes (Fig. 4B) wererecorded in both the cortex and medullary region of the kidney;additionally, considerable congestion in the blood vessels aroundthe tubules coupled to varying degrees of hemorrhage was ob-served. Previous studies have reported similar histopathologicalalterations in mice kidney treated with CCl4, including tubular epi-thelial cells alterations [58], renal tubular dilatation with cell vac-uolization which appeared foamy [59], tubular degeneration andnecrosis [5], as well as alterations present in both the cortex and

medullary regions [60]. The vasoconstriction induced by CCl4 pro-duced a local ischemic environment, which lead to multiple cellu-lar damages, including the deterioration of membrane integrity[60]. In our study, the NGN + CCl4 treated group showed almostnormal morphology and normal architecture of the kidney(Figs. 2C1–2, 3C1–2 and 4C). Other reports indicated renal struc-tural protection of naringenin against cadmium [50], whereasnaringin showed nephroprotection against glycerol action in rats[61].

3.3. Protective effects of naringenin against TGF-b1 renaloverexpression induced by CCl4

The protective effects of NGN on the TGF-b1 expression, ele-vated by CCl4 profibrotic activity, are shown in Fig. 5. The kidneyslides of the control mice did not show substantial TGF-b1 immu-nopositivity (Fig 5A1–2). The TGF-b1 expression in the groupreceiving NGN alone was similar to the controls (Fig. 5D1–2). Bycontrast, strong TGF-b1 expression was observed for the CCl4 group(Fig. 5B1–2). TGF-b1 immunoreactivity was observed mainly in themesangial cells of the glomeruli and tubulointerstitial areas, show-ing as a predominantly brown staining. The former decreased inthe kidney of CCl4-intoxicated mice pretreated with NGN(Fig. 5C1–2).

3.4. Protective effects of naringenin against TIM-1 renaloverexpression induced by CCl4

TIM-1 – T-cell immunoglobulin (TIM-1) or kidney injury mole-cule-1 (KIM-1) is considered a renal injury biomarker [62]. In ourstudy, TIM-1 expression was located in cortical area, slightly inthe proximal tubules, for the toxic group, in accordance with otherfindings [63,64]. Immunopositivity was absent in the pre-treatedNGN group, as well as flavonoid alone group (Fig. 6).

Fig. 7. Transmission electron micrograph of the kidney. (A1) Control-groupshowing proximal convoluted tubules with the normal aspect of the cuboidalepithelial cells lining with euchromatic nuclei (N), apical long brush border (BB),basal enfolding (arrowheads), elongated vertical mitochondria (M) and a lysosomalcompartment that includes endosomes (⁄) and lysosomes. (A2) Control-groupshowing the distal convoluted tubules cells with euchromatic nuclei (N), longmitochondria (M) oriented vertical among the numerous deep enfolding of thebasolateral plasma membrane (arrowhead), short apical plasma membrane micro-projection (MM) and a small population of apical cytoplasmic vesicles (V). (B1–4)CCl4-treated group showing proximal convoluted tubular cells with numerouslysosomes, endosomes (⁄), ‘‘myeloid bodies’’ (MB), dilated basal enfolding (arrow-head), collagen accumulation (c) and damaged mitochondria (M). (B5) Sometubules contain numerous lipid drops (L). (B6) CCl4-treated group showing distaltubular cells with dilated basal enfolding (arrowhead). (C1–2) NGN + CCl4 groupshowing a marked improvement of the ultrastructural aspect of the proximal anddistal tubular cells, which is comparable to that of the control group. (D1–2) NGNalone group showing the normal ultrastructural aspect of the proximal and distaltubular cells which is comparable to that of the control group.

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3.5. Protective effects of naringenin against ultrastructural renalinjuries induced by CCl4

Electron microscopic examination of the renal cortex of thecontrol group revealed a normal aspect of the proximal convolutedtubules, where the epithelial cells lining appeared with euchro-matic nuclei and an apical long brush border, deep basolateralplasma membrane infoldings and basal vertical mitochondria(Fig. 7A1). The cells of the distal convoluted tubules exhibitedeuchromatic nuclei, extensive basolateral plasma membraneinfoldings, short apical plasma membrane microprojection and asmall population of apical cytoplasmic vesicles (Fig. 7A2).

As far as we know, no report about ultrastructural injuries inthe kidneys of mice exposed to CCl4 exists. Our ultrastructuralinvestigation of the CCl4-treated group revealed multiple altera-tions of the proximal tubular epithelial cells (Fig. 7B1–5). Further-more, the tubules were dilated with an increase in theinvagination of the dilated basal cell membrane, resulting in largeirregular spaces lined by the plasma membrane (Fig. 7B2). Alteredmitochondria were also observed (Fig. 7B3–4). Additionally, therewas a marked increase of electron dense lysosomes-like struc-tures with the presence of numerous lysosomes and dense gran-ular bodies which varied in size and shape (Fig. 7B1); the lattercould be dense secondary lysosomes or a mass formed by the fu-sion of such lysosomes [65]. Large ‘‘myeloid bodies’’, indicative ofan inhibited or altered function of the intralysosomal enzymaticmachinery, were also present [66]. Such ‘‘myeloid bodies’’ wereobserved in renal biopsies of patients exposed to gentamicin, vio-mycin or chromium [67] as well as in kidneys of Swiss ICR micetreated with 2-bromoethylamine hydrobromide [68]. Also, mye-loid bodies formation was noticed in hepatocytes after CCl4

administration [69]. It seems that these appear as a result ofthe impairment of biodegradation of polar lipids associated withthe decrease of lysosomal enzymes activity which cause the lyso-somes enlargement and finally their breakdown with the releaseof myeloid bodies into the tubular lumen with concomitant tubu-lar damage [70].

Moreover, some tubules are characterized by lipid drops accu-mulation (Fig. 7B5), whereas a similar effect was reported for hepa-tocytes following CCl4 action [71]. The distal tubules are alsoaffected, with epithelial lining cells presenting basal enfolding dil-atations (Fig. 7B6). These results are in accordance with other re-ports concluding that the first segments of proximal tubules arethe renal cells types most frequently involved in nephrotoxic pro-cesses, followed later by distal tubuli [66]. The group benefitingfrom a pre-treatment with naringenin (NGN + CCl4) showed amarked improvement of the ultrastructural aspect of the proximaland distal tubular cells, which was comparable to that of the con-trol group (Fig. 7C1–2).

To summarize, our results (Fig. 8) indicate an overall improve-ment of the structural and ultrastructural integrity of the kidneyand renal antioxidant defenses upon pre-administration ofnaringenin to CCl4 treated mice.

4. Conclusions

The present study suggests that the flavonoid naringenin has arenal protective potential. It effectively counteracts the CCl4-in-duced oxidative damage and morphological injury at kidney levelby conserving the endogenous antioxidant mechanism and scav-enging free radicals. As a result, consumption of fruit or vegetablesenriched in this flavonoid can protect humans against nephrotox-ins and help maintain healthy kidneys. Additionally, it appears thatnaringenin efficiently counteracts the oxidative stress induced byseveral toxins.

Fig. 8. CCl4 induced injury in renal morphology and kidney antioxidant mechanism and its prevention by flavonoid naringenin administration: A summary.

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Conflict of interest statement

The authors declare that there are no conflicts of interest.

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