Modulation Effects of Eugenol on Nephrotoxicity Triggered by Silver Nanoparticles in Adult Rats

Abstract

The use of silver nanoparticles (AgNPs) is expanding. This study evaluates the modulator effect of eugenol (Eug) on AgNP-induced nephrotoxicity in rats. Sixty male rats were separated into six groups: control, Eug, AgNPs low-dose, AgNPs high-dose, Eug + AgNPs low-dose, and Eug + AgNPs high-dose. After 30 days, kidney function, antioxidative and proinflammatory status, histopathological, histomorphometrical, and immunohistochemical assessments were performed. AgNPs markedly induced oxidative stress in renal tissues, characterized by increased levels of blood urea nitrogen, creatinine, uric acid, kidney injury molecule-1, the total oxidant capacity, malondialdehyde, tumor necrosis factor-alpha (TNF-α), and interleukin-6, as well as decreased levels of the total antioxidant capacity, superoxide dismutase, catalase, reduced glutathione, and glutathione peroxidase. Moreover, the normal renal architecture was destroyed, and the thickness of the renal capsules, cortex, and medulla, alongside the diameter and quantity of the normal Malpighian corpuscles and the proximal and distal convoluted tubules were decreased. Immunoreactivity for P53, caspase-3, and TNF-α reactive proteins were significantly increased; however, Bcl-2 immunoreactivity was decreased. Eug reversed most biochemical, histological, histomorphometrical, and immunohistochemical changes in AgNP-treated animals. This study demonstrated that nephrotoxicity in AgNP-treated rats was mitigated by an Eug supplementation. Eug's antioxidant, antiapoptotic, and anti-inflammatory capabilities were the key in modulating AgNPs nephrotoxicity.

Keywords: eugenol; histology; histopathology; immunohistochemistry; kidney; oxidative stress; silver nanoparticles.

Figure 1

Schematic representation of the study design and main results.

Figure 2

Levels of kidney injury molecule-1 (KIM-1) in renal tissues of the control and treated animal groups. The data are presented as mean ± SEM (n = 6). Values in the same row that are followed by different superscript letters differ significantly at 5% (p ≤ 0.05) level of significance according to ANOVA test and the TUKEY test. Eug, eugenol; AgNPs, silver nanoparticles.

Figure 3

Kidney sections of control and treated animal groups stained with H&E displaying (A,B) orderly renal architecture with outstandingly organized renal corpuscles (RC) forming of Bowman’s capsules (BC) enclosing normal Bowman’s spaces (blue asterisks) surrounding the glomeruli (G), in addition to the proximal (PCT) and distal (DCT) convoluted tubules in the cortical zone, as well as normal descending (DLH) and ascending (ALH) limps of the loops of Henle, and collecting tubules (CT) in medullary area of control rats; (C,D) regular cortical and medullary renal structure in Eug-treated rats; (E,F) hazardous effects on renal cortical portion represented by altered renal corpuscles (RC) which have decreased Bowman’s spaces (arrowheads) with expansion of their glomeruli (G), and focal tubular necrosis where the proximal (PCT) and distal (DCT) convoluted tubules are filled with hyaline casts or cellular debris (yellow asterisks), as well as their epithelia showed nuclear pyknosis (Pk), karyorrhexis (Kh), or karyolysis (Kl). Severely deteriorated inter-tubular blood vessels with hemorrhagic blood masses (green arrows). Additionally, renal medulla appeared with conspicuous damage of the lining cells of the descending (DLH) and ascending (ALH) limbs of the loops of Henle and collecting tubules (CT), as well as extravasated hemolyzed blood (green asterisks) were seen in AgNPs low-dose-treated rats; (G,H) severely deteriorated renal cortex with malformed renal corpuscles showing hypertrophied glomeruli (HG) and increased Bowman’s spaces (blue asterisks), as well as the proximal (PCT) and distal (DCT) convoluted tubules appeared with pyknotic (Pk) or karyorrhexed (Kh) nuclei. The inter-tubular spaces were filled with infiltrated inflammatory cells (IC) and stagnant hemolyzed blood (SHB). Additionally, deteriorated renal medulla with stagnant hemolyzed blood (SHB) in the inter-tubular spaces and marked coagulative necrosis of the lining cells of the descending (DLH) and ascending (ALH) limbs of the loops of Henle, and the collecting tubules (CT) represented with nuclear pyknosis (Pk) were seen in AgNPs high-dose-treated rats; (I,J) remarkable improvement in the renal tissues’ histological structure, with regular cortical and medullary regions were recorded in Eug + AgNPs low-dose-treated rats; (K,L) restoration of the kidney structure was noticed in the renal cortex and medulla in Eug + AgNPs high-dose-treated rats.

Figure 4

Histomorphometrical evaluation of renal tissue components in the control and treated animal groups showing (A) the thickness of fibrous renal capsules (μm²), (B) the thickness of renal cortex (μm²), (C) the thickness of renal medulla (μm²), (D) the diameter of Malpighian corpuscles (μm²), (E) the diameter of proximal convoluted tubules (μm²), and (F) the diameter of distal convoluted tubules (μm²). Each column represents mean ± SEM, n = 6. Means with different superscript letters differ significantly at 5% (p ≤ 0.05) level of significance according to ANOVA test and the TUKEY test. Eug, eugenol; AgNPs, silver nanoparticles.

Figure 5

Histomorphometrical evaluation of renal tissue components in the control and treated animal groups showing the number of (A) normal Malpighian corpuscles, (B) normal proximal convoluted tubules, (C) normal distal convoluted tubules, (D) damaged Malpighian corpuscles, (E) damaged proximal convoluted tubules, and (F) damaged distal convoluted tubules. Each column represents mean ± SEM, n = 6. Means with different superscript letters differ significantly at 5% (p ≤ 0.05) level of significance according to ANOVA test and the TUKEY test. Eug, eugenol; AgNPs, silver nanoparticles.

Figure 6

Immunohistochemical evaluation of Bcl-2 expression in renal tissues of the control and treated animal groups showing (A) no staining in the negative control, (B) an intense immunostainability in the control group, (C) a strong immunoreaction in the Eug-treated group, (D) a moderate immunoreaction in the low-dose AgNPs-treated group, (E) a weak immunostainability in the high-dose AgNPs-treated group, (F) a moderate immunostaining in Eug + AgNPs low-dose-treated group; (G) a modest immunostainability in the group treated with Eug + AgNPs at a high dosage.

Figure 7

Immunohistochemical evaluation of P53 expression in renal tissues of the control and treated animals groups showing (A) a negative immunostaining in the negative control, (B) a weak immunostainability in the control group, (C) a weak immunostainability in the Eug-treated group, (D) a moderate immunoreaction in low-dose AgNPs-treated group, (E) a strong immunostainability in the AgNPs high-dose-treated group, (F) a weak affinity for P53 in the Eug + ANPs low-dose-treated group, and (G) a modest immunostainability in the group treated with Eug + AgNPs at a high dosage.

Figure 8

Immunohistochemical analysis of Caspase-3 (Cas3) expression in renal tissues of the control and treated animal groups showing (A) no stainability in the negative control, (B) a weak immunostaining in the control group, (C) a weak immunoreaction in the Eug-treated groups, (D) a high positive immunostainability in the low-dose AgNPs-treated group, (E) a strong immunoreaction in the AgNPs high-dose-treated group, and (F,G) a modest immunostainability in both groups treated with Eug paralleled with either the low or high dose of AgNPs.

Figure 9

Immunohistochemical analysis of TNF-α expression in renal tissues the control and treated animal groups showing (A) a negative immunoreactivity in negative control, (B) a mild immunoreaction in the control group, (C) a week immunoreaction in the Eug-treated group, (D) an intense immunostainability in low-dose AgNPs-treated group, (E) a strong immunoreaction in the AgNPs high-dose-treated group, (F) a modest immunostainability in the Eug + AgNPs low-dose-treated group, and (G) a moderate immunostaining in the group treated with Eug + AgNPs at a high dosage.

Figure 10

Immunohistochemical image inspection of the area percentage of the immunoexpression of the reactive proteins (A) Bcl-2, (B) P53, (C) Cas3, and (D) TNF-α in the renal tissues of the control and treated animal groups. Each column represents mean ± SEM, n = 6. Means with different superscript letters differ significantly at 5% (p ≤ 0.05) level of significance according to ANOVA test and the TUKEY test. Eug: eugenol; AgNPs: silver nanoparticles.