Nanoparticles were found to reach the systemic circulation and disseminate to several organs such as the liver, spleen and kidneys [3
]. Kidneys play an important role in eliminating xenobiotics from the body and NPs absorbed in the systemic circulation can be excreted by renal clearance [35
]. Few studies have described the potential toxicity of NPs to renal tubular and glomerular targets [7
Kidney damage with morphological, pathological, and cellular changes leading to kidney dysfunction after exposure to NPs has been studied [12
In the present study, low and high doses of ZnO NPs significantly elevated inflammatory cytokine levels, including TNF α-, IL-6, and CRP in rat serum compared with the normal control group. Levels of VEGF, NO and IgG were also significantly elevated. These findings were accompanied with a dose dependent increase in serum urea and creatinine levels in n-ZnO intoxicated rats compared with normal control rats. Tosu et al. [39
elucidated the inflammatory effects of 50- and 100-nm ZnO particles on human umbilical vein endothelial cells (HUVECs). Nano-ZnO concentrations of
μg/ml resulted in increased cell proliferation, while those of
μg/ml caused dose-dependent increases in oxidized glutathione levels. Nano-ZnO particles induced a dose-dependent increase in the expression of the intercellular adhesion molecule (ICAM-1) protein, an indicator of vascular endothelium inflammation, and caused marked increases in NF-κB activity. Additionally, TNF-α, a typical inflammatory cytokine, induced ICAM-1 expression in an NF-κB-dependent manner. ZnO also synergistically enhanced TNF-α-induced ICAM-1 expression. Moreover, Fine et al. [40
] found that exposures to low concentrations of zinc oxide elevated circulating levels of different cytokines, which could account for the symptoms of the metal fume fever syndrome. This is in accordance with the present study as low and high doses of ZnO NPs significantly elevated cytokines (TNF-α and IL-6) levels in rat serum compared with normal control group.
Growing evidence indicates the beneficial role of a nutrient mixture of L-Arg and Qur in inhibiting the inflammatory response by down-regulating pro-inflammatory cytokine protein expression levels [41
]. Moreover, Mostafavi et al. [42
] found that Qur and Arg plays a protective role against the imbalance elicited between the production of free radicals and antioxidant defense systems, and suggested that a combination of these two antioxidants may find clinical application where cellular damage is a consequence of reactive oxygen species.
The current study shows that cytokine (TNF-α and IL-6) levels were reduced post-Qur treatment compared with the n-ZnO treated group. This was in accordance with the results of Behling et al. [43
] who found that cisplatin-treated rats presented a transitory increase in plasma creatinine levels, tubular cell necrosis and increased immunostaining for vimentin, alpha-SM-actin, fibronectin, and NF-κB in the renal cortex and outer medulla. Meanwhile, Qur treatment attenuated renal functional, histological and immunohistochemical alterations induced by cisplatin [43
]. Other studies showed that NF-κB plays a pivotal role in progressive kidney diseases by regulating the accumulation of macrophages [44
]. Qur potently inhibited NF-κB activation in cultured rat proximal tubular cells (PTCs), because NF-κB regulates inflammatory signaling and adhesion molecules in PTCs. Additionally, Qur is known to modulate the action of several inflammatory cytokines that are of particular concern to transplant recipients, including IL-1β, IL-2, IL-6, IL-15 and TNF-α [45
]. Hushmendy et al.[48
] found that Qur significantly inhibited cytokine levels and T-cell proliferation, suggesting that it may be effective in reducing transplant rejection. These findings may explain earlier findings that administration of Qur inhibited tubular injury and the elevation in inflammatory cytokine levels.
In the present study Arg significantly reduced the elevated levels of inflammatory cytokines (TNF-α and IL-6) compared with n-ZnO treated group. Moreover, Arg alone or in combination with Qur significantly decreased serum creatinine level as compared with rats intoxicated with a high dose of n-ZnO particles. This was attributed to the fact that Arg has various metabolic and immunologic effects and has been considered to be conditionally essential, particularly under inflammatory and oxidative stress [49
]. It has protective effects against oxidative stress and inflammation in different pathological conditions [42
]. It can modulate the inflammatory response by modulating the production of inflammatory mediators, such as C-reactive protein as well as cytokine release from activated immune competent cells which play a crucial role in the progression of the pathology [50
Elevation of CRP level post_n-ZnO administration compared with the normal control group in the current work was in accordance with the work of Kim et al.
who clarify that CRP is related to the incidence of many pathological condition as coronary heart disease, hypertension, and inflammation. Both Qur and/or Arg treatments reduced CRP level compared with n-ZnO intoxicated group. This is in agreement with Kleemann et al.
] who clarified that dietary intake of flavonoids such as Qur was associated with lower CRP levels. Moreover, Wu et al.
] found that Arg can modulate inflammatory response by modulating the production of inflammatory mediators, such as C-reactive protein.
The increase in the circulating IgG in rat serum intoxicated with both doses of ZnO-NPs is an immune response induced by NPs toxicity. It is suggested that the increase in the circulating antibodies are the result of the production of different inflammatory cytokines, including TNF-α, with potential impact on immunoglobulin production during inflammation. These results may indicate tha tZnO-NPs induced inflammatory kidney injury through production of the inflammatory mediators [52
The IgG level was reduced by Arg and Qur treatments. This may explain the role of these agents to suppress the release of inflammatory mediators.
There was a significant increase in serum nitrite/nitrate level in n-ZnO intoxicated rats. High amounts of NO are released from the inducible NO synthase (iNOS) isoform in response to inflammatory stimuli from a variety of cell types [53
Renal proximal tubule and inner medullary collecting duct cells can produce NO via expression of an inducible isoform of nitric oxide synthase [55
]. Mesangial cells and invading immune cells are capable of expressing iNOS upon stimulation with TNF-α and IL-1b. The release of large amounts of NO in the glomerulus may lead to the progression of renal failure during several forms of glomerulonephritis [56
]. Increased tissue factor expression is thought to play a significant role in the development of multiorgan system failure in acute injury [57
]. Vascular endothelial growth factor (VEGF), which is a potent mitogen for endothelial cells, has been reported to be expressed in several tissues, including the kidney. The expression of various tissue factors, cytokines, and chemokines in response to inflammatory tissue injury stimulates VEGF synthesizing cells such as platelets, immune cells, and inflammatory cells [58
]. Besides its mitogenic properties, VEGF is able to promote angiogenesis and increase vascular leakage [61
]. However, it was found that stimulation of angiogenesis may contribute to the transition from acute to chronic inflammation. Some investigations have revealed that new vessels can significantly contribute to perpetuation of the inflammatory response by expressing chemokines and adhesion molecules promoting the recruitment of inflammatory cells [63
]. This suggests the possibility that TNF-α and VEGF might act synergistically to potentiate kidney injury and/or systemic organ dysfunction [64
and explain the elevation in TNF-α, NO, and VEGF levels post-n-ZnO treatment compared with the control group.
The use of anti-angiogenic agents may then represent an attractive alternative therapeutic tool to prevent or significantly reduce fibrosis progression .The protective ingestion of the agents used alone or in combination, markedly reduced the dramatic increase in this angiogenic biomarker in sera of n-ZnO intoxicated rats, suggesting their anti-angiogenic beneficial action. Previous investigations revealed the role of Arg in reducing VEGF expression [65
Serum nitrite/nitrate was significantly decreased upon treatment of intoxicated animals with Qur or with the Qur and Arg combination but not with Arg alone. However, the increase in NO level in the Arg-treated group was accompanied by a significant increase in renal GSH content of n-ZnO intoxicated rats. The n-ZnO treated group showed a significant increase in free radical generation after n-ZnO treatment with resultant damaging effect on the kidney tissue as evidenced from histopathological examination (Figure ). Data of Arg-treated group suggests that pharmacological increases in NO levels did not exacerbate the increase in free radical formation. High levels of NO or Arg in the Arg-treated group may be protective, probably due to their property of scavenging free radicals as well as inhibiting xanthenes oxidase (XO) enzyme [66
In the present study, GSH levels were significantly reduced after n-ZnO treatment, while treatment with both Arg and Qur increased renal GSH content when compared with the n-ZnO treated group. This was attributed to the antioxidant properties of both agents. The Qur antioxidant effect is based on its ability to quench hydrogen peroxide [14
]. Qur reduces platelet aggregation and adhesion by reducing hydrogen peroxide production [14
]. Qur attenuated cyclosporine-induced oxidative stress by restoring the activity of the antioxidative enzymes glutathione peroxidase and catalase, preventing its nephrotoxic action and kidney damage [67
]. Arg significantly reduced lipid peroxidation and increased GSH content in the heart tissue of exhaustively exercised rats [68
These findings augment the current results in which the combination of both agents significantly decreased kidney damage by synergistically increasing cellular levels of GSH.
These biochemical findings were supported by the histopathological examination of kidney tissues, which clarified that animals received either low or high doses of n-ZnO particles showed moderate to massive atrophy and fragmentation of numerous glomeruli. The renal tubules showed epithelial desquamation, degeneration and necrosis. Some renal tubules showed casts in their lumina. Sever congestion was also observed in renal interstitium. These hazardous effects were dose-dependent. Co-treatment of n-ZnO_intoxicated rats with Qur or Arg, significantly improved most of the deviated histopathological parameters. The combination of Qur and Arg nearly prevented the damaging effect of n-ZnO intoxication that was manifested microscopically by extensive improvements in the histological features of kidney cells in the form of fragmentation of few glomeruli and epithelial exfoliation in few renal tubules as well as marked reduction in deposition of collagen fibers in the interstitial tissues.