Impact of Pulmonary Exposure to Cerium Oxide Nanoparticles on Experimental Acute Kidney Injury
Abderrahim Nemmara Suhail Al-Salamb Zinab Al Ansaria Zainab A. Alkharasa
Rauda M. Al Ahbabia Sumaya Beegama Priya Yuvarajua Javed Yasinc
Badreldin H. Alid
aDepartments of Physiology, College of Medicine and Health Sciences, United Arab Emirates University, Al Ain, United Arab Emirates, bDepartment of Pathology, College of Medicine and Health Sciences, United Arab Emirates University, Al Ain, United Arab Emirates, cDepartment of Internal Medicine, College of Medicine and Health Sciences, United Arab Emirates University, Al Ain, United Arab Emirates, dDepartment of Pharmacology, College of Medicine & Health Sciences, Sultan Qaboos University, Al-Khod, Sultanate of Oman
Key Words
Cerium oxide nanoparticles • Lung inflammation • Acute renal failure
Background/Aims: Cerium oxide nanoparticles (CeO2 NPs) are released from diesel engines that use cerium compounds as a catalytic agent to decrease the diesel exhaust particles, leading to human exposure by inhalation to CeO2 NPs. We have recently demonstrated that pulmonary exposure to CeO2 NPs induces lung inflammation, thrombosis, and oxidative stress in various organs including kidneys. It is well known that particulate air pollution effects are greater in patients with renal diseases. The aim of this study is to investigate the effects of pulmonary exposure to CeO2 NPs in a rat model of acute kidney injury (AKI). Methods: AKI was induced in rats by a single intraperitoneal injection of cisplatin (CP, 6 mg/kg). Six days later, the rats were intratracheally (i.t.) instilled with either CeO2 NPs (1 mg/kg) or saline (control), and various renal and pulmonary endpoints were assessed 24 h afterward using histological, colorimetric assay, enzyme-linked immunosorbent assay and Comet assay techniques. Results: CP alone decreased body weight, and increased water intake, urine volume and relative kidney weight. CP also increased the plasma concentrations urea and creatinine, and decreased creatinine clearance. In the kidneys, CP significantly increased renal injury molecule-1, interleukin-6 (IL-6), tumor necrosis factor α (TNFα) and glutathione concentrations, and caused renal tubular necrosis, and DNA injury assessed by Comet assay. All these actions were significantly aggravated in rats given both CP and CeO2 NPs. Histopathological changes in lungs of CeO2 NPs-treated rats included marked interstitial cell infiltration and congestion. These were aggravated by the combination of CP + CeO2 NPs. Moreover, this combination exacerbated the increase in the concentrations of TNFα and IL-6, and the decrease in the activity of pulmonary catalase and total nitric oxide concentration, and lung DNA damage. Conclusion: We conclude that the presence of CeO2 NPs in the lung exacerbated the renal and lung effects of CP-induced AKI.
Introduction
There has been an accelerated increase in the use of different types of nanomaterials as a result of their unique physicochemical and bioreactive characteristics. Nanomaterials possess different physicochemical and biological properties than larger materials of identical chemical composition. They are used in industries including sunscreens, food, paints, textile, electronics, sports, and biomedical application and imaging [1]. Nonetheless, extensive use of nanomaterials may lead to exposure of humans by various routes including inhalation, dermal and oral routes, raising concerns about their potential toxicity [1, 2].
Cerium oxide nanoparticles (CeO2 NPs) have been used in Europe, Asia and North America as fuel additive, which serves as a combustion catalyst leading to enhanced fuel efficiency [3-6]. Although this addition enhances the ability of diesel engines and reduces the emission of diesel exhaust particles (DEP), it leads, however, to direct emission of CeO2 NPs in the environment leading to human exposure by inhalation [7]. The latter could have adverse health effects [7].
It has been shown that exposure to CeO2 NPs causes inflammation and oxidative stress leading to apoptosis and autophagy in vitro [8] and lung inflammation and fibrosis after their pulmonary exposure in vivo [9, 10]. Furthermore, it has been demonstrated that following their pulmonary deposition, CeO2 NPs are able to pass through the air-blood barrier and reach the systemic circulation, and from there distribute to various sites in the body [11]. We have recently demonstrated that pulmonary exposure to CeO2 NPs induces lung inflammation and thrombotic events, and cause inflammation and oxidative stress in various extrapulmonary sites, including the kidneys [12, 13].
It is well-established that there is a crosstalk between the lung and the kidney, and that their functions are tightly associated under physiological and pathophysiological situations [14]. In fact, it is well-known that different diseases that may affect one organ can have direct consequences on the other one [14]. It has been shown that lung injury can worsen kidney failure [14]. Moreover, it has been reported that patients with pre-existing diseases such as kidney failure are more susceptible to the adverse health effect of particulate air pollution [15]. Actually, it has been demonstrated that living near a major roadway contributes to the decline in renal function [16]. We have also recently provided experimental evidence that pulmonary exposure to diesel exhaust particles (DEPs) aggravate both acute and chronic renal failure [17, 18]. However, as far as we are aware, little is known about the impact of lung exposure to CeO2 NPs in animal model of acute renal failure. As far as we know, only one study has reported that CeO2 NPs attenuate acute kidney injury induced by intra-abdominal infection in Sprague-Dawley rats [19]. The latter findings are in complete disagreement with various publications reporting that CeO2 NPs induce inflammation, oxidative stress and DNA damage in various organs including the kidneys [8-10, 12, 13].
Consequently, the purpose of this study was to evaluate whether and to what extent pulmonary exposure to CeO2 NPs may have an exacerbating effects in an animal model of acute kidney injury (AKI) induced by cisplatin (CP, [cis-diaminedichloroplatinum II]), an anticancer drug, which induces nephrotoxicity through inflammation, apoptosis and generation of reactive oxygen species [20], by assessing various renal and pulmonary variables.
Materials and Methods
Particles
CeO2 NPs, 10 wt % in water with average diameter at ~20 nm, were obtained from Sigma-Aldrich (St Louis, MO, USA). CeO2 NPs samples diluted in saline were used for rat exposures. To minimize aggregation, particle suspensions were always sonicated for 5 min (Clifton Ultrasonic Bath, Clifton, New Jersey, USA). Particle suspensions were prepared promptly before use and were vortexed to offer well mixed suspension prior each instillation. The same particles from the same source were characterized and used recently by Ma and co-workers [10, 21].
The endotoxin concentration in the CeO2 NPs and saline used was quantified, as described by the manufacturer, by chromogenic Limulus Amebocyte Lysate (Pierce, Rockford, IL) test. The concentrations were lower than the detection limit (0.1 EU/ml) in the saline, and CeO2 NPs solutions.
Animals and i.t. instillation
This project was reviewed and approved (ERA_2016_4408) by the Institutional Review Board of the United Arab Emirates University, College of Medicine and Health Sciences, and experiments were performed in accordance with protocols approved by the Institutional Animal Care and Research Advisory Committee.
Male Wistar rats (Taconic Farms Inc., Germantown, New York, USA), aged 10–12 weeks and initially weighing 223 ± 13 g, were given a standard laboratory chow and water ad libitum. They were randomly divided into four groups and individually housed in metabolic cages, to facilitate urine collection, at a temperature of 23 ± 2°C, relative humidity of 50–60% and a 12-h dark–light cycle. An acclimatization period of four days was allowed for the rats before any experimentation. The rats were weighed at the beginning of the experiment and just before sacrifice. Rats were cared for under a protocol approved by the Animal Research Ethics Committee of our college, and according to the NIH Guide for the Care and Use of Laboratory Animals, NIH publication no. 85-23, 1985.
Treatments
The AKI in rats was induced by a single intraperitoneal (i.p.) injection of CP (David Bull Laboratories, PTY Ltd, Victoria, Australia) at a dose of 6 mg/kg [22, 23]. Control animals received similar volume of normal saline i.p. On day 6 of treatment, the animals were anesthetized by isoflurane inhalation, and placed supine with extended neck on an angled board. A Becton Dickinson 18 Gauge cannula was inserted via the mouth into the trachea. CeO2 NPs suspension (150 µl) or saline-only were instilled (150 µl) via a sterile syringe and followed by an air bolus of 100 µl.
The four groups were treated as follows:
Group 1: single normal saline (control, 500 μl /rat) given i.p., and on day 6 of the treatment, a single i.t. administration of saline (150 μl per rat);
Group 2: single normal saline (control, 500 μl /rat) given i.p., and on day 6 of the treatment, a single i.t. administration of CeO2 NPs (1 mg/kg);
Group 3: single CP (6 mg/kg) given i.p., and on day 6 of the treatment, a single i.t. administration of saline (150 μl per rat);
Group 4: single CP (6 mg/kg) given i.p., and on day 6 of the treatment, a single i.t. administration of CeO2 NPs (1mg/kg);
On day 6, immediately after i.t. administration of saline or CeO2 NPs, rats were placed in metabolic cages, and urine of each rat was collected over a 24-h period and the volume measured.
Blood collection, histology and biochemical analysis
Rats were anesthetized with sodium pentobarbital (60 mg/kg, i.p.), and blood was drawn from the inferior vena cava in ethylenediaminetetraacetic acid (4%). The collected blood was centrifuged at 4°C for 15 min at 900× g, and the plasma samples were stored at −80°C pending analysis.
The animals were sacrificed with an overdose of anesthesia. Right kidneys were excised, washed with ice-cold saline, blotted with filter paper and weighed. Each kidney was cassetted and fixed directly in 10% neutral formalin for 24 hours, which was followed by dehydration in increasing concentrations of ethanol, clearing with xylene and embedding with paraffin. Four-μm sections were prepared from paraffin blocks and stained with hematoxylin and eosin. The stained sections were evaluated by the histopathologist (S.A.), using light microscopy. The microscopic scoring of the kidney sections was carried out in a blinded fashion by our histopathologist who was unaware of the treatment groups and assigned a score which represents measurement of extent of acute tubular injury area in the cortical and medullary tubules on a scale of 0–4 (0, no acute tubular injury; 1, a few focal acute tubular injury areas of ≤25% of the kidney; 2, acute tubular injury area was about 26-50% of kidney; 3, acute tubular injury area was 51-75% of kidney; 4, showing acute tubular injury in 76-100% of kidney) [24]. Image J software (NIH, USA) was used to measure the extent of necrosis.
Right lungs were excised, washed with ice-cold saline, blotted with filter paper and weighed. Then they were fixed by with 10% buffered formalin. Each lung was sectioned, casseted and dehydrated in increasing concentrations of ethanol, cleared with xylene and embedded with paraffin. Three-μm sections were prepared from paraffin blocks and stained with haematoxylin and eosin. The stained sections were evaluated using light microscopy.
The concentrations of urea in plasma, and creatinine in both plasma and urine were spectrophotometrically measured using commercial kits (Roche Diagnostics, Indianapolis, IN, USA).
Left kidneys and left lungs of rats from the different groups were quickly collected and rinsed with ice-cold PBS (pH 7.4) before homogenization, as described before [13, 25]. The homogenates were centrifuged for 10 min at 3000 x g to remove cellular debris, and the supernatants were used for further analysis [25]. Protein content was measured by Bradford’s method. The concentrations of tumor necrosis factor α (TNFα) (Duo Set, R & D systems, Minneapolis, MN, USA), interleukin 6 (IL-6) (Duo Set, R & D systems, Minneapolis, MN, USA), kidney injury molecule 1 (KIM) (Duo Set, R & D systems, Minneapolis, MN, USA) and glutathione (GSH) (Sigma-Aldrich Fine Chemicals, St Louis, MO, USA) were determined in kidney homogenates using commercial Kits.
In lung homogenates, IL-6 and TNFα, as well as catalase activity were measured using a kit from Cayman Chemicals Company (Ann Arbor, MI, USA) and the determination of NO was performed with a total NO assay kit from R & D systems (Minneapolis, MN, USA) which measures the more stable NO metabolites NO2- and NO3- [26, 27].
Assessment of DNA damage by COMET assay
Immediately after sacrifice, the lung and kidneys were removed from each animal. Single-cell suspensions of the different lungs and kidneys were obtained and analyzed according to the method described in our previous publications [28-31]. Each collected organ was washed in a chilled medium (RPMI 1640, 15% DMSO, 1.8% (w/v) NaCl). The lung and kidney tissues were put in 1.5 ml medium and cut finely into pieces in a Petri dish. The slices were allowed to deposit and the supernatant was collected in a 15 ml tube. The collected cell suspension was centrifuged at 1000 rpm for 5 min at 4°C. The supernatant was removed and the pellets were suspended in 0.5 ml of the medium. The cell suspensions were mixed with low melting point agarose solution (0.65%) and spread onto agarose (1.5%)–precoated microscope slides. For each group, five slides were prepared and incubated in ice cold lysis buffer (2.5M NaCl, 10mM Tris, 100mM EDTA, 1% Triton X-100 and 10% DMSO) at 4°C for at least one hour to remove the cell membranes. Following incubation, slides were placed in a horizontal electrophoresis unit and incubated in electrophoresis buffer (0.2M EDTA, 5M NaCl, pH 10) for 20 min for DNA unwinding and the expression of alkali labile sites. Then, electrophoresis was conducted for 20 min at 25V and 300mA. After that, the slides were neutralized with Tris buffer (0.4M Trizma base, pH 7.5) for 5 min and washed with methanol. Then the slides were stained with propidium iodide, as previously described [29, 32]. All these steps were performed in darkness to prevent additional DNA damage. The slides were mounted on a fluorescent microscope and cell scoring was performed. The measurement of length of the DNA migration (i.e. diameter of the nucleus plus migrated DNA) was calculated using the image analysis Axiovision 3.1 software (Carl Zeiss, Canada) [29, 33].
Statistics
All data were analyzed with GraphPad Prism Version 4.01 for Windows software (Graphpad Software Inc., San Diego, USA). Data were analyzed for normal distribution using using the D'Agostino and Pearson omnibus normality test. Data are expressed as means ± SEM. Comparisons between groups were performed by one way analysis of variance (ANOVA), followed by Newman Keuls test for comparing treated with control data. P values ≤0.05 are considered significant.
Results
Body weight, relative kidney weight, water intake and urine volume
Fig. 1A displays no difference in body weight between saline and CeO2 NPs groups. Nevertheless, rats treated concomitantly with CP and saline lost about 17.2 % (P<0.001 versus saline group), while those administered with CP and CeO2 NPs lost about 20.5 % (P<0.001 versus DEP group), respectively. Fig. 1B shows that the relative kidney weight was significantly augmented in rats given CP + saline compared with those administered with saline only (P<0.01). Moreover, the relative kidney weight was significantly increased in rats administered with CP + CeO2 NPs compared with those given either CeO2 NPs only (P<0.001) or those given saline + CP (P<0.001). Fig. 1C shows that water intake was significantly increased in the CP + saline group compared with the saline alone (P<0.05), and the CP + CeO2 NPs group compared with either CP + saline group (P<0.05) or CeO2 NPs alone (P<0.001). Similarly, Fig. 1D shows that urine volume was significantly augmented in the CP + saline group compared with the saline group (P<0.001), and the CP + CeO2 NPs group compared with either CP + saline group (P<0.01) or CeO2 NPs group (P<0.001).
Plasma creatinine and urea concentration and creatinine clearance
Fig. 2A shows no difference in urea concentration has been observed between saline and CeO2 NPs groups. However, rats treated with CP + saline showed a significant increase in the concentration of urea compared with the saline group (P<0.001). Remarkably, CP + CeO2 NPs treatment significantly augmented the concentration of urea more than in rats given either CP + saline (P<0.05) or CeO2 NPs alone (P<0.001). Likewise, as shown in Fig. 2B, the concentration of creatinine in plasma significantly increased in the CP + saline group compared with the saline one (P<0.01), and in the CP + CeO2 NPs group compared with the CP + saline (P<0.05) or CeO2 NPs group (P<0.01). Fig. 2C displays that creatinine clearance was significantly reduced in rats treated with CP + saline compared with those treated with saline (P<0.001), and in rats treated with CP + CeO2 NPs compared with those treated with either CP + saline (P<0.05) or those treated CeO2 NPs alone (P<0.001).
KIM-1, IL-6, TNFα and GSH concentrations in kidney homogenates
Fig. 3A illustrates that treatment with CP + saline significantly increased the concentration of KIM-1 compared with that of the saline-treated group (P<0.01). Simultaneous treatment with CP and CeO2 NPs significantly augmented the concentration of KIM-1 in kidney homogenates compared with either CP + saline group (P<0.05) or CeO2 NPs group (P<0.001). Figures 3B and 3C show that the concentrations of IL-6 and TNFα in kidney homogenates were significantly increased in CeO2 NPs group compared with saline group. Moreover, the concentrations of IL-6 and TNFα were significantly increased in CP + saline group compared with saline group, and in CP + CeO2 NPs compared with either CP + saline or CeO2 NPs group. Fig. 3D shows that compared with saline group, treatment with CeO2 NPs induced a significant increase in GSH levels. Moreover, the levels of GSH were also significantly augmented in CP + saline group versus saline group and, in CP + CeO2 NPs group versus either CP + saline group or CeO2 NPs group.
DNA damage in the kidney
Fig. 4 depicts the evaluation of DNA injury in the kidney by the Comet assay. Compared with saline group, pulmonary exposure to CeO2 NPs induced a significant increase in DNA damage (P<0.05). DNA damage was significantly augmented in CP + saline group compared with saline group (P<0.05). Furthermore, the DNA damage observed in CeO2 NPs + CP group was significantly higher compared with either CP + saline (P<0.01) or CeO2 NPs alone (P<0.01).
Kidney histopathology
Fig. 5 and Table 1 illustrate kidney histopathology and the percentage and scores for tubular injury in the four studied groups. Saline-treated group showed normal kidney architecture and histology and was given a score of 0 (Fig. 5A-B). In CP + saline group (Fig. 5C-D), there was an acute tubular injury in 37.50 ± 2.49 % of examined tissue areas (score 2) which showed the presence of tubular distention with necrotic material involving loss of brush border, tubular dilatation, tubular cells necrosis, tubular nuclear pyknosis, tubular nuclear enlargement with hyperchromasia, tubular cells flattening, macrophages within the lumen, epithelial cells within the lumen and intra-luminal eosinophilic material. In CeO2 NPs group (Fig. 5E-F), there were focal areas of acute tubular injury accounting 7.37 ± 0.94 of total examined areas involving proximal convoluted tubules characterized by mild tubular dilatation, intracytoplasmic aggregates of giant lysosomes within tubular cells, epithelial cells falling within the lumen of tubular cells and intratubular secretion. In CP + CeO2 NPs group (Fig. 5G-H), there was an increased frequency of acute tubular injury when compared with either CP + saline group or CeO2 NPs group. The acute tubular injury observed CP + CeO2 NPs involved 58.63 ± 6.27% of examined areas (score 3). The latter showed the presence of tubular distention with necrotic material involving, loss of brush border, tubular dilatation, tubular cells necrosis, tubular nuclear pyknosis, tubular nuclear enlargement with hyperchromasia, tubular cells flattening, macrophages within the lumen, epithelial cells within the lumen and intra-luminal eosinophilic material.
Lung histopathology
Saline-treated group showed normal lung architecture with unremarkable changes (Fig. 6A). In CP + saline group, there was a moderate widening of interalveolar interstitial spaces with mixed inflammatory cells consisting of neutrophil polymorphs, lymphocytes and macrophages (Fig. 6B). In CeO2 NPs (Fig. 6C), there was focal mild widening of interalveolar interstitial spaces with mixed inflammatory cells consisting of neutrophil polymorphs, lymphocytes and macrophages. In CP + CeO2 NPs (Fig. 6D), there was a severe widening of interalveolar interstitial spaces with mixed inflammatory cells consisting of neutrophil polymorphs, lymphocytes and macrophages.
IL-6, TNFα, catalase and total NO levels in lung homogenates
Fig. 7A-B illustrates that treatment with CP + CeO2 NPs significantly increased the concentration of IL-6 and TNFα compared with CP + saline group or CeO2 NPs group. On the other hand, the activity of catalase was significantly reduced in CP + CeO2 NPs compared with either CP + saline group or CeO2 NPs group (Fig. 7C). Moreover, the catalase activity decreased significantly in rats treated with CeO2 NPs compared with those given saline, and in rats treated with saline + CP compared with those treated with saline (Fig. 7C). The total NO activity was significantly reduced in CP + CeO2 NPs compared with either CP + saline group or CeO2 NPs group (Fig. 7C-D).
DNA damage in the lung
As shown in Fig. 8, compared with saline group, treatment with CeO2 NPs induced a significant increase in DNA damage (P<0.001). DNA damage was significantly increased in CP + saline group compared with saline group (P<0.001). Additionally, the DNA damage seen in CeO2 NPs + CP group was significantly higher compared with either CP + saline (P<0.05) or CeO2 NPs alone (P<0.001).
Discussion
CP is one of the most effective anticancer drugs used in the therapy of a variety of solid tumours [34], mostly owing to its high efficacy and low cost. CP efficacy increases with dose, but high doses are associated with many severe adverse effects, particularly nephrotoxicity, including apoptosis, inflammation, necrosis and death in proximal tubules and collecting ducts [34]. The mechanism underlying the side effects induced by CP is not fully understood but it has been suggested to be related to oxidative stress and inflammation [35]. Several studies have established the occurrence of renal failure with a single i.p. dose of 6 mg/kg in rats [17, 22, 36]. The dose of CeO2 NPs tested here has been chosen from earlier studies using animal models of i.t. or oropharyngeal instillation of CeO2 NPs and evaluating their impact on lung inflammation, oxidative stress, thrombosis and ischemia-reperfusion injury in mice and rats [10, 12, 13, 37]. Here, the exposure of rats to CeO2 NPs was performed by i.t. instillation technique which offers a more accurate way of administration, since rats are obligate nose breathers that filter most inhaled particles [38].
There is discrepancy in the literature regarding the effects of CeO2 NPs. In fact, while some studies reported that these nanoparticles are capable of abrogating inflammation and oxidative stress in vitro and in vivo [8, 39], several others studies reported their ability to induce inflammation, oxidative stress, DNA damage and apoptosis in various organs [9, 10, 12, 13].
It has been shown that inhaled nanoparticles (<30 nm in diameter) can cross the alveolar capillary barrier and reach the blood [40-42]. The latter have been reported to be filtered and excreted by the kidneys and preferentially deposit at sites of vascular inflammation in both experimental animals and healthy subjects [40]. Moreover, we have recently showed that pulmonary exposure to CeO2NPs induce inflammation, oxidative stress and DNA damage in different organs including the kidney [12, 13], thus, in the preset study, we thought it was important to assess if pulmonary exposure to CeO2NPs is capable of aggravating acute renal injury induced by CP. This approach is particularly relevant since it is well-established that patients with renal disease develop states of increased inflammation and oxidative stress which can make them more susceptible to the effects of inhaled nanoparticles [43].
Our data show a decrease in body weight by around 17 % after CP administration and that the combination of CP and CeO2 NPs further reduced it to 20% but the difference between CP + saline and CP + CeO2 NPs groups was statistically insignificant. Moreover, we found that the relative kidney weight, water intake and urine volume were all increased in CP + saline compared with saline and that the concomitant administration of CP + CeO2 NPs has aggravated these 3 variables compared with either CP + saline or CeO2 NPs. The reduction of body weight and increase in kidney relative weight can be ascribed to the renal tubular damage, and subsequent deterioration of the capacity of tubular cells to reabsorb water, causing polyuria leading to dehydration, which can explain the ensuing increase of water intake. In the present study, the worsening of kidney function in rats exposed to CP and CeO2 NPs was confirmed by the significant increase in the concentrations of creatinine and urea in plasma and significant decline of creatinine clearance compared with rats treated with either CP + saline or CeO2 NPs. Such findings have not been reported before. It has been previously shown that the combination of CP + DEPs (1 mg/kg) aggravated the serum concentration of urea and creatinine compared with either CP + saline or DEPs [17]. However, no aggravating effect was observed for urine volume in CP + DEPs compared with either CP + saline or DEPs [17].
In order to investigate the mechanisms by which the combination of CP and CeO2 NPs exerts their toxic effects, we measured, in kidney homogenates, several traditional and novel markers of kidney injury (KIM-1), inflammation (TNFα and IL-6) and oxidative stress (GSH). KIM-1 is very sensitive (95%) biomarker of early renal injury expressed by renal tubular epithelium and its expression has been reported to persist until kidney recovery [44]. The increase of KIM-1 has been reported in rats treated with CP [45]. Our data show that the concentration of KIM-1 was increased in kidney homogenate of rats given CP and saline compared with saline-treated ones. Interestingly, we found that the administration of both CP and CeO2 NPs significantly elevated the concentrations of KIM-1 in kidney homogenates compared with rats given either CP and saline or CeO2 NPs. Similarly, the concentrations of the pro-inflammatory cytokines IL-6 and TNFα were both significantly higher in rats treated with CP and CeO2 NPs compared with those given either CP and saline or CeO2 NPs. Along with the increase in KIM-1, IL-6 and TNFα, our data show that the levels of the antioxidant GSH were also significantly increased in CP + CeO2 NPs compared with either CP + saline or CeO2 NPs, indicating the occurrence of adaptive reaction in the kidney to counterbalance the potentially damaging action of oxygen radicals induced by the concomitant administration of CP and CeO2 NPs. The latter findings suggest the aggravating effects of CP and CeO2 NPs involve inflammation and oxidative stress. It has been reported that oxidative stress and inflammation are considered to be the driving factors in CP-induced nephrotoxicity and the use of natural antioxidants and anti-inflammatory agents such as emodin or ellagic acid alleviate these effects [20, 45]. We have recently demonstrated in mice that pulmonary exposure to CeO2 NPs induces oxidative stress and DNA injury in the kidney [13]. Here, we confirmed the occurrence of DNA damage in rats given CeO2 NPs and those treated with both CP and saline. Interestingly, we found that the DNA damage is exacerbated in rats given both CP and CeO2 NPs compared with either CP + saline group or CeO2 NPs group. The latter result could be explained by the potentiation of injury, inflammation and oxidative stress exerted by the combination of CP and CeO2 NPs. Histopathological analysis of the kidneys of rats given CeO2 revealed the presence of focal areas of acute tubular injury (7% of total examined areas; score 1) involving proximal convoluted tubules. The treatment with CP and saline induced significantly more acute tubular injury (37% of total examined areas; score 2). Remarkably, the association of CP and CeO2 NPs caused a much higher increase in extent of acute tubular injury (58% of total examined areas; score 3). Previously, in a rat model of AKI induced by CP administration, we showed no effect on kidney histology of pulmonary exposure to DEPs (1 mg/kg), and the administration of CP + saline or CP + DEPs induced comparable degree of acute tubular necrosis [17]. Thus, the current data suggest that at the same dose, i.e. 1 mg/kg, CeO2 NPs alone or in combination with CP has more nephrotoxic effects than DEP alone or in combination with CP. The latter could be explained by the small homogenous size of CeO2 NPs (20 nm) as compared with DEP (geometric mean aerodynamic diameter of 215 nm) [46]. The small size and large surface area of CeO2 NPs allow them to cross different biological barriers and exert more toxicity [2, 47, 48].
Various studies have shown the presence of a close association between kidney injury and lung diseases [14]. This relationship takes place as a result of the occurrence of systemic inflammation and oxidative stress which subsequently trigger lung damage [14]. Primary kidney disease can induce inflammation and oxidative stress which can affect the lung, and vice versa kidney dysfunction can happen as a result of lung injury and the release of mediators of inflammation and reactive oxygen species in the blood which can reach the kidneys [14, 49]. Our lung histology data show that compared with saline group, the rats given either CeO2 NPs alone or CP + saline displayed the presence of lung inflammation. Moreover, rats given both CP + CeO2 NPs revealed a substantial potentiation of pulmonary inflammation. Likewise, the concentrations of markers of inflammation including IL-6 and TNFα in lung homogenates were significantly elevated by the combination of CP and CeO2 NPs compared with either CP + saline or CeO2 NPs. It has been shown that oxidative stress induces a decrease of bioactivity of NO [50]. Here, we show that the levels of NO were significantly decreased in lung homogenates of CP + CeO2 NPs compared with either CP + saline or CeO2 NPs. Moreover, the measurement of the antioxidant enzyme catalase exhibited its depletion in CeO2 NPs-treated group and CP + saline group compared with saline group. A significantly higher decline in catalase activity was seen in rats given both CP and CeO2 NPs. The latter finding indicates that this antioxidant has been consumed as a result of oxidative stress [51]. Additionally, compared with saline group, we found the presence of lung DNA damage in rats treated CeO2 NPs and those given both CP and saline. Remarkably, the DNA damage was found to be worsened in rats given both CP and CeO2 NPs. Such finding has never been reported before. Lung DNA damage has been reported in adenine-induced chronic kidney disease in mice, and was explained by the occurrence of oxidative stress [51]. The results obtained presently in the lung confirm previous clinical and experimental studies that kidney injury cause pulmonary pathophysiological effects [14, 17, 43, 51], and hence indicate that pulmonary exposure to CeO2 NPs exacerbates the kidney injury.
Conclusion
Our data provide novel experimental evidence that pulmonary exposure to CeO2 NPs in animal model of renal failure exacerbates both kidney and lung injury, and highlight the importance of performing a thorough evaluation of the toxicity of nanoparticles, particularly in animal models of human diseases.
Acknowledgements
This work was supported by funds of Summer Undergraduate Research Experiences Program, the College of Medicine and Health Sciences grant, United Arab Emirates University (UAEU) and by a joint UAEU-SQU grant.
Disclosure Statement
The authors declare to have no competing interests.
References
|
1
Radomska A, Leszczyszyn J, Radomski MW: The Nanopharmacology and
Nanotoxicology of Nanomaterials: New Opportunities and Challenges. Adv Clin
Exp Med 2016;25:151-162. |
|
|
|
|
|
2
Oberdorster G: Safety assessment for nanotechnology and nanomedicine:
concepts of nanotoxicology. J Intern Med 2010;267:89-105. |
|
|
|
|
|
3
Park B, Donaldson K, Duffin R, Tran L, Kelly F, Mudway I, Morin JP, Guest R,
Jenkinson P, Samaras Z, Giannouli M, Kouridis H, Martin P: Hazard and risk
assessment of a nanoparticulate cerium oxide-based diesel fuel additive - a
case study. Inhal Toxicol 2008;20:547-566. |
|
|
|
|
|
4 Health Effect Institute (HEI): Evaluation of human health risk from cerium added to diesel fuel. Health Effects Institute, Boston, MA. Communication 9, 2001. URL: https://www.healtheffects.org/system/files/Cerium.pdf. |
|
|
|
|
|
5 Wakefield G, Wu X, Gardener M, Park B, Anderson S: EnviroxTM fuel-borne catalyst: developing and launching a nano-fuel additive. Tech Anal Strat Manag 2008; DOI:10.1080/09537320701726825. |
|
|
|
|
|
6 Selvan AM, Anand V, Udayakumar MRB: Effects of cerium oxide nanoparticle addition in diesel and diesel-biodiesel-ethanol blends on the performance and emission characteristics of a CI engine. ARPN J Eng Appl Sci 2009;4:1-6. |
|
|
|
|
|
7
Cassee FR, van Balen EC, Singh C, Green D, Muijser H, Weinstein J, Dreher K:
Exposure, health and ecological effects review of engineered nanoscale cerium
and cerium oxide associated with its use as a fuel additive. Crit Rev Toxicol
2011;41:213-229. |
|
|
|
|
|
8
Hussain S, Al-Nsour F, Rice AB, Marshburn J, Yingling B, Ji Z, Zink JI,
Walker NJ, Garantziotis S: Cerium dioxide nanoparticles induce apoptosis and
autophagy in human peripheral blood monocytes. ACS Nano 2012;6:5820-5829. |
|
|
|
|
|
9
Snow SJ, Mcgee J, Miller DB, Bass V, Schladweiler MC, Thomas RF, Krantz T,
King C, Ledbetter AD, Richards J, Weinstein JP, Conner T, Willis R, Linak WP,
Nash D, Wood CE, Elmore SA, Morrison JP, Johnson CL, Gilmour MI et al.:
Inhaled diesel emissions generated with cerium oxide nanoparticle fuel
additive induce adverse pulmonary and systemic effects. Toxicol Sci 2014;142:403-417. |
|
|
|
|
|
10
Ma JY, Zhao H, Mercer RR, Barger M, Rao M, Meighan T, Schwegler-Berry D,
Castranova V, Ma JK: Cerium oxide nanoparticle-induced pulmonary inflammation
and alveolar macrophage functional change in rats. Nanotoxicology
2011;5:312-325. |
|
|
|
|
|
11
He X, Zhang H, Ma Y, Bai W, Zhang Z, Lu K, Ding Y, Zhao Y, Chai Z: Lung
deposition and extrapulmonary translocation of nano-ceria after intratracheal
instillation. Nanotechnology 2010;21:285103. |
|
|
|
|
|
12
Nemmar A, Al-Salam S, Beegam S, Yuvaraju P, Ali BH: The acute pulmonary and
thrombotic effects of cerium oxide nanoparticles after intratracheal
instillation in mice. Int J Nanomedicine 2017;12:2913-2922. |
|
|
|
|
|
13 Nemmar A, Yuvaraju P, Beegam S, Fahim MA, Ali BH: Cerium Oxide Nanoparticles in Lung Acutely Induce Oxidative Stress, Inflammation, and DNA Damage in Various Organs of Mice. Oxid Med Cell Longev 2017;2017:9639035. |
|
|
|
|
|
14
Domenech P, Perez T, Saldarini A, Uad P, Musso CG: Kidney-lung
pathophysiological crosstalk: its characteristics and importance. Int Urol
Nephrol 2017; 49:1211-1215. |
|
|
|
|
|
15
Xu X, Wang G, Chen N, Lu T, Nie S, Xu G, Zhang P, Luo Y, Wang Y, Wang X,
Schwartz J, Geng J, Hou FF: Long-Term Exposure to Air Pollution and Increased
Risk of Membranous Nephropathy in China. J Am Soc Nephrol 2016;27:3739-3746. |
|
|
|
|
|
16
Lue SH, Wellenius GA, Wilker EH, Mostofsky E, Mittleman MA: Residential
proximity to major roadways and renal function. J Epidemiol Community Health
2013;67:629-634. |
|
|
|
|
|
17
Nemmar A, Al Salam S, Zia S, Yasin J, Al H, I, Ali BH: Diesel exhaust
particles in the lung aggravate experimental acute renal failure. Toxicol Sci
2010;113:267-277. |
|
|
|
|
|
18
Nemmar A, Karaca T, Beegam S, Yuvaraju P, Yasin J, Hamadi NK, Ali BH:
Prolonged Pulmonary Exposure to Diesel Exhaust Particles Exacerbates Renal
Oxidative Stress, Inflammation and DNA Damage in Mice with Adenine-Induced
Chronic Renal Failure. Cell Physiol Biochem 2016;38:1703-1713. |
|
|
|
|
|
19
Manne ND, Arvapalli R, Nepal N, Shokuhfar T, Rice KM, Asano S, Blough ER:
Cerium oxide nanoparticles attenuate acute kidney injury induced by
intra-abdominal infection in Sprague-Dawley rats. J Nanobiotechnology
2015;13:75. |
|
|
|
|
|
20
Ali BH, Al-Salam S, Al Husseini IS, Al-Lawati I, Waly M, Yasin J, Fahim M,
Nemmar A: Abrogation of cisplatin-induced nephrotoxicity by emodin in rats.
Fundam Clin Pharmacol 2013;27:192-200. |
|
|
|
|
|
21
Ma JY, Young SH, Mercer RR, Barger M, Schwegler-Berry D, Ma JK, Castranova V:
Interactive effects of cerium oxide and diesel exhaust nanoparticles on
inducing pulmonary fibrosis. Toxicol Appl Pharmacol 2014;278:135-147. |
|
|
|
|
|
22
Ali BH, Al Moundhri MS, Tag EM, Nemmar A, Tanira MO: The ameliorative effect
of cysteine prodrug L-2-oxothiazolidine-4-carboxylic acid on
cisplatin-induced nephrotoxicity in rats. Fundam Clin Pharmacol
2007;21:547-553. |
|
|
|
|
|
23
Ali BH, Al Moundhri M, Eldin MT, Nemmar A, Al Siyabi S, Annamalai K:
Amelioration of cisplatin-induced nephrotoxicity in rats by
tetramethylpyrazine, a major constituent of the Chinese herb Ligusticum
wallichi. Exp Biol Med (Maywood) 2008;233:891-896. |
|
|
|
|
|
24
Zhao H, Qin HY, Cao LF, Chen YH, Tan ZX, Zhang C, Xu DX: Phenylbutyric acid
inhibits epithelial-mesenchymal transition during bleomycin-induced lung
fibrosis. Toxicol Lett 2015;232:213-220. |
|
|
|
|
|
25
Nemmar A, Al-Salam S, Yuvaraju P, Beegam S, Ali BH: Emodin mitigates diesel
exhaust particles-induced increase in airway resistance, inflammation and oxidative
stress in mice. Respir Physiol Neurobiol 2015;215:51-57. |
|
|
|
|
|
26
Tsikas D: Methods of quantitative analysis of the nitric oxide metabolites
nitrite and nitrate in human biological fluids. Free Radic Res
2005;39:797-815. |
|
|
|
|
|
27
Wennmalm A, Benthin G, Edlund A, Jungersten L, Kieler-Jensen N, Lundin S,
Westfelt UN, Petersson AS, Waagstein F: Metabolism and excretion of nitric
oxide in humans. An experimental and clinical study. Circ Res
1993;73:1121-1127. PMID:8222083 |
|
|
|
|
|
28
Al Suleimani YM, Al Mahruqi AS, Al ZM, Shalaby A, Ashique M, Nemmar A, Ali
BH: Effect of diesel exhaust particles on renal vascular responses in rats
with chronic kidney disease. Environ Toxicol 2017;32:541-549. |
|
|
|
|
|
29
Nemmar A, Yuvaraju P, Beegam S, Yasin J, Kazzam EE, Ali BH: Oxidative stress,
inflammation, and DNA damage in multiple organs of mice acutely exposed to
amorphous silica nanoparticles. Int J Nanomedicine 2016;11:919-928. |
|
|
|
|
|
30
Nemmar A, Al-Salam S, Yuvaraju P, Beegam S, Yasin J, Ali BH: Chronic Exposure
to Water-Pipe Smoke Induces Alveolar Enlargement, DNA Damage and Impairment
of Lung Function. Cell Physiol Biochem 2016;38:982-992. |
|
|
|
|
|
31
Nemmar A, Beegam S, Yuvaraju P, Yasin J, Tariq S, Attoub S, Ali BH:
Ultrasmall superparamagnetic iron oxide nanoparticles acutely promote
thrombosis and cardiac oxidative stress and DNA damage in mice. Part Fibre
Toxicol 2016;13:22. |
|
|
|
|
|
32
Olive PL, Banath JP, Fjell CD: DNA strand breakage and DNA structure
influence staining with propidium iodide using the alkaline comet assay.
Cytometry 1994;16:305-312. |
|
|
|
|
|
33
Hartmann A, Speit G: The contribution of cytotoxicity to DNA-effects in the
single cell gel test (comet assay). Toxicol Lett 1997;90:183-188. PMID:9067486 |
|
|
|
|
|
34
Yao X, Panichpisal K, Kurtzman N, Nugent K: Cisplatin nephrotoxicity: a
review. Am J Med Sci 2007;334:115-124. |
|
|
|
|
|
35
Chirino YI, Pedraza-Chaverri J: Role of oxidative and nitrosative stress in
cisplatin-induced nephrotoxicity. Experimental and Toxicologic Pathology 2009;61:223-242. |
|
|
|
|
|
36
Mohan IK, Khan M, Shobha JC, Naidu MU, Prayag A, Kuppusamy P, Kutala VK:
Protection against cisplatin-induced nephrotoxicity by Spirulina in rats.
Cancer Chemother Pharmacol 2006;58:802-808. |
|
|
|
|
|
37
Wingard CJ, Walters DM, Cathey BL, Hilderbrand SC, Katwa P, Lin S, Ke PC,
Podila R, Rao A, Lust RM, Brown JM: Mast cells contribute to altered vascular
reactivity and ischemia-reperfusion injury following cerium oxide
nanoparticle instillation. Nanotoxicology 2011;5:531-545. |
|
|
|
|
|
38
Driscoll KE, Costa DL, Hatch G, Henderson R, Oberdorster G, Salem H,
Schlesinger RB: Intratracheal instillation as an exposure technique for the
evaluation of respiratory tract toxicity: uses and limitations. Toxicol Sci
2000;55:24-35. |
|
|
|
|
|
39
Niu J, Wang K, Kolattukudy PE: Cerium oxide nanoparticles inhibit oxidative
stress and nuclear factor-kappaB activation in H9c2 cardiomyocytes exposed to
cigarette smoke extract. J Pharmacol Exp Ther 2011;338:53-61. |
|
|
|
|
|
40
Miller MR, Raftis JB, Langrish JP, McLean SG, Samutrtai P, Connell SP, Wilson
S, Vesey AT, Fokkens PHB, Boere AJF, Krystek P, Campbell CJ, Hadoke PWF,
Donaldson K, Cassee FR, Newby DE, Duffin R, Mills NL: Inhaled Nanoparticles
Accumulate at Sites of Vascular Disease. ACS Nano 2017;11:4542-4552. |
|
|
|
|
|
41
Nemmar A, Hoet PH, Vanquickenborne B, Dinsdale D, Thomeer M, Hoylaerts MF,
Vanbilloen H, Mortelmans L, Nemery B: Passage of inhaled particles into the
blood circulation in humans. Circulation 2002;105:411-414. |
|
|
|
|
|
42
Pery AR, Brochot C, Hoet PH, Nemmar A, Bois FY: Development of a
physiologically based kinetic model for 99m-Technetium-labelled carbon
nanoparticles inhaled by humans. Inhal Toxicol 2009;21:1099-1107. |
|
|
|
|
|
43
Xu X, Nie S, Ding H, Hou FF: Environmental pollution and kidney diseases. Nat
Rev Nephrol 2018;14:313-324. |
|
|
|
|
|
44
Waring WS, Moonie A: Earlier recognition of nephrotoxicity using novel
biomarkers of acute kidney injury. Clin Toxicol (Phila) 2011;49:720-728. |
|
|
|
|
|
45
Al-Kharusi N, Babiker HA, Al-Salam S, Waly MI, Nemmar A, Al-Lawati I, Yasin
J, Beegam S, Ali BH: Ellagic acid protects against cisplatin-induced
nephrotoxicity in rats: a dose-dependent study. Eur Rev Med Pharmacol Sci
2013;17:299-310. |
|
|
|
|
|
46
Saber AT, Bornholdt J, Dybdahl M, Sharma AK, Loft S, Vogel U, Wallin H: Tumor
necrosis factor is not required for particle-induced genotoxicity and
pulmonary inflammation. Arch Toxicol 2005;79:177-182. |
|
|
|
|
|
47
Radomska A, Leszczyszyn J, Radomski MW: The Nanopharmacology and
Nanotoxicology of Nanomaterials: New Opportunities and Challenges. Adv Clin
Exp Med 2016;25:151-162. |
|
|
|
|
|
48 Nemmar A, Holme JA, Rosas I, Schwarze PE, Alfaro-Moreno E: Recent advances in particulate matter and nanoparticle toxicology: a review of the in vivo and in vitro studies. Biomed Res Int 2013;2013:279371. https://doi.org/10.1155/2013/279371 PMID:23865044 |
|
|
|
|
|
49
Visconti L, Santoro D, Cernaro V, Buemi M, Lacquaniti A: Kidney-lung
connections in acute and chronic diseases: current perspectives. J Nephrol 2016;29:341-348. |
|
|
|
|
|
50
Forstermann U: Nitric oxide and oxidative stress in vascular disease.
Pflugers Arch 2010;459:923-939. |
|
|
|
|
|
51
Nemmar A, Karaca T, Beegam S, Yuvaraju P, Yasin J, Ali BH: Lung Oxidative
Stress, DNA Damage, Apoptosis, and Fibrosis in Adenine-Induced Chronic Kidney
Disease in Mice. Front Physiol 2017;8:896. |
|