Nano Research & Applications

Keywords

Lipopolysaccharide (LPS); Sepsis; CeO₂ nanoparticles; Acute Kidney Injury (AKI); Acute Lung Injury (ALI)

Introduction

Sepsis, the leading cause of mortality in critically ill patients, is characterized by multiple-organ dysfunction syndrome (MODS) [1,2]. Sepsis is known to impair both pulmonary and renal function, and the combination of acute lung injury (ALI) with acute kidney injury (AKI) from sepsis is associated with a >70% mortality rate % [2-4]. While the pathogenesis of sepsis has not yet been fully elucidated, it is understood the elevations of serum lipopolysaccharide (LPS) at least partly mediate the systemic inflammatory response syndrome (SIRS) that precedes the development of organ injury [4]. The development of SIRS is thought to be due, in large part, by the “over-activation” of the liver macrophages (Kupffer cells) which causes massive increases in the amount of circulating TNF-α, interleukin (IL)-1β, and IL-6 [5-11] which in turn are associated with increased renal and pulmonary oxidative stress and damage [12,13]. Because of the damaging effects that SIRS has on cellular ROS levels it has been postulated that anti-oxidant drugs may be effective in sepsis [14]. Recently, several nanoparticle compounds have been developed to scavenge reactive oxygen species (ROS) [15-18]. One compound that is potentially useful for biomedical applications is cerium oxide nanoparticles (CeO₂ NP) which may have efficacy in pathologies involving oxidative stress [18,19] and inflammation [20,21]. CeO₂ NPs are unique as they can effectively scavenge ROS by cycling between reduced (Ce+3) and oxidized (Ce⁺⁴) states [22]. In addition, CeO₂ NP act as superoxide dismutase [23] and catalase [24] mimetics depending on the particle redox state.

Current sepsis treatment is largely supportive in nature and is centered on the utilization of antibiotics, NSAIDS, intravenous fluids, and mechanical ventilator support where warranted [25]. Previously, our group showed that a single intravenous administration of 0.5 mg/kg CeO₂ NP significantly increased survival and reduced hepatic damage, serum cytokines and chemokines, and inflammatory related signaling in LPS-induced septic rats [26]. With the same animals and tissues utilized in our previous work [26], we hypothesized that a diminished SIRS response observed in these animals would result in decreased measurements of lung and kidney injury.

Material and Methods

CeO₂ NP preparation, characterization and administration

CeO₂ NP were acquired from Sigma (Manhattan, KS, USA) and prepared as described previously [26]. A Vibra Cell Sonicator (Sonics & Materials, Inc.) was used to prepare stock suspensions in dd H₂0 (3.5 mg/ml, 2 min at 600 W, room temperature) prior to characterization. A Hitach-H-7000 electron microscope (50,000X magnification) was used for imaging. ImageJ software was used to calculate particle size. A particle size analyzer (HORIBA, Model- LB-550) equipped with a He-Ne laser (633 nm) determined the hydrodynamic size and size distribution of CeO₂ NP using back scattered light. Freshly prepared samples were analyzed in triplicate once daily over three days.

Animal care and experimental design

Male (300 - 350 g) Sprague-Dawley rats were purchased from Charles River Laboratories International Inc. Rats were dual housed with a 12/12- hr dark/light cycle at 22 ± 2° C with ad libitum food and water. Rats acclimated for at least two weeks prior to experiments. All procedures were performed as described in the guide for the care and use of laboratory animals as approved by the council of the American Physiological Society and the institutional animal use review board of Marshall University. Rats were randomized into four groups: 1) The control group (n=6); 2) CeO₂ NPs group (n=6) that were administered 1.5 ml of endotoxin free water by the intraperitoneal (I.P.) route; 3) LPS (n=12) and 4) LPS + CeO₂ NP treatment groups were administered 1.5 ml of LPS (055-B5 from Sigma, 40 mg/kg) in sterile water by the I.P. route. Immediately after the injection of water or LPS, 200 μl of sterile distilled water was administered intravenously (tail vein) to the (1) control and (3) sepsis groups. Similarly, CeO₂ NP (0.5 mg/kg in 200 μl of sterile distilled water) was administered intravenously to the (2) CeO₂ NP and (4) sepsis + CeO₂ NP groups.

Tissue collection, estimation of CeO₂ NP content in kidney and lung and histology

Rats were humanely euthanized under anesthesia for collection of the kidneys and lungs at 6 or 24 hr after study initiation. Organs were cleaned with Krebs–Ringer bicarbonate buffer (KRB) to wash away any blood prior to flash freezing in liquid nitrogen and storage at -80°C until analysis. CeO₂ NP content in organs was determined by Elemental Analysis Inc. (Lexington, KY) with induction coupled plasma-mass spectrometry (ICP-MS, described previously [27]). The kidney and lung were embedded in paraffin, and stained with hematoxylin and eosin (H&E) for histopathological analysis. Embedded samples were viewed using an EVOSfl fluorescence (Fisher Scientific, Pittsburgh, PA, USA). Another set of paraffin embedded kidney sample were stained with periodic acid Schiff (PAS) for the examination of basement membrane damage.

Evaluation of kidney damage markers and serum biochemical analysis

Blood, 6 or 24 h after the start of the study, was collected. It was then centrifuged (5,000xg for 10 min at room temperature) and stored as serum at -80°C until analysis. Serum from each group (n=6) were pooled. Pooled serum was screened for biomarkers of kidney damage (β-2 microglobulin, calbindin, clusterin, cystatin-C, epidermal growth factor rat (EGF-Rat), kidney injury molecule-1 (KIM-1), neutrophil gelatinase-associated lipocalin (NAGL), osteopontin, tissue inhibitory of metalloproteinase-1 (TIMP-1), and vascular endothelial growth factor A (VEGF-A)) and renal function (cystatin-C)) by Myriad RBM (Austin, TX) in triplicate. An Abaxis VetScan® analyzer (Abaxis, Union City, CA, USA) was used to quantitate serum levels of BUN, creatinine and potassium.

Assessment of apoptosis in kidney by TUNEL staining

Sections of paraffin embedded tissue (5 μm, prepared as per manufacturer’s specifications) were analyzed with a TUNEL staining kit (Roche Applied Science, Indianapolis, IN). As a positive control, identical experiments were performed with DNase I. Experiments omitting the labeling reagent were used as a negative control. An EVOSfl fluorescence microscope (Fisher Scientific, Pittsburgh, PA, USA, 200x magnification) was used to capture images for counting of TUNEL-positive nuclei. Data was reported as the number of TUNEL-positive nuclei/500 nuclei.

Renal and lung superoxide level assessment

Superoxide levels in the renal and lung sections were estimated using dihydroethidium staining after deparafinization and rehydration of tissue sections. This involved room temperature incubation of sections with 5 mM dihydroethidium for 1 h in a dark room. An Evos FL microscope (Life technologies, Grand Island, NY) was used to image each section after it was washed with PBS (3 × 5 min). Mean fluorescence intensity was determined across various groups using image J analysis software as a measure for superoxide levels.

SDS-PAGE and immunoblotting

Sections of frozen kidney and lung (~100 mg) were pulverized to a fine powder. The powder was then added to 900 μl of T-PER (Pierce, Rockford, IL, USA) which contained 1% protease and phosphatase inhibitors. The organ mixture was then homogenized prior to centrifugation (10,000×g for 10 min, 4°C) and the supernatant was collected. The protein in each sample was quantified by a 660 nm protein assay (Pierce, Rockford, IL, USA). Each sample was identically diluted with Laemilli buffer (4x) and loaded onto a 10% PAGEr Gold Tris-Glycine Precast gel (Lonza, Rockland, ME), subjected to SDS-PAGE, and then transferred to a nitrocellulose membrane as previously described [28]. Milk (5%) in TBST was used as a blocking agent and incubated with the membrane for 1 h and prior to probing with the proper primary antibody (Cell Signaling Technology, Danvers, MA). Afterward, TBST was used to wash the membranes (3 × 5 min) before 1 h of incubation with secondary antibody (anti-rabbit, Cell Signaling Technology, Danvers, MA). Super-signal West Pico Chemiluminiscent substrate (Pierce, Rockford, IL, USA) was used to determine immunoreactivity and Fluorchem 9900 software (Protein Simple, Santa Clara, CA) was used to quantitate this property.

Measurement of inflammatory cytokines and chemokines in lung homogenate

The relative concentrations of 12 proinflammatory cytokines/ chemokines in the lung tissue lysate (n = 6/group at 6 h and 24 h) were determined with a Rat Inflammatory Cytokines Multi- Analyte ELISArray kit (Qiagen, CA, USA, catalogue number MER004A) as suggested by the manufacturer.

Myeloperoxidase activity (MPO) in lung homogenate

Myeloperoxidase activity, a general peroxidase activity, was performed as previously described [29]. Frozen lung samples were homogenized and sonicated in 50 mmol/l potassium phosphate buffer containing 0.5% hexadecyltrimethyammonium bromide (HTAB) and 5 mmol/l ethylene diamine tetracetic acid (EDTA). Afterward, these samples were centrifuged (10000xg for 10 min, 4°C) prior to the collection of the supernatant. Supernants were incubated in a 50 mmol/l potassium phosphate buffer containing H2O2 (1.5 mol/l) and O-dianisidine dihydrochloride (167 μg/ml). The enzyme activity was determined spectrophotometically by measuring the change in absorbance at 460 nm over 3 minutes in a 96-well plate reader. Values represent the change in OD/min/ gram tissue weight.

Statistical analysis

Data from these studies was reported as mean ± standard error of the mean (SEM). A one way ANOVA was performed in SigmaStat (Aspire Software International, Auburn VA) to analyze dependent variables; post-hoc tests were performed when appropriate. Significance was set at p <0.05.

Results

Characterization of CeO₂ NP

Scanning electron microscopy (SEM, Figure 1) and transmission electron microscopy (Figure 1) analysis estimated each nanoparticle to be approximately 40 - 60 nm long. Experiments using dynamic light scattering were used to estimate CeO₂ NP mean hydrodynamic diameter to be approximately 63 ± 11 nm (Figure 1).

Figure 1: CeO₂ NP characterization and the effect of the agent on LPS- induced acute kidney injury. A (i) SEM image of CeO₂ NP, A (ii) TEM image of CeO₂ NP, B (i) Dynamic light scattering (DLS) size histogram, B (ii) blood urea nitrogen (BUN) level in serum, C (i) creatinine level in serum, C (ii) phosphorous level in serum (n=6/group). Values are mean ± SEM (≥ 3 independent analyses). Representative micrographs of kidney tissue (n=3/group) 24 h after sepsis induction as visualized by light microscopy, (D) visual inspection of the kidney sections by H & E staining demonstrated evidence of increased Bowman’s space (*) and renal tubular necrosis, (E) PAS staining showed the basal membrane damage and irregularities. *p < 0.05 compared to the control group, # p < 0.05 compared to LPS group.

CeO₂ NP administration provided protection against renal damage

Kidney ceria content was not increased by nanoparticle treatment relative to untreated animals (Table 1). The LPS treatment decreased kidney weight while the CeO₂ NP treatment was increased kidney weight (Table 1, p<0.05).

Table 1: Cerium oxide accumulation in different organs and their weight.

Compared to control animals, serum blood urea nitrogen (BUN) and creatinine (SCr) levels were significantly higher at 6 and 24 h in the septic animals (p<0.05). The amount of serum phosphorus was lower at 6 h in septic animals (p<0.05). CeO₂ nanoparticle treatment decreased BUN and creatinine (SCr) at 24 h and increase the serum phosphorus level at 6 h (Figure 1, p<0.05). In the H & E kidney tissue in the sepsis group, there was structural deterioration of the glomerulus, decreased Bowman's space, loss of brush border, and tubular cell sloughing (Figure 1). Similarly, as seen in the PAS stained kidney tissue, sepsis was also linked to the loss of brush border and thickening of the parietal Bowman’s capsule (Figure 1). CeO₂ nanoparticle treatment appeared to attenuate the deleterious effects of sepsis on renal structure (Figure 1).

CeO₂ NP attenuate sepsis induced biomarkers for the early detection of AKI

Sepsis elevated the levels of β-2 microglobulin, osteopontin, NAGA, VEGT-A and TIMP-1 rat at 6 h, and calbindin, β-2 microglobulin, KIM-1 rat, cystatin-C, osteopontin, NGAL, VEGT-A and TIMP-1 rat at 24 h time points relative to control animals. Treatment with CeO₂ nanoparticles attenuated sepsis induced increases in calbindin, KIM-1 rat, cystatin-C and osteopontin at 24 h (Figure 2, p<0.05).

Figure 2: CeO₂ NP effect on LPS-induced elevations in biomarkers of AKI. Markers: A (i) calbindin, A (ii) β-2 microglobulin, B (i) kidney injury molecule-1 (KIM-1), B (ii) cystatin-C, C (i) osteopontin, C (ii) neutrophil gelatinase-associated lipocalin (NAGL), D (i) vascular endothelial growth factor-A (VEGF-A) and D (ii) tissue inhibitory of metalloproteinase-1 (TIMP-1). Values are mean ± SEM of at least three independent experiments. *p < 0.05 compared to control group, # p < 0.05 compared to LPS group.

CeO₂ NP attenuate sepsis induced oxidative stress and renal apoptosis

Relative to controls, animals in the septic group appeared to exhibit increased superoxide levels. CeO₂ NP treatment, however, resulted in decreased superoxide levels (Figure 3). Similarly, sepsis associated increases in GRP-78, phosphorylated elf-2, and HSP-90 were decreased with CeO₂ nanoparticles treatment (Figure 3, (p<0.05)).

Figure 3: CeO₂ NP effects on LPS induced renal superoxide level and inflammatory markers. Representative images of kidney tissue (n=3/ group) stained with dihydroethidium (DHE) 24 h after sepsis induction as visualized by fluorescent microscopy and imaged at 200x (Scale bar=100 μm). B (i) Quantification of superoxide levels indifferent groups. Both total and phosphorylated proteins were quantitated by immunoblotting and normalized to GAPDH, B (ii) GRP-78, C (i) p-elF-2, and C (ii) HSP-90. Values are mean ± SEM of at least three independent experiments. *p < 0.05 compared to control group, # p < 0.05 compared to sepsis group.

The TUNEL staining procedure was employed to quantitate the number of nuclei containing fragmented DNA. This analysis was used to determine if kidney damage was associated with elevated cellular apoptosis. It was found that sepsis resulted in an increase in the count of TUNEL positive nuclei relative to control, but nanoparticle treatment resulted in a reduction in these counts relative to control (Figure 4, p<0.05). Consistent with these data, sepsis-induced increases in cleaved caspase-3 (19 and 17-kDa fragment) and the Bax/Bcl-2 ratio were also decreased following nanoparticle treatment (Figure 4, p<0.05).

Figure 4: Effect of CeO₂ NP on apoptosis and its marker proteins changes induced by LPS. A. Mitochondrial dependent renal apoptosis was measured by a TUNEL method (n=3 scale bar=100 μm), B (i) measurement of TUNEL positive nuclei. Western blotting (normalized to GAPDH) was used to measure apoptotic mediator protein expression levels: B (ii) cleaved caspase-3, and B (iii) Bax/Bcl-2 ratio. Values are mean ± SEM of at least three independent experiments. *P <0.05 compared to control group, # p<0.05 compared to sepsis group.

CeO₂ NP administration increases lung ceria content and decreases lung damage

Relative to untreated control rats, lung ceria content was elevated in animals receiving nanoparticle injections (Table 1). Sepsis increased and nanoparticle treatment decreased lung weight (Table 1), p<0.05). in the control and cerium oxide groups, the lung morphology was unremarkable (Figure 5). In contrast, lung tissues from septic animals exhibited extensive morphological damage, including evidence of hemorrhage, interstitial edema, thickening of the alveolar wall, and increased tissue infiltration with leukocytes (Figure 5). As with lung weight, we found that CeO₂ np administration prevented the occurrence of visible sepsis related lung injury (Figure 5).

Figure 5: Effect of CeO₂ NP on sepsis- induced lung injury and superoxide level and MPO activity. A. Representative images of lung morphology (n=3/group) 24 h after sepsis induction as visualized by light microscopy. Septic related changes in the lung were associated with changes in alveolar structure, evidence of pulmonary inflammation, neutrophil infiltration and a thickening of the alveolar septum. B. Representative micrographs of lung tissue (n=3/group) stained with dihydroethidium (DHE) 24 h after sepsis induction and visualized by fluorescent microscopy and imaged at 200 X (Scale bar=100 μm). C (i) Superoxide quantification by group. C (ii) levels of myeloperoxidase activity in lung homogenate from control (n=6), CeO₂NP (n=6), sepsis (n=6), sepsis+CeO₂ NP (n=6): analyzed in triplicate. *Significantly different from control (P ˂ 0.05). # Significantly different from LPS treatment (P ˂ 0.05).

CeO₂ NP attenuate sepsis induced increases in MPO activity and inflammatory signaling in the lungs

Infiltration of neutrophils into the lung is an important feature of LPS-induced ALI [30]. To evaluate the effects of the CeO₂ nanoparticle treatment on lung neutrophil levels, we determined the effect of sepsis and CeO₂ NP treatment on lung MPO activity. Consistent with our histological data, sepsis increased and the nanoparticle treatment decreased MPO activity (Figure 5, p<0.05). Similarly, lung sections collected from septic animals exhibited increased in superoxide levels compared to control rats (Figure 5, p<0.05). The superoxide levels were decreased by CeO₂ NP administration (Figure 5, p<0.05). Compared to control animals, sepsis increased and the nanoparticle treatment decreased the expression of TNF-α, IL-6, IL-1α and IL-1β (Figure 6, p<0.05). Following the lung histology, sepsis increased the amount of phosphorylated ERK1/2 and Bax, while CeO₂ NP administration decreased these measurements (Figure 6).

Figure 6: Protective effect of CeO₂ NP on LPS induced cytokines and apoptotic related markers. Cytokines and chemokine levels were determined in lung homogenates: A (i) TNF-α, A (ii) IL-6, B (i) IL-1 α, and B (ii) IL-1β. Homogenates were pooled in control (n=6), CeO₂ NP (n=6), Sepsis (n=6), Sepsis+CeO₂NP (n=6). Pooled homogenate was analyzed in triplicate. C (i) ERK-P-44/42 marker protein, C (ii) apoptotic and inflammatory related marker proteins were determined by immunoblotting. *Significantly different from control (P ˂ 0.05). #Significantly different from LPS treatment (P ˂ 0.05).

Discussion

Sepsis induced AKI and ALI are two common side effects of sepsis which are associated with increased patient morbidity and mortality [31,32]. The standard treatment for AKI and ALI is primarily supportive in nature [2,33]. Herein, we examine if CeO₂ NP are effective in protecting the kidney and lung following a sepsis. As found previously, sepsis was associated with both AKI and ALI (Figures 1-5). In the renal studies, sepsis resulted in increased serum blood urea nitrogen, creatinine along with several biomarkers of renal injury including KIM-1 and β-2 microglobulin (Figure 1). From a functional perspective, we also found that sepsis appeared to decrease the renal glomerular filtration rate as evidence by increased serum cystatin-C levels [34]. Importantly, we found that these changes were attenuated with a one-time administration of the CeO₂ NP (Figure 2).

It is thought that the increased oxidative stress caused by SIRS is one of the primary causes of sepsis associated organ damage [35]. Recent data suggests that CeO₂ NP can function as highly active ROS scavengers in addition to acting as catalase and SOD mimetics [36]. As predicted, in the current study CeO₂ NP treatment significantly reduced sepsis-induced increases in renal superoxide oxide measurements (Figure 3). In addition to increased ROS, elevations in ER stress have also been suggested in the pathogenesis of acute kidney diseases [37]. To examine this, we next determined how sepsis and the nanoparticle treatment affected the expression of the ER stress makers, GRP- 78 and elF2α. Relative to control animals, the GRP-78 and elF2α expression was significantly elevated with sepsis and importantly, attenuated by the CeO₂ NP treatment (Figure 3).

In addition to increases in cellular ROS and ER stress, it is also posited that renal cell apoptosis may also play a role in AKI [38]. To investigate this, we tested the effectiveness of CeO₂ NP in reducing cellular apoptosis in the kidney. As per our histological analysis and measurement of renal damage, we found that sepsis increased the counts of TUNEL positive cells, elevated the amount of cleaved caspase-3, and increased the Bax/Bcl-2 ratio which was reduced by CeO₂ NP administration (Figure 4).

Sepsis-induced acute lung injury (ALI) in animals is characteristic of excessive infiltration of neutrophils, pro-inflammatory cytokine release, and loss of vascular barrier integrity [39]. It is thought that activated neutrophils can induce the extensive lung inflammation that oftentimes increases the permeability of alveolar capillary membrane [40]. Indeed, migrating neutrophils cause mechanical damage to alveolar structure and worsen the influx of fluid into alveolar space [41]. In addition, neutrophils can also release free radicals that cause oxidative stress which play an important role in structural, functional and inflammatory responses that characterized ALI [42]. In the current study, LPS resulted in significant alteration in the pulmonary structure that was accompanied by neutrophil infiltration in the lung interstitial space and enhanced MPO activity (Figure 5). Similar to the kidneys, the CeO₂ NP reduced visible lung injury relative to control (Figure 5).

Inflammatory cytokines are believed to be crucially involved in the lung injury/damage in observed in septic animals. Among these cytokines, TNF-α, IL-1β and IL-6 are thought to play critical roles in early phase of ALI. It has been reported that resident alveolar macrophages and recruited neutrophils release TNF-α and IL- 1β in early phase of inflammation in response to the pathogen stimulus, resulting in the subsequent inflammatory cascade and lung injury [43]. In the current study, the expression of TNF-α, IL- 1β, IL-1α and IL-6 were markedly induced by LPS challenge, which was blocked by CeO₂ NP administration (Figure 6).

In addition to elevation in cytokine concentrations, excessive iNOS and COX-2 expression may contribute to the pathophysiology of sepsis-induced ALI [44]. In the current study, it was found that LPS increased iNOS and COX-2 expression in lung homogenates, while CeO₂ NP treatment decreased iNOS and COX-2 protein levels. As with the renal studies, and consistent with our histological analysis, we also found that CeO₂ NP can attenuate LPS-induced elevations in the Bax/Bcl-2 ratio and caspase-3 cleavage (Figure 4).

Conclusion

In summary, this study demonstrated that a single dose 0.5 mg/kg of intravenous CeO₂ NP to rats with LPS-induced sepsis significantly decreased the severity of AKI and ALI. In addition to improvements in renal structure, the CeO₂ NP treatment also decreased circulating NGAL and Kim-1 levels, ER stress and renal apoptosis. Similarly, the CeO₂ NP also resulted in improved in lung morphology, and decreased neutrophil infiltration, MPO activity, and pulmonary cytokine expression (TNF-α, IL-1β, IL-6). Therefore, we propose that CeO₂ NP acts on reducing sepsisinduced oxidative stress, and hence exerts a protective effect on acute kidney and lung injuries (Figure 7). However, much indepth work remains. For example, how CeO₂ NP can diminish pulmonary neutrophil infiltration? How CeO₂ NP can change critical biomarkers (such as HSP-90, elF2α, GRP-78, caspase-3, and Bax) in terms of translational or post-translational mechanisms? With more thorough investigations, we will be able to understand better the mechanism of CeO₂ NP protective effects in sepsis as a whole. Based on our current data, CeO₂ NP could be a potential pharmacotherapy for sepsis-associated AKI and ALI.

Figure 7: Proposed mechanism of CeO2 NP in attenuation of acute kidney injury (AKI) and acute lung injury (ALI). Lipopolysaccharide (LPS) can trigger cellular and inflammatory responses, which result in tissue excessive oxidative stress and organ injuries. CeO₂ NP exerts protective effects, at least in part, by attenuating oxidative stress and damages. Red arrow indicates “triggering/stimulating/ increasing”, and blue line suggests “inhibiting/decreasing”.

Acknowledgements

This work was supported in part from DOE grant (DE-PS02- 09ER-01 to E.R.B).

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