Diesel Exhaust Particles in the Lung Aggravate Experimental Acute

TOXICOLOGICAL SCIENCES 113(1), 267–277 (2010)

doi:10.1093/toxsci/kfp222

Advance Access publication October 1, 2009

Diesel Exhaust Particles in the Lung Aggravate Experimental AcuteRenal Failure

Abderrahim Nemmar,*,1 Suhail Al-Salam,† Shaheen Zia,* Javed Yasin,‡ Isehaq Al Husseni,§ and Badreldin H. Ali§

*Department of Physiology, †Department of Pathology, and ‡Department of Internal Medicine, Faculty of Medicine and Health Sciences, United Arab Emirates

University, Al Ain, United Arab Emirates 17666; and §Department of Pharmacology and Clinical Pharmacy, College of Medicine & Health Sciences, Sultan

Qaboos University, Muscat, 123 Sultanate of Oman

1 To whom correspondence should be addressed at Department of Physiology, Faculty of Medicine and Health Sciences, United Arab Emirates University,

PO Box 17666, Al Ain, United Arab Emirates. Fax: þ9713 7671966. E-mail: [email protected].

Received May 11, 2009; accepted September 14, 2009

Inhaled particles are associated with pulmonary and extrapulmo-

nary effects. Also, acute renal failure (ARF) is associated with

increased mortality, related to pulmonary complications. Here, we

tested the possible potentiating effect of diesel exhaust particles

(DEP) in an animal model of ARF induced by a single ip injection of

cisplatin (CP, 6 mg/kg) in rats. Six days later, the rats were

intratracheally instilled with either DEP (0.5 or 1 mg/kg) or saline

(control) and renal, systemic, and pulmonary variables were studied

24 h thereafter. CP increased the serum concentrations of urea and

creatinine and reduced glutathione (GSH) concentration and

superoxide dismutase activity in renal cortex. CP caused renal

tubular necrosis; increased urine volume, protein concentrations,

and N-acetyl-b-D-glucosaminidase (NAG) activity; and decreased

urine osmolality. The combination of DEP and CP aggravated the

CP-induced effects on serum urea and creatinine, urine NAG

activity, and renal GSH. The arterial O2 saturation and PO2 were

significantly decreased in CP1 DEP versus CP1 saline and CP1DEP versus DEP. The number of platelets was reduced in DEP

compared to saline-treated rats andCP1DEP versus DEP alone or

CP 1 saline. Increases in macrophage and neutrophils numbers in

bronchoalveolar lavage were found in DEP versus saline group and

CP 1 DEP versus CP. Histopathological changes in lungs of DEP-

treated rats were aggravated by the combination of CP 1 DEP.

These included marked interstitial cell infiltration and congestion.

We conclude that the presence of DEP in the lung aggravated the

renal, pulmonary, and systemic effects of CP-induced ARF.

Key Words: air pollution; diesel exhaust particles; lung

inflammation; acute renal failure.

Acute renal failure (ARF) is increasingly becoming more

frequent and is associated with high costs and adverse clinical

outcomes, including excess mortality, increased length of

hospital stay, and the requirement for chronic dialysis in

survivors (Hoste and Schurgers, 2008; Pannu et al., 2008).

Several studies have reported consistent association between

ARF and dysfunction of extrarenal organs, particularly the

lungs (Hoke et al., 2007; Pierson, 2006;). Experimentally, ARF

resulting from either ischemia or bilateral nephrectomy has

been reported to cause lung inflammation (Hoke et al., 2007).

Furthermore, it has been recently demonstrated that the kidney

plays an important role in the production and elimination of

mediators of pulmonary injury and that prolonged exposure to

these mediators contributes to pulmonary injury (Grigoryev

et al., 2008; Hoke et al., 2007).

Ischemia and toxicity are considered the main pathophysi-

ological factors that lead to the development of ARF (Ali and

Al Moundhri, 2006; Ali et al., 2007; Grigoryev et al., 2008;

Hoke et al., 2007). A substance that is well known to induce

toxic kidney injury is cisplatin (CP). CP is a potent anticancer

drug that is commonly used against multiple solid human

cancers, including testicular, cervical, ovarian, head, and neck

malignancies. The drug is bioactivated to a nephrotoxicant and

is also known to produce proximal tubular injury, which is

thought to be due to a combination of direct cytotoxicity,

intrarenal vasoconstriction, and oxidative stress (Ali and Al

Moundhri, 2006; Ali et al., 2007, 2008).

Inhaled particulate air pollution with particle diameter less than

2.5 lm contributes to respiratory and cardiovascular morbidity

and mortality (Kunzli et al., 2005; Pekkanen et al., 2002; Peters

et al., 2001; Pope et al., 2002). Diesel exhaust particles (DEP),

which are the major contributors to PM2.5 and ultrafine particles

(diameter� 0.1 lm) in cities, have been identified in a number of

epidemiological studies to cause adverse health effects, including

cardiorespiratory diseases, particularly in individuals with

preexisting disease (Atkinson et al., 2001; Pope et al., 1992).

Experimental exposure to DEP causes systemic and inflamma-

tory response in the airways and impairs the regulation of

vascular tone and endogenous fibrinolysis in healthy human

volunteers (Mills et al., 2005, 2007; Salvi et al., 1999). Moreover,

we (Nemmar and Inuwa, 2008; Nemmar et al., 2003a,b, 2004a,

2007) and others (Inoue et al., 2005, 2006) have reported that

exposure to DEP cause pulmonary inflammation and thrombotic

complication in hamsters and mice.

� The Author 2009. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved.For permissions, please email: [email protected]

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Interest in the nonpulmonary targets of particulate air

pollutants has been increasing since the demonstration that

inhaled ultrafine particles are able to translocate directly from

the lungs to extrapulmonary tissues and cause the release of

soluble inflammatory mediators into the systemic circulation,

which affect other organs, such as the liver, the heart, and even

the brain (Nemmar et al., 2004b; Oberdorster et al., 2005;

Peters et al., 2006; Vermylen et al., 2005). However, as far as

we are aware, the effect of particulate air pollution on ARF has

not been yet investigated.

Therefore, the aim of this study was to investigate, in vivo,

the possible aggravating effect of pulmonary exposure to DEP

in an animal model of ARF induced by CP, by measuring some

commonly used renal, systemic, and pulmonary variables.

MATERIALS AND METHODS

Animals. Male Wistar rats (Taconic Farms Inc., Germantown, NY), aged

10–12 weeks and initially weighing 258 ± 6 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 4 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

National Institutes of Health Guide for the Care and Use of Laboratory

Animals, NIH publication no. 85-23, 1985.

Intratracheal instillation. We used DEP (SRM 2975) from the National

Institute of Standards and Technology (Gaithersburg, MD). We have recently

(Nemmar et al., 2007) analyzed the size of DEP used in the present study by

transmission electron microscopy and found a substantial amount of ultrafine

(nano)-sized particle aggregates and larger particle aggregates (< 1 lm in

largest diameter). DEP were suspended in sterile normal saline (NaCl 0.9%)

containing Tween 80 (0.01%). To minimize aggregation, particle suspensions

were always sonicated (Clifton Ultrasonic Bath, Clifton, NJ) for 15 min and

vortexed before their dilution and prior to intratracheal (i.t.) administration.

Control animals received normal saline containing Tween 80 (0.01%).

Treatments. The ARF in rats was induced by a single ip injection of CP

(David Bull Laboratories, PTY Ltd, Victoria, Australia) at a dose of 6 mg/kg

(Ali et al., 2007, 2008). Control animals received similar volume of normal

saline ip. On day 6 of treatment, the animals were anesthetized with ip injection

of ketamine (75 mg/kg) and xylazine (10 mg/kg) and placed supine with

extended neck on an angled board. A Becton Dickinson 18 Gauge cannula

(Franklin Lakes, NJ) was inserted via the mouth into the trachea. DEP

suspension (0.5 or 1 mg/kg in 150 ll) or vehicle only were instilled (150 ll) via

a sterile syringe and followed by an air bolus of 100 ll.

The six groups were treated as follows (n ¼ 6–8 in each group):

� Group 1: single normal saline (control, 500 ll/rat) given ip, and on day 6

of the treatment, a single i.t. administration of saline (150 ll per rat);


of the treatment, a single i.t. administration of DEP (0.5 mg/kg);


of the treatment, a single i.t. administration of DEP (1 mg/kg);

� Group 4: single CP (6 mg/kg) given ip, and on day 6 of the treatment,

a single i.t. administration of saline (150 ll per rat);


a single i.t. administration of DEP (0.5 mg/kg); and


a single i.t. administration of DEP (1 mg/kg).

On day 6, immediately after i.t. administration of saline or DEP, rats were

placed in metabolic cages and urine of each rat was collected over a 24-h period

and the volume measured.

Blood collection and bronchoalveolar lavage. Twenty-four hour after the

i.t. administration of saline or DEP, the rats were anesthetized as described

above and blood was drawn from the inferior vena cava in EDTA (4%).

A sample was used for hematocrit measurement and platelets and white blood

cells counts using an ABX Micros 60 counter (ABX Diagnostics, Montpellier,

France). The remaining blood was left at room temperature for 2 h before it was

centrifuged at 900 3 g at 4�C for 15 min to separate serum. The serum obtained

was stored frozen at �80�C to await biochemical analyses.

The rats were then sacrificed with an overdose of ketamine. Bronchoalveolar

lavage (BAL) was then performed by cannulating the trachea, the left bronchus

was clamped. The bronchi and right lung were lavaged three times with 5 ml

sterile 0.9% NaCl. The BAL fluid was pooled in a plastic tube on ice. No

difference in the amount of recovered fluid was observed between the different

groups. BAL fluid was centrifuged (1000 3 g 3 10 min, 4�C). Cell counting

was performed in a hemocytometer after resuspension of the pellets and

staining with 1% gentian violet. The cell differentials were performed on

cytocentrifuge preparations fixed in methanol and stained with Diff Quick

(Dade Behring, Marburg, Germany). The supernatant was stored at �80�Cuntil further analysis.

Biochemical analysis and histopathology. Lungs and kidneys were

excised, washed with ice-cold saline, blotted with filter paper, and weighed.

Small pieces from the left lung and left kidney were fixed in 10% neutral

formalin, dehydrated in increasing concentrations of ethanol, cleared with

xylene, and embedded in paraffin. The cortex of the right kidney was excised

from the medulla and rapidly homogenized in ice-cold saline to produce

10 (wt/vol) tissue homogenate.

The concentrations of urea and creatinine in serum were spectrophotomet-

rically measured using commercial kits (BioMerieux, Marcy-l’Etoile, France).

Urine osmolality was measured by the freezing point depression method

(�70�C) using an osmometer (Roebling, Berlin, Germany) and N-acetyl-b-D-

glucosaminidase (NAG) activity by kits from Diazyme, General Atomics, San

Diego, CA. Urine protein concentration was measured spectrophotometrically

using a kit from BioMerieux. In renal cortex homogenates, glutathione (GSH)

concentration and superoxide dismutase (SOD) activity were measured

spectrophotometrically (Randox, Antrim, UK). The concentration of CP (as

platinum) in cortical tissue was measured by flameless atomic absorption

spectrophotometry (Perkin-Elmer, Vernon Hills, IL; 3300 DV ICP-OES

equipped with a cross-flow nebulizer, in addition to an ultrasonic nebulizer).

The procedure involved mineralization of the kidney cortex tissue with

a mixture of concentrated HNO3 and H2O2, followed by determination of

platinum in the extract, using inductively coupled plasma optical emission

spectrometry, at an emission wavelength of 265.945 nm.

Five-micrometer sections were prepared from left lung and left kidney

paraffin blocks and stained with hematoxylin and eosin. Staining for apoptosis

in the kidney sections has been performed using signal stain cleaved caspase-3

Immunohistochemical detection Kit (Cell Signaling Technology, Boston, MA).

This kit was used to detect the activation of caspase using avidin-biotin

immunoperoxidase method to detect intracellular caspase-3 protein. Staining

was performed on 5-lm paraffin sections from left kidney by standard

technique using rabbit anti-cleaved caspase-3 (clone Asp175, 1:50) (Vielhauer

et al., 2005). A known positive control sections for apoptosis were used. For

negative control, primary antibody was replaced with normal rabbit serum.

Blood gas measurements. Arterial blood gases were measured in separate

animals following the protocol described above. Immediately after the

anesthesia, arterial blood was obtained via cardiac puncture in EDTA. Analysis

was performed immediately after collection with a Roche blood gas analyzer

(Mannheim, Germany).

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Statistics. All data were analyzed with GraphPad Prism Version 4.01 for

Windows software (Graphpad Software Inc., San Diego, CA). Data were analyzed

for normal distribution using the D’Agostino and Pearson omnibus normality test.

Data are expressed as means ± SD. Comparisons between groups were performed

by one-way ANOVA, followed by Newman-Keuls test for comparing treated with

control data. The p values of � 0.05 are considered significant.

RESULTS

Effect of CP and DEP on Renal Variables

To induce ARF, rats were treated with a single CP injection

at a dose of 6 mg/kg and this resulted in ARF, similar to

previously reported studies (Ali et al., 2007; Mohan et al.,2006). In the present work, rats given saline gained about 2.6%

(p < 0.05), while those receiving DEP 0.5 or 1 mg/kg gained

about 6 and 0.7% (p: not significant) of their initial body

weight. However, rats treated concomitantly with CP and

saline lost about 6.4% (p < 0.05), whereas those administered

with CP and DEP 0.5 and 1 mg/kg lost more weight, i.e., 8.6

and 9.3% (p < 0.05), respectively.

The kidney weights were slightly but not significantly

increased in DEP 0.5 mg/kg-exposed (7.1 ± 0.3 g/kg body

weight) and DEP 1 mg/kg rats (7.2 ± 0.4 g/kg body weight)

compared to saline-treated (6.6 ± 0.2 g/kg) rats. However, CP þsaline treatment significantly increased kidney weight (9.6 ± 0.9

g/kg, p < 0.01) compared to the saline group. Rats treated with

CP þ DEP 0.5 mg/kg significantly increased kidney weight

(8.7 ± 0.7 g/kg, p < 0.05) compared to DEP 0.5 mg/kg alone.

Similarly, the combination of CP þ DEP 1 mg/kg treatment

increased the weight of kidneys (8.8 ± 0.5 g/kg, p ¼ 0.05)

compared to the DEP 1 mg/kg group. No statistical difference

has been observed between CP þ saline and CP þ DEP.

Figure 1 illustrates the effects of saline and CP with or

without DEP, on the concentrations of creatinine and urea in

serum. DEP did not significantly affect the concentrations of

urea or creatinine compared to the saline-treated group.

However, CP þ saline significantly increased the concentration

of urea and creatinine compared to the saline-treated rats.

Interestingly, CP þ DEP 0.5 and 1 mg/kg treatment

significantly and consistently increased the concentration of

urea and creatinine in serum more than in rats treated with DEP

0.5 or 1 mg/kg alone or CP þ saline.

DEP 1 mg/kg alone caused a significant decrease of renal

GSH compared to the saline group. On the other hand, CP þsaline decreased the level of renal GSH compared to the saline-

treated rats. A more pronounced effect was seen in the CP þDEP 1 mg/kg group in which the concentrations of renal GSH

was significantly reduced compared to both DEP 1 mg/kg and

saline þ CP-treated groups (Fig. 2A). However, the renal SOD

concentration was only slightly and insignificantly decreased

after DEP 1 mg/kg exposure. Treatment with both CP þ saline

and CP þ DEP 1 mg/kg reduced the activity of renal SOD

compared to values from rats treated with saline and DEP,

respectively (Fig. 2B).

Figure 3 depicts the effect of treatment with saline and CP

with or without DEP on urinalyses. Compared with saline- or

DEP-treated animals, CP þ saline and CP þ DEP treatments

increased the 24-h urine volume (Fig. 3A). Urinary NAG

activity was significantly increased in CP þ saline compared

with saline-treated controls (p < 0.001) and in rats treated with

CP þ DEP compared to the DEP-treated group (p < 0.001).

Interestingly, the combination of CP þ DEP 1 mg/kg

significantly enhanced (p < 0.01) the NAG activity compared

to CP þ saline (Fig. 3B). Urine protein concentrations were not

affected by saline or DEP alone. However, CP þ saline and

CP þ DEP treatments significantly increased the urine protein

concentration to a similar level when compared to saline- and

DEP-treated groups, respectively (Fig. 3C). CP þ saline and

CP þ DEP treatments decreased urine osmolality when compared

to saline- and DEP-treated groups (p < 0.001), respectively

(Fig. 3D). No significant difference in urine osmolality was

observed between rats treated with CP þ saline and CP þ DEP.

Figure 4 shows representative micrographs of renal cortex

from the six groups used. The kidney architecture was not

FIG. 1. Serum urea (A) and creatinine (B) concentrations in Wistar rats

treated with saline (control), DEP 0.5 mg/kg, DEP 1 mg/kg, CP þ saline, CP þDEP 0.5 mg/kg, or CP þ DEP 1 mg/kg (n ¼ 6–8). Mean ± SD. Statistical

analysis by Newman-Keuls test.

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affected by saline (Fig. 4A), DEP 0.5 mg/kg (Fig. 4B), or DEP

1 mg/kg (Fig. 4C) treatments. However, in CP þ saline

(Fig. 4D), CP þ DEP 0.5 mg/kg (Fig. 4E), and CP þ DEP 1

mg/kg (Fig. 4F), the renal cortex showed the presence of acute

tubular necrosis with the presence apoptotic cells, tubular

distention with eosinophilic material, interstitial edema, and

congestion. No accumulation of DEP in the different kidney

sections has been observed in animal exposed to DEP. The

staining for the detection of apoptotic cells showed no evidence

of apoptotic cells in the kidneys of saline or DEP groups

(Figs. 5A–C). Apoptotic cells were seen in the kidneys of CP þsaline (Fig. 5D), CP þ DEP 0.5 mg/kg (Fig. 5E), and CP þDEP 1 mg/kg (Fig. 5F). However, there was no significant

difference in the number of apoptotic cells between these three

groups.

The concentration of platinum in the renal cortex of rats

given CP þ saline (3.95 ± 0.17 ppm) was not significantly

different from that in rats treated with CP þ DEP 0.5 mg/kg

(4.07 ± 0.3 ppm) or CP þ DEP 1 mg/kg (4.08 ± 0.09 ppm). No

platinum has been found in the kidneys of rats treated with

saline, DEP 0.5 mg/kg, or DEP 1 mg/kg.

Effect of CP and DEP on Systemic Variables

The exposure of rats to DEP 1 mg/kg slightly but

insignificantly decreased the PaO2 (Fig. 6A). Pretreatment of

rats with CP þ DEP 0.5 mg/kg significantly decreased PaO2

compared to DEP 0.5 mg/kg alone (Fig. 6A). Interestingly, we

found that treatment with CP þ DEP 1 mg/kg significantly

decreased the PaO2 compared to treatment with CP þ saline

and DEP 1 mg/kg alone (Fig. 6A). Nevertheless, no significant

effect on PaCO2 has been observed between saline (39.5 ± 2.8),

DEP 0.5 mg/kg (39.0 ± 4), DEP 1 mg/kg (41.2 ± 1.9), CP þsaline (41.4 ± 1.5), CP þ DEP 0.5 mg/kg (41.0 ± 2.3), or

DEP þ CP (41.2 ± 0.9).

Figure 6B illustrates that rats treated with DEP 1 mg/kg

slightly but insignificantly decreased the arterial O2 saturation

(SaO2) (Fig. 6B). However, we found that treatment with CP þDEP 1 mg/kg significantly decreased the SaO2 compared to

treatment with CP þ saline or to DEP 1 mg/kg alone (Fig. 6B).

Similarly, the hematocrit in group treated with CP þ DEP

1 mg/kg slightly but significantly increased compared to

treatment with CP þ saline or to DEP 1 mg/kg alone (Fig. 6C)

Figure 7 depicts the effect of treatment with saline and CP

with or without DEP on the numbers of leukocytes and

platelets in whole blood. Although the level of significance was

only reached in group treated with CP þ DEP 1 mg/kg (p <0.05), a dose-dependent increase in leukocytes numbers was

observed after exposure of rats to CP þ DEP 0.5 and 1 mg/kg

compared to CP þ saline (Fig. 7A). The numbers of platelets

was affected by the different treatments (Fig. 7B). Both doses

of DEP caused a significant decrease of platelet number

compared to the saline group. On the other hand, treatment

with CP þ saline decreased the platelet number compared to

saline-treated rats. An aggravating effect was seen in CP þDEP 1 mg/kg group in which the number of platelets was

significantly reduced compared to both DEP 1 mg/kg and CP þsaline-treated groups. The different treatments had no signif-

icant effect on the number of red blood cells and hemoglobin

concentrations (data not shown).

Effect of CP and DEP on Pulmonary Variables

Depending on the treatment performed, the cells found in

BAL were primarily macrophages and polymorphonuclear

leukocytes (PMN) (Fig. 8) and no lymphocytes or other cells

were observed microscopically. The pulmonary administration

of DEP resulted in a marked influx of macrophage and PMN in

the lung compared to saline-treated rats. Similarly, CP þ DEP

significantly and dose-dependently increased the numbers of

macrophage (0.5 mg/kg, p < 0.05 and 1 mg/kg, p < 0.005) and

PMN (0.5 mg/kg, p < 0.05 and 1 mg/kg, p < 0.01) compared to

CP þ saline group. Although the numbers of macrophage and

PMN in CP þ DEP group were higher compared to DEP-

treated rats, this difference did not reach statistical significance.

FIG. 2. Reduced GSH (A) and SOD (B) activity in renal cortex, in Wistar

rats treated with saline (control), DEP 1 mg/kg, CP þ saline, or CP þ DEP 1

mg/kg (n ¼ 6). Mean ± SD. Statistical analysis by Newman-Keuls test.

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Figure 9 shows representative histological micrographs of

lungs from the six groups studied. The histopathology findings

described below were uniform within the same lung tissue and

from rat to rat. Sections from the saline-treated rats showed

normal lung architecture (Fig. 9A). In DEP-treated lungs (0.5

and 1 mg/kg), particles engulfed by macrophages in the

interalveolar interstitium associated with increased interstitial

cellularity and widening of the interalveolar spaces have been

observed (Figs. 9B and 9C). CP þ saline caused an increase in

cellularity of interalveolar interstitium and mild congestion

(Fig. 9D). Importantly, these effects were aggravated by the

concomitant administration of CP þ DEP, which showed the

presence of aggregates of DEP in the interalveolar interstitium

and marked interstitial cellular expansion (Figs. 9E and 9F).

Moreover, in CP þ DEP 1 mg/kg, congestion and severe

interstitial and intra-alveolar edema were observed (Fig. 9F).

DISCUSSION

In this study, we provide the first experimental evidence that

DEP deposited in the lungs can aggravate experimental ARF.

Our study shows that a number of renal, systemic, and

pulmonary end points were potentiated by the concomitant

administration of CP and DEP.

ARF remains a common and critical clinical entity affecting 5–

7% of all hospitalized patients. It carries a significant morbidity

and a 20–70% mortality rate (Singri et al., 2003). The number of

people affected by air pollution is extremely large, and as

a consequence, estimates of the public health burden of ambient

air pollution are substantial. A tri-national European study

attributed some 6% of all deaths and 2% of hospital admissions to

ambient air pollution (Kunzli et al., 2000). Lung and kidney

function are intimately related in both health and disease. Several

studies have reported consistent association between ARF and

pulmonary dysfunction (Hoke et al., 2007; Pierson, 2006).

Indeed, it is well established that lung injury can aggravate ARF

and vice versa (Pierson, 2006). When ARF and acute lung injury

are combined, the mortality rate exceeds 80% (Chien et al.,2004). Experimentally, ARF has been reported to cause lung

inflammation (Grigoryev et al., 2008; Hoke et al., 2007).

Recently, Brook (2008) pointed out in an editorial that

exposure to particulate air pollution play some role in variety of

FIG. 3. Urine volume (A), N-acetyl-b-D-glucosaminidase (NAG; B), proteins (C), and osmolality (D) in Wistar rats treated with saline (control), DEP

0.5 mg/kg, DEP 1 mg/kg, CP þ saline, CP þ DEP 0.5 mg/kg, or CP þ DEP 1 mg/kg (n ¼ 6–8). Mean ± SD. Statistical analysis by Newman-Keuls test.


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human diseases, including cardiovascular, neurological, and

renal. Particulate air pollution influences susceptibility to hard

events and may be particularly harmful to high-risk groups

such as people with diabetes, elderly people, and people with

hypertension (Brook et al., 2004). All of which are known to

be associated with a high risk of developing complications,

such as ARF (Leblanc et al., 2005). However, there is little

information regarding renal effects of particulate air pollution,

although one study (Hendryx, 2009) reported a higher kidney,

heart, respiratory, disease, and mortality in coal mining areas

due to environmental exposure to particulate matter and toxic

agents. While the interest in extrapulmonary effects of parti-

culate air pollution and animal model of enhanced susceptibil-

ity is increasing, the possible aggravating effect of particulate

air pollution on animal model of ARF has not been reported so

far. Thus, in the present study, we tested the possibility of

aggravating effect of DEP on animal model of CP-induced

renal failure.

Despite the notion that the i.t. instillation of a bolus of

particles as a model of exposure to particulate air pollution may

not be an ideal model, this method of delivery has been shown

to be a reliable, convenient, and valid mode of administration

of foreign compounds into the airways as it permits the

accurate introduction of a range of doses to the lungs within

a short time (Driscoll et al., 2000). Nevertheless, additional

studies using inhalation exposure are needed to verify our

findings. The dose of particles we used in our study (0.5 and

1 mg/kg) are comparable to the doses of DEP we previously

used in hamster (Nemmar et al., 2003a,b, 2004a) and lower

than the 5 mg/kg used in rats by others (Castranova et al.,2001; Yokota et al., 2005).

To induce ARF, rats were given CP, which is a potent

anticancer drug that is commonly used to treat multiple solid

human cancers (Zhang et al., 2006). In the present study,

reduction in body weight following CP treatment may be due to

injury to the renal tubules and the subsequent loss of the tubular

cells to reabsorb water, resulting in dehydration and thus loss of

body weight. The further loss of body weight following

pulmonary exposure to DEP is a reflection of the aggravating

effects of DEP on CP-induced ARF. The deterioration of

kidney function when rats were exposed to both DEP and CP is

supported by the results of the kidney function tests. Serum

creatinine and urea concentrations were significantly higher in

rats treated with CP þ DEP than those treated with CP þ saline.

Compared to CP or DEP alone, the treatment of animals with

CP þ DEP or CP þ saline increased the urine volume and

FIG. 4. Representative light microscopy sections of renal tissue of rats

given saline (control, A), DEP 0.5 mg/kg (B), DEP 1 mg/kg (C), CP þ saline

(D), CP þ DEP 0.5 mg/kg (E), and CP þ DEP 1 mg/kg (F). The micrograph

showing acute tubular necrosis with apoptotic cells (arrows), tubular distention

with eosinophilic material (arrow heads), and interstitial edema and congestion.

FIG. 5. Immunohistochemical analysis of the renal tissue sections.

Staining for the detection of apoptotic cells showed no evidence of apoptotic

cells in the kidneys of saline (control, A), DEP 0.5 mg/kg (B), and DEP 1 mg/

kg (C) groups. Apoptotic cells with dark brown granular cytoplasmic staining

for caspase-3, streptavidin-biotin complex immunohistochemistry were only

seen in the kidneys of CP þ saline (D), CP þ DEP 0.5 mg/kg (E), and CP þDEP 1 mg/kg (F) where acute tubular necrosis with apoptotic cell (arrows) and

tubular distention with necrotic material (arrow heads) have been observed.

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protein and decreased its osmolality. However, no aggravating

effect was observed in rats given CP þ DEP when compared

to those that have been given CP þ saline. Nevertheless, the

increase in activity of NAG was significantly potentiated in

rats treated with CP þ DEP 1 mg/kg when compared to

those given CP þ saline. Increased enzyme activity in the urine

is generally regarded as a reliable indicator of renal tubular

dysfunction (Bosomworth et al., 1999). The lysosomal enzyme

NAG is one of the most important marker of tubular damage

most commonly used, primarily because NAG assays are

sensitive enough to allow dilution of the urine, thus over-

coming any enzyme inhibition (Bosomworth et al., 1999).

Moreover, we found that DEP caused slight but insignificant

hypoxemia. We measured arterial blood gas in anesthetized

animals; therefore, a possible effect of anesthesia on arterial

PO2 cannot be excluded. However, the comparison between

FIG. 6. Arterial blood PO2 (A), SaO2 (B), and hematocrit (C) in Wistar

rats treated with saline (control), DEP 0.5 mg/kg, DEP 1 mg/kg, CP þ saline,

CP þ DEP 0.5 mg/kg, or CP þ DEP 1 mg/kg (n ¼ 6–8). Statistical analysis by

Newman-Keuls test.

FIG. 7. Numbers of white blood cells (WBC; A) and platelets (B) in

whole blood in Wistar rats treated with saline (control), DEP 0.5 mg/kg, DEP

1 mg/kg, CP þ saline, CP þ DEP 0.5 mg/kg, or CP þ DEP 1 mg/kg (n ¼ 6–8).

Mean ± SD. Statistical analysis by Newman-Keuls test.


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the different groups, which were anesthetesized the same way,

showed statistically significant differences. We found that

CP þ DEP 1 mg/kg significantly decreased the PaO2 and SaO2

compared to CP þ saline or DEP 1 mg/kg alone. It has been

suggested that pollution may results in hypoxemia and that

these effects might be most relevant in older and sicker

individuals (DeMeo et al., 2004; Pope et al., 1999). These

effects are compatible with histopathological findings observed

in the lungs where marked interstitial and intra-alveolar edema

was observed (Fig. 9F). This effect could explain the decrease

of PaO2 and SaO2 by the impairment of O2 transport through

the alveolar capillary barrier. Moreover, we found slight but

significant increase in hematocrit in CP þ DEP (1 mg/kg)

versus DEP 1 mg/kg alone and CP þ saline. An increase of

hematocrit levels following exposure to particulate matter has

been reported in men (Riediker et al., 2004) and rats (2 mg/kg

but not at lower dose) (Rivero et al., 2005). Additional studies

are needed to establish the effect of a decrease in PaO2 in the

lung on the level of gas exchange in the tissue. The PaCO2 did

not significantly change in different groups probably because to

its easier diffusion as compared to O2.

The kidneys are the major site for CP accumulation, and this

results in necrosis of the terminal portion of the proximal renal

tubules and apoptosis in the distal nephron (Ali et al., 2007).

The concentrations of CP in the renal cortex in rats treated with

CP þ DEP were similar to those treated with CP þ saline. This

observation could explain the absence of further necrosis of the

renal tissues seen in the slides of kidneys from rats treated with

CP þ DEP compared to CP þ saline.

Our data confirm that CP decreases renal GSH concentra-

tion and SOD activity, leaving the renal tissues vulnerable to

damage by oxygen free radicals that are responsible for the

induction of tubular epithelial cell death. The SOD activity

was not affected by DEP administration, and no potentiating

effect was observed in CP þ DEP compared to CP þ saline.

Interestingly, DEP alone significantly reduced the GSH

levels, and this effect was enhanced by the combination

of CP þ DEP. This may suggest that SOD was a less sensi-

tive marker for the generation of free radicals than GSH

(Ajith et al., 2007). Several studies have reported that

GSH has a protective effect against CP nephrotoxicity,

and recently, CP has been shown to be metabolized to

FIG. 8. Macrophages (A) and PMN (B) numbers in BAL fluid in Wistar

rats treated with saline (control), DEP 0.5 mg/kg, DEP 1 mg/kg, CP þ saline,

CP þ DEP 0.5 mg/kg, or CP þ DEP 1 mg/kg (n ¼ 6–8). Mean ± SD. Statistical

analysis by Newman-Keuls test.

FIG. 9. Representative light microscopy sections of lung tissues of rats

given saline (control, A), DEP 0.5 mg/kg (B), DEP 1 mg/kg (C), CP þ saline

(D), CP þ DEP 0.5 mg/kg (E), and CP þ DEP 1 mg/kg (F). The micrograph

shows DEP engulfed by macrophages or free in the interalveolar interstitium

(thick arrows) and the presence of marked interstitial cellular infiltration (thin

arrows) and intra-alveolar edema (arrow heads).

274 NEMMAR ET AL.

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a nephrotoxicant through a GSH-conjugate intermediate

(Hanigan and Devarajan, 2003; Zhang et al., 2006). Oxidative

stress in kidney plays an important role in CP-induced renal

damage, and several antioxidants and thiol compounds have

been shown to protect against CP nephrotoxicity (Ali and Al

Moundhri, 2006; Ali et al., 2007).

Studies in humans and in animal models have demonstrated

that ARF has a significant effect on the function of extrarenal

organs (Grigoryev et al., 2008; Hoke et al., 2007). In-

flammation is a major component of the initiation and

exacerbation of kidney injury, and local inflammation of

kidney tissues could be a source of the development of

inflammation and injury in extrarenal organs (Grigoryev et al.,2008). Therefore, in the present study, we sought to count the

number of circulatory cells, as a marker of systemic

inflammation. Although not statistically significant, we found

an increase of leukocyte numbers in DEP-treated group (1 mg/

kg) compared to saline group. We made a similar observation

in mice exposed to DEP (Nemmar et al., 2009). The fact that

CP þ DEP dose-dependently and significantly (at 1 mg/kg)

increased leukocyte numbers compared to CP þ saline

suggests that ARF exacerbates systemic inflammation. This

finding is in agreement with the concept that particulate air

pollution effects are aggravated in people with preexisting

diseases (Brook et al., 2004). We have shown that the

systemic administration of DEP increases the number of

leukocytes and decreases both the number of erythrocytes and

the hemoglobin concentration (Nemmar and Inuwa, 2008). In

contrast, DEP alone caused a significant decrease of platelet

number compared to the saline-treated group. Moreover,

a more pronounced reduction in platelet number was seen in

the CP þ DEP group, suggesting the occurrence of platelet

aggregation in vivo. These results are in agreement with

animal and human studies, which have reported a decrease in

platelet number following exposure to particulate air pollution

(Nemmar et al., 2008; Ruckerl et al., 2007). Moreover, we

have recently demonstrated a prothrombotic effect of DEP in

hamsters as well as an activation of platelets (Nemmar et al.,2003a,b, 2004a). It remains to be established whether these

effects are aggravated by direct effect of DEP that have

presumably translocated into blood and/or through pulmonary

release of proinflammatory cytokines, such as interleukin (IL)-

6, IL-8, or tumor necrosis factor-a. The fact that DEP could

not be found in kidney sections does not totally exclude the

direct effects of DEP on the kidneys. Recently, L’azou et al.(2008) reported that carbon black nanoparticles exert a cyto-

toxic effect on renal cells in vitro and suggested involvement

of particle internalization as well as activation of intracellular

mechanisms that might include generation of reactive oxygen

species.

Our study showed that exposure to DEP caused lung

inflammation characterized by an increase in the number of

macrophage and PMN in BAL. This finding corroborate with

our previous studies in hamsters, which showed influx of

inflammatory cells in BAL after pulmonary exposure to DEP

(0.05–5 mg/kg) (Nemmar et al., 2003a,b, 2004a). Moreover,

Rao et al. (2005) reported in rats a dose-dependent increase in

PMN in BAL 1, 7, and 30 days following the i.t. instillation

of three concentrations of DEP (5, 35, and 50 mg/kg body

weight). The histologic findings revealed the presence of

increased interstitial cellularity and widening of the inter-

alveolar spaces. These findings are in agreement with

previous studies, which reported the direct effect of DEP

on lung inflammation (Nemmar et al., 2004a). CP pre-

treatment did not affect the number of BAL macrophages

and PMN, but histologic examination showed an increase in

cellularity of the interalveolar interstitium and mild conges-

tion. A major finding of the present work is that the combined

administration of DEP and CP aggravated pulmonary

inflammation and caused interstitial congestion and severe

interstitial and intra-alveolar edema (observed at 1 mg/kg).

These results confirm earlier studies that showed that ARF

resulting from either ischemia or bilateral nephrectomy

causes lung inflammation (Grigoryev et al., 2008; Hoke

et al., 2007) and thus suggest that inhaled particulate air

pollution can potentiate ARF.

DEP consists of an elemental carbonaceous core onto which

various organic compounds are adsorbed. Consequently,

additional studies are needed to establish that constituents of

DEP are responsible for the observed effects and the potential

effect of cytochrome P450 activity in lung, liver, and kidney on

circulating levels of chemical constituents associated with

particles on kidney toxicity (Pratibha et al., 2006).

Our data provide novel evidence that pulmonary deposition

of DEP potentiates the renal, systemic, and pulmonary effects

of CP-induced ARF and highlight the importance of environ-

mental factors such as particulate air pollution in aggravating

ARF. Our findings provide a plausible explanation for both the

extrarenal effect of ARF and the extrapulmonary effects of

particulate air pollution.

FUNDING

Faculty of Medicine and Health Sciences grant (NP/09/04);

United Arab Emirates individual grant (02-05-8-11/09).

ACKNOWLEDGMENTS

We thank Mr M. H. Mansour (College of Agriculture, Sultan

Qaboos University), Ms M. Sudhadevi (Department of

Pathology, Faculty of Medicine and Health Sciences, UAE

University), and Mr S. Dhanasekaran (Department of Physi-

ology, Faculty of Medicine and Health Sciences, UAE

University) for their technical assistances. We are grateful to

Professor David Cook, Ontario, Canada, for his critical reading

of the manuscript.


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Diesel Exhaust Particles in the Lung Aggravate Experimental Acute