Early kidney damage induced by subchronic exposure to PM 2.5 in rats
Aztatzi-Aguilar et al. Particle and Fibre Toxicology
Early kidney damage induced by subchronic exposure to PM in rats 2.5
O. G. Aztatzi-Aguilar 0
M. Uribe-Ramírez 0
J. Narváez-Morales 0
A. De Vizcaya-Ruiz 0
O. Barbier 0
0 Departamento de Toxicología, Centro de Investigaciones y de Estudios Avanzados del Instituto Politécnico Nacional, Avenida Instituto Politécnico Nacional , No. 2508, Col San Pedro Zacatenco, Ciudad de Mexico C.P. 07360 , Mexico
Background: Particulate matter exposure is associated with respiratory and cardiovascular system dysfunction. Recently, we demonstrated that fine particles, also named PM2.5, modify the expression of some components of the angiotensin and bradykinin systems, which are involved in lung, cardiac and renal regulation. The endocrine kidney function is associated with the regulation of angiotensin and bradykinin, and it can suffer damage even as a consequence of minor alterations of these systems. We hypothesized that exposure to PM2.5 can contribute to early kidney damage as a consequence of an angiotensin/bradykinin system imbalance, oxidative stress and/or inflammation. Results: After acute and subchronic exposure to PM2.5, lung damage was confirmed by increased bronchoalveolar lavage fluid (BALF) differential cell counts and a decrease of surfactant protein-A levels. We observed a statistically significant increment in median blood pressure, urine volume and water consumption after PM2.5 exposure. Moreover, increases in the levels of early kidney damage markers were observed after subchronic PM2.5 exposure: the most sensitive markers, β-2-microglobulin and cystatin-C, increased during the first, second, sixth and eighth weeks of exposure. In addition, a reduction in the levels of specific cytokines (IL-1β, IL-6, TNF-α, IL-4, IL-10, INF-γ, IL-17a, MIP-2 and RANTES), and up-regulated angiotensin and bradykinin system markers and indicators of a depleted antioxidant response, were also observed. All of these effects are in concurrence with the presence of renal histological lesions and an early pro-fibrotic state. Conclusion: Subchronic exposure to PM2.5 induced an early kidney damage response that involved the angiotensin/ bradykinin systems as well as antioxidant and immune imbalance. Our study demonstrates that PM2.5 can induce a systemic imbalance that not only affects the cardiovascular system, but also affects the kidney, which may also overall contribute to PM-related diseases.
Kidney biomarkers; Inflammation; Antioxidant response; Angiotensin and bradykinin systems; Cardiovascular diseases
Substantial epidemiological evidence obtained through
multi-city and meta-analysis studies has indicated that
medium and long-term exposure to particulate matter of
less than 2.5 μm (PM2.5) is associated with an increase in
the incidence of adverse respiratory and cardiovascular
The health effects reported as a consequence of PM2.5
exposure are associated with cellular and molecular
inflammation and oxidative stress responses, which are
considered to be the underlying mechanisms that drive
the cardiopulmonary effects [2–5]. We recently
demonstrated that subchronic exposure to coarse, fine and
ultrafine particles increases the expression of
angiotensin receptor type-1 (AT1R) in the lungs and heart.
Other genes of the angiotensin and bradykinin
endocrine systems, RAS (renin angiotensin system) and
KKS (kalikrein kinin system), which are known to be
regulated by the kidney, were also up-regulated .
The kidneys regulate blood pressure, fluid and sodium
homeostasis. These organs are controlled by the
sympathetic nervous system . However, renal dysfunction and
the development of cardiovascular diseases (CVD) are
closely associated. The prevalence of CVD, such as
congestive heart failure, coronary artery disease, peripheral vascular
disease, and myocardial infarction, amongst others, has
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been reported in conditions of renal insufficiency and in
patients undergoing dialysis, which indicates that there is
cross-talk between the kidney and the cardiovascular
system [8, 9]. In contrast, the contribution of the CVD to
renal dysfunction is poorly understood and has not been
adequately studied at a cellular and molecular levels,
although there is evidence that diseases such as
atherosclerosis  and hypertension  can contribute to the
development of renal dysfunction. The relationship
between CVD and renal dysfunction could be considered to
be bidirectional given that both factors are independently
associated as prognostic indicators. Amann et al. postulated
that cardiovascular dysfunction and renal diseases share, as
a potential pathogenic mechanism, impaired endothelial
function . However, the most important causes of
mortality in end-stage kidney disease are CVD and infections,
where the infections are thought to be associated with
disorders of the innate and adaptive immune responses .
Currently, the use of new early molecular biomarkers
to establish kidney dysfunction has improved the
prognosis for kidney diseases, including acute renal failure
. These new markers are proteins that are present in
the serum, pass through the glomeruli and can be
reabsorbed by the proximal tubules. These proteins include
albumin, α-1-glycoprotein (AGP), cystatin-C (Cys-C)
and β-2-microglobulin (β2M). Other markers that can
be over-expressed after damage to tubular cells include
neutrophil gelatinase-associated lipocain (NGAL) and
kidney-injury-molecule type-1 (KIM-1). In addition, the
presence of these proteins in urine provides a new tool
to determine the nephrotoxicity of toxicants such as
drugs and inorganic elements as cadmium [14–17].
Few epidemiological approaches have been reported
associating PM exposure with a decline of renal function.
Estimated glomerular filtration rate (eGFR) reduction has
been reported in a one-year study in elderly men from
Boston, Massachusetts , exposed to PM in a cross
sectional study of residents living near a major roadway .
There are some experimental studies on rodents that
suggest that kidney could be a toxicological target of PM.
Nemmar et al., (2009) in a Wistar rat model of acute renal
failure, induced by the nephrotoxic cisplatin drug, observed
that the intratracheal exposure to diesel exhaust
particulates (DEP) enhanced the cisplatin induced kidney damage
manifested in serum urea and creatinine, the augment of
N-acetyl-β-D-glucosaminidase (NAG) activity, and the
depletion of GSH content. Also in this study a decrease in
blood of the PO2, saturation of O2 and changes in
hematologic parameters were observed. These results
suggest that exposure to particles aggravates renal,
pulmonary and systemic effects of cisplatin toxicity, DEP alone did
not induced kidney damage .
Moreover, Yan et al., (2014) in a diabetic type-1 rat
model induced with streptozotocin drug after subchronic
exposure to 16 weeks to PM2.5, observed an increment in
the glycated hemoglobin A1c, IL-6 and fibrinogen,
without changes in blood parameters associated with kidney
function such as creatinine, microalbumin, NAG, β2M,
and blood ureic nitrogen (BUN). Histological analysis
showed that PM2.5 exposure increased myocarditis, aortic
medial thickness, advanced glomerulosclerosis, and a
punctual tubular damage of kidney in the diabetic rat
Clearly, PM2.5 exposure exacerbates the complication
on diagnosed or induced diseases such as renal failure
and diabetes, for that reason pre-existing illnesses and
elderly individuals are vulnerable groups to the toxic
effects of PM2.5.
For these reasons, we hypothesized that exposure to PM
triggers an initial endocrine response in the lungs that
may affect the heart, and consequently other organs.
Cardiovascular effects have been partially attributed to the
soluble fraction of particles, in addition cytokines and
oxidative stress metabolites, and translocation of the smallest
particles also contribute to heart and endothelial damage
. On this basis, it is possible to suggest that cytokines
and oxidative stress metabolites that circulate throughout
the blood stream can reach other organs, such as the
kidneys. Moreover, alterations in the vascular tone and a
subsequent endothelial dysfunction are factors that
contribute to the deterioration of kidney function, and have
also been related to PM exposure [3, 21–24]. More
knowledge on the possible impact of PM exposure on the renal
function is needed.
The goals of the present study were to evaluate the
blood pressure status after continuous exposure to PM2.5
for a period of eight weeks and to assess renal function
using serum creatinine levels and conventional urine
testing as well as measuring early kidney damage biomarkers
in the urine. In addition, we determined the effects of
exposure to PM2.5 on the immune and antioxidant responses
as well as the effects on the endocrine system by
examining the expression of components of the angiotensin and
bradykinin systems. Finally, we performed a histological
evaluation of the kidney tissue at the end of exposure to
demonstrate that the subchronic exposure to PM2.5
contributes to the renal response during the pulmonary and
cardiovascular toxicity of PM2.5.
Sprague–Dawley male rats were purchased from Harlan
México Laboratories (Mexico city, Mexico). The animals
were maintained in a freestanding clean room with a
changing station docking port (bioBubble®, Colorado,
USA). The rats were provided with filtered water and
food ad libitum and maintained in a light:dark
photoperiod of 12:12 h. All the animals were acclimated and
trained for approximately two months for metabolic
cage allocation and blood pressure measurements in a
rat-holder for blood pressure measurement recording.
The procedures were performed in a controlled
environment within a bioBubble® station.
The particle exposures were performed in whole body
chambers associated with a particle concentrator located at
CINVESTAV. The particulate concentrator had a particle
cutoff size of 2.5 μm. The acute exposure consisted in
3 days, 5 h per day. The subchronic exposure consisted of
repeated periods of inhalation for five hours per day, four
days per week for eight consecutive weeks. The schedule of
exposure was in the morning (8:00 to 13:00 h) from
Monday to Thursday in the rainy season (June-August) of 2013.
To evaluate enrichment by the particle concentrator and
estimate the particulate chamber concentration, we
performed PM2.5 ambient air monitoring using a 47-mm teflon
filter in a MiniVol samplers with an 5 L∙min−1 air flow: the
air ambient monitoring period followed the same schedule
as the animal exposures. The particulate concentration in
the chambers was estimated using a 47-mm teflon filter
allocated in a holder inlet which received a 2.5 L min−1
constant airflow, which was the same air flow supplied in
each chamber. Body mass was considered among control
and PM2.5 group as air volume displacement, it was
adjusted to 1.1 ± 0.2 kg per chamber at the beginning of each
weekly experiment, each chamber had a volume of 18 L.
The concentrator system was allocated within an enclosed
laboratory with controlled air atmosphere (air conditioning
and ventilation system), thus temperature and humidity
were constant in the exposure chambers. Filters from each
week were used for gravimetric analysis. To calculate the
particulate enrichment factor, all data were adjusted for the
air flow and the ratio between ambient air and the chamber
particulate concentrations from each week.
In parallel with the ambient air monitoring, and
according to Alfaro-Moreno et al.  we collected PM2.5,
at the same schedule of animal exposure, in
cellulosenitrate membranes with home-made modifications using
HiVol samplers, in order to obtain large quantities of
PM2.5 to determine endotoxin content and reactive
oxidant activity using the DTT assay in the PM2.5 samples.
We housed the animals three per chamber with four
chambers per group for a total of twelve animals each
for the filtered air (FA) control group and the PM2.5 test
group. The animals were randomly selected according to
the analyses that were to be performed. For histology
analysis, we selected four animal per group; for blood
pressure and biochemical analysis, we selected eight per
group. From the latter group, we randomly selected six
animals for the metabolic cage evaluation to obtain urine
and water consumption data.
Twenty-four hours after the last exposure, the animals
were anesthetized with 20 mg/kg of sodium pentobarbital
and sacrificed by abdominal aorta puncture (terminal
exsanguination). Serum samples were obtained to
determinate the creatinine concentration using a commercial kit
(RANDOX laboratories Ltd, Ardmore, Diamond Road,
UK). The kidneys were removed and stored at −70 °C
Bronchoalveolar lavage fluid (BALF)
In an anaesthetized animal, a transversal incision between
the beginning of the rib cage and the head was performed;
afterwards the muscle was removed to expose the trachea.
Then a cannula was allocated into the trachea until the
carina trachea, and fixed to perform a lavage (3×) with a
syringe with an isotonic saline solution (at 37 °C) in a 1:15
(volume:body weight) relation. The recovered solution
was centrifuged at 2,000 rpm for 5 min, and the cell pellet
was suspended in a final volume of 0.5 ml to perform cell
counting with trypan blue solution (0.4%; Sigma Aldrich,
St. Louis, MO. USA).
Cells for differential cell count (0.1 ml cell suspension)
were prepared using cytospin slides and centrifuged
(600 rpm, 5 min), and stained with Wright’s stain
protocol. One hundred cells per slide (two slides by sample)
were scored and a double blind determination was
performed. Differential counting was adjusted by the total
Dithiothreitol assay and endotoxin levels
To demonstrate the reactive oxidative ability of PM2.5,
we performed the “dithiothreitol (DTT) assay” as
described in De Vizcaya-Ruiz et al. (2006), this assay
provides the intrinsic oxidative activity of particulates
integrates organic and inorganic components and their
redox capability. We combined 10 μg of scraped PM2.5
from each week with DTT (Sigma Aldrich, St. Louis,
MO. USA), followed by the addition of a DTNB Sigma
Aldrich, St. Louis, MO. USA) solution with which the
remaining thiol was allowed to react to generate
5mercapto-2-nitrobenzoic acid, and the absorption at
412 nm was measured. Briefly, the PM2.5 samples were
incubated at 37 °C with 0.5 M PBS, pH 7.4, in double
deionized water with 1 mM DTT for 0 –45 min. The
incubation mixture was then mixed with 10%
trichloroacetic acid to stop the reaction; a portion of the mixture
was then dissolved in a solution of Tris buffer at pH 8.9
containing 20 mM EDTA and 10 mM DTNB. As an
internal control, we used the standard reference material
NIST-1649a (U.S. Department of Commerce, Washington.
D.C., USA). The redox activity was expressed as the
difference between the rate of DTT (nmol) consumed per
minute per microgram of sample and the activity observed
in the absence of PM.
The endotoxin levels were determined with the Limulus
Amebocyte Lysate Pyrochrome Chromogenic Test Kit
(Pyrochrome Associates of Cape Cod Incorporated,
Falmouth, MA, USA) as recommended by the commercial
manufacturer. We used lyophilized endotoxin (Escherichia
coli; Control Standard Endotoxin, O113:H10). We used
25 μg of scraped PM2.5, and the assay was performed in
triplicate for each sample.
Blood pressure measurement
To record the blood pressure, animals were warmed
(29 ± 1 °C) for a period of 5–10 min prior each
measurement to ensure adequate vasodilatation.
Afterwards, the animals were placed in restraints and five
blood pressure measurements using a cutoff ring and
a transducer were performed (PanLab Harvard Apparatus,
Letica 5002. Cornellà de Llobregat, Barcelona, Spain).
Acute exposure blood pressure was evaluated at the last
day of exposure. The blood pressure in the subchronic
exposure was evaluated one day before the initiation of the
8-week exposure (basal measurement) and on the fourth
day after every weekly exposure (post-exposure), with an
intermediate resting period of two hours for the animals
to eat and hydrate to minimize the effects of stress.
PanLab Harvard Apparatus calculated the mean blood
pressure (MBP) as follow:
MBP ¼ diastolic pressure
þ 0:33 ðsystolic pressure – diastolic pressureÞ:
During twelve-hour period, rats were placed in the
metabolic cages (Harvard Apparatus, Hollistone, Massachusetts,
USA.), which were used to harvest urine, estimate the water
intake, and calculate the urinary flow. These data were
adjusted for body weight. Food consumption measurements
were not performed to avoid contamination of the urine.
The samples were centrifuged at 1,000 rpm for 10 min at
4 °C and stored at −70 °C until used. Aliquots of the urine
samples from the 8th week were sent to a veterinary
laboratory without previous centrifugation (DIVET S.A. de C.V.)
for general urine examination (GUE). Serum and urine
creatinine were used to estimate glomerular filtration rate
(eGFR), which was calculated using the following equation:
eGFR ¼ ðUrine filow rate μl= min=100 g of body weightÞ
½Urine Creatinine ðmg=mlÞ=Serum Creatinine ðmg=mlÞ
Early kidney damage biomarkers and cytokines
determination by Luminex technology
The early kidney damage biomarkers and cytokines were
determinate in rat urine and total protein homogenates of
the kidney cortex, respectively, using Milliplex magnetic
bead-based multi-analyte profiling via Luminex
technology, which is a high throughput immunoassay.
We used the rat kidney toxicity magnetic bead panel 2
(RKTX2MAG-37 K, from EMD Millipore, Darmstadt,
Germany) to evaluate albumin, α-1 acid glycoprotein
(AGP), β-2-microglobulin (β2M), cystatin-C (Cys-C),
epidermal growth factor (EGF) and neutrophil gelatinase
associated with lipocalin (NGAL). The determination of
the early kidney damage biomarkers was performed in
duplicate on the urine samples from the first, second,
fourth, sixth and eighth weeks. Biomarker analysis was
carried out using a Magpix® System (EDM-Millipore,
Darmstadt, Germany). The data obtained were adjusted
for the urinary volume.
Furthermore, cytokine concentration of IL-1β, IL-6 and
TNFα were determined in urine of the PM2.5 subchronic
exposure using the rat cytokine/chemokine magnetic bead
panel (RECYTMAG-65 K, three-plex from EDM-Millipore®,
Darmstadt, Germany). Moreover, the concentration of
various cytokines (IL-1β, IL-6, TNF-α, IL-4, IL-10, INF-γ,
IL-17a) and chemokines (MIP-2 and RANTES) were
determined in kidney cortex protein extracts (see protein
extraction in Western blot section), with the rat
cytokine/chemokine magnetic bead panel (RECYTMAG-65 K, nine-plex
from EDM-Millipore®, Darmstadt, Germany). To perform
cytokine analysis, the total protein concentration was
quantified using the Bradford assay and diluted to a final protein
concentration of 1 μg/μl.
Total protein from the kidney cortex was obtained by
homogenization of the tissue in Nonidet-P40 buffer
(150 mM NaCl, 1% NP40, 50 mM Tris–HCl pH 8.0 and
protease inhibitors), which is used to study cytosolic,
membrane-bound or whole cell protein extracts The
homogenates were centrifuged at 10,000 rpm 4 °C, and
the supernatant was collected and stored at −70 °C until
use. A microplate-based Bradford protein assay was used
to determine the protein concentrations. An aliquot of
15 μg of protein was loaded onto SDS-polyacrylamide
gels and transferred to a PVDF membrane. Then, the
membranes were blocked for 1 h with 5% not-fat milk
and incubated overnight with primary antibodies to HO-1
(1:1000, rabbit polyclonal, ADI-SPA-895 from Enzo Life
Science, NY, USA) or TGF-β (1:800, rabbit, polyclonal,
ab66043, Abcam, Cambridge, UK) or from Santa Cruz
Biotechnology (Delaware Ave, Santa Cruz, CA, USA):
AT1R (1:600, rabbit polyclonal AT1R 306 antibody,
Sc579), ACE (1:2000; goat polyclonal ACE N-20 antibody,
Sc-12184), B1R (1:600, goat polyclonal B1R M-19 antibody,
Sc-15048), KLK-1 (1:800, goat polyclonal KLK-1 V-14
antibody, Sc-23800), γ-GCSc (1:1000, rabbit polyclonal
γGCSc H-338 antibody, Sc-22755), SOD-2 (1:2000, goat
polyclonal SOD-2 N-20 antibody, Sc-18503), or Nrf-2
(1:800, rabbit polyclonal Nrf-2 C-20, sc-722). The blots
were then incubated with HRP-labeled secondary
antibodies (Bio-Rad Laboratories, Hercules, CA, USA) for 1 h
at a dilution of 1:15000. The immunoreactivity was
detected using Luminata Western blotting detection
reagent (Millipore). The bands were visualized by exposure
to X-ray films and photodocumented with a UVP EC3
imaging system (UVP Inc., USA). We used α-actin (a
donation of Dr. Hernández-Hernández, CINVESTAV-IPN)
as an internal control to correct for protein loading.
Real time-polymerase chain-reaction
Total RNA was isolated from kidney cortex using the
phenol-chloroform method (TRIzol reagent, Invitrogen™,
Life Technologies, Thermo Fisher Scientific, Carlsbad,
CA, USA). An aliquot of 10 μg of total RNA was
subjected to DNAse treatment (Ambion, Turbo
DNAsefreeTM, Life Technologies, Carlsbad, CA, USA). cDNA
synthesis was performed with 3 μg of total RNA
according to the manufacturer’s instructions (SuperScript II,
Invitrogen™, Life Technologies, Thermo Fisher Scientific,
Carlsbad, CA, USA).
Real-time PCR was performed with a final concentration
of 15 ng of cDNA using SYBR Green (Maxima SYBR
Green/ROX qPCR master mix, Thermo Fisher Scientific)
in an Applied Biosystem StepOne™ instrument.
Specific oligonucleotides (Table 1) were used to evaluate
the mRNA levels for the RAS genes At1r and Ace and for
the KKS genes B1r and Klk-1. To evaluate the antioxidant
response, we measured Hmox1, Sod2 and Nrf2. Finally, we
evaluated the expression of pro-collagen-III (Col3a1) as a
marker of the fibrotic process. We used 18S as the
Table 1 Oligonucleotides used for PCR amplification
Fw 5'-TGCCTTCCTCTGCTGCCATTAG -3'
Rv 5'-ATGCTCGGCTGGGACTTGTG -3'
The gene name, the sequence of the forward (Fw) and reverse (Rv)
oligonucleotides, and the Genbank ID or commercial source are shown
housekeeping gene. The oligonucleotides were synthesized
commercially by Sigma Aldrich or Applied Biosystems.
PCR cycling conditions were optimized for each
oligonucleotide set as follows: 95 °C for 10 min, followed by
40 cycles at 95 °C for 10 s, and 60 °C for 1 min. The data
were analyzed using the 2-ΔΔCt method, the calculation for
mRNA expression of each gene was performed by
correcting the values using 18 s as the housekeeping gene.
To perform the histological analysis, the animals were
anesthetized and whole-body perfused through the
jugular vein with saline solution and then fixed with 4%
buffered paraformaldehyde. The kidneys were excised and
embedded in paraffin. Slides of 5-μm sections of the
tissues were stained with Hematoxylin/Eosin stain and
Masson’s Trichrome stain (HT15, Sigma Aldrich, USA).
For histological analyses two slides per animal were
analyzed. Tubular lesions were determined by the loss of
morphological features (e.g. tubular epithelial height of
the tubular cuboidal epithelium).
Tubular height was analyzed; five randomized
photographs from the kidney cortex were taken with a light
microscopy with a 10× objective for each group (four
animals per group). Also, we performed twenty
randomized measurements of tubular height for each picture.
The median was obtained for each microscopy field and
Collagen deposit was quantified in five randomized
fields per sample. Photographs were taken with a 10×
objective and analyzed using Image J software. Median
was obtained for each kidney sample and used for
Statistical analyses were performed using SigmaPlot version
11.0. We performed descriptive statistical analysis and
produced box-plots to show the medians and interquartile
25–75 ranges. To compare two groups, we performed the
U-Mann Whitney test on the basis of the non-normal data.
Repeated measures ANOVA was performed for the early
kidney biomarkers analysis, water consumption, and
urinary flow rate. A Pearson correlation analysis was performed
between PM mass, endotoxin, PM2.5 redox activity and
urine early kidney damage biomarkers. A p value ≤0.05 was
considered statistically significant. All comparisons were
performed relative to the filtered air control group.
The animals were exposed in the months of June to August
of 2013. These months are considered the rainy season in
Mexico City. However, during the acute and subchronic
exposure periods, ambient air parameters remained constant:
scant rainfall occurred, median percent humidity was 50%
with a maximum and minimum percentages of 84 and 32,
respectively, and the median temperature was of 21 °C with
a range of 11 to 28 °C (Fig. 1a).
During both exposure periods, we determined PM2.5
levels in ambient air. Our outdoor air monitoring
measurements indicated a median mass concentration of
23.5 μg/m3 in the acute exposure period. Moreover,
during the subchronic exposure period the median mass
concentration was 28 μg/m3 (25–30 μg/m3), the 25–75
interquartile range is shown in parentheses (Fig. 1b).
The gravimetric analysis of the particle concentrator
filters indicated a median mass concentration in the
exposure chambers for the acute exposure of 445 μg/m3,
and for the subchronic exposure of 375 μg/m3 (300 –
494 μg/m3) the 25–75 interquartile range is shown in
parentheses. The particulate enrichment for the acute
exposure was 19 times and the average for the
subchronic exposure was 16 times (9.5 times the lowest and
20.7 times the highest); the lowest enrichment was
Fig. 1 Particulate exposure description. Animal exposure was performed in the raining season in Mexico City. Ambient parameters such as relativity
humidity and temperature was monitored by a weather station, it was not observed raining during the schedule exposure. We report the median and
the range for each week (a). During eight weeks animals exposure (input concentrator) and ambient air were monitored simultaneous, each week was
defined as 4 days/week, 5 h/ day. The particulate concentrator enrichment have a minimum enrichment of 9.5 and a maximum of 20.7 times (b).
Particulate scraped dust from each week was used to determinate the endotoxin content and the oxidative capability of particles by DTT oxidation
assay, our data showed that Endotoxin and DTT have the same pattern and the weeks 2, 3 and 8 have the highest values during the exposure (c).
Each graph point represent the triplicate average ± standar desviation
observed in the last week, in which the lowest ambient
air concentration was also recorded (Fig. 1b).
In addition, PM2.5 were collected weekly
simultaneously with the animal exposures and used to determine
the intrinsic particulate oxidative activity with the DTT
assay as well as the endotoxin content. We observed an
intrinsic oxidative activity of PM2.5 in the acute exposure
(0.043 nmol DTT/min*μg), however, in the subchronic
exposure high oxidative activity on the second, third and
the last weeks at approximately 0.08 nmol DTT/min*μg,
and the lowest oxidative activities, 0.2 nmol DTT/
min*μg, were observed on the fourth and fifth weeks
(Fig. 1c). Moreover, we observed in acute exposure an
endotoxin concentration of 1 ± 0.08 ng/mg, and in the
subchronic exposure observed the highest particulate
endotoxin content, up to 8 ng/mg, in the second and
third weeks, but during the first, fourth, fifth, sixth and
seventh week, the values were approximately 0.1 ng/mg,
and during the eighth week, the endotoxin levels were
approximately 1 ng/mg (Fig. 1c).
To demonstrate that the PM2.5 was able to induce a lung
response, we evaluated the bronchoalveolar lavage fluid
(BALF) cell counts and determined the protein
concentration of surfactant protein-A (SPA) as an indicator of
molecular damage (Fig. 2). At the end of the subchronic
PM2.5 exposure, a significant increase in the macrophage
(MΦ) counts was observed, and monocytes and
lymphocytes were not observed (Fig. 2b). To establish lung
inflammation in response to PM2.5 differential cell count
in lung BALF was performed, we included an
independent experiment to establish the acute response of the
lung to PM2.5 (3 days; 5 h/day exposure). We observed a
decrease in the MΦ counts and an increase in monocyte
and lymphocyte counts in the BALF of the acutely
exposed animals (Fig. 2a).
With respect to the SPA levels, a marginal reduction
(p = 0.06) was observed in animals acutely exposed to
PM2.5 (Fig. 2c). This decrease in SPA was statistically
significant in the PM2.5 eight-week exposure group
compared to the FA group (p < 0.05; Fig. 2d).
Lung inflammation and an increment in blood
pressure was observed after the acute exposure to PM2.5,
however no effects were observed in kidney
parameters: urinary flow, kidney relative weight, plasma
creatinine, urine pH, urine specify gravity, and hematuria
(Additional file 1: Table S1), thus we excluded the analysis
of urinary kidney biomarkers of the acute exposure.
Blood pressure measurement
In this study, we report the mean blood pressure (MBP) as
a physiological parameter of vascular tone on the basis that
it is indicative of the perfusion pressure of organs, which
could be affected by exposure to PM2.5. In subchronic
Fig. 2 PM2.5 exposure induces inflammatory response and a reduction in SPA levels in rat lungs. Rats were acutely (3 days, 5 h/day) and
subchronically exposed to concentrated PM2.5 (8 weeks, 4 days/week, 5 h/day), and filtered air (FA) as a control group. Subcellular population
counts of macrophagues (Mφ), monocytes and lymphocytes from PM2.5 bronchialveolar lavage showed an augment after the acute exposure (a);
however, in subchronic exposure only Mφ count augment in the PM2.5 group was observed (b). Surfactant protein type-A (SPA) showed a marginal
down regulation in the acute exposure to PM2.5 (c), on the other hand, SPA levels after subchronic exposure to PM2.5 decrease statistically (d). *in
boxplot graphyc indicates statistical significant differences (p < 0.05)
exposure blood pressure was measured before the
beginning the exposure and after every weekly exposure. We
analyzed MBP using two different approaches: 1) we
compared the weekly MBP measurements from the PM2.5
group after exposure with the FA group and 2) we
compared all the measurements with the basal MBP
measurement to evaluate changes in the blood pressure as a result
of the experimental exposure procedure (Table 2).
We observed increases in the MBP after the first, fifth
and eighth week of exposure to PM2.5 compared to the
FA control group. The same differences were observed
in the comparison of the PM2.5 group measurements to
the initial basal measurements in this group. The
increased MBP values in the first, fifth and eighth week
were driven by increases in the diastolic blood pressure,
which showed the same differences as the median (data
not shown). Thus, the blood pressure data indicate that
exposure to PM2.5 can affect the vascular tone and
probably the perfusion of organs.
During the eight weeks of exposure, both groups were
weighed at the end of every week. We did not observe
differences between the body weights of the FA and PM2.5
Table 2 Mean blood pressure measurements after subchronic
exposure to PM2.5 and filtered air (FA)
Basal 109.3 109.6
groups (Fig. 3a). In addition, water consumption and the
urinary flow rates were measured during the twelve-hour
period in the metabolic cages. Subchronic exposure
results in a significant increases in the water intake in the
second, third, fifth, sixth and eighth weeks (p < 0.05;
Fig. 3b). On the first, fourth and seventh weeks, the
median water consumption in the PM2.5 groups was
increased, but the difference between the groups was not
statistically significant. In addition, the urinary flow rate
was increased in the PM2.5 group compared to the FA
group for all eight weeks (p < 0.05; Fig. 3c).
Urine and kidney function parameters
At the end of the exposure period, no significant
differences in the kidney relative weights were observed.
However, plasma creatinine increased slightly significantly in
the PM2.5 group compared to the FA group (p < 0.05).
The eGRF a decrement in the PM2.5 group compared to
the FA group (488 vs. 643 μl/min/100 g of body weight).
These data suggested kidney damage after subchronic
exposure (Table 3).
The urine parameters from the GUE performed on the
week 8 from subchronic exposure, such as pH and urine
specific gravity, did not show any statistically significant
differences. However, a moderate presence of hemoglobin
and erythrocytes in the urine from the PM2.5 group was
observed, whereas these findings were absent in the urine
of the FA group. In addition, we observed a statistically
significant frequency of high hematuria in the sediment of
the urine from the PM2.5 group compared to the FA group
Early kidney damage markers
We collected twelve-hour urine from animals after the
last day of each exposure, six early kidney damage
biomarkers were measured after the first, second, fourth,
sixth and eighth weeks of subchronic exposure (Fig. 4).
Albumin was detected in urine of both groups after the
subchronic exposure, and no statistical difference was
observed (Fig. 4a). The AGP (Fig. 4b), EGF (Fig. 4e) and
NGAL (Fig. 4f ) markers showed statistically significant
differences between PM2.5 and FA groups only in the
second week of exposure. On the other hand, the
markers that showed the greatest significant differences
were β2M (Fig. 4c) and Cys-C (Fig. 4d), which showed
increases in the first, second, sixth and eighth week in
the PM2.5-exposed groups, with the greatest increase
occurring in the second week.
Pearson correlation analysis between endotoxin and
intrinsic redox activity of PM2.5 and urine early kidney
biomarkers was performed (Additional file 1: Table S2),
statistical analysis showed a significant correlation of
endotoxin with renal biomarkers. DTT assay also showed
Fig. 3 The PM2.5 continuous exposure affects the hydric state. Rats were subchronically exposed to concentrated PM2.5 (8 weeks, 4 days/week,
5 h/ day). Particulate exposure does not modify the body weight of animals during the eight weeks (a). However, the water consumption (b) and
the urinary flow rate (c) increase significantly weekly at the end each exposure week. On boxplot graphyc* indicates statistical differences (p < 0.05)
a positive and significant correlation with early kidney
damage biomarkers, although not as strong.
RAS/KKS endocrine response
RAS/KKS have been reported to be important regulators
of the physiopathology of the kidney and the
cardiovascular system. For the RAS, we evaluated the expression
of At1r and Ace in the kidney because these genes are
involved in the endocrine pathway and the production
of angiotensin, respectively. The mRNA levels of both
genes were decreased in the kidney cortex in the group
exposed to PM2.5 after eight weeks of exposure
compared to the FA group (Table 4). However, the protein
levels of AT1R (Fig. 5a) and ACE (Fig. 5b) were
significantly greater in the PM2.5 group than in the FA group.
From the KKS, we evaluated B1r and Klk-1, two
important mediators of vascular tone and the inflammatory
response. After eight weeks of exposure to PM2.5, we
observed a 4-fold increase in the B1r mRNA levels compared
to the FA group (Table 4), as well as an increase in the
protein levels in the PM2.5 group (Fig. 5c). On the other hand,
Fig. 4 Exposure to PM2.5 induces the release of kidney early damage markers in urine. Rats were exposed to concentrated PM2.5 (8 weeks, 4 days/
week, 5 h/ day). At the end of each week twelve hours-urine was collected by metabolic cage. Six early kidney markers were evaluated by
Luminex technology. Albumin does not show statistical differences at any time evaluated (a). α-1-glycoprotein (b), β-2-Microglobulin (c), Cistatin-C (d),
epithermal growth factor (e) and lipocalin-2/NGAL (f) have the highest increment in urine levels in the second week. The most sensitive early markers
a long to the particle exposure were β-2-Microglobulin and Cistatin-C showed increments on the first, second, sixth, and eighth week. At week 4 any
early damage kidney marker showed statistical differences. On boxplot graphyc * indicate statistical differences (p < 0.05)
the Klk-1 mRNA levels in the PM2.5 group were reduced to
half the value of those of the FA group (Table 4), and the
same decrease in the KLK-1 protein levels was observed in
the PM2.5 group compared to the FA group (Fig. 5d).
The toxicity of PM2.5 can be largely explained by the
induction of oxidative stress and the inflammatory
response. To evaluate the role of oxidative stress, we
Table 4 PM2.5 subchronic exposure modulates the mRNA
expression of endocrine Angiotensin and Bradykinin system
components and the antioxidant response in rat kidney
evaluated the mRNA of Nrf-2, Hmox1 and Sod2 as
molecular elements that respond to oxidative stress.
We observed a trend toward an increase in Nrf-2 mRNA
with a marginal significance (p = 0.055); however, the
mRNA levels of the antioxidant enzymes Sod2 and Hmox1
decreased significantly in the PM2.5 group compared to the
FA group (Table 4). To confirm these results, the protein
Fig. 5 Proteins of angiotensin-, and bradykinin- systems respond at the end of eight weeks to PM2.5 exposure. After eight exposure weeks
(4 days/week, 5 h/ day) in kidney rats, we observed an augment in angiotensin-converter enzyme (ACE) and angiotensin-receptor type-I (AT1R),
(a) and (b) respectively. Bradykinin receptor type-1 (B1R) increase and tissue kallikrein (KLK-1) decrease, (c) and (d) respectively. On boxplot graphyc *
indicates statistical differences (p < 0.05)
levels were evaluated. The Nrf-2 protein levels (Fig. 6a) as
well as those of SOD-2 (Fig. 6b) and HO-1 (Fig. 6c) were
reduced in the kidney cortex of the PM2.5 group. In
addition, to confirm a possible antioxidant response, we
decided to evaluate the protein levels of the catalytic
subunit of the heavy chain of gamma-glutamyl cysteine ligase
(γ-GCLc), which is involved in the synthesis of glutathione.
We observed a modest statistically significant increase in
the γ-GCLc levels in the PM2.5 group compared to the FA
group (Fig. 6d).
Inflammatory cytokine evaluation
Urine cytokine determination was performed to evaluate
pro-inflammatory response (IL-1β, IL-6, and TNFα) of the
kidney. We observed that urine cytokine concentrations
were below the LUMINEX assay detection limit in PM2.5
exposed group. To confirm the inflammatory response to
PM2.5 subchronic exposure was determined by evaluating
a panel of pro- and anti-inflammatory cytokines (IL-6,
IL1β, TNFα, IL-4, IL-10, INF-γ and IL-17a) and chemokines
(MIP-2 and RANTES) in kidney cortex homogenates.
IL6 and RANTES were the most abundant cytokines by
nanograms per μl concentrations, whereas the remaining
cytokines were observed in picograms per μl (Table 5).
The levels of all of the cytokines in the kidney cortices
of the PM2.5 group were decreased, with the exception of
RANTES, which was slightly increased. All of these
Fig. 6 Kidney antioxidants decreased at the end of the eight weeks of PM2.5 exposure. After eight exposure weeks (4 days/week, 5 h/ day) in kidney rats
we observed a decrement of the antioxidant nuclear transcription factor (Nrf-2) (a), and antioxidant enzymes such as mitochondrial superoxide dismutase
(SOD-2) and hemoxygenase type-1 (HO-1), (b) and (c) respectively. However, the gamma-glutamil cysteine ligase heavy chain the catalytic subunit (γ-GCLc)
was up-regulated showed the kidney dependence of glutathione resource (d) On boxplot graphyc * indicates statistical differences (p < 0.05)
Table 5 Subchronic exposure to PM2.5 decreased the cytokine
levels in the total protein of the kidney cortex
differences of the levels in the FA group were statistically
significant. Specifically, compared to those of the FA
group, the levels of IL-6 in PM2.5 kidney cortical
homogenates were seven-fold lower; IL-1β was 1.6-fold lower;
TNFα was 4.8-fold lower; IL-4 was 2.5-fold lower; IL-10
was 2.4-fold lower; IL-17a was 5.3-fold lower; MIP-2 was
2.6-fold lower; and INF-γ was slightly lower (Table 5).
Histology and pro-fibrotic state
After eight weeks of continuous PM2.5 exposure, the
histology of the H&E-stained kidney samples demonstrated
the presence of tubular lesions with alterations in the
tissue structure including a statistical difference (p < 0.05) in
the reduction in height of the cuboidal epithelium of the
tubules; FA 15.4 μm (25–75 quartile 15.2 –16.7) versus
PM2.5 9.3 μm (25 –75 quartile 8.1 –10.3) and an
intertubular immune cell infiltration (Fig. 7a).
On the basis of the histology, we evaluated the levels
of KIM-1 in the total protein extracts of the kidney
cortex using Western blotting to confirm the damage in
proximal tubules. We observed a statistically significant
induction of KIM-1 in the kidney cortex of the
PM2.5exposed group; the levels were 4-fold higher than those
of the FA group (Fig. 7b).
We also evaluated whether PM2.5 induced a
profibrogenic state at the end of the eight weeks of
exposure. TGF-β expression was analyzed as a response
Fig. 7 Exposure to PM2.5 induces early kidney lesions with the presence of tubular deterioration. After eight exposure weeks (4 days/week, 5 h/ day) in
kidney histology we observed tubular lesion of hematoxylin and eosin staining slides (a). Tubular damage was corroborate by the immunodetection of
Kidney injury molecule-1 (KIM-1) (b). On boxplot graphic * indicates statistical differences (p < 0.05)
to the resolution of inflammation and as an inducer
of fibrosis. We observed an approximately three-fold
higher level of TGF-β in the total protein extracts of
the kidney cortex of the PM2.5 group than those of
the FA group (Fig. 8a). Finally, we evaluated the
expression of Col3a1 and the presence of collagen in
the kidney tissue using Masson’s staining to
demonstrate the pro-fibrogenic state. The level of Col3a1 in
the kidney samples from the PM2.5 group was lower
than that of the FA group (Fig. 8b). However,
Masson’s staining indicated the presence of collagen in
kidney cortices in the PM2.5 group, mainly in the
tubular zone and the glomeruli (Fig. 8c). Collagen
quantification showed a statistical significant
increment in the PM2.5 group of 3.2% (1.5 –4.3) in the
occupied area by collagen with respect to the FA group
0.55% (0.36 –0.6).
Epidemiological data indicate that PM2.5 is one of the
main air environmental xenobiotics associated with
respiratory and cardiovascular disease mortality and
morbidity [1, 5, 26, 27].
The present study provides new evidence that PM2.5
exposure can also induce changes in kidney physiology.
There are few studies that suggest that PM2.5 exposure
in human populations or in in vivo rodent models can
alter kidney physiological parameters [19, 20]. However,
kidney physiology could be compromised as a
secondary target organ given the physiological functions of the
kidneys, including: 1) blood pressure control; 2)
hydration state equilibrium; 3) acid-alkaline homeostasis; 4)
endocrine function; and 5) excretion/reabsorption of
molecules during urine formation to maintain the body
To investigate whether kidney physiology is affected by
PM2.5 exposure, we first evaluated the lung damage
induced by PM2.5 exposure. The data indicated differences
between the acute and subchronic inflammatory
responses. In the PM2.5 acute exposure, cellular recruitment
to the BALF was observed, which was characterized by
increases in monocytes and lymphocytes accompanied by a
reduction in Mφ counts. The latter observation could be
explained by the induction of cell death of alveolar Mφ,
the presence of PM in high concentrations in the alveolar
space and their surface components can activate scavenger
receptors from neighboring cells and mediate Mφ
apoptosis [28, 29]. On the other hand, during the subchronic
exposure, an increase in the number of Mφ in the BALF
of the PM2.5 exposed group compared with the FA group
was observed, suggesting a recovery and activation of the
immune response and the recruitment of Mφ by the end
of the eight-week exposure. The subchronic exposure to
PM10 and DEP in rabbits and mice, resulted in an increase
in alveolar macrophages was observed as the result of the
recruiting of circulating monocytes that differentiated to
macrophages [30, 31].
The observed increase in the Mφ cellular population
in the BALF after subchronic exposure demonstrated
that the repeated PM2.5 exposure compromised the lung
physiology. This observation was supported by evidence
of an alteration in the surfactant system, which is
necessary for the regulation of surface tension and contributes
to the innate immunological barrier. Surfactant
proteinA is a good candidate as a lung damage biomarker
because it is down-regulated in lung pathological states,
Fig. 8 Exposure to PM2.5 induces an early pro-fibrosis state. After eight exposure weeks (4 days/week, 5 h/ day) in kidney rat we observed
the promotion of fibrosis by the detection of beta- transforming growth factor (TGF-β) (a) and the evaluation of pro-collagen-III mRNA (Col3a1) by
qPCR, (b) Premature deposit of collagen in tubular and glomerulli by the Masson’s trichromic staining (c). Yellow and red arrows indicate deposit of
colagen in control (FA) and PM2.5 groups, respectivetly. On boxplot graphic *indicates statistical differences (p < 0.05)
such as idiopathic pulmonary fibrosis , adult
respiratory distress syndrome, asbestosis and silicosis . In
our experimental model, PM2.5 exposure decreased lung
SPA protein levels after the subchronic exposure. These
data suggest that PM2.5 can adversely affect pulmonary
surface tension and that the antioxidant and innate
immune functions of the lung would also be affected
because SPA participates in the mechanical functions of
the lung, modulates oxidation of the phospholipids by
serving as an antioxidant , and acts as a type of
pulmonary host defense molecule, called a “collectin”
(collagen-lectin), which protects against viruses, fungi
and bacteria .
It has been reported that lung damage may contribute
to, or be coincident with, the cardiovascular effect induced
by PM2.5 exposure. For this reason, we evaluated the MBP
as a cardiovascular response to PM2.5 on the basis that it
represents the peripheral blood pressure and blood
perfusion of the organs. The MBP showed an augment after
subchronic exposure to PM2.5. These changes were
confirmed when we compared the measurements with the
initial measurements of the PM2.5 and FA groups. As a
consequence of an increased MBP, organ blood perfusion
could be affected. The systemic blood pressure and
kidneys have a close physiological relationship.
Approximately 20% of the total renal blood flow supplies the
glomeruli and 80% supplies the tubules . According to
these data, the peritubular capillaries could be affected
and suffer damage due to the MBP increase.
In the kidneys, autonomic control of blood pressure is
maintained as a constant perfusion flow along
peritubular capillaries by the sympathetic nervous system (SNS),
which innervates the three major renal neuro-effectors,
the juxtaglomerular granular cells, the tubular epithelial
cells, and the renal vasculature [7, 37]. It has been
observed that the SNS is over-activated in hypertensive
states and exposure to PM2.5 could stimulate its activity
[38, 39]. Stimulation of the renal sympathetic nerve
activity (RSNA) leads to an increase in renin secretion,
and the increased renal tubular sodium reabsorption
decreases the renal blood flow . As an alternative
hypothesis, PM2.5 exposure may impact the RSNA by
affecting the circumventricular organs within the central
nervous system because these brain structures lack a
normal blood–brain barrier and are sensitive to circulating
Ang-II . In the context of the effect of PM on the RAS
and KKS described in our previous report , systemic
stimulation of the circumventricular organs by Ang-II
may contributes to the stimulation of RSNA through its
connections with sympathetic preganglionic neurons in
the intermediolateral column of the spinal cord to the
efferent sympathetic fibers that innervate the kidneys.
Eight weeks of exposure to PM2.5 or FA did not affect
the body weights or kidney relative weights in the rat
experimental model used in this study. However, exposure
to PM2.5 modified water consumption as well as the
urinary flow rate. The water consumption data suggested
a possible dehydration of the animals in the PM2.5
group. The possible roles of airflow and the time in the
hermetic chamber in these changes were excluded
because both, the PM2.5 and FA groups, were exposed
under the same experimental conditions of airflow
(2.5 L min−1) and time (5 h). For this reason, it can be
suggested that: 1) exposure to PM2.5 could induce a loss
of water within the airways through the hydroscopic
properties of the PM2.5 that stimulated thirst and water
intake, and both phenomena could have contributed to
the increase of urinary flow; 2) neurohormonal
stimulation (angiotensin-aldosterone-vasopressin) could promote
increased water consumption and/or increase urine
production through the autonomous nervous system and the
central nervous system (CNS). The last elements of this
pathway to be affected are the circumventricular organs,
which are sensitive to osmolality fluctuations and produce
the sense of thirst . A dehydrated state or a
vulnerability to this condition has been observed in diseases such as
cystic fibrosis, bronchitis and asthma. The pulmonary
water lining is necessary for adequate pulmonary
clearance and is involved in the hydration and protection of
the pulmonary epithelium [43–45]. A dehydrated state
resulting from exposure to PM2.5 may contribute to lung
susceptibility to infection and diseases.
Although the urinary volume increased, the general
urine parameters, such as pH and specific gravity, were
not modified at the end of the subchronic exposure.
However, surprisingly, the presence of hemoglobin in
the urine from the PM2.5 group was observed. These
data were consistent with the analysis of the urinary
sediment, which showed the presence of erythrocytes
was indicative of an isolated microscopic hematuria in
the PM2.5-exposed group. This could have been the
result of an effect on the renal microvascular circulation.
However, there are many different causes of hematuria,
including urinary infections, hypertension, and cancer,
among other pathological states . Further analysis
must be performed to determine the origin of the red
blood cells present in the urine and their association
with particulate exposure.
The hydration state and the urinary sediment analysis
suggest that the kidney function was compromised after
exposure to PM2.5. The data for the plasma creatinine and
eGFR levels support our hypothesis. Specifically, the
plasma creatinine increased modestly and the eGFR
decreased (approximately 15% compared to the FA group)
after eight weeks of PM2.5 exposure. These results indicate
that glomerular filtration could be compromised.
Moreover, the early kidney biomarkers demonstrated a
dysfunction in the reabsorption of proteins. Of the six proteins
measured, the five non-albumin proteins were altered in
the second week, which indicated that this particular week
was a vulnerable stage for kidney physiological balance
after PM2.5 exposure. Moreover, the most sensitive
biomarkers were β2M and Cys-C, which showed differences
beginning in the first week and continuing to the second,
sixth and eight weeks. The presence of chronic renal
disease is associated with the progression of CVD, and this
relationship is exacerbated in terminally ill patients.
However, changes in early kidney biomarkers, such as
Cys-C in non-kidney diseases, have been associated with
an increased rate of mortality from both CVD and
nonCVD (pulmonary diseases, cancer, infections), as indicated
by the quartiles of kidney function . On the other
hand, β2M, also used as an early kidney biomarker, has
been reported to be independently associated with the
mortality from heart attacks and strokes in patients with
asymptomatic coronary atherosclerosis .
The use of serum creatinine and its depuration as an
accurate measure of eGFR is unreliable. As reported by Endre
and Westhuyzen (2008), for renal diseases such as acute
kidney injury, serum creatinine is not a real-time marker
during rapidly changing renal function and can vary widely
with age, gender, and muscle metabolism and hydration
status. Also, the tubular secretion of creatinine, which can
contribute up to 50% as renal function declines, limits eGFR
use as a kidney injury marker. The use of new molecular
biomarkers increases the prognosis sensitivity of renal
damage and also helps to specify the site where the damage
occurs [49, 50]. The low association of renal damage and
PM exposure could be related with the poor prognosis
when creatinine parameters are used [18, 19]. New
molecular kidney damage biomarkers used in the present study
contribute to establish the nephrotoxic effect of PM2.5.
Previously, we reported that PM2.5 induced up-regulation
of AT1R in lung and heart tissues. This receptor is involved
in cardiovascular diseases, such as hypertension. The
elements of the RAS are expressed constitutively and are
over-expressed in the pathological states of kidney. As
expected, we observed the up-regulation of the levels of the
AT1R, and ACE proteins; however, discrepancies in the
mRNA levels and proteins were observed. Protein and
mRNA differences of AT1R and ACE levels were observed;
the discrepancy could be explained by other molecular
mechanism involved in transcription and translational
cellular processes. Cis-acting sequences and
glucocorticoidresponsiveness elements control AT1R gene mRNA
translation, also, the AUG codon in the 5′-leader of AT1R
transcript is involved in AT1R protein increment without the
induction of mRNA .
Based on this background, the mRNA and proteins for
AT1R and ACE, the RAS genes expressed in the kidneys,
were modulated after a subchronic exposure to PM2.5,
which suggested that PM2.5 was able to induce RAS
upregulation in organs, including the kidneys, that are
distal to the deposition of the particulates.
Concerning the KKS, an endocrine system that
counterbalances RAS, a decrease in the kallikrein released into the
urine of hypertensive patients with primary aldosteronism
has been previously reported , but in hypertensive rat
models, a decrease in the expression of this enzyme has
been observed [53, 54]. The KLK-1 decrease is associated
with renal dysfunction, and the administration or the
transfection of KLK-1 reverses the fibrosis and glomerular
hypertrophy; reduces inflammatory cell infiltration,
apoptosis, and TGF-β expression; and also decreases oxidative
stress due to the inhibition of NADPH oxidase activity
and the increase in the nitric oxide concentration. Thus,
renal function recovery contributes to cardiovascular
improvement. All of these effects were abolished by the
administration of icatibant, a B2R inhibitor, indicating that
the beneficial effects of KLK-1 are mediated by the
generation of bradykinin and the B2R pathway [55, 56].
The measurements of the kallikrein mRNA and protein
levels were down-regulated in the kidney tissue after PM2.5
exposure; this decrease could be explained by a release of
the enzyme into the urine as previously reported . On
the other hand, the KLK-1 gene has been reported to
contain an intron-III retention splice variant that has a high
GC content sequence. This feature of intron-III might
indicate that this region of the gene is more susceptible to
transcription factor binding, resulting in higher transcriptional
activity, which may depend on epigenetic regulation .
The role of bradykinin is to mediate vessel
vasodilatation through the ligand-dependent activation of the
bradykinin receptor type-2 (B2R); however, B1R can be induced
by cytokines during inflammatory processes. PM2.5
exposure increased the B1R mRNA and protein levels in the
kidney, which suggested that this organ may suffer from
an inflammatory process and continuous oxidative stress
on the basis that B1R stimulates the production of
inducible nitric oxide enzyme [55–60].
In this context, to demonstrate oxidative stress involved
in kidney damage after PM2.5 exposure, we analyzed the
mRNA induction of genes associated with the antioxidant
response and their protein levels. Our results showed an
induction of Nrf2 and a down-regulation of Hmox1 and
Sod2. The protein levels of all of the analyzed genes were
decreased. To demonstrate that glutathione synthesis was
also affected by PM2.5 exposure, we evaluated the catalytic
subunit of the γ-GCL protein and observed a marginal
induction. Our results show low levels of enzymatic
antioxidants after the subchronic exposure, which could be due
to: 1) the possible regulation of enzymatic antioxidants by
secondary mediators, such TGF-β, which down-regulate
catalase, glutathione reductase and SOD enzymes in the
kidney ; 2) the susceptibility of antioxidant proteins to
oxidation by oxygen and nitrogen radical molecules ;
and 3) the loss of Nrf-2 activity due to severe kidney
damage without de novo synthesis and the restoring of
antioxidant levels .
One of the most important hypothesis of PM2.5 toxicity is
the induction of inflammatory process in lung tissue with
the release of cytokines, such as IL-6, TNFα, IL-8, amongst
others [2, 3, 61, 62]. We evaluated kidney inflammation by
measuring the cytokine levels in protein extracts of the
kidney cortex, lower levels were observed in the PM2.5
exposed group, and in urine levels were lower than the assay
detection limit, in contrast with PM-toxicity hypothesis.
This results could be explained by the fact that cytokines
are removed from the tubular lumen by
endocytosis/metabolism along the proximal tubules . Moreover, it has
been reported that isolated proximal tubules have the
ability to reduce the human recombinant IL-6 concentration
under normoxic or hypoxic conditions .
In contrast to the other cytokines, the expression of
RANTES increased. This effect probably occurred because
this chemokine has the longest half-life among the
cytokines and chemokines evaluated or because the induction
of RANTES occurred in a more delayed manner. In
human mesangial cell lines under a pro-inflammatory
condition obtained by stimulation with TNFα or a combination
of TNFα and IL-1β, a slow mRNA expression of RANTES
after 12 h of stimulation and increased protein expression
after 36 h was observed [65, 66].
The renal cytokine levels of the PM2.5 group were lower
than in the FA group. The expression of chemokine genes
can be inhibited by glucocorticoids as well as by cytokines,
such as TGF-β and prostaglandins . We observed an
increase in the TGF-β protein levels in the kidney cortex
extracts. This observation could explain part of the effect
on the cytokines as well as on the expression of the
The present evidence indicates that inhalation of PM2.5
is able to induce physiological and molecular responses in
the kidney. We were also interested to determine whether
exposure to PM2.5 could cause tissue lesions. Through the
histological study using H&E staining, we observed
intertubular infiltration and a reduction in the height of
the tubular simple cuboidal epithelium. The significant
changes observed in tubular height suggest tubular
damage, this type of tubular lesions has been
described in human acute kidney injury . This last
observation was supported by the evaluation of
KIM1, a specific biomarker of proximal tubule damage
that is undetectable in healthy kidneys but is induced
during renal injury . The analysis showed a 4-fold
increase in KIM-1 in the PM2.5 group compared to the FA
group. Thus, exposure to PM2.5 induced injury to the
proximal tubule epithelium.
Our data indicated that during the eight weeks of
exposure to PM2.5, the kidneys undergo constant damage, but it
is probable that a response to repair the damage is equally
triggered. As mentioned earlier, an increase in TGFβ in
the PM2.5 group was observed at the end of exposure.
TGFβ is known to be involved in tissue repair after injury
in which it promotes the deposit of components of the
extracellular matrix, such as collagen. However,
continuous uncontrolled TGFβ production can increase the
extracellular matrix and generate fibrotic tissue . We
observed a probable repair process in the kidneys,
although the Col3a1 levels decreased, Masson’s Trichromic
staining showed an increment in the collagen deposit area
on renal tissue. This evidence supports the idea that the
subchronic exposure to PM2.5 induces kidney injury, and
consequently promotes a repair process, by the presence
of TGF-β and the deposit of collagen, however, the
persistent exposure to PM2.5 could promote a pro-fibrotic state
and a kidney dysfunction.
Changes in urine biomarkers fluctuate along the
eight-week exposure period, particle concentration in
the exposure chambers do not correlate with the
urinary early kidney damage biomarkers. Despite having
enriched the PM2.5 concentration between nine—and
twenty-fold, we did not observe correlation of the
highest PM2.5 concentration corresponding to the greatest
increase in early kidney damage biomarkers. However,
the changes or fluctuation in the intrinsic redox activity
of PM2.5 and the endotoxin content did show a
statistical correlation (Additional file 1: Table S2). Endotoxin
data correlated with a 60% (plus/minus) of the urinary
kidney damage biomarkers (AGP, β2M, Cys-C, EGF and
NGAL) evaluated and the redox activity to a 30% (plus/
minus) with AGP and Cys-C. Thus, PM mass does not
entirely explain the observed kidney effects, PM
components and their biological and chemical stimulation are
relevant factors in PM toxicity. Analysis of the endotoxin
content and the redox activity (DTT assay) of PM2.5, as
general parameters of particulate reactivity, to better
understand the relation of PM2.5 concentration exposure
and biomarker results was performed. It has previously
been reported that acute intraperitoneal or intravenous
administration of endotoxin (lipopolysaccharide) in rodent
models can induce acute renal failure [71, 72] through
stimulation of toll-like receptor-type 4 and tumor necrosis
factor. Thus, it is possible that in our model the PM2.5
endotoxin content, after the deposit and accumulation of
PM2.5 in lung, contributed to the deleterious effect in the
kidneys through circulation. Endotoxin exposure can
induce an inflammatory response as a consequence of an
acute exposure and contribute to the development of
tolerance after repeated doses . Nevertheless, kidney
damage biomarkers showed a significant correlation with
PM2.5 endotoxin content indicating kidney’s sensitivity to
endotoxin and suggesting that kidney injury could, in part,
be explained by it. However, we do not discard that other
components of PM, inorganic or organic, play a role as
enhancers or coadjuvants in kidney-induced damage, also
the time of exposure can influence this kidney response,
and other factors intrinsic of particulates (such source and
composition) and of population (age and gender).
Moreover, based on our data, acute PM2.5 exposure could be
the starting point of the kidney injury but would not
compromise kidney function. However, repeated and
longer term exposure induces kidney injury exceeding the
capability of organ defense against pathological
conditions, that in a condition of pre-existing diseases, related
with cardiovascular or renal systems, could accelerate the
In summary, exposure to PM2.5 induced its pulmonary
toxic effect mainly through oxidative stress and
inflammatory processes due to the interaction between the particles
and lung tissue. This initial response can trigger the
release of secondary mediators (cytokines and oxygen free
radicals) that may modulate the expression of two of the
major endocrine systems involved in the regulation of
vascular tone, the RAS and KKS. This modulation produces a
second level of damage in which the impaired balance of
these endocrine systems promotes the over-expression of
AT1R . We have demonstrated kidney damage through
the analysis of early kidney biomarkers (KIM-1, Cys-C,
β2M), histological changes (tubular height, collagen
deposit) and the induction of RAS/KKS endocrine genes
(AT1R, B1R), and blood pressure increment, after the
subchronic exposure to PM2.5. Under the reasoning that
exposure to PM2.5 triggers lung inflammation that can be
translocated to circulation and implicates other organs,
we tested cytokine release in serum and kidney tissue.
Cytokine levels in serum were under the test detection
limit, and cytokines in kidney tissue were below control
group levels. Further analysis to demonstrate if the release
of secondary mediators from the lung into blood
circulation are involved in kidney damage are needed; also
temporality of the immunologic modulation of cytokines
should be considered. Furthermore, kidney damage is a
consequence that could be triggered beyond the
inflammatory response and other non-immunological
mechanisms could be implicated such as: oxidative stress,
endocrine disruption, peritubular microvascular damage
from blood pressure changes, and ANS misbalance.
Finally, we propose that the kidney can subsequently
exacerbate the initial effects. Several hypotheses can be
established to determine how the effects on the kidneys
contribute to the biological effects of PM2.5. These include
modulation of the autonomic nervous control, the ionic
balance and the hydration state, among other effects.
Renal alterations induced by exposure to PM2.5 might
contribute to the cardiopulmonary PM2.5 toxicity through
feedback of the initial effect in several ways. The strongest
evidence for this hypothesis is that patients with acute
kidney injury have an increased incidence of respiratory
failure; the details of this pathological process are not
completely understood. However, in acute ischemic
kidney injury or bilateral nephrectomy in IL-6
knockout mice, it has been established that neither surgical
procedure induces a pulmonary effect compared to the
wild-type mice [74, 75]. Thus, damage to the lungs and
heart generated by PM2.5 could contribute to renal
The repeated exposure to PM2.5 not only induced
biochemical and physiological responses associated
with cardiopulmonary toxicity but also induced early
kidney dysfunction, which was characterized by a reduced
level of eGFR, presence of hematuria, increased urinary
early kidney damage biomarkers, imbalance in the RAS/
KKS response, the impairment of the antioxidant response
with a reduction of enzymatic antioxidants, suppression of
cytokine expression and finally, histological lesions with
the presence of intertubular cell infiltration and the
presence of collagen, which indicated a pro-fibrotic
damage. The early kidney damage could be associated
with the intrinsic PM2.5 oxidant reactivity and the
endotoxin content of PM2.5. The present study
demonstrates the effects of subchronic PM2.5 exposure on
the physiology of the kidney. Furthermore, the data
presented here suggests that the kidneys are a novel target
organ that is involved in cardiopulmonary responses and
could aggravate systemic effects. This process requires
Additional file 1: Table S1. Blood pressure, kidney and urine
parameters after acute exposure to PM2.5. Table S2. Pearson correlation
of particulate endotoxin content and intrinsic oxidative particulate activity
(DTT assay) with early kidney damage biomarkers. (DOCX 15 kb)
Ace: Angiotensin converting enzyme mRNA; ACE: Angiotensin converting
enzyme; AGP: α-1-glycoprotein; AT1R: Angiotensin receptor type-1;
At1r: Angiotensin receptor type-1 mRNA; B1R: Bradykinin receptor type-1;
B1r: Bradykinin receptor type-1 mRNA; B2R: Bradykinin receptor type-2;
BALF: Bronchoalveolar lavage fluid; Col3a1: Pro-collagen-III mRNA;
CVD: Cardiovascular diseases; Cys-C: Cystatin-C; DTT: Dithiothreitol;
EGF: Epidermal growth factor; eGFR: Estimated glomerular filtration rate;
FA: Filtered air; GUE: General urine exam; Hmox1: Heme oxygenase type-1
mRNA; HO-1: Heme oxygenase type-1; KIM-1: Kidney-injury-molecule type-1;
KKS: Kalikrein kinin system; KLK-1: Kallikrein-1; Klk-1: Kallikrein-1 mRNA;
MBP: Mean blood pressure; MΦ: Macrophage; NGAL: Neutrophil
gelatinaseassociated lipocain; Nrf-2: Nuclear factor (erythroid-derived 2)-like 2;
Nrf2: Nuclear factor (erythroid-derived 2)-like 2 mRNA; PM2.5: Particulate
matter of less than 2.5 μm; RAS: Renin angiotensin system;
SOD2: Superoxide dismutase type-2; Sod2: Superoxide dismutase type-2 mRNA;
SPA: Surfactant protein-A; TGF-β: Transforming growth factor-beta; β2M:
β-2microglobulin; γ-GCSc: Heavy chain of gamma-glutamyl cysteine ligase
Availability of data and materials
All data generated or analysed during this study are included in this
published article [and its supplementary information files].
OGAA performed the animal exposure, proposed the molecular targets, carried
out the molecular biology, analyzed the data and wrote the manuscript. TMUR
participated in the animal exposure and urine analysis. JNM participates on the
metabolic cage experiments, Western Blot and PCR analysis. OB participated in
the animal histological preparation, data analysis, study supervision, and
supervised the draft of the manuscript. ADVR devised and developed the
animal exposure, carried out its design and coordination, and supervised the
draft of the manuscript. All authors read and approved the final manuscript.
Consent for publication
Ethics approval and consent to participate
All experimental procedures described in this study were carried out
according to the “Principles of Laboratory Animal Care” (NIH publication
#8523, revised 1985) guidelines and to the “Norma Oficial Mexicana de la
Secretaría de Agricultura, Ganadería, Desarrollo Rural, Pesca y Alimentación”
(SAGARPA), subsection: “Especificaciones técnicas para la producción,
cuidado y uso de los animales de laboratorio” (Clave NOM-062- ZOO-1999).
Animal handling and exposure was described in protocol ID. 0038–13, which
was approved by the Institutional Internal Committee for the Use and Care
of Laboratory Animals (Comité Interno para el Cuidado y Uso de los
Animales de Laboratorio), Cinvestav.
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