Animals and experimental design: Female NZBWF1 mice, 75-day-old, were purchased from Jackson Laboratory (Bar Harbor, USA) and utilized as SLE model (n = 10). Female C57/Bl6 mice of the same age were obtained from University of Campinas and utilized as controls (n = 16). After 15 days of acclimatization, each strain was randomly subdivided into two groups, one exposed to filtered air (FA) and one exposed to concentrated ambient particles (CAP). In total, animals were divided into four groups: control mice exposed to FA (C57-FA) (n = 8); control mice exposed to CAP (C57-CAP) (n = 8); lupus-prone mice exposed to FA (NZBW-FA) (n = 10); lupus-prone mice exposed to CAP (NZBW-CAP) (n = 10). Exposure to FA or CAP occurred for four months (starting from 90-day-old until 210-day-old). During the exposure period, animals were weighed, and urine samples were collected monthly. Also, the health status of the animals was checked daily, and survival was evaluated. Experimental design is shown in Fig. 1. Animals were allocated in cages (4–5 animals/cage) lined with pine wood shavings, and cardboard tubes and cotton balls for nesting were used as environmental enrichment strategies. Food and water were provided ad libitum. Except during CAP exposures, cages were put into closed ventilated hacks supplied with HEPA filtered air, controlled temperature of 21–23ºC and under light/dark cycle of 12 h/12 h. Animal procedures were approved by the Ethical Committee for Animal Research (CEUA) of the School of Medicine of University of São Paulo (FMUSP), under protocol number 095/17.
Daily exposure to PM2.5
Animal exposure to PM2.5 was performed in the Harvard Ambient Particle Concentrator (HAPC), located at FMUSP (Sao Paulo, Brazil). Exposures were performed in whole-body inhalation chambers (CAP or FA) as described in previous studies [28, 29, 30]. Before the initiation of the exposures, animals were also acclimatized to the HAPC for 5 days to reduce the distress of handling. C57-FA and NZBW-FA groups were exposed only to FA. C57-CAP and NZBW-CAP groups were exposed daily to 600 µg/m³ of CAP. This concentration represents the adjusted air concentration that São Paulo residents are exposed to [30]. This adjusted air concentration (Cair−adj) of 600 µg/m³ in 1 day/24hs was determined following current methodology of Environmental Protection Agency (EPA) [31], as follows
Cair−adj = Cair x ET x EF x ED/AT x 1 day/24 hours
Where:
Cair = Concentration of contaminant in air (mg/m3) = 25 µg/m³ (São Paulo)
ET = Exposure time (hours/day) = 24 h/day
EF = Exposure frequency (days/year) = 365 days/year
ED = Exposure duration (years) = 1 year
AT = Averaging time (days) = 365 days
PM2.5 characterization
Samples of ambient PM2.5 were collected for characterization of PM2.5 elemental composition. For this, polycarbonate filter samples were coupled into Harvard Virtual Impactor (a device that permits only the passage of particles with diameter less than 2,5 µm) for 24hs. A flux of 10L/min was generated by a vacuum pump for retention of ambient PM2.5 in sample filters. Characterization of PM2.5 elemental composition was performed by energy dispersive X-ray spectroscopy (EDX) as previously described [32]. 20 filter samples were analyzed by EDX.
Proteinuria analysis: Isolated urine samples were collected monthly by stimulation of perineal area as previous described [33]. Protein/creatinine ratio (PCR) and albumin/creatinine ratio (ACR) of urine samples were assessed. Dosage of total proteinuria was performed using Sensiprot kit (Labtest Diagnosis, Brazil). Procedures were conducted according to the manufacturer. Samples were diluted at 1:5 and incubated with color reagent at 37ºC/5 min. Absorbance was read at wavelength 600 nm. Quantification of proteinuria (mg/dL) was determined based on the absorbance of the sample (Asa) and standard (Ast) through the equation “[(Asa ÷ Ast) x 50] x 5”, where 50 corresponds to the protein standard concentration in mg/dL and 5 to the dilution factor. Dosage of creatininuria was performed following modified Jaffe method (Labtest Diagnosis, Brazil). Samples were diluted at 1:125 and incubated with a solution of 80% picric acid, 1% m/v + 20% sodium hydroxide, 10% m/v at 25ºC/20 min. Absorbance was read at wavelength 520 nm. Quantification of creatininuria (mg/dL) was determined based on the absorbance of the sample (Asa) and standard (Ast) through the equation “[(Asa) ÷ (Ast) x 5] x 25”, where 5 corresponds to the standard concentration in mg/dL and 25 to the dilution factor (1:125) corrected by the creatinine standard concentration (5 mg/dL). Albuminuria quantification was performed using electrophoresis gel. Firstly, urine samples were treated with laemmli buffer (5% β-mercaptoethanol) and heat (95ºC/5 min) for protein denaturation. Proteins were run on a 10% SDS-polyacrylamide electrophoresis gel and stained with Coomassie Blue R-259. After the staining, gels were immersed in a destain solution (7.5% acetic acid + 25% methanol + 67.5% distilled water) and gel images were obtained from Amersham Imager 600 (GE Healthcare, USA). Quantification of albuminuria (µg/mL) was performed by ImageJ software (Media Cybernetics, USA). Briefly, stained albumin in the samples was observed in bands at 66.5 kDa and the pixels of each band were measured and compared to a standard band (corresponding to 125 µg/mL of BSA). Note that for each gel there was a standard band control. Then, albuminuria were normalized by their creatinine concentration previously measured in the respective urine samples.
Euthanasia procedures
Animals were euthanized right after the fourth month of exposure (at 210 day-old) through overdose inhalation of the anesthetic isoflurane (Cristalia, Brazil). Blood samples were collected for hemogram and serologic analyses. Kidneys, spleen, liver, heart and thymus were collected, weighed and stored for histological and molecular analyses.
Hemogram analysis
Complete blood count was performed for blood cells screening. Red blood cells, white blood cells (including total count and percentage of neutrophils, eosinophils, basophils, monocytes and lymphocytes) and platelets were counted. Analysis was conducted using the automated hematologic counter pocH 100iV (Sysmex, Brazil).
Anti-DNA quantification: Blood samples were centrifuged (3500 rpm/10 min) for plasma collection. Quantification of anti-DNA antibodies was performed by indirect immunofluorescence using NOVA Lite® dsDNA Crithidia luciliae kit (Inova Diagnostics, USA) following manufacturer protocol. Goat anti-Mouse IgG (H + L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 (Invitrogen, A-11001) was utilized at 1:100 for conjugation.
Histological analyses of kidneys
After euthanasia, kidneys were stored in buffered formaldehyde (10%) for 24hs. Then, kidneys were processed and embedded in paraffin. For stereological analysis kidneys were sliced (5 µm thick) in seriated sections spaced by 100 µm and stained with hematoxylin-eosin (HE). In the interval between serial sections, sets of sections were collected for immunohistochemistry and histopathology. HE slides were scanned and digitalized using Pannoramic SCAN (3DHistech, Hungary). Volume estimation of kidney compartments was determined using Cavalieri method [34]. Using ImageJ software (Media Cybernetics, USA), a grid was superposed over the digitalized images (x10 magnification) and the incident points were counted. Relative volumes of cortex and medulla were calculated by dividing the sum of incident points of each compartment by the sum of total points of the kidney. Absolute volumes of compartments were calculated by multiplying relative volumes by total kidney volume. Total kidney volume was estimated by dividing kidney weight by kidney density (d = 1,06 g/cm³). For histopathologic analysis kidneys slices were randomly selected and stained using Periodic acid-Schiff (PAS). PAS slides were scanned and digitalized as previously described. Case viewer software (3DHistech, Hungary) was utilized for quantification (attribution of histopathological scores [35]) of tubular and glomerular alterations (x400 magnification) and for estimation of glomerular volume by measuring average diameter of glomeruli [36]. For analysis of collagen deposition another set of randomly selected were stained by picrosirius-hematoxylin method [37]. Slides were scanned and digitalized as previously described. Ten pictures per animal were taken. Threshold tool of ImageJ software (Media Cybernetics, USA) was utilized for estimation of the area stained with picrosirius (x100 magnification).
Immunohistochemistry: Immunohistochemistry protocol followed the manufacturer instructions. Briefly, sections were hydrated, antigen retrieval was performed in sodium citrate buffer (pH = 6) at 96ºC/20 min, endogen peroxidase was blocked with methanol (95%) + peroxide hydrogen (5%) for 30 min and non-specific bounds were blocked with bovine serum albumin (2%) for 60 min. Then we incubated sections with primary antibodies anti-C3 (1:2000 - ab200999), anti-IgG (1:250 - ab190475) and macrophage marker (1:10 - sc-101447) at 4ºC/overnight. Next, slices were incubated with HRP-polymer anti-rabbit (ab214880) or HRP-polymer anti-rat (ab214882) for 90 min and the chromogen 3,3’-diaminobenzidine (DAB) was added. Counterstaining was performed with hematoxylin. Slides were scanned and digitalized as described previously. Ten pictures per animal were taken. Threshold tool of ImageJ software (Media Cybernetics, USA) was utilized for estimation of area stained with DAB (x100 magnification).
Gene expression
Total RNA from kidneys was extracted with Trizol reagent (Invitrogen, USA). Kidneys were immersed in trizol and macerated in Precellys (Bertin Instruments, France). Chloroform was added, the homogenate was centrifuged, and the supernatant (RNA) was collected. RNA precipitation was performed with isopropanol. After centrifugation RNA pellet was washed with ethanol 70% twice and diluted in DEPC H2O. Quantification of total RNA (ng/µL) and quality of extraction (260/230) was performed using Nanodrop (Thermo Scientific, USA). Synthesis of cDNA was performed with 2000 ng of total RNA. First step was the degradation of contaminant DNA with RNAase-free DNase (Promega, USA). Second step was the addition of oligo-dT (Promega, USA). Last step was the reaction with dNTP and M-MLV Reverse Transcriptase (Promega, USA). All steps were performed in Mastercycler (Eppendorf, Germany). Relative gene expression of NFκB and TGF-β was determined by quantitative real-time PCR following 2−ΔΔCT method [38]. HPRT was utilized as endogenous gene (Additional file 1). For each gene, 4 µL cDNA sample + 0,5 µL forward primer + 0,5 µL reverse primer + 5 µL mastermix SYBR (Invitrogen, USA) were assembled. Samples were run in duplicates. qRT-PCR was performed at Quantstudio 12K Flex (Applied Biosystems, USA).
Oxidative stress
Pro-oxidant (NADPH oxidase and hydrogen peroxide) and antioxidant (FRAP) assays were performed for oxidative stress analysis of kidneys following procedures described elsewhere [39]. Firstly, kidneys were homogenized in an Ultra80 Turrax Blender (UltraStirrer, Switzerland) with 120 mM KCl and 20 nM sodium phosphate buffer (1 g tissue/4 mL solution) and centrifuged at 600 rpm/10 min. Activity of NADPH oxidase enzyme was determined by ELISA based on superoxide anion production [40]. The assay was performed with 50 mM phosphate buffer containing 2 mM EDTA and 150 mM sucrose, 1.3 mM NADPH and 10 µL of homogenate samples. Superoxide production was determined using a spectrometer (SpectraMax2, Molecular Devices) at 340 nm wavelength. Hydrogen peroxide dosage was determined by oxidation of phenol red by radish peroxidase [41]. 70 µL of homogenate samples were added in 180 µL of radish peroxidase solution (dextrose buffer + phenol red + radish peroxidase type II). After 25 min of incubation, 5 µL of sodium hydroxide were added to stop the reaction and absorbance was determined in a spectrometer (SpectraMax2, Molecular Devices) at 630 nm wavelength. Non-enzymatic antioxidant activity was determined by ferric reducing antioxidant power (FRAP) [42]. 10 µL of homogenate samples were added in 290 µL of FRAP reactive (sodium acetate and acetic acid buffer + 10 mM TPTZ + 20 mM hexahydrate ferric chloride). After 5 min of incubation, absorbance was determined in a spectrometer (SpectraMax2, Molecular Devices) at 593 nm wavelength.
Statistical analyses
SPSS software version 17.0 (IBM, USA) was utilized for statistical analyses. Firstly, descriptive data were obtained, and normality was verified by Kolmogorov-Smirnov test. Variables with normal distribution were analyzed through the parametric test ANOVA and post-hoc tests of Tukey HSD, Gabriel or Games Howell. Test-T was performed for comparison between 2 groups. Variables with non-normal distribution were ranked and analyzed through ANOVA. Kruskal-Wallis (4 groups) and Mann-Whitey test (2 groups) were also applied for non-normal parameters. Survival analysis was performed with Kepler-Maier test. Differences between groups were considered statistically significant when p value was less than 0.05 (p < 0.05).