Background
Chronic kidney disease (CKD) has become a growing epidemic that not only puts a substantial burden on global healthcare resources[
1‐
5], but is also an important cause of mortality and disability worldwide[
6,
7]. The risk factors of CKD have been extensively investigated in many previous epidemiologic and clinical observational studies[
1,
3‐
5,
8‐
11]. Although age, diabetes mellitus, and hypertension have been well reported to be the prevalent CKD risk factors[
1,
11‐
13], other risk factors including genetic, racial or familial predisposition[
14,
15], “western” lifestyle[
1], infectious diseases with infection-related glomerulonephritis[
16], and chronic exposure to certain chemicals[
17,
18], may also play an important role in the initiation and propagation of CKD. However, the potential impact of chronic exposure to environmental contaminants on the initiation and aggregation of CKD has scarcely been reported.
Nonylphenol (NP), an environmental organic compound derived from alkylphenols, has a chemical structure mimicking that of natural estrogen in the animal/human body[
19‐
21]. Since previous studies have reported that NP interferes with the development and function of endocrine-reproductive systems through binding to the estrogen receptors[
19‐
23], NP and other environmental contaminants have been recently categorized into endocrine-disrupting chemicals[
24]. Interestingly, a study using animal model has revealed that chronic exposure to low-dose of NP can induce polycystic kidney disease in rats[
25]. The underlying mechanisms, however, has not been fully investigated. Besides, previously animal model study has shown that chronic exposure to adenine (AD), an important component of DNA, can cause crystal formation in kidney parenchyma and also CKD[
26,
27]. Thus, AD-induced CKD in rats has long been accepted as an experimental model for investigating the pathologies and mechanisms of CKD[
26‐
28].
Although inflammatory reaction, oxidative stress, and the generation of reactive oxygen species (ROS) have been reported to be the principal mechanisms involved in various etiologies of CKD[
1,
17,
18,
29‐
32], whether similar contributors are also responsible for environmental organic compound-induced CKD remains unclear. Beside its action in lowering the serum cholesterol, Rosuvastatin has also been reported to possess anti-inflammatory and anti-oxidant properties[
33]. Accordingly, this study tested the hypothesis that chronic exposure to NP will aggravate AD-induced CKD in a rat model that could be attenuated by rosuvastatin therapy. The underlying mechanisms were also investigated.
Methods
Ethics
All animal experimental procedures were approved by the Institute of Animal Care and Use Committee at our hospital and performed in accordance with the Guide for the Care and Use of Laboratory Animals (NIH publication No. 85–23, National Academy Press, Washington, DC, USA, revised 1996). All the technicians who performed the bench work blinded in the treatment protocol.
Animal model and blood sampling for levels of serum creatinine and blood urea nitrogen after CKD induction
Fifty pathogen-free, 8-week-old male Wistar rats, weighing about 250 g (Charles River Technology, BioLASCO Taiwan Co., Ltd., Taiwan) were equally divided into group 1 (control), group 2 [AD in fodder at a concentration of 0.25% (i.e. 1 gm of AD in 100 gm of chow)], group 3 [NP (2.0 mg/kg/day) by gavage], group 4 (combined AD + NP), and group 5 [combined AD + NP + rosuvastatin (20 mg/kg/day) by gavage]. The dosage of rosuvastatin for the animals was based on our previous study[
34] and the dosage of AD for the animals was according to previous reports[
27‐
29] with some modifications. The AD was administered in fodder rather than by gavage due to the pharmacokinetic half life of AD was rather shorter.
AD, NP, and rosuvastatin were given to the animals for totally 24 weeks. The body weight, concentrations of serum creatinine and blood urea nitrogen (BUN) were measured every four weeks during the course of the experiment.
Blood sampling, collection of 24-hour urine for renal function assessment and collection of kidney tissue
By the end of 24 weeks of treatment, the animals were sacrificed. The kidneys were collected for individual study, while the blood samples were collected for serum levels of BUN and creatinine. Quantification of BUN and creatinine level was performed using standard laboratory equipment at our hospital. For further determining the renal function, the ratio of protein to creatinine which was also utilized was based on our recent report[
35].
Hematoxylin and eosin staining, histopathology scoring and Periodic Acid-Schiff (PAS) staining
Kidney specimens from all animals were fixed in 10% buffered formalin before embedding in paraffin. Tissue was sectioned at 5 μm before being stained with hematoxylin and eosin (H & E) for light microscopic analysis. Histopathology scoring was applied as described previously[
36] in a blind fashion. The score was given based on grading of tubular necrosis, loss of brush border, cast formation, and tubular dilatation in 10 randomly chosen, non-overlapping fields (200x) as follows: 0 (none), 1 (≤10%), 2 (11–25%), 3 (26–45%), 4 (46–75%), and 5 (≥76%).
Additionally, to determine the architecture of basement membrane and the structure of renal tubular border, Periodic Acid Schiff (PAS) staining was utilized in the present study.
Immunofluorescence (IF) and Immunohistochemical (IHC) studies
IF staining was performed for the examinations of CD68 (macrophage surface marker)-positive cells using respective primary antibodies. Additionally, IHC labeling technique was adopted for identifying glutathione reductase (GR)-positive cells using respective primary antibody. Irrelevant antibodies were used as controls in the current study.
Western Blot Analyses for Nuclear Factor (NF)-κB, Intercellular Adhesion Molecule (ICAM)-1, Heme Oxygenase (HO)-1, NAD(P)H Quinone Oxidoreductase (NQO) 1, NADPH oxidase (NOX)-1 and NOX-2 in Kidney.
Equal amounts (10–30 μg) of renal protein extracts were loaded and separated by SDS-PAGE using 8–10% acrylamide gradients. Following electrophoresis, the separated proteins were transferred electrophoretically to a polyvinylidene difluoride (PVDF) membrane (Amersham Biosciences). Nonspecific proteins were blocked by incubating the membrane in blocking buffer (5% nonfat dry milk in T-TBS containing 0.05% Tween 20) overnight. The membranes were incubated with the indicated primary antibodies (NQO- 1, 1: 1000, Abcam; GPx, 1: 2000, Abcam; HO-1, 1: 250, Abcam; ICAM-1, 1: 2000, Abcam; NF-κB, 1: 200, Santa Cruz; Actin 1: 10000, Chemicon; NOX-1, 1:1500, Sigma; NOX-2, 1:500, Sigma) for 1 hour at room temperature. Horseradish peroxidase-conjugated anti-rabbit immunoglobulin IgG (1: 2000, Cell signaling) was used as a second antibody for 1 hour at room temperature. The washing procedure was repeated eight times within one hour.
Western blot analysis for oxidative stress in kidney
The Oxyblot Oxidized Protein Detection Kit was purchased from Chemicon (S7150). The procedure of 2,4-dinitrophenylhydrazine (DNPH) derivatization was carried out on 6 μg of protein for 15 minutes according to the manufacturer’s instructions. One-dimensional electrophoresis was carried out on 12% SDS/polyacrylamide gel after DNPH derivatization. Proteins were transferred to nitrocellulose membranes which were then incubated in the primary antibody solution (anti-DNP 1: 150) for two hours, followed by incubation with second antibody solution (1:300) for one hour at room temperature. The washing procedure was repeated eight times within 40 minutes.
Immunoreactive bands were visualized by enhanced chemiluminescence (ECL; Amersham Biosciences) which was then exposed to Biomax L film (Kodak). For quantification, ECL signals were digitized using Labwork software (UVP). For oxyblot protein analysis, a standard control was loaded on each gel.
Lysis/binding buffer (High Pure RNA Tissue Kit, Roche, Germany) 400 μL and an appropriate amount of frozen kidney was added to a nuclease-free 1.5 mL microcentrifuge tube, followed by disruption and homogenization of the tissue by using a rotor-stator homogenizer (Roche). The lysate in the microcentrifuge tube was then centrifuged for two minutes at 13,000 g. Only the superficially collected supernatant was utilized for subsequent steps. Absolute ethanol 200 μL was added to the lysate supernatant and mixed well. The entire sample in the upper reservoir was pipetted into a High Pure Filter Tube (Roche) that was placed in the Collection Tube (Roche). This sample was then centrifuged for 30 seconds at 13,000 g in a standard tabletop microcentrifuge. After that, the Filter Tube was removed from the Collection Tube and the flowthrough liquid was discarded. For each isolation, 90 μL DNase incubation buffer was pipetted into a sterile 1.5 mL reaction tube, 10 μL DNase I working solution was then added, mixed and incubated for 15 minutes at 25 °C. Wash buffer I 500 μL was then added to the upper reservoir of the filter tube, which was then centrifuged for 15 seconds at 8,000 g. The filter tube was removed from the Collection Tube and the flowthrough liquid was then discarded. Wash Buffer II 500 μL was added to the upper reservoir of the Filter Tube, which was then centrifuged for 15 seconds at 8,000 g and the flowthrough was discarded. Wash buffer II 300 μL was added to the upper reservoir of the filter tube, which was centrifuged for 2 minutes full-speed at approximately 13,000 g. The column was then carefully removed from the collection tube such that the column did not contact the flowthrough to avoid ethanol carryover. The filter tube was then inserted into a 1.5 mL nuclease-free and sterilized microcentrifuge tube. Elution Buffer 100 μL was added to the upper reservoir of the filter tube; the tube assembly was then centrifuged for 1 minute at 8,000 g resulting in eluted RNA in the microcentrifuge tube.
Reverse transcription qPCR analysis
Quantitative reverse transcription-polymerase chain reaction (RT-qPCR) was conducted using LightCycler TaqMan Master (Roche, Germany) in a single capillary tube according to the manufacturer’s guidelines for individual component concentrations as we previously reported[
37,
38]. Forward and reverse primers were each designed based on individual exons of the target gene sequence to avoid amplifying genomic DNA.
During PCR, the probe was hybridized to its complementary single-strand DNA sequence within the PCR target. As amplification occurred, the probe was degraded due to the exonuclease activity of Taq DNA polymerase, thereby separating the quencher from reporter dye during extension. During the entire amplification cycle, light emission increased exponentially. A positive result was determined by identifying the threshold cycle value at which reporter dye emission appeared above background.
Statistical analysis
Quantitative data are expressed as means ± SD. Statistical analysis was adequately performed by ANOVA followed by Bonferroni multiple-comparison post hoc test. Statistical analysis was performed using SAS statistical software for Windows version 8.2 (SAS institute, Cary, NC). A probability value <0.05 was considered statistically significant.
Discussion
This study, which used a rodent model to investigate the therapeutic impact of rosuvastatin therapy on AD-NP-induced CKD, provided several striking implications. First, the major contributors to AD-NP-induced CKD were found to be inflammatory reaction, ROS generation, and cellular apoptosis. Second, rosuvastatin treatment significantly preserved architectural integrity of renal parenchyma and attenuated the deterioration of renal function after AD-NP-induced kidney damage. Third, rosuvastatin therapy consistently initiated anti-inflammatory, anti-apoptotic, and anti-oxidative effects.
Aggravation of AD-Induced renal dysfunction by NP
Although AD-induced CKD animal model has been validated in previous studies[
27‐
29], the authors of those studies used a relatively higher dose of AD (i.e. 0.75%) compared with that used in the present study (i.e. 0.25%). Our results, therefore, demonstrated that AD successfully induced CKD in a rodent model even at one-third of the reported dose. Furthermore, the novel finding of the current study is that NP feeding aggravated the deterioration of AD-induced renal function impairment. This finding implicates that not only is NP an endocrine-disrupting organic compound in the environment[
25], but it also participates in deteriorating the mammalian renal function in subjects with pre-existing chronic renal disease.
One important finding in the present study is that both gross anatomical and histological findings showed irregular appearance and crystal depositions in kidneys after AD treatment. Consistently, previous studies[
27‐
29] have also shown similar findings. Besides, another finding of interest in the current study is that despite the lack of significant difference in renal functions (i.e. serum BUN and creatinine levels) between the normal controls and NP-fed animals by the end of study period, immense differences in renal parenchyma including the histological pictures, mRNA, and protein expressions of various inflammation- and apoptosis-related molecules were noted between the two groups. Our findings, therefore, may suggest that NP, an environmental organic compound[
25], may play an essential role in aggravating mammalian CKD on chronic exposure.
The possible mechanisms of AD-NP-induced CKD
Interestingly, although AD-induced CKD in rodents is a well-established model[
27‐
29], the mechanistic basis of AD-induced CKD has seldom been investigated[
27‐
29]. Additionally, the mechanism of AD-NP-induced CKD in the rat model has not been reported. Another important finding in the present study is that the expressions of inflammatory markers including CD68-positive cells, TNF-1α, MMP-9, ICAM-1, and NF-κB were notably higher in NP-treated animals (group 2), and further increased in the AD-treated group (group 3), and highest in the AD-NP-treated group (group 4) compared with those in the normal controls (group 1). The principal findings of the present study are that ROS production (NOX-1, NOX-2), oxidized protein expression (protein carbonyls), and endothelial damage index (ET-1 gene expression) were notably increased in group 2, further increased in group 3, and substantially elevated in group 4 compared with group 1. Numerous previous studies have shown that CKD elicits rigorous inflammatory reaction, oxidative stress, and the generation of ROS[
1,
18,
19,
30‐
33], which, in turn, cause further damage to renal parenchyma and destroy the architectural integrity of the kidney. In this way, our findings, in addition to reinforcing those of previous studies[
1,
18,
19,
30‐
33], further showed that AD, NP, and combined AD-NP treatment caused different degrees of renal damage in a rodent model of CKD.
Of importance is that, as compared with group 4 animals, the expressions of these inflammatory and oxidative biomarkers at both gene and protein levels were significantly suppressed in group 5 animals following rosuvastatin administration. Moreover, the generations of anti-oxidants at IHC stain (GR-positive cells), protein (HO-1 and NQO1) and mRNA (HO-1, NQO1, GR, and GPx) levels were also markedly enhanced in group 5 animals compared with those in group 4. Besides, at both protein and mRNA levels, the apoptotic biomarkers (caspase 3 and Bax) were markedly reduced, whereas the anti-apoptotic biomarker (Bcl-2) and anti-inflammatory biomarkers at gene level (IL-10, eNOS) were significantly enhanced in group 5 animals compared with those in group 4. These results, at least in part, may suggest the involvement of the apoptotic signaling pathway in CKD. Our findings further support those of previous studies showing that cellular apoptosis/death is always present in CKD[
39,
40].
A body of evidence has demonstrated that statin has distinctive properties of being anti-inflammatory[
33], inhibiting ROS production[
41,
42] and reducing oxidant/free radical generations[
43,
44]. Using an animal model of AD-NP-induced CKD, the results of the present study also revealed that rosuvastatin also possesses all of the above-mentioned distinctive properties[
33,
41‐
43]. Our findings, therefore, in addition to corroborating those of previous studies[
33,
41‐
44], suggest the potential benefit of early statin use in the setting of CKD.
Study limitations
This study, however, has its limitations. First, although multifaceted signaling pathways were found to be involved in this AD-NP-induced CKD animal model, we remain uncertain how many signaling pathways exactly contribute to our observations. Second, the results of this study did not specify the most important mechanism involved in AD-NP-induced CKD. Third, we remain uncertain for why a bit different association between antioxidants and detoxifying enzymes and oxidative-stress biomarkers were found in the current study. Interestingly, a similar result has also been found recently in a rat animal model of acute kidney ischemia-reperfusion injury with stem cell therapy[
36]. We suggest that the findings in the current study may implicate that inherent anti-oxidant system can be initiated by CKD in response to NP, AD, and AD + NP stress and that this effect could be further enhanced by rosuvastatin therapy.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
CHY, CKS, SL, YTC, and HKY designed the experiment, drafted and performed animal experiments. YCL, LTC, YLC, THT, YHK, PLS were responsible for the laboratory assay and troubleshooting. CYH, CGW, SL and HKY participated in refinement of experiment protocol and coordination and helped in drafting the manuscript. All authors read and approved the final manuscript.