- Email: [email protected]
6 mice
Journal Pre-proof Peptidome profiles in melamine diet-induced bladder stones in C57BL/6 mice
Liying Liu, Mai Luo, Xiaofan Xiong, Lingyu Zhao, Xiaofe Wang, Lei Ni, Juan Yang, Chen Huang PII:
S0041-008X(19)30394-1
DOI:
https://doi.org/10.1016/j.taap.2019.114786
Reference:
YTAAP 114786
To appear in:
Toxicology and Applied Pharmacology
Received date:
4 April 2019
Revised date:
4 October 2019
Accepted date:
21 October 2019
Please cite this article as: L. Liu, M. Luo, X. Xiong, et al., Peptidome profiles in melamine diet-induced bladder stones in C57BL/6 mice, Toxicology and Applied Pharmacology (2018), https://doi.org/10.1016/j.taap.2019.114786
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
© 2018 Published by Elsevier.
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Peptidome Profiles in Melamine Diet-Induced Bladder Stones in C57BL/6 Mice Liying Liua,1, Mai Luob,1, Xiaofan Xiongc, Lingyu Zhaoc, Xiaofe Wanga, Lei Nic, Juan Yanga,c,**, Chen Huanga,c,* a
The Center Laboratory for Biomedical Research, Xi'an Jiaotong University Health
Science Center, Xi’an, Shaanxi Province710061, People's Republic of China
of
b
Core Research Laboratory, The Second Affiliated Hospital, School of Medicine,
ro
Xi’an Jiaotong University, Xi’an, Shaanxi Province710004, People's Republic of
Department of Cell Biology and Genetics, School of Basic Medical Sciences, Xi'an
re
c
-p
China.
*
Correspondence to:
na
People's Republic of China
lP
Jiaotong University Health Science Center, Shaanxi, Xi'an, Shaanxi Province710061,
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Prof. Chen Huang, The Center Laboratory for Biomedical Research, Environment and Genes Related to Diseases Key Laboratory of Education Ministry, Xi'an Jiaotong University; Department of Cell Biology and Genetics, School of Basic Medical Sciences, Xi'an Jiaotong University Health Science Center, Shaanxi, Xi'an, Shaanxi Province710061, People's Republic of China E-mail: [email protected]
**Correspondence to: Prof. Juan Yang, The Center Laboratory for Biomedical Research, Environment and 1
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Genes Related to Diseases Key Laboratory of Education Ministry, Xi'an Jiaotong University; Department of Cell Biology and Genetics, School of Basic Medical Sciences, Xi'an Jiaotong University Health Science Center, Shaanxi, Xi'an, Shaanxi Province710061, People's Republic of China
ro
These authors contributed equally to this work.
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Abbreviations:
lP
re
-p
1
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E-mail:[email protected]
spectrometry
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MALDI -TOF-MS: matrix-assisted laser desorption ionization- time of flight- mass
MB–WCX: magnetic bead-based weak cation exchange chromatography LC-ESI-MS/MS: liquid chromatography with electrospray ionization and tandem triple quadrupole mass spectrometry Ndufα8: NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 8 Bsg: Basigin
Abstract The aim of this research was to detect potential serum biomarkers of melamine diet-induced bladder stones in C57BL/6 mice. Magnetic bead-based weak 2
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cationexchange chromatography (MB-WCX) and matrix-assisted laser desorption ionization-time of flight-mass spectrometry (MALDI-TOF-MS) were employed to detect serum biomarkers in 10 mice fed a melamine diet and 10 control mice. Seventeen peaks (fold change>1.5) with a mass to charge (m/z) value of 1,000– 10,000 Da were detected in the two groups. Among the significant peaks, five were
of
upregulated and the other 12 were downregulated in the model group. Among the upregulated peaks, 2954.49 and 1710.49 were found to correspond to the peptide
ro
regions of NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 8(Ndufα8)
-p
and basigin, respectively, by liquid chromatography with electrospray ionization and
re
tandem triple quadrupole mass spectrometry(LC-ESI-MS/MS). Western blot analysis
lP
was used to detect the expression of Ndufα8 and basigin in another 10 model mice
na
and 10 control mice. The western blot results confirmed the LC-ESI-MS/MS data. The expression of serum basigin and Ndufα8 was partly dependent the concentration
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of melamine, but no time dependence. In conclusion, Ndufα8 and basigin may be potential serum biomarkers for the detection of melamine diet-induced bladder stones in C57BL/6 mice.
Key Words: Melamine, Bladder stone, serum biomarkers, Ndufα8, Basigin
1. Introduction For many years, assays of serum biomarkers have played an important role in the diagnosis and prognosis of a variety of diseases including lung cancer (Tang et al., 2017),
hepatocellular
carcinoma
(Peng 3
et
al.,
2017),
pancreatic
ductal
Journal Pre-proof adenocarcinoma (Tian et al., 2008), Alzheimer’s disease (Lue et al., 2017), diabetes (Freitas et al.,2017), sepsis and bacterial infections (Tan and Goh, 2017)and ovarian cancer (Chatterjee et al., 2017). In this study, we aimed to identify a serum biomarker for melamine-induced bladder stones. Melamine (1,3,5-triazine-2,4,6-triamine, C3H6N6) is an industrially synthesized
of
chemical used for the production of laminates, coatings, glues, heat-tolerant polymers and plastics(Cheng et al.,2014; Skinner et al.,2010; Suchý et al., 2009; Zhang et al.,
ro
2012), and melamine contamination has been reported in products such as milk, infant
-p
formula, frozen yogurt, petfood, biscuits, candy and coffee (Mauer et al., 2009).The
re
nitrogen content of food products increases when melamine is present (Chan et al.，
lP
2008).
na
A well-documented case of melamine contamination occurred in China in 2008. Infant formula contaminated with melamine resulted in cases of infant urolithiasis
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(Chen, 2009;Wu et al.,2009). Xu and his research team established a standardized melamine-induced cystolith mouse model to study this phenomenon (Xu et al., 2011). They determined the most appropriate daily dose of dietary melamine required for cystolith formation, and revealed that bladder epithelial hyperplasia caused by melamine-induced bladder calculus formation can occur at concentrations over this dose. They also investigated the recovery of bladder epithelial hyperplasia after melamine withdrawal (Sun et al., 2014). Furthermore, they found that urinary alkalinization can be used to prevent and treat melamine-induced cystoliths in mice (Ren et al., 2014). Taken together, these studies confirmed that the mouse model was 4
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an appropriate model with which to study melamine-induced cystolith formation. However, there have been no previous reports of a biomarker to detect melamine-induced cystoliths or bladder stones induced by urinary system disease. The aim of the current study was therefore to identify a sensitive serum biomarker in the melamine-induced cystolith mouse model, and use this biomarker not only to
of
diagnose melamine-induced cystoliths in mice at an early stage, but also to reveal the
ro
molecular mechanisms of melamine-induced cystolith formation.
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2. Materials and methods
re
2.1 Chemicals
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Melamine [2, 4, 6-triamino-s-triazine, CAS No. 108-78-1], produced by
na
Ker-mel Chemical Agent Ltd. Co., Tianjin, China (Batch No. 20060828;analytical purity P99.5%), was a gift from Professor Xu of Xi’an Jiaotong University Health
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Science Center.
2.2 Animal model
Three-week-old C57BL/6 specific-pathogen-free mice were obtained from, and housed in, the Central Laboratory of Animal, Xi’an Jiaotong University Health Science Center. The animals were housed at a temperature of 18–22℃, with relative humidity of 40%–60%, a light/dark cycle of 12 h/12 h, and free access to sterile water (pH 6.0). All animal experiments were approved by the Institutional Animal Care and Use Committee of Xi'an Jiaotong University and were performed according to the 5
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institution's guidelines for the use of laboratory animals.
2.3 Establishment of a bladder stone model in mice fed a diet containing melamine A total of 20 C57BL/6 mice were randomly divided into two groups, one fed a
of
diet containing 9373 ppm of melamine and a control group fed the same diet with no melamine. There were five female and five male mice in each group. All mice ate and
ro
drank freely. The bladder stone mouse model was established according to a previous
-p
publication (Xu et al., 2011), and on day 15, blood was collected and the mice were
re
euthanized. Blood samples were centrifuged at 3000 g for 10 min at 4 °C, and then
lP
serum was collected and stored at ˗80 °C for further investigation. In addition, the
na
bladder of each mouse was opened and examined for stones. Stones were carefully
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removed and photographed.
2.4 Serum sample purification and MALDI-TOF MS analysis Magnetic bead-based purification (MB-WCX, Bremen, Germany) was employed to purify the serum samples, according to the manufacturer’s instructions. To prepare the MALDI target,1μl of a mixture containing 10μl of 0.3g/lα-cyano-4-hydroxy cinnamic acid in 2:1 ethanol/acetone (v/v) and 1 μl of the eluted peptide fraction was spotted onto a MALDI AnchorChipTM (Bruker Daltonics, Bremen,Germany) sample target platform (384 spots). To evaluate reproducibility, each serum sample was spotted in triplicate. 6
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Air-dried targets were measured immediately using a calibrated Autoflex III matrix-assisted
laser
desorption/ionization
time-of-flight
mass
spectrometry(MALDI-TOF MS, Bruker Daltonics) with FlexControl software (version 3.0, Bruker Daltonics), and optimized measuring protocols were established. Matrix suppression up to1,000 Da, with a mass range of 1,000–10,000 Da, was set as
of
the default. Instrument calibration parameters were determined using standard peptide
ro
and protein mixtures.
-p
2.5 Data analyses
re
Data analyses were performed using the programs Flex Analysis v3.0 and
lP
ClinProTools v2.2 (Bruker Daltonics).ClinProTools v2.2 uses a standard data
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preparation workflow including spectra pretreatment, peak picking and peak calculation operations, and was applied for the recognition of peptide patterns in this
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study. For statistical analyses, a k-nearest neighbor genetic algorithm, as implemented in the software suite, was used to identify statistically significant differences in protein peaks among the groups analyzed.
2.6 Peptide identification by LC-ESI-MS/MS After completing the statistical analysis, differentially expressed peptides were identified. The peptide sequences of potential m/z peaks were first determined by LC-ESI-MS/MS, and the identified sequence data were then subjected to a Mascot database search to identify the corresponding full-length protein matches. 7
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2.7 Western blot validation of the identified proteins To validate the Ndufα8 and basigin in the bladder stone model mice, a second mouse model was established using the same methods. Serum samples from the melamine fed (n=10) and control (n=10) mice were diluted 20 times before being
of
separated by SDS-PAGE. The protein bands were electrophoretically transferred to polyvinylidene difluoride membrane (Millipore). Then, rabbit polyclonal anti-Ndufα8
ro
antibody (Proteintech, 15064-1-AP; 1:1000), rabbit monoclonal anti-CD147 antibody
-p
(Basigin; Abcam, ab108317; 1:1000), and rabbit polyclonal anti-albumin antibody
re
(Proteintech, 16475-1-AP; 1:5000) were used for detection in the enhanced
lP
chemiluminescence detection system (Pierce). Albumin was also analyzed as an
na
internal control protein. The blots were scanned (UVP BioSpectrum 410, USA) and
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the band density was measured using Quantity One imaging software.
2.8 The time-course and dose-response of melamine effects on the expression of biomarker candidate genes In order to analyze the time-course and dose-response of melamine effects on the expression of biomarker candidate genes，the time-course groups and dose-response groups were detected individually. In time-course experiment, the model group mice were fed a diet containing 9373 ppm of melamine. The model group was divided two groups: melamine-diet for two weeks, melamine-diet for three weeks. The control group （ normal diet ） was divided into 2-week group and 3-week group. In 8
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dose-gradient of melamine experiments, the experiment group were divided four groups: control group (normal diet), 9373 ppm of melamine diet group, 1.5×9373 ppm of melamine diet group, 2×9373 ppm of melamine diet group. Each concentration group was fed with melamine diet for 2 weeks. In time-course and dose-gradient experiment, there were 6 C57BL/6 mice in each group. Methods of
ro
biomarker was detected by western blot analysis.
of
collecting serum and stone from experimental mice were described as 2.3. Serum
2.9 Statistical analysis
-p
Statistical analyses were conducted using GraphPad Prism v7.0 software
re
(GraphPad Software, Inc., San Diego, CA, USA). T-test and one-way analysis
lP
variance (ANOVA) were used. All data were expressed as the mean ± SD. The results
na
were considered statistically significant if P<0.05. Correlations between the mean expression of all detected m/z peaks were evaluated using the Spearman rank-order
3 Results
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correlation coefficient.
3.1 Bladder stones were induced by a melamine diet in the mouse model In this study, no fatalities occurred in any of the experimental groups. Bladder stones were observed in two of the experimental groups (70% prevalence), but not in the control mice. The number of stone granules in a single bladder was between one and two (Fig. 1). 9
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3.2 The detection of significant m/z peaks in the sera from the model mice compared with the control mice Twenty serum samples (10 from controls and 10 from model mice) were purified by MB-WCX and then detected by MALDI-TOF MS. There were 17 peaks (fold
of
change>1.5) at a mass to charge (m/z) value of 1,000–10,000 in the two groups. Among the significant peaks, five were upregulated and the other 12 were
ro
downregulated in the model group (Table 1). The control group (green) and the model
-p
group (red) had protein profiles from 1 to 10 kDa (Fig. 2A). In component analysis, a
re
bivariate plot of the model (red) and control (green) groups showed few overlapping
lP
regions (Fig. 2B). The two groups showed protein profiles ranging from 1 to 10 kDa
na
(Fig. 2C). Within this mass range, two significant peaks at 2954.49 and 1710.49 were
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detected.
3.3 Peak selection
ClinProTools analysis revealed the two most significant peaks，2954.49 and 1710.49 were chosen to be analyzed. Receiver operating characteristic (ROC) curves of control group (blue) and the model group (red) are shown in Fig. 3. The area under the curve (AUC) values of these two peaks were 0.9079 (m/z: 2954.49) and 0.7762 (m/z: 1710.49) (Fig. 3B and D). Compared with the control group, the average expression levels of the two analyzed peaks in the model group were significantly higher (P<0.001; Fig.3E and 3F). 10
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3.4 Identification of two serum biomarkers LC-ESI-MS/MS and the Mascot database (Fig. 4 and 5) were used for MS/MS fragmentation of the two identified peptides (m/z: 2954.49 and m/z: 1710.49), resulting in the identification of the sequences SHCAEPFTEYWTcLDYSNMQLFR and SGEYSCIFLPEPVGR. These two peptides were found to be located within the
of
Ndufα8 and basigin sequences, respectively, as shown in Table 2.
ro
3.5 Protein expression of Ndufα8 and basigin was upregulated in the serum of
-p
the model mice
re
In the second mouse model experiments, we tested the expression of Ndufα8 and
lP
basigin in serum samples by western blot analysis, as shown in Fig.6. The expression of Ndufα8 was upregulated in the model group, for both male and female mice,
na
confirming the LC-ESI-MS/MS data. However, the expression of basigin was
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upregulated only in the male mice in the model group. There was no significant difference in expression of this protein between female mice in the model and control groups.
3.6 The expression of serum basigin and Ndufα8 was partly dependent the concentration of melamine, but no time dependence In this study, one mouse died in the three-week model group. Bladder stones were observed in dose-response and time-course model groups. The number of stone granules in a single bladder was between one and three (Fig. 7A, B). In the concentration group, the stone formation rates of 9373ppm, 1.5×9373ppm and 2 11
Journal Pre-proof ×9373ppm were 50% (3/3), 83% (5/6) and 83% (5/6) respectively. In the time-course group, the stone formation rates of two-week model group and three-week model group were 33% (2/6) and 60% (3/5), respectively. The serum expression of basigin and Ndufα8 was detected by western blot analysis. The results of dose-response groups as showed in Fig.7A, C, E, G, and the time-course groups as showed in Fig.7B, D, F, H. Compared with the control group,
of
Ndufα8 was highly expressed in the model group. With the increase of melamine
ro
concentration, the expression of serum Ndufα8 was also up-regulated (Fig.7G, P <
-p
0.05), but the expression level of 2 ×9373ppm group was lower than that of 9373ppm
re
group and 1.5×9373ppm group. The group with the highest expression was
lP
1.5×9373ppm group. In time-course group, the expression of serum Ndufα8 in the
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two weeks and three weeks model group were also up-regulated. However, compared with the expression in two-week model group, the expression in three-week model
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group was down-regulated, and there no significant difference between control group and model group.so the expression of Ndufα8 was not time-dependent (Fig.7H). Similarly, the expression of basigin in different melamine concentration groups was higher than that in the control group (Fig.7E, P < 0.05), although the expression of basigin was gradually down-regulated as the concentration increased. In time-course group, the expression of serum basigin was not time-dependent (Fig. 7F), also similar with Ndufα8. 4 Discussion In 2008, milk formula contaminated with melamine caused adverse renal 12
Journal Pre-proof outcomes in a number of infants worldwide, after which melamine became a public health issue (Guan et al., 2009). However, the data on acute and chronic exposure to melamine in humans remain limited and most information has been extrapolated from animal studies. The clinical signs and symptoms associated with melamine exposure in infants are nonspecific. The bladder stones formed in response to exposure may be radiolucent and are not consistently seen on ultrasound (Skinner et al., 2010). It is therefore necessary to find new diagnostic methods for detecting melamine toxicity in
of
the urinary system and bladder stone formation. In this study, we investigated serum
ro
biomarkers in a bladder stone mouse model induced by a melamine-contaminated diet.
-p
We employed MALDI-TOF MS analysis as this technique is widely applied in the
re
analysis of serum samples for the diagnosis of human diseases and is proven to be an
Schwamborn et al., 2009).
lP
effective method for the identification of potential serum biomarkers (Yang et al.,2014;
na
In this study, we used MB-WCX fractionation followed by MALDI-TOF MS
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techniques combined with ClinProTools software to analyze the serum proteomic profiles of model and control C57BL/6mice, and generated 17 discriminating m/z peaks
that could accurately distinguish the model sera from the control sera. Two candidate peaks (m/z: 2954.49 and m/z: 1710.49), which were upregulated in the model group, were identified by LC-ESI-MS/MS. These two potential serum biomarkers were identified as peptide regions of the Ndufα8 and basigin proteins, respectively. The results of western blot analysis confirmed the results of MALDI-TOF MS and LC-ESIMS/MS analysis. Through the analysis of the time-course and dose-response of 13
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melamine effects on the expression of biomarker candidate genes, we found that the expression of serum basigin and Ndufα8 was partly dependent the concentration of melamine, but no time dependence. It is may be suggested that concentration of melamine is the main factor affecting the expression of serum Ndufα8 and basigin. Ndufα8 is an accessory subunit of the mitochondrial membrane respiratory chain NADH dehydrogenase (Complex I), which is believed to not be involved in catalysis.
of
Complex I functions in the transfer of electrons from NADH to the respiratory chain.
ro
The immediate electron acceptor for the enzyme is believed to be ubiquinone (http://www.uniprot.org/uniprot/Q9DCJ5). Ndufα8 mRNAs were found in a wide
-p
range of human tissues. The highest Ndufα8 mRNA content was observed in tissues
re
with a high metabolic energy demand. In descending order, NDUFA8 mRNA was
lP
mostly expressed in human heart, skeletal muscle, and fetal heart (Triepels et al., 1998). LHX6-NDUFA8, as a chimeric fusion RNA, was detected exclusively in
na
cervical cancer tissues and CIN patient Pap smears samples, and its expression may
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be an ideal biomarkers in cervical cancer (Wu et al., 2018). In addition to the above report, Ndufα8 was related to other diseases has not been reported. However, in our study Ndufα8 was upregulated in the model group, suggesting that Ndufα8 is related to the formation of bladder stones induced by melamine in mice. Basigin (also known as CD147 and EMMPRIN (Muramatsu and Miyauchi, 2003)) is a transmembrane glycoprotein, encoded by the Bsg gene, that belongs to the Ig superfamily and has two Ig domains. Its protein portion is 27 kDa and its glycosylated form is 43–66 kDa (Naruhashi et al., 1997). Basigin has reported functions in neurobiology, tumorigenesis, immunity and inflammation (Muramatsuand Miyauchi, 2003;Kosugi et al., 2015; Naruhashi et al., 1997). CD147 is frequently overexpressed 14
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in human cancers, such as head and neck squamous cell carcinoma, breast, gastric, colorectal, prostate, ovarian and renal cell cancer, along with hepatocellular carcinoma, glioma, laryngeal squamous cell carcinoma and skin carcinoma (Yu et al., 2019; Muramatsu, 2016; Wu et al., 2011; Xin et al., 2016; Sameshima et al., 2000; Rosenthal et al., 2003; Liang et al., 2009; Chen, et al., 2001).
of
Unlike Ndufα8, basigin is a multifunctional protein. In this study, basigin was identified as a serum biomarker in bladder stone mice fed a melamine diet.
ro
Upregulated basigin expression in this model may be related to inflammation because
-p
urinary bladder infection is a predisposing factor for stone formation. Furthermore,
re
urinary stones can promote further infection of other adjacent organs such as the
lP
prostate and seminal vesicles (Senapati et al.,2016).Secondly, pediatric urolithiasis
na
remains endemic in low-resource countries where infants constitute 17–40% of all children with urolithiasis(Zafar, et al., 2017). Yoshiko Mori1 et al. found plasma
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CD147 levels in patients with inflammation-related kidney diseases such as IgAN and HSPN showed a strong correlation with eGFR (Yoshiko Mori1, et al., 2018). Basigin may therefore be a potential serum biomarker in patients with bladder stone. For example, in infants with bladder stones, basigin and Ndufα8 could be used to investigate whether the formula milk powder is contaminated with melamine. In future studies, the molecular mechanism that links bladder stone formation with the upregulation of basigin and Ndufα8 in melamine-induced cystoliths will be investigated.
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5. Conclusions The results obtained in this study demonstrated that basigin and Ndufα8 are potential serum biomarkers of melamine diet-induced bladder stones in C57BL/6mice. These markers could be used to diagnose infants with bladder stones induced by milk
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formula contaminated with melamine.
Funding
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This study was supported by Shaanxi Province Natural Science Foundation (grant
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number: 2014JM2-3032); Shaanxi Health Scientific Research Project (grant
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Acknowledgements
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number:2014D75).
We thank Prof. Changfu Xu, Vice Prof. Shuting Ren and Xiaoli Gao of the
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Department of Pathology, School of Basic Medical Sciences, Xi'an Jiaotong University Health Science Center. They provided the melamine used in this study and provided valuable advise on establishing the mouse model. We thank International Science Editing for editing this manuscript.
Conflicts of interest The authors declare that have no conflict of interest.
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recovery of bladder epithelial hyperplasia caused by a melamine diet-induced bladder calculus in mice. Food Chem. Toxicol. 64, 378–382. Schwamborn, K., Krieg, R.C., Grosse, J., Reulen, N., Weiskirchen, R.,Knuechel, R., Jakse, G., &Henkel, C., 2009. Serum proteomic profiling in patients withbladdercancer. Eur. Urol.56, 989–996. Sameshima, T., Nabeshima, K., Toole, B.P., Yokogami, K., Okada, Y., Goya, T., Koono, M., &Wakisaka, S., 2000. Expression of emmprin (CD147), a cell surface inducer of matrix metalloproteinases, in normal human brain and gliomas. Int. J. Cancer 88, 21-27.
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Senapati, S., Suklabaidya, S., Mallik, H., Panda, S., Hota, D., &Baisakh, M.R., 2016. Urinary bladder stone associated with seminal vesicle and prostate infection in a Copenhagen rat. J. Nat. Sci., Biol. Med. 7,193-197. Tian, M., Cui, Y.Z., Song, G.H., Zong, M.J., Zhou, X.Y., Chen, Y., Han, J.X., 2008. Proteomic analysis identifies MMP-9, DJ-1 and A1BG as overexpressed proteins
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in pancreatic juice from pancreatic ductal adenocarcinoma patients. BMC Cancer 8, 241.
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Tan, T.L., &Goh, Y.Y., 2017. The role of group IIA secretory phospholipase A2
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(sPLA2-IIA) as a biomarker for the diagnosis of sepsis and bacterial infection in
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adults—A systematic review. PLoSone 12, e0180554.
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Tang, Z. M., Ling Z. G., Wang C.M., Wu Y. B., Kong, J. L., 2017. Serum
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tumor-associated autoantibodies as diagnostic biomarkers for lung cancer: A systematic review and meta-analysis. PLoS One 12, e0182117.
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Triepels, R., van den Heuvel, L., Loeffen, J., Smeets, R., Trijbels, F., &Smeitink, J., 1998. The nuclear-encoded human NADH:ubiquinone oxidoreductase NDUFA8 subunit: cDNA cloning, chromosomal localization, tissue distribution, and mutation detection in complex-I-deficient patients. Hum Genet 103, 557–563. UniProtKB - Q9DCJ5 (NDUA8_MOUSE). http://www.uniprot.org/uniprot/Q9DCJ5. Wu, Y.N., Zhao, Y.F., Li, J.G., &Melamine Analysis Group, 2009. A survey on occurrence of melamine and its analogues in tainted infant formula in China. Biomed. Environ. Sci. 22, 95–99. Wu, J., Ru, N.Y., Zhang, Y., Li, Y., Wei, D., Ren, Z., Huang, X.F., Chen, Z.N., &Bian, 20
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H., 2011. HAb18G/CD147 promotes epithelial-mesenchymal transition through TGF-β signaling and is transcriptionally regulated by Slug. Oncogene 30, 4410– 4427. Wu, P., Yang, S., Singh, S., Qin, F, Kumar, S., Wang, L., Ma, D., Li, H., 2018. The Landscape and Implications of Chimeric RNAs in Cervical Cancer.
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EBioMedicine 37,158-167. Xu, C.F., Gao, X.L., Du, Y.X., &Ren, S.T., 2011. The rapid establishment and
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Food Chem. Toxicol. 49, 3013–3017.
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implications of a melamine-induced standardized bladder stone model in mice.
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Xin, X., Zeng, X., Gu, H., Li, M., Tan, H., Jin, Z., Hua, T., Shi, R., &Wang, H., 2016.
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CD147/EMMPRIN overexpression and prognosis in cancer: A systematic review
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and meta-analysis. Sci. Rep. 6, 32804.
Yang, J., Yang, J., Gao, Y., Zhao, L.Y., Liu, L.Y., Qin, Y.N., Wang, X.F., Song, T.S.,
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&Huang, C., 2014. Identification of Potential Serum Proteomic Biomarkers for Clear Cell Renal Cell Carcinoma. PLoS One 9, e111364. Yoshiko Mori, Tomohiro Masuda, Tomoki Kosugi, Tomoki Yoshioka, Mayuko Hori, Hiroshi Nagaya, Kayaho Maeda, Yuka Sato, Hiroshi Kojima, Noritoshi Kato, Takuji Ishimoto,
Takayuki Katsuno,
Yukio Yuzawa,
Kenji Kadomatsu,
Shoichi Maruyama, 2018. The clinical relevance of plasma CD147/basigin in biopsy-proven kidney diseases. Clin. Exp. Nephrol. 22, 815–824. Yu B. B., Zhang Y., Wu K.L., Wang L.Z., Jiang Y. Y., Chen W. T., &Yan M.,2019. CD147 promotes progression of head and neck squamous cell carcinoma via 21
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NF‐kappa B signaling. J Cell Mol Med. 23, 954-966. Zhang, Q., Gamboa da Costa, G., Von Tungeln, L.S., Jacob ,C.C., Brown, R.P., &Goering, P.L., 2012. Urinary Biomarker Detection of Melamine- and Cyanuric Acid-Induced Kidney Injury in Rats. Toxicol. Sci. 129, 1–8. Zafar, M.N., Ayub, S., Tanwri, H., Naqvi, S.A.A., &Rizvi, S.A.H., 2017. Composition
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of urinary calculi in infants: a report from an endemic country. Urolithiasis. doi:
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10.1007/s00240-017-1010-1.
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Legends:
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Fig.1 The incidence of bladder stones in the model group. The incidence of bladder
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stones was 7/10 (4/5 female, 3/5 male).
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Fig.2 Comparative profiling of serum peptides between the model group (red) and
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control group (green). A. Mass spectra (1,000–10,000 Da) of serum samples from the model group (red) and control group (green). B. Bivariate plot of the model group (red) and control group (green). C. Representative mass spectra (1,000–10,000 Da) of serum samples from the model group (red) and control group (blue).Each sample represents three replications.
Fig.3 Representative spectra of two selected serum protein peaks from the model group (red) and control group (blue). A, C. The spectra of peaks 2794.04 and 1710.49 in the control group and model group separately. B, D. Receiver operating characteristic (ROC) curves for the two selected peaks are indicated together with 22
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their area under the curve (AUC) values. E, F. Average expression levels of two selected peaks in the model and control groups. **P< 0.000001.
Fig. 4.
LC-ESI-MS/MS spectrum of peptides with m/z of 2954.49 Da
Fig. 5 LC-ESI-MS/MS spectrum of peptides with m/z of 1710.49 Da Fig.6 Expression of Ndufα8 and basigin in serum samples as determined by western
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blot. A.Detection of Ndufα8 and basigin expression in the two groups. B. Relative
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protein levels of Ndufα8 (Ndufα8/Albumin). Expression of Ndufα8 was upregulated
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in the model group, including female and male mice. C. Relative protein levels of
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basigin (Basigin /Albumin). Expression of basigin was upregulated in model male
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mice; however, there was no significant difference in female mice in the model and control groups. Data represent mean±SD. Statistical significance was determined
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using the t-test, *P<0.05, **P<0.01.
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Fig.7 The time-course and dose-response of melamine effects on the expression of biomarker candidate genes. A and B was the incidence of bladder stones in the dose-response group and time-course group individually. In the concentration group, the stone formation rates of 9373ppm, 1.5×9373ppm and 2 ×9373ppm were 50% (3/3), 83% (5/6) and 83% (5/6) respectively. In the time-course group, the stone formation rates of two-week model group and three-week model group were 33% (2/6) and 60% (3/5), respectively. C. Detection the expression of basigin and Ndufα8 in the dose-response group. D. Detection the expression of basigin and Ndufα8 in the time-course group. E. Relative protein levels of basigin (basigin/Albumin) in 23
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dose-response group. The expression of basigin in different melamine concentration groups was higher than that in the control group (P=0.027 < 0.05), although the expression of basigin was gradually down-regulated as the concentration increased. F. Relative protein levels of basigin (basigin/Albumin) in time-course group. The expression of serum basigin in the two weeks and three weeks model group were up-regulated, but there no significant difference between control group and model
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group. G. Relative protein levels of Ndufα8 (Ndufα8/Albumin) in dose-response
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group. With the increase of melamine concentration, the expression of serum Ndufα8
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was also up-regulated (P=0.0331 < 0.05), but the expression level of 2 ×9373ppm
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group was lower than that of 9373ppm group and 1.5×9373ppm group. The group
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with the highest expression was 1.5×9373ppm group. H. Relative protein levels of
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Ndufα8 (Ndufα8/Albumin) in time-course group. The expression of serum Ndufα8 in the two weeks and three weeks model group were up-regulated, but there no
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significant difference between control group and model group.
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Fig. 1
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Fig. 2
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Fig. 3
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Fig. 4
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Fig. 5
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Fig. 6
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Fig. 7
Table 1 Mean level of differentially expressed proteins between the control and model 32
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groups. Control m/z
P-value
Trend of Model group
Fold change variation
< 0.000001
2.2
0.9
2.44
↓
4685.54
< 0.000001
1.64
0.54
3.04
↓
4094.64
< 0.000001
2.69
0.95
2.83
↓
1710.49
< 0.000001
19.16
45.50
2.37
↑
5805.92
< 0.000001
4.99
1.44
3.47
↓
5518.07
< 0.000001
2.94
0.48
6.1
↓
5885.82
< 0.000001
2.89
0.54
5.4
↓
3855.01
< 0.000001
3.11
7.31
2.35
↑
3642.27
< 0.000001
2.89
5.62
1.94
↑
2970.68
0.00908
7.6
14.1
1.86
↑
1730.63
< 0.000001
4.82
1.77
2.72
↓
2085.12
< 0.000001
5.6
2.2
2.55
↓
1958.42
< 0.000001
3.04
1.61
1.89
↓
5581.59
< 0.000001
1.73
0.69
2.51
↓
2954.49 5562.86
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5761.36
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4148.6
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group
< 0.000001
1.26
0.65
1.94
↓
< 0.000001
2.92
5.83
2.00
↑
0.00745
1.67
0.9
1.86
↓
Table2 Sequence identification of the two serum protein biomarkers. 33
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Peptide sequence (m/z)
Gene Name
UniProtKB ID NADH Q9DCJ5
2954.49
dehydrogenase Ndufα8
SHCAEPFTEYWTcLDYSNMQLFR (NDUA8_MOUSE)
[ubiquinone] 1 alpha subcomplex subunit 8
P18572 Basigin
SGEYSCIFLPEPVGR
na
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(BASI_MOUSE)
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1710.49
34
Bsg
Journal Pre-proof Declaration of interests ■ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:
35
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1. Peptidome profiles in melamine diet-induced bladder stones mice were generated. 2. Serum biomarkers were identified in the melamine diet-induced bladder stones mice. 3. Ndufα8 may be potential biomarker for the melamine diet-induced bladder stones. 4. Basigin may be potential biomarker for the melamine diet-induced bladder stones.
36
Liying Liu, Mai Luo, Xiaofan Xiong, Lingyu Zhao, Xiaofe Wang, Lei Ni, Juan Yang, Chen Huang PII:
S0041-008X(19)30394-1
DOI:
https://doi.org/10.1016/j.taap.2019.114786
Reference:
YTAAP 114786
To appear in:
Toxicology and Applied Pharmacology
Received date:
4 April 2019
Revised date:
4 October 2019
Accepted date:
21 October 2019
Please cite this article as: L. Liu, M. Luo, X. Xiong, et al., Peptidome profiles in melamine diet-induced bladder stones in C57BL/6 mice, Toxicology and Applied Pharmacology (2018), https://doi.org/10.1016/j.taap.2019.114786
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
© 2018 Published by Elsevier.
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Peptidome Profiles in Melamine Diet-Induced Bladder Stones in C57BL/6 Mice Liying Liua,1, Mai Luob,1, Xiaofan Xiongc, Lingyu Zhaoc, Xiaofe Wanga, Lei Nic, Juan Yanga,c,**, Chen Huanga,c,* a
The Center Laboratory for Biomedical Research, Xi'an Jiaotong University Health
Science Center, Xi’an, Shaanxi Province710061, People's Republic of China
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b
Core Research Laboratory, The Second Affiliated Hospital, School of Medicine,
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Xi’an Jiaotong University, Xi’an, Shaanxi Province710004, People's Republic of
Department of Cell Biology and Genetics, School of Basic Medical Sciences, Xi'an
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c
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China.
*
Correspondence to:
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People's Republic of China
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Jiaotong University Health Science Center, Shaanxi, Xi'an, Shaanxi Province710061,
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Prof. Chen Huang, The Center Laboratory for Biomedical Research, Environment and Genes Related to Diseases Key Laboratory of Education Ministry, Xi'an Jiaotong University; Department of Cell Biology and Genetics, School of Basic Medical Sciences, Xi'an Jiaotong University Health Science Center, Shaanxi, Xi'an, Shaanxi Province710061, People's Republic of China E-mail: [email protected]
**Correspondence to: Prof. Juan Yang, The Center Laboratory for Biomedical Research, Environment and 1
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Genes Related to Diseases Key Laboratory of Education Ministry, Xi'an Jiaotong University; Department of Cell Biology and Genetics, School of Basic Medical Sciences, Xi'an Jiaotong University Health Science Center, Shaanxi, Xi'an, Shaanxi Province710061, People's Republic of China
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These authors contributed equally to this work.
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Abbreviations:
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1
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E-mail:[email protected]
spectrometry
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MALDI -TOF-MS: matrix-assisted laser desorption ionization- time of flight- mass
MB–WCX: magnetic bead-based weak cation exchange chromatography LC-ESI-MS/MS: liquid chromatography with electrospray ionization and tandem triple quadrupole mass spectrometry Ndufα8: NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 8 Bsg: Basigin
Abstract The aim of this research was to detect potential serum biomarkers of melamine diet-induced bladder stones in C57BL/6 mice. Magnetic bead-based weak 2
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cationexchange chromatography (MB-WCX) and matrix-assisted laser desorption ionization-time of flight-mass spectrometry (MALDI-TOF-MS) were employed to detect serum biomarkers in 10 mice fed a melamine diet and 10 control mice. Seventeen peaks (fold change>1.5) with a mass to charge (m/z) value of 1,000– 10,000 Da were detected in the two groups. Among the significant peaks, five were
of
upregulated and the other 12 were downregulated in the model group. Among the upregulated peaks, 2954.49 and 1710.49 were found to correspond to the peptide
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regions of NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 8(Ndufα8)
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and basigin, respectively, by liquid chromatography with electrospray ionization and
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tandem triple quadrupole mass spectrometry(LC-ESI-MS/MS). Western blot analysis
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was used to detect the expression of Ndufα8 and basigin in another 10 model mice
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and 10 control mice. The western blot results confirmed the LC-ESI-MS/MS data. The expression of serum basigin and Ndufα8 was partly dependent the concentration
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of melamine, but no time dependence. In conclusion, Ndufα8 and basigin may be potential serum biomarkers for the detection of melamine diet-induced bladder stones in C57BL/6 mice.
Key Words: Melamine, Bladder stone, serum biomarkers, Ndufα8, Basigin
1. Introduction For many years, assays of serum biomarkers have played an important role in the diagnosis and prognosis of a variety of diseases including lung cancer (Tang et al., 2017),
hepatocellular
carcinoma
(Peng 3
et
al.,
2017),
pancreatic
ductal
Journal Pre-proof adenocarcinoma (Tian et al., 2008), Alzheimer’s disease (Lue et al., 2017), diabetes (Freitas et al.,2017), sepsis and bacterial infections (Tan and Goh, 2017)and ovarian cancer (Chatterjee et al., 2017). In this study, we aimed to identify a serum biomarker for melamine-induced bladder stones. Melamine (1,3,5-triazine-2,4,6-triamine, C3H6N6) is an industrially synthesized
of
chemical used for the production of laminates, coatings, glues, heat-tolerant polymers and plastics(Cheng et al.,2014; Skinner et al.,2010; Suchý et al., 2009; Zhang et al.,
ro
2012), and melamine contamination has been reported in products such as milk, infant
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formula, frozen yogurt, petfood, biscuits, candy and coffee (Mauer et al., 2009).The
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nitrogen content of food products increases when melamine is present (Chan et al.，
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2008).
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A well-documented case of melamine contamination occurred in China in 2008. Infant formula contaminated with melamine resulted in cases of infant urolithiasis
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(Chen, 2009;Wu et al.,2009). Xu and his research team established a standardized melamine-induced cystolith mouse model to study this phenomenon (Xu et al., 2011). They determined the most appropriate daily dose of dietary melamine required for cystolith formation, and revealed that bladder epithelial hyperplasia caused by melamine-induced bladder calculus formation can occur at concentrations over this dose. They also investigated the recovery of bladder epithelial hyperplasia after melamine withdrawal (Sun et al., 2014). Furthermore, they found that urinary alkalinization can be used to prevent and treat melamine-induced cystoliths in mice (Ren et al., 2014). Taken together, these studies confirmed that the mouse model was 4
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an appropriate model with which to study melamine-induced cystolith formation. However, there have been no previous reports of a biomarker to detect melamine-induced cystoliths or bladder stones induced by urinary system disease. The aim of the current study was therefore to identify a sensitive serum biomarker in the melamine-induced cystolith mouse model, and use this biomarker not only to
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diagnose melamine-induced cystoliths in mice at an early stage, but also to reveal the
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molecular mechanisms of melamine-induced cystolith formation.
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2. Materials and methods
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2.1 Chemicals
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Melamine [2, 4, 6-triamino-s-triazine, CAS No. 108-78-1], produced by
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Ker-mel Chemical Agent Ltd. Co., Tianjin, China (Batch No. 20060828;analytical purity P99.5%), was a gift from Professor Xu of Xi’an Jiaotong University Health
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Science Center.
2.2 Animal model
Three-week-old C57BL/6 specific-pathogen-free mice were obtained from, and housed in, the Central Laboratory of Animal, Xi’an Jiaotong University Health Science Center. The animals were housed at a temperature of 18–22℃, with relative humidity of 40%–60%, a light/dark cycle of 12 h/12 h, and free access to sterile water (pH 6.0). All animal experiments were approved by the Institutional Animal Care and Use Committee of Xi'an Jiaotong University and were performed according to the 5
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institution's guidelines for the use of laboratory animals.
2.3 Establishment of a bladder stone model in mice fed a diet containing melamine A total of 20 C57BL/6 mice were randomly divided into two groups, one fed a
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diet containing 9373 ppm of melamine and a control group fed the same diet with no melamine. There were five female and five male mice in each group. All mice ate and
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drank freely. The bladder stone mouse model was established according to a previous
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publication (Xu et al., 2011), and on day 15, blood was collected and the mice were
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euthanized. Blood samples were centrifuged at 3000 g for 10 min at 4 °C, and then
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serum was collected and stored at ˗80 °C for further investigation. In addition, the
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bladder of each mouse was opened and examined for stones. Stones were carefully
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removed and photographed.
2.4 Serum sample purification and MALDI-TOF MS analysis Magnetic bead-based purification (MB-WCX, Bremen, Germany) was employed to purify the serum samples, according to the manufacturer’s instructions. To prepare the MALDI target,1μl of a mixture containing 10μl of 0.3g/lα-cyano-4-hydroxy cinnamic acid in 2:1 ethanol/acetone (v/v) and 1 μl of the eluted peptide fraction was spotted onto a MALDI AnchorChipTM (Bruker Daltonics, Bremen,Germany) sample target platform (384 spots). To evaluate reproducibility, each serum sample was spotted in triplicate. 6
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Air-dried targets were measured immediately using a calibrated Autoflex III matrix-assisted
laser
desorption/ionization
time-of-flight
mass
spectrometry(MALDI-TOF MS, Bruker Daltonics) with FlexControl software (version 3.0, Bruker Daltonics), and optimized measuring protocols were established. Matrix suppression up to1,000 Da, with a mass range of 1,000–10,000 Da, was set as
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the default. Instrument calibration parameters were determined using standard peptide
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and protein mixtures.
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2.5 Data analyses
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Data analyses were performed using the programs Flex Analysis v3.0 and
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ClinProTools v2.2 (Bruker Daltonics).ClinProTools v2.2 uses a standard data
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preparation workflow including spectra pretreatment, peak picking and peak calculation operations, and was applied for the recognition of peptide patterns in this
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study. For statistical analyses, a k-nearest neighbor genetic algorithm, as implemented in the software suite, was used to identify statistically significant differences in protein peaks among the groups analyzed.
2.6 Peptide identification by LC-ESI-MS/MS After completing the statistical analysis, differentially expressed peptides were identified. The peptide sequences of potential m/z peaks were first determined by LC-ESI-MS/MS, and the identified sequence data were then subjected to a Mascot database search to identify the corresponding full-length protein matches. 7
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2.7 Western blot validation of the identified proteins To validate the Ndufα8 and basigin in the bladder stone model mice, a second mouse model was established using the same methods. Serum samples from the melamine fed (n=10) and control (n=10) mice were diluted 20 times before being
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separated by SDS-PAGE. The protein bands were electrophoretically transferred to polyvinylidene difluoride membrane (Millipore). Then, rabbit polyclonal anti-Ndufα8
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antibody (Proteintech, 15064-1-AP; 1:1000), rabbit monoclonal anti-CD147 antibody
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(Basigin; Abcam, ab108317; 1:1000), and rabbit polyclonal anti-albumin antibody
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(Proteintech, 16475-1-AP; 1:5000) were used for detection in the enhanced
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chemiluminescence detection system (Pierce). Albumin was also analyzed as an
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internal control protein. The blots were scanned (UVP BioSpectrum 410, USA) and
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the band density was measured using Quantity One imaging software.
2.8 The time-course and dose-response of melamine effects on the expression of biomarker candidate genes In order to analyze the time-course and dose-response of melamine effects on the expression of biomarker candidate genes，the time-course groups and dose-response groups were detected individually. In time-course experiment, the model group mice were fed a diet containing 9373 ppm of melamine. The model group was divided two groups: melamine-diet for two weeks, melamine-diet for three weeks. The control group （ normal diet ） was divided into 2-week group and 3-week group. In 8
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dose-gradient of melamine experiments, the experiment group were divided four groups: control group (normal diet), 9373 ppm of melamine diet group, 1.5×9373 ppm of melamine diet group, 2×9373 ppm of melamine diet group. Each concentration group was fed with melamine diet for 2 weeks. In time-course and dose-gradient experiment, there were 6 C57BL/6 mice in each group. Methods of
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biomarker was detected by western blot analysis.
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collecting serum and stone from experimental mice were described as 2.3. Serum
2.9 Statistical analysis
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Statistical analyses were conducted using GraphPad Prism v7.0 software
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(GraphPad Software, Inc., San Diego, CA, USA). T-test and one-way analysis
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variance (ANOVA) were used. All data were expressed as the mean ± SD. The results
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were considered statistically significant if P<0.05. Correlations between the mean expression of all detected m/z peaks were evaluated using the Spearman rank-order
3 Results
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correlation coefficient.
3.1 Bladder stones were induced by a melamine diet in the mouse model In this study, no fatalities occurred in any of the experimental groups. Bladder stones were observed in two of the experimental groups (70% prevalence), but not in the control mice. The number of stone granules in a single bladder was between one and two (Fig. 1). 9
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3.2 The detection of significant m/z peaks in the sera from the model mice compared with the control mice Twenty serum samples (10 from controls and 10 from model mice) were purified by MB-WCX and then detected by MALDI-TOF MS. There were 17 peaks (fold
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change>1.5) at a mass to charge (m/z) value of 1,000–10,000 in the two groups. Among the significant peaks, five were upregulated and the other 12 were
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downregulated in the model group (Table 1). The control group (green) and the model
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group (red) had protein profiles from 1 to 10 kDa (Fig. 2A). In component analysis, a
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bivariate plot of the model (red) and control (green) groups showed few overlapping
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regions (Fig. 2B). The two groups showed protein profiles ranging from 1 to 10 kDa
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(Fig. 2C). Within this mass range, two significant peaks at 2954.49 and 1710.49 were
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detected.
3.3 Peak selection
ClinProTools analysis revealed the two most significant peaks，2954.49 and 1710.49 were chosen to be analyzed. Receiver operating characteristic (ROC) curves of control group (blue) and the model group (red) are shown in Fig. 3. The area under the curve (AUC) values of these two peaks were 0.9079 (m/z: 2954.49) and 0.7762 (m/z: 1710.49) (Fig. 3B and D). Compared with the control group, the average expression levels of the two analyzed peaks in the model group were significantly higher (P<0.001; Fig.3E and 3F). 10
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3.4 Identification of two serum biomarkers LC-ESI-MS/MS and the Mascot database (Fig. 4 and 5) were used for MS/MS fragmentation of the two identified peptides (m/z: 2954.49 and m/z: 1710.49), resulting in the identification of the sequences SHCAEPFTEYWTcLDYSNMQLFR and SGEYSCIFLPEPVGR. These two peptides were found to be located within the
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Ndufα8 and basigin sequences, respectively, as shown in Table 2.
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3.5 Protein expression of Ndufα8 and basigin was upregulated in the serum of
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the model mice
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In the second mouse model experiments, we tested the expression of Ndufα8 and
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basigin in serum samples by western blot analysis, as shown in Fig.6. The expression of Ndufα8 was upregulated in the model group, for both male and female mice,
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confirming the LC-ESI-MS/MS data. However, the expression of basigin was
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upregulated only in the male mice in the model group. There was no significant difference in expression of this protein between female mice in the model and control groups.
3.6 The expression of serum basigin and Ndufα8 was partly dependent the concentration of melamine, but no time dependence In this study, one mouse died in the three-week model group. Bladder stones were observed in dose-response and time-course model groups. The number of stone granules in a single bladder was between one and three (Fig. 7A, B). In the concentration group, the stone formation rates of 9373ppm, 1.5×9373ppm and 2 11
Journal Pre-proof ×9373ppm were 50% (3/3), 83% (5/6) and 83% (5/6) respectively. In the time-course group, the stone formation rates of two-week model group and three-week model group were 33% (2/6) and 60% (3/5), respectively. The serum expression of basigin and Ndufα8 was detected by western blot analysis. The results of dose-response groups as showed in Fig.7A, C, E, G, and the time-course groups as showed in Fig.7B, D, F, H. Compared with the control group,
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Ndufα8 was highly expressed in the model group. With the increase of melamine
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concentration, the expression of serum Ndufα8 was also up-regulated (Fig.7G, P <
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0.05), but the expression level of 2 ×9373ppm group was lower than that of 9373ppm
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group and 1.5×9373ppm group. The group with the highest expression was
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1.5×9373ppm group. In time-course group, the expression of serum Ndufα8 in the
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two weeks and three weeks model group were also up-regulated. However, compared with the expression in two-week model group, the expression in three-week model
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group was down-regulated, and there no significant difference between control group and model group.so the expression of Ndufα8 was not time-dependent (Fig.7H). Similarly, the expression of basigin in different melamine concentration groups was higher than that in the control group (Fig.7E, P < 0.05), although the expression of basigin was gradually down-regulated as the concentration increased. In time-course group, the expression of serum basigin was not time-dependent (Fig. 7F), also similar with Ndufα8. 4 Discussion In 2008, milk formula contaminated with melamine caused adverse renal 12
Journal Pre-proof outcomes in a number of infants worldwide, after which melamine became a public health issue (Guan et al., 2009). However, the data on acute and chronic exposure to melamine in humans remain limited and most information has been extrapolated from animal studies. The clinical signs and symptoms associated with melamine exposure in infants are nonspecific. The bladder stones formed in response to exposure may be radiolucent and are not consistently seen on ultrasound (Skinner et al., 2010). It is therefore necessary to find new diagnostic methods for detecting melamine toxicity in
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the urinary system and bladder stone formation. In this study, we investigated serum
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biomarkers in a bladder stone mouse model induced by a melamine-contaminated diet.
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We employed MALDI-TOF MS analysis as this technique is widely applied in the
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analysis of serum samples for the diagnosis of human diseases and is proven to be an
Schwamborn et al., 2009).
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effective method for the identification of potential serum biomarkers (Yang et al.,2014;
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In this study, we used MB-WCX fractionation followed by MALDI-TOF MS
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techniques combined with ClinProTools software to analyze the serum proteomic profiles of model and control C57BL/6mice, and generated 17 discriminating m/z peaks
that could accurately distinguish the model sera from the control sera. Two candidate peaks (m/z: 2954.49 and m/z: 1710.49), which were upregulated in the model group, were identified by LC-ESI-MS/MS. These two potential serum biomarkers were identified as peptide regions of the Ndufα8 and basigin proteins, respectively. The results of western blot analysis confirmed the results of MALDI-TOF MS and LC-ESIMS/MS analysis. Through the analysis of the time-course and dose-response of 13
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melamine effects on the expression of biomarker candidate genes, we found that the expression of serum basigin and Ndufα8 was partly dependent the concentration of melamine, but no time dependence. It is may be suggested that concentration of melamine is the main factor affecting the expression of serum Ndufα8 and basigin. Ndufα8 is an accessory subunit of the mitochondrial membrane respiratory chain NADH dehydrogenase (Complex I), which is believed to not be involved in catalysis.
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Complex I functions in the transfer of electrons from NADH to the respiratory chain.
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The immediate electron acceptor for the enzyme is believed to be ubiquinone (http://www.uniprot.org/uniprot/Q9DCJ5). Ndufα8 mRNAs were found in a wide
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range of human tissues. The highest Ndufα8 mRNA content was observed in tissues
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with a high metabolic energy demand. In descending order, NDUFA8 mRNA was
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mostly expressed in human heart, skeletal muscle, and fetal heart (Triepels et al., 1998). LHX6-NDUFA8, as a chimeric fusion RNA, was detected exclusively in
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cervical cancer tissues and CIN patient Pap smears samples, and its expression may
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be an ideal biomarkers in cervical cancer (Wu et al., 2018). In addition to the above report, Ndufα8 was related to other diseases has not been reported. However, in our study Ndufα8 was upregulated in the model group, suggesting that Ndufα8 is related to the formation of bladder stones induced by melamine in mice. Basigin (also known as CD147 and EMMPRIN (Muramatsu and Miyauchi, 2003)) is a transmembrane glycoprotein, encoded by the Bsg gene, that belongs to the Ig superfamily and has two Ig domains. Its protein portion is 27 kDa and its glycosylated form is 43–66 kDa (Naruhashi et al., 1997). Basigin has reported functions in neurobiology, tumorigenesis, immunity and inflammation (Muramatsuand Miyauchi, 2003;Kosugi et al., 2015; Naruhashi et al., 1997). CD147 is frequently overexpressed 14
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in human cancers, such as head and neck squamous cell carcinoma, breast, gastric, colorectal, prostate, ovarian and renal cell cancer, along with hepatocellular carcinoma, glioma, laryngeal squamous cell carcinoma and skin carcinoma (Yu et al., 2019; Muramatsu, 2016; Wu et al., 2011; Xin et al., 2016; Sameshima et al., 2000; Rosenthal et al., 2003; Liang et al., 2009; Chen, et al., 2001).
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Unlike Ndufα8, basigin is a multifunctional protein. In this study, basigin was identified as a serum biomarker in bladder stone mice fed a melamine diet.
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Upregulated basigin expression in this model may be related to inflammation because
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urinary bladder infection is a predisposing factor for stone formation. Furthermore,
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urinary stones can promote further infection of other adjacent organs such as the
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prostate and seminal vesicles (Senapati et al.,2016).Secondly, pediatric urolithiasis
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remains endemic in low-resource countries where infants constitute 17–40% of all children with urolithiasis(Zafar, et al., 2017). Yoshiko Mori1 et al. found plasma
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CD147 levels in patients with inflammation-related kidney diseases such as IgAN and HSPN showed a strong correlation with eGFR (Yoshiko Mori1, et al., 2018). Basigin may therefore be a potential serum biomarker in patients with bladder stone. For example, in infants with bladder stones, basigin and Ndufα8 could be used to investigate whether the formula milk powder is contaminated with melamine. In future studies, the molecular mechanism that links bladder stone formation with the upregulation of basigin and Ndufα8 in melamine-induced cystoliths will be investigated.
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5. Conclusions The results obtained in this study demonstrated that basigin and Ndufα8 are potential serum biomarkers of melamine diet-induced bladder stones in C57BL/6mice. These markers could be used to diagnose infants with bladder stones induced by milk
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formula contaminated with melamine.
Funding
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This study was supported by Shaanxi Province Natural Science Foundation (grant
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number: 2014JM2-3032); Shaanxi Health Scientific Research Project (grant
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Acknowledgements
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number:2014D75).
We thank Prof. Changfu Xu, Vice Prof. Shuting Ren and Xiaoli Gao of the
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Department of Pathology, School of Basic Medical Sciences, Xi'an Jiaotong University Health Science Center. They provided the melamine used in this study and provided valuable advise on establishing the mouse model. We thank International Science Editing for editing this manuscript.
Conflicts of interest The authors declare that have no conflict of interest.
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&Huang, C., 2014. Identification of Potential Serum Proteomic Biomarkers for Clear Cell Renal Cell Carcinoma. PLoS One 9, e111364. Yoshiko Mori, Tomohiro Masuda, Tomoki Kosugi, Tomoki Yoshioka, Mayuko Hori, Hiroshi Nagaya, Kayaho Maeda, Yuka Sato, Hiroshi Kojima, Noritoshi Kato, Takuji Ishimoto,
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Shoichi Maruyama, 2018. The clinical relevance of plasma CD147/basigin in biopsy-proven kidney diseases. Clin. Exp. Nephrol. 22, 815–824. Yu B. B., Zhang Y., Wu K.L., Wang L.Z., Jiang Y. Y., Chen W. T., &Yan M.,2019. CD147 promotes progression of head and neck squamous cell carcinoma via 21
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NF‐kappa B signaling. J Cell Mol Med. 23, 954-966. Zhang, Q., Gamboa da Costa, G., Von Tungeln, L.S., Jacob ,C.C., Brown, R.P., &Goering, P.L., 2012. Urinary Biomarker Detection of Melamine- and Cyanuric Acid-Induced Kidney Injury in Rats. Toxicol. Sci. 129, 1–8. Zafar, M.N., Ayub, S., Tanwri, H., Naqvi, S.A.A., &Rizvi, S.A.H., 2017. Composition
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10.1007/s00240-017-1010-1.
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Legends:
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Fig.1 The incidence of bladder stones in the model group. The incidence of bladder
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stones was 7/10 (4/5 female, 3/5 male).
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Fig.2 Comparative profiling of serum peptides between the model group (red) and
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control group (green). A. Mass spectra (1,000–10,000 Da) of serum samples from the model group (red) and control group (green). B. Bivariate plot of the model group (red) and control group (green). C. Representative mass spectra (1,000–10,000 Da) of serum samples from the model group (red) and control group (blue).Each sample represents three replications.
Fig.3 Representative spectra of two selected serum protein peaks from the model group (red) and control group (blue). A, C. The spectra of peaks 2794.04 and 1710.49 in the control group and model group separately. B, D. Receiver operating characteristic (ROC) curves for the two selected peaks are indicated together with 22
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their area under the curve (AUC) values. E, F. Average expression levels of two selected peaks in the model and control groups. **P< 0.000001.
Fig. 4.
LC-ESI-MS/MS spectrum of peptides with m/z of 2954.49 Da
Fig. 5 LC-ESI-MS/MS spectrum of peptides with m/z of 1710.49 Da Fig.6 Expression of Ndufα8 and basigin in serum samples as determined by western
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blot. A.Detection of Ndufα8 and basigin expression in the two groups. B. Relative
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protein levels of Ndufα8 (Ndufα8/Albumin). Expression of Ndufα8 was upregulated
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in the model group, including female and male mice. C. Relative protein levels of
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basigin (Basigin /Albumin). Expression of basigin was upregulated in model male
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mice; however, there was no significant difference in female mice in the model and control groups. Data represent mean±SD. Statistical significance was determined
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using the t-test, *P<0.05, **P<0.01.
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Fig.7 The time-course and dose-response of melamine effects on the expression of biomarker candidate genes. A and B was the incidence of bladder stones in the dose-response group and time-course group individually. In the concentration group, the stone formation rates of 9373ppm, 1.5×9373ppm and 2 ×9373ppm were 50% (3/3), 83% (5/6) and 83% (5/6) respectively. In the time-course group, the stone formation rates of two-week model group and three-week model group were 33% (2/6) and 60% (3/5), respectively. C. Detection the expression of basigin and Ndufα8 in the dose-response group. D. Detection the expression of basigin and Ndufα8 in the time-course group. E. Relative protein levels of basigin (basigin/Albumin) in 23
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dose-response group. The expression of basigin in different melamine concentration groups was higher than that in the control group (P=0.027 < 0.05), although the expression of basigin was gradually down-regulated as the concentration increased. F. Relative protein levels of basigin (basigin/Albumin) in time-course group. The expression of serum basigin in the two weeks and three weeks model group were up-regulated, but there no significant difference between control group and model
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group. G. Relative protein levels of Ndufα8 (Ndufα8/Albumin) in dose-response
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group. With the increase of melamine concentration, the expression of serum Ndufα8
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was also up-regulated (P=0.0331 < 0.05), but the expression level of 2 ×9373ppm
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group was lower than that of 9373ppm group and 1.5×9373ppm group. The group
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with the highest expression was 1.5×9373ppm group. H. Relative protein levels of
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Ndufα8 (Ndufα8/Albumin) in time-course group. The expression of serum Ndufα8 in the two weeks and three weeks model group were up-regulated, but there no
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significant difference between control group and model group.
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Table 1 Mean level of differentially expressed proteins between the control and model 32
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groups. Control m/z
P-value
Trend of Model group
Fold change variation
< 0.000001
2.2
0.9
2.44
↓
4685.54
< 0.000001
1.64
0.54
3.04
↓
4094.64
< 0.000001
2.69
0.95
2.83
↓
1710.49
< 0.000001
19.16
45.50
2.37
↑
5805.92
< 0.000001
4.99
1.44
3.47
↓
5518.07
< 0.000001
2.94
0.48
6.1
↓
5885.82
< 0.000001
2.89
0.54
5.4
↓
3855.01
< 0.000001
3.11
7.31
2.35
↑
3642.27
< 0.000001
2.89
5.62
1.94
↑
2970.68
0.00908
7.6
14.1
1.86
↑
1730.63
< 0.000001
4.82
1.77
2.72
↓
2085.12
< 0.000001
5.6
2.2
2.55
↓
1958.42
< 0.000001
3.04
1.61
1.89
↓
5581.59
< 0.000001
1.73
0.69
2.51
↓
2954.49 5562.86
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5761.36
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4148.6
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< 0.000001
1.26
0.65
1.94
↓
< 0.000001
2.92
5.83
2.00
↑
0.00745
1.67
0.9
1.86
↓
Table2 Sequence identification of the two serum protein biomarkers. 33
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Peptide sequence (m/z)
Gene Name
UniProtKB ID NADH Q9DCJ5
2954.49
dehydrogenase Ndufα8
SHCAEPFTEYWTcLDYSNMQLFR (NDUA8_MOUSE)
[ubiquinone] 1 alpha subcomplex subunit 8
P18572 Basigin
SGEYSCIFLPEPVGR
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(BASI_MOUSE)
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1710.49
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Bsg
Journal Pre-proof Declaration of interests ■ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:
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1. Peptidome profiles in melamine diet-induced bladder stones mice were generated. 2. Serum biomarkers were identified in the melamine diet-induced bladder stones mice. 3. Ndufα8 may be potential biomarker for the melamine diet-induced bladder stones. 4. Basigin may be potential biomarker for the melamine diet-induced bladder stones.
36