The incidence of chronic renal failure (CRF) is increasing annually on a global scale [
FuShengong Decoction (FSGD) is summarized by Professor of Chinese medicine master Ziguang Guo, who added and deducted some herbs based on the classic formula “jisheng shenqi pills” with 60 years of clinical experience [
Isobaric tags for relative and absolute quantitation (iTRAQ) is a technology that can be used to simultaneously measure protein amounts in a multitude of test samples. This method significantly reduces the variability caused by multiple tests, thereby improving the accuracy of qualitative and quantitative protein analyses. iTRAQ has the ability to produce highly accurate and comprehensive information on hundreds to thousands of proteins. In a Pubmed search we found only 173 papers that used iTRAQ labeling to detect serum differential proteins; of these articles none reported on CRF.
In this study, we performed proteomic analysis using iTRAQ technology coupled with nanoscale liquid chromatography tandem mass spectrometry (nanoLC-MS/MS) to unveil molecular mechanism and identify potential biomarkers of FSGD. At the same time, we elucidated potential pathogenesis and the key pathway of these proteins through pathway analysis and protein networks. Furthermore, via the ELISA method, Western blot, and RT-qPCR we verified two dysregulated proteins (HP and AAT) that are of much interest as these two proteins were able to distinguish the CRF levels between model group and FSGD group, and they may act as biomarkers of FSGD.
FSGD ingredients were selected according to the “Chinese Pharmacopoeia” 2010 Edition. FSGD were soaked for 30 minutes with purified water and boiled three times every 30 minutes for a total of 90 minutes; then the boiling liquid was collected, filtered, concentrated to crude drug with the amount of 1 g/ml, and stored at 4°C for use.
A total of 33 male Sprague-Dawley rats (SYXK (Chongqing) 2012-0001), weighting
To increase accuracy and reduce variability in measures of protein concentration, the same amount of blood from each group was mixed into one sample [
Strong cation exchange (SCX) chromatography was performed to separate protein with the LC-20AB HPLC Pump system (Shimadzu, Kyoto, Honshu, Japan) with Gemini-NX C18 column (Phenomenex, Torrance, CA, US) (4.6 × 250 mm, 5
The fractions were centrifuged at 12,000
Peptide and protein identification were performed using the ProteinPilot™ software (version 4.2; Applied Biosystems, USA) and searching an automated database against the rat database (IPI_rat_v3.87) with the Mascot search engine (version 2.3.02; Matrix Science, London, UK). To screen the differential proteins, the threshold was applied as follows: the unused ProtScore > 1.3 and at least one peptide with a 95% confidence level [
Gene ontology (GO) analysis and the Kyoto encyclopedia of genes and genomes (KEGG) database were used to enrich and cluster the differential proteins. Each protein was represented by its cellular components, molecular function, and biological process by the GO database; meanwhile the pathway analysis was performed using the KEGG database. Functional networks were determined by STRING protein-protein interaction networks. All of the above analyses were conducted with Omicsbean software (Geneforhealth, Shanghai, China).
Rat HPT ELISA kit (Abcam, Cambridge, MA, USA, SwissProt: P06866) and rat
The renal samples stored at −80°C were uniformized in TRIzol (Tiangen Biotech, Beijing, China) and the RNA extraction was performed according to the manufacturer’s directions. Then the RNA was transcribed into cDNA (Toyobo, Shanghai, China) according to the manufacturer’s protocols. The PCR reaction was submitted to CFX96 Touch Real Time PCR (Bio Rad, Hercules, CA, USA) with the following primers: rat
The proteins were separated from frozen renal tissues and the protein concentration was measured by bicinchoninic acid (BCA) assay kit (Thermo Fisher Scientific, Rockford, IL, USA). The protein samples were resolved and transferred onto polyvinylidene fluoride (PVDF) membranes. After blocking with 5% nonfat milk at room temperature for 2 h, the membranes were performed using specific primary antibody as follows: haptoglobin and alpha-1-antitrypsin (Abcam, Cambridge, MA, USA). The blots were incubated with horseradish peroxidase-conjugated secondary antibodies (Abcam, Cambridge, MA, USA) and then exposed with an ECL kit (GE Healthcare, Chicago, IL, USA).
Statistical analyses were performed with GraphPad Prism software version 5.01 (GraphPad Software, Inc., San Diego, CA, USA). Variables in each group were tested to determine if they were normally distributed. Multiple comparisons of sample means were used for analysis of variance. The SNK method was used for pairwise comparison.
The overall proteins were compared among the three groups. In total, 417 proteins were confirmed with 5% local false discovery rate (FDR) and > 95% confidence score. Nineteen proteins with differential expression were found using stringent criteria. Among these proteins, twelve were found to be upregulated in the models compared with the controls and these same proteins were found to be downregulated in the FSGD group compared with the models. Additionally two downregulated proteins were then shown to be upregulated in the FSGD group, and five proteins exhibited no significant differences between groups. In addition, the levels of five proteins showed no difference between the controls and FSGD group. It is worth mentioning that the fold changes of HP were striking after FSGD treatment (Table
Differentially expressed proteins among control, model, and FSGD groups obtained by iTRAQ-nanoHPLC-MS/MS. 113: control; 114: model; 115: FSGD; —: ratio between 0.83 and 1.20.
Protein ID | Protein name | 115:114 | 114:113 | 115:113 |
---|---|---|---|---|
sp∣P06866 | Haptoglobin | 0.10 | 12.62 | 1.74 |
sp∣P04639 | Apolipoprotein A-I | 0.17 | 4.90 | — |
sp∣Q6P734 | Plasma protease C1 inhibitor | 0.19 | 1.80 | 0.32 |
sp∣P02651 | Apolipoprotein A-IV | 0.23 | 2.28 | 0.52 |
sp∣P02764 | Alpha-1-acid glycoprotein | 0.25 | 1.87 | 0.45 |
sp∣P09006 | Serine protease inhibitor A3N | 0.28 | 1.71 | 0.48 |
tr∣D4A183 | Protein Vnn3 | 0.35 | — | 0.31 |
sp∣Q03626 | Murinoglobulin-1 | 0.35 | — | 0.40 |
sp∣Q63207 | Coagulation factor X | 0.36 | — | 0.37 |
sp∣D3ZTE0 | Coagulation factor XII | 0.43 | 1.84 | 0.79 |
tr∣Q68FY4 | Group specific component | 0.45 | 2.22 | — |
sp∣P17475 | Alpha-1-antiproteinase | 0.46 | 2.74 | 1.25 |
tr∣A9CME3 | Complement component 4 binding protein, alpha | 0.49 | 2.20 | — |
sp∣P01015 | Angiotensinogen | 0.49 | 2.72 | 1.46 |
tr∣G3V8B1 | Glycosylphosphatidylinositol specific phospholipase D1, isoform CRA_a | 0.58 | — | 0.67 |
tr∣F1M6Z1 | Apolipoprotein B-100 | 0.73 | 4.60 | 3.30 |
sp∣P02770 | Serum albumin | 1.28 | 0.79 | — |
sp∣P31211 | Corticosteroid-binding globulin | 2.24 | 1.79 | 3.48 |
sp∣Q01177 | Plasminogen | 2.90 | 0.35 | — |
Differentially expressed proteins of the FSGD and model groups were catalogued based on GO enrichment analysis. It was revealed that most of the proteins were involved in the response to external stimulus (12, 63.16%), inflammatory response (9, 47.37%), and negative regulation of hydrolase activity (7, 36.84%). In addition, the subcellular proteins were distributed in the extracellular region (17, 89.47%), extracellular region part (16, 84.21%), and extracellular space (16, 84.21%) and associated with molecular function regulators (9, 47.37%), enzyme regulator activity (8, 42.11%), and enzyme inhibitor activity (7, 36.84%) (Figure
GO analysis of differentially expressed proteins.
The KEGG pathway mapping indicated that complement and coagulation cascades (6 proteins) were the predominant pathways. Vitamin digestion and absorption (3 proteins) and fat digestion and absorption (3 proteins) were also verified; these proteins are associated with the immune, endocrine, and digestive systems (Figure
KEGG pathway mapping of differentially expressed proteins.
The interactions among the differentially proteins were analyzed by the STRING network (Figure
The network of differentially expressed proteins obtained by STRING analysis.
Based on the central role in the STRING network and the fold changes, we selected HP and AAT for further analysis. The results revealed that both HP and AAT levels were significantly increased (
The expressions of HP and AAT among the control, model, and FSGD groups obtained by the ELISA, Western blot, and RT-qPCR methods. ((a) and (b)) The result of ELISA method. (c) The expression of each gene by RT-qPCR. (d) Western blot analysis for HP and AAT. (e) Densitometric analysis for HP and AAT. A
We examined the therapeutic effect of FSGD in the adenine-induced CRF rats. In traditional Chinese medicine, compounds of Chinese herbs have long-standing and widespread clinical applications. Multiple components of different herbs can concurrently attack multiple targets involved in the pathogenesis of the diseases. Thus, compounds are more important than a single herb [
We initially examined the mechanisms of the adenine-induced CRF. CRF is similar to chronic renal insufficiency and chronic kidney disease. Several previous studies have showed that diabetic nephropathy [
By function and pathway analysis, our study demonstrated that complement and coagulation cascade pathways and inflammatory response have a striking response on protein structure. The complement system is composed of over 30 serum proteins and cell membrane proteins [
Haptoglobin (HP), as an acute phase protein, exists as two major alleles: HP1 and HP2. The main efficacy of HP1 is antioxidant and anti-inflammatory, while HP2 plays an important role in antagonism. The main function of HP is to combine with free hemoglobin (Hb) and bind to monocytic cells and lymphocytes, thereby avoiding the loss of the Hb and heme iron from the kidney and damage to the kidney [
Alpha-1-antitrypsin (AAT), as the serine proteinase inhibitor, can prevent pathological damage of tissue, inhibit infection and inflammation, and organize and maintain the internal environment of body [
In conclusion, we succeeded in finding differentially expressed proteins in the adenine-induced CRF. According to the function and pathway analyses, it was demonstrated that these proteins are involved in multiple pathways and biological processes, but mainly in the inflammatory response. The results are consistent with the multitarget way of traditional Chinese medicine. Interestingly, HP and AAT exhibited significantly changes and located in key positions. This finding was verified by ELISA and the results were consistent with serum proteomics. We presumed that HP and AAT could be applicable as markers in the progression of CRF and may be the candidate biomarkers of FSGD. Further research is needed to explore the role of these protein functions in pathogenesis.
Yu Yang and Junmeng Wei are co-first authors.
The authors have no conflicts of interest to disclose.
This study was supported by the Natural Science Foundation of Chongqing (Grant no. Cstc2011jjA10058) and the Science and Technology Project of Jiulongpo, Chongqing (Grant nos. 62