Research Article | Open Access
Yu Yang, Junmeng Wei, Xuekuan Huang, Mingjun Wu, Zhenbing Lv, Pan Tong, Rui Chang, "iTRAQ-Based Proteomics of Chronic Renal Failure Rats after FuShengong Decoction Treatment Reveals Haptoglobin and Alpha-1-Antitrypsin as Potential Biomarkers", Evidence-Based Complementary and Alternative Medicine, vol. 2017, Article ID 1480514, 9 pages, 2017. https://doi.org/10.1155/2017/1480514
iTRAQ-Based Proteomics of Chronic Renal Failure Rats after FuShengong Decoction Treatment Reveals Haptoglobin and Alpha-1-Antitrypsin as Potential Biomarkers
Background. Chronic renal failure (CRF) has become a global health problem and bears a huge economic burden. FuShengong Decoction (FSGD) as traditional Chinese medicine has multiple pharmacological effects. Objectives. To understand the underlying molecular mechanism and signaling pathway involved in the FSGD treatment of CRF and screen differentially expressed proteins in rats with CRF treated with FSGD. Methods. Thirty-three male Sprague-Dawley rats were randomly divided into control group, CRF group, and FSGD group. Differentially expressed proteins were screened by iTRAQ coupled with nanoLC-MS/MS, and these identified proteins were later analyzed by GO, KEGG, and STRING. Additionally, haptoglobin (HP) and alpha-1-antitrypsin (AAT) were finally verified by ELISA, Western blot, and real time PCR. Results. A total of 417 proteins were identified. Nineteen differentially expressed proteins were identified in the FSGD group compared with the model group, of which 3 proteins were upregulated and 16 proteins were downregulated. Cluster analysis indicated that inflammatory response was associated with these proteins and complement and coagulation cascade pathways were predominantly involved. The validation methods further confirmed that the levels of HP and AAT were significantly increased. Conclusions. HP and AAT may be the important biomarkers in the pathogenesis of CRF and FSGD therapy.
The incidence of chronic renal failure (CRF) is increasing annually on a global scale , thus placing enormous burden on the medical system of many countries . Renal fibrosis characterized a common endpoint of different kidney diseases which resulted in kidney functional impairment ultimately leading to terminal renal failure. Tubulointerstitial fibrosis and glomerulosclerosis were closely associated with diverse action mechanisms such as abnormality of gene and protein expressions [3–6] as well as their downstream low-molecular-weight metabolite dysregulations [7–10]. The relationships of abnormal gene or protein expressions and endogenous metabolite disturbance were demonstrated in diverse chronic renal diseases [11–16]. Current clinical therapies for CRF are scarce and often ineffective . Traditional Chinese medicine is potentially a meaningful alternative therapy for CRF [18–22].
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 . FSGD is composed of Radix Astragali, Rehmannia glutinosa, Dioscorea opposita, Fructus Corni, Semen Plantaginis, Radix Achyranthis Bidentatae, Cortex Moutan Radicis, Rhizoma Alismatis, Poria, Rhizoma Atractylodis, Cortex Eucommiae, Hirudo, and Cortex Phellodendri. Research indicates that Radix Astragali, which is the dominate component, plays a role in improving the immunity and renal function . Rehmannia glutinosa has been shown as an effective constituent that can suppress inflammation and enhance renal function . Dioscorea opposita has been used to strengthen bone and tonify the kidney . It has been reported that Rhizoma Alismatis showed dual effect including promotion and inhibition of diuretic activity on renal function and antihyperlipidemia effect [27–29]. Several studies have demonstrated that Poria possessed nephroprotective activities including diuretic activity and treatment of CRF [30–34] and antihyperlipidemia effect [35, 36]. Although the clinical application of FSGD in the treatment of CRF has been verified, the underlying molecular mechanisms of its effect remain unknown.
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.
2. Materials and Methods
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.
2.2. Animals and Sample Collection
A total of 33 male Sprague-Dawley rats (SYXK (Chongqing) 2012-0001), weighting g, were fed adaptively for 1 week and then randomly divided into 3 groups: control group, model group, and FSGD group (11 in each group). The control group was fed standard chow, while the other two groups were fed 0.5% adenine (Sigma-Aldrich, St. Louis, MO, USA) chow for 3 weeks to induce chronic renal failure [37, 38]. After the models were successfully made, rats in control and model groups received saline in the amount of 20 ml/kg/d, while those in FSGD group received 16 g/kg/d, administered by gastric irrigation, respectively, for 30 days. All rats were starved for 12 h and anesthetized by 3% pentobarbital (Beijing Propbs Biotechnology, Beijing, China) at a dose of 5 ml/kg at 30 days, and blood samples were later acquired by cardiac puncture. Blood was placed at room temperature for 0.5 hours and was subsequently centrifuged at 4°C, 1300 for 15 min, and the supernatant was obtained and stored at −80°C for further analysis. Kidneys were immediately washed by phosphate buffer saline and stored at −80°C for histological study. The experimental animals were disposed according to the “Guide for the Care and Use of Laboratory Animals” approved by the Committee of Chongqing Medical University.
2.3. iTRAQ Labeling
To increase accuracy and reduce variability in measures of protein concentration, the same amount of blood from each group was mixed into one sample [39, 40]. High-abundance proteins such as albumin and IgG were depleted by using the Multiple Affinity Removal System (Agilent, Palo Alto, CA, USA) according to the manufacturer’s instructions. Next, proteins were concentrated and desalted . A total of 200 μg proteins were soaked in 6 μl dithiothreitol (Amresco, Solon, OH, USA) and 2 μl indole-3-acetic acid (Amresco, Solon, OH, USA) for 1 h at 37°C and then centrifuged. The deposit was subsequently removed. The samples were then digested with trypsin (AB Sciex, Framingham, MA, USA) with the ratio of protein : trypsin = 50 : 1 at 37°C overnight. The peptides were labeled with iTRAQ reagent (AB Sciex, Framingham, MA, USA) (each reagent was dissolved in 70 μl of ethanol) and incubated at room temperature for 2 h. The samples were labeled as follows: the control group, 113; the model group, 114; the FSGD group, 115; then they were mixed and dried by vacuum centrifugation. To avoid the labeling bias, two independent biological replicates were performed.
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 μm 110A). The peptide mixture was eluted with a liner gradient of buffer A (AB Sciex, Framingham, MA, USA) and 5% buffer B (AB Sciex, Framingham, MA, USA) for 30 min, 15%–90% buffer B for 25 min, and 5% buffer B for 10 min at a flow of 0.8 ml/min. A total of 50 components were collected and vacuum-dried.
2.4. Mass Spectrometry (MS) Analyses
The fractions were centrifuged at 12,000 for 8 min and supernatant was collected. The samples were analyzed with nanoHPLC-MS/MS (Thermo Scientific, Waltham, MA, USA). Specific parameters were as follows: ion spray voltage, 2.3 kv; Curtain gas, 35 psi; survey scan, 300–1800 for MS scans; dynamic exclusion duration of 25 s; survey scan, 100–1500 for MS/MS scans.
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 , ratios with fold change > 1.2 (or <0.83), and values < 0.05 were considered to be significant.
2.5. Bioinformatics Analysis
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).
2.6. ELISA Methods
Rat HPT ELISA kit (Abcam, Cambridge, MA, USA, SwissProt: P06866) and rat α-1-AT ELISA kit (Abcam, Cambridge, MA, USA, SwissProt: P17475) were used to detect protein levels in the serum. The protein concentration analysis of each group was performed according to the manufacturer’s protocols. Concentrations in each group were compared with Student’s -tests after logarithmic transformation.
2.7. Real Time Quantitative PCR
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 HP: sense, 5′-TGTGCCGTAGCTGAGTATGGTGTG-3′, antisense, 5′-GAATTGCCCTGCCCCACTGT-3′, rat Serpina1: sense, 5′-CCCTTGGCGACCCTCCTCTT-3′, antisense, 5′-CCCCACCGAAGAACCAGGATATA-3′, and β-actin: sense, 5′-ACCCCGTGCTGCTGACCGAG-3′, antisense, 5′-TCCCGGCCAGCCAGGTCCA-3′ according to the manufacturer’s instructions. Afterwards, the expression of genes was calculated from standard curve with the expression of β-actin gene as reference.
2.8. Western Blot Analysis
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).
2.9. Statistical Analysis
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. was considered to be significant.
3.1. Comparative Analysis of Serum Proteomic Changes in Each Group
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 1).
3.2. GO Analysis, KEGG Pathway, and STRING
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 1).
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 2).
The interactions among the differentially proteins were analyzed by the STRING network (Figure 3). STRING analysis showed that Alb and Serpina1 played a central role in the network.
3.3. The Validation of ELISA, Western Blot, and RT-qPCR
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 (, , resp.) in the control group compared with the model group. HP and AAT levels were significantly decreased (, , resp.) in the model group compared to the FSGD group. In addition, there were no significant differences in HP and AAT levels between the model group and the FSGD group (, , resp.) (Figure 4).
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 [19, 43]. Previous studies have verified that FSGD is effective in the treatment of CRF. Specifically, levels of serum creatinine (SCr) and blood urea nitrogen (BUN) decreased significantly; renal function and nephridial tissues were improved after treatment of FSGD [44, 45]. We found that possible mechanisms may be the inhibition of Sonic Hedgehog (SHH) signaling pathway and/or reduced levels of α-SMA in nephridial tissue by detecting renal tissues. To characterize the effect of FSGD, we conducted the present experiment.
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 [46, 47], hypertensive nephropathy [48, 49], and lupus nephritis  are the primary causes of CRF. Inagi  found that the accumulation of advanced glycation end product (AGE) produces glycative stress closely associated with kidney disease. Various signaling pathways are involved in process of chronic kidney disease, such as Wnt/β-catenin, TGF-β/Smads, JNK/STAT3, and MAPKs . The common pathway of these renal diseases is tubulointerstitial fibrosis, which is characterized by the superfluous deposition of extracellular matrix, infiltration of lymphocytes, dendritic cells, macrophages , and fibroblast proliferation/differentiation.
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 , appears congenitally and/or is an acquired immune effector, and is one of the most powerful barriers against invading pathogens. The deposition of immune complexes mediated inflammatory response causing tissue injury . In addition, coagulation factors are activated after the interaction of platelets and endotheliocyte, thus enhancing coagulation and inflammation . Keir and Langman  also indicated that complement factors are related to kidney function. Further, activation of the complement system may aggravate kidney damage. Hence, we assume that the development of CRF accompanied activation of the complement system and inflammatory infiltration.
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 [57, 58]. Several studies have demonstrated that higher levels of HP are correlated with liver fibrosis , various cancers [60–62], and cardiovascular disease . HP is mainly produced in the liver but also expressed in kidney. The common mechanism of these diseases indicates that HP is associated with inflammation and the immune response . In addition, several previous studies have showed that HP can predict various kidney diseases [64–66]. Specifically, previous reports show that HP2 is more closely related to kidney disease. Elevated concentrations of HP2 in the serum have been shown to increase deterioration of renal function . HP response to injury or improved oxidative stress could be expressed in the renal tubules. This phenomenon may contribute to development of haptoglobin-hemoglobin complex that cannot be filtered from the glomerulus and, along with iron, accumulates in renal proximal tubule. This is a possible mechanism of CRF. Our proteomic result further demonstrates that HP is critical in the progress of CRF.
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 . AAT has multiple activities, such as cytoprotective, immunomodulatory activity and downregulation of neutrophil elastase during the inflammatory processes. Accumulating studies have showed that AAT may lead to lung pathologies , type 1 diabetes , arthritis , and lupus . Kwak et al.  found that the expression of AAT is elevated in renal biopsies, while other investigators detected that it is overexpressed in the urine of some renal diseases [73–75]. This phenomenon was consistent with our result in which AAT was upregulated. Upregulation of AAT could lead to inhibition of elastase, which can contribute to regulating inflammation and the accumulation of mesangial matrix, as well as maintaining the elasticity of blood vessels and glomerular integrity. AAT is located in the cytoplasm of podocytes and it is possibly related to epithelial dysfunction and podocyte stress and results in renal fibrosis.
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.
Conflicts of Interest
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 and 37 ).
- Y. Teerawattananon, A. Luz, S. Pilasant et al., “How to meet the demand for good quality renal dialysis as part of universal health coverage in resource-limited settings?” Health Research Policy and Systems, vol. 14, no. 1, p. 21, 2016.
- W. C. Tsai, H. Y. Wu, Y. S. Peng et al., “Risk factors for development and progression of chronic kidney disease: a systematic review and exploratory meta-analysis,” Medicine, vol. 95, no. 11, Article ID e3013, 2016.
- R. J. Tan, D. Zhou, L. Zhou, and Y. Liu, “Wnt/β-catenin signaling and kidney fibrosis,” Kidney International Supplements, vol. 4, no. 1, pp. 84–90, 2014.
- Y.-Y. Zhao and R.-C. Lin, “UPLC-MSE application in disease biomarker discovery: the discoveries in proteomics to metabolomics,” Chemico-Biological Interactions, vol. 215, pp. 7–16, 2014.
- J. Klein, P. Kavvadas, N. Prakoura et al., “Renal fibrosis: insight from proteomics in animal models and human disease,” Proteomics, vol. 11, no. 4, pp. 805–815, 2011.
- M. Edeling, G. Ragi, S. Huang, H. Pavenstädt, and K. Susztak, “Developmental signalling pathways in renal fibrosis: the roles of Notch, Wnt and Hedgehog,” Nature Reviews Nephrology, vol. 12, no. 7, pp. 426–439, 2016.
- Y.-Y. Zhao, X.-L. Cheng, F. Wei et al., “Serum metabonomics study of adenine-induced chronic renal failure in rats by ultra performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry,” Biomarkers, vol. 17, no. 1, pp. 48–55, 2012.
- Y. Y. Zhao, “Metabolomics in chronic kidney disease,” Clinica Chimica Acta, vol. 422, pp. 59–69, 2013.
- H. Chen, L. Chen, D. Liu et al., “Combined clinical phenotype and lipidomic analysis reveals the impact of chronic kidney disease on lipid metabolism,” Journal of Proteome Research, vol. 16, no. 4, pp. 1566–1578, 2017.
- Y.-Y. Zhao, J. Liu, X.-L. Cheng, X. Bai, and R.-C. Lin, “Urinary metabonomics study on biochemical changes in an experimental model of chronic renal failure by adenine based on UPLC Q-TOF/MS,” Clinica Chimica Acta, vol. 413, no. 5-6, pp. 642–649, 2012.
- H. Chen, G. Cao, D. Q. Chen et al., “Metabolomics insights into activated redox signaling and lipid metabolism dysfunction in chronic kidney disease progression,” Redox Biology, vol. 10, pp. 168–178, 2016.
- Y.-Y. Zhao, X.-L. Cheng, F. Wei et al., “Intrarenal metabolomic investigation of chronic kidney disease and its TGF-β1 mechanism in induced-adenine rats using UPLC Q-TOF/HSMS/MS E,” Journal of Proteome Research, vol. 12, no. 2, pp. 692–703, 2013.
- D.-Q. Chen, H. Chen, L. Chen et al., “The link between phenotype and fatty acid metabolism in advanced chronic kidney disease,” Nephrology Dialysis Transplantation, 2017.
- Y.-Y. Zhao, H.-L. Wang, X.-L. Cheng et al., “Metabolomics analysis reveals the association between lipid abnormalities and oxidative stress, inflammation, fibrosis, and Nrf2 dysfunction in aristolochic acid-induced nephropathy,” Scientific Reports, vol. 5, Article ID 12936, 2015.
- Z. H. Zhang, H. Chen, N. D. Vaziri et al., “Metabolomic signatures of chronic kidney disease of diverse etiologies in the rats and humans,” Journal of Proteome Research, vol. 15, no. 10, pp. 3802–3812, 2016.
- D.-Q. Chen, G. Cao, H. Chen et al., “Gene and protein expressions and metabolomics exhibit activated redox signaling and wnt/β-catenin pathway are associated with metabolite dysfunction in patients with chronic kidney disease,” Redox Biology, vol. 12, pp. 505–521, 2017.
- Y. Liu, “Cellular and molecular mechanisms of renal fibrosis,” Nature Reviews Nephrology, vol. 7, no. 12, pp. 684–696, 2011.
- Y. Zhong, M. C. Menon, Y. Deng, Y. Chen, and J. C. He, “Recent advances in traditional chinese medicine for kidney disease,” American Journal of Kidney Diseases, vol. 66, no. 3, pp. 513–522, 2015.
- Z.-H. Zhang, N. D. Vaziri, F. Wei, X.-L. Cheng, X. Bai, and Y.-Y. Zhao, “An integrated lipidomics and metabolomics reveal nephroprotective effect and biochemical mechanism of Rheum officinale in chronic renal failure,” Scientific Reports, vol. 6, Article ID 22151, 2016.
- Y. Zhong, Y. Deng, Y. Chen, P. Y. Chuang, and J. Cijiang He, “Therapeutic use of traditional Chinese herbal medications for chronic kidney diseases,” Kidney International, vol. 84, no. 6, pp. 1108–1118, 2013.
- Z.-H. Zhang, F. Wei, N. D. Vaziri et al., “Metabolomics insights into chronic kidney disease and modulatory effect of rhubarb against tubulointerstitial fibrosis,” Scientific Reports, vol. 5, 2015.
- Y. Chen, G. Cai, X. Sun, and X. Chen, “Treatment of chronic kidney disease using a traditional Chinese medicine, Flos Abelmoschus manihot (Linnaeus) Medicus (Malvaceae),” Clinical and Experimental Pharmacology and Physiology, vol. 43, no. 2, pp. 145–148, 2016.
- X. K. Huang, “FuShengong decoction,” in Clinical Experiences of Ziguang Guo, X. K. Huang, Ed., People's Medical Publishing House, Beijing, China, 2009.
- C. Zhu, Y. Gao, T. Jiang, C. Hao, Z. Gao, and Y. Sun, “Meta-analysis of Huangqi injection for the adjunctive therapy of aplastic anemia,” International Journal of Clinical & Experimental Medicine, vol. 8, no. 7, pp. 10256–64, 2015.
- G.-H. Baek, Y.-S. Jang, S.-I. Jeong et al., “Rehmannia glutinosa suppresses inflammatory responses elicited by advanced glycation end products,” Inflammation, vol. 35, no. 4, pp. 1232–1241, 2012.
- N. Han, J. Xu, F. Xu, Z. Liu, and J. Yin, “The in vivo effects of a fraction from Dioscorea spongiosa on glucocorticoid-induced osteoporosis,” Journal of Ethnopharmacology, vol. 185, pp. 53–59, 2016.
- Y.-L. Feng, H. Chen, T. Tian, D.-Q. Chen, Y.-Y. Zhao, and R.-C. Lin, “Diuretic and anti-diuretic activities of the ethanol and aqueous extracts of Alismatis rhizoma,” Journal of Ethnopharmacology, vol. 154, no. 2, pp. 386–390, 2014.
- D.-Q. Chen, Y.-L. Feng, T. Tian et al., “Diuretic and anti-diuretic activities of fractions of Alismatis rhizoma,” Journal of Ethnopharmacology, vol. 157, no. 1, pp. 114–118, 2014.
- H. Miao, L. Zhang, D. Q. Chen, H. Chen, Y. Y. Zhao, and S. C. Ma, “Urinary biomarker and treatment mechanism of Rhizoma Alismatis on hyperlipidemia,” Biomedical Chromatography, vol. 31, no. 4, p. e3829, 2017.
- Y.-Y. Zhao, Y.-L. Feng, X. Du, Z.-H. Xi, X.-L. Cheng, and F. Wei, “Diuretic activity of the ethanol and aqueous extracts of the surface layer of Poria cocos in rat,” Journal of Ethnopharmacology, vol. 144, no. 3, pp. 775–778, 2012.
- Y.-L. Feng, P. Lei, T. Tian et al., “Diuretic activity of some fractions of the epidermis of Poria cocos,” Journal of Ethnopharmacology, vol. 150, no. 3, pp. 1114–1118, 2013.
- Y.-Y. Zhao, Y.-L. Feng, X. Bai, X.-J. Tan, R.-C. Lin, and Q. Mei, “Ultra performance liquid chromatography-based metabonomic study of therapeutic effect of the surface layer of poria cocos on adenine-induced chronic kidney disease provides new insight into anti-fibrosis mechanism,” PLoS ONE, vol. 8, no. 3, Article ID e59617, 2013.
- Y.-Y. Zhao, H.-T. Li, Y.-L. Feng, X. Bai, and R.-C. Lin, “Urinary metabonomic study of the surface layer of Poria cocos as an effective treatment for chronic renal injury in rats,” Journal of Ethnopharmacology, vol. 148, no. 2, pp. 403–410, 2013.
- Y.-Y. Zhao, P. Lei, D.-Q. Chen, Y.-L. Feng, and X. Bai, “Renal metabolic profiling of early renal injury and renoprotective effects of Poria cocos epidermis using UPLC Q-TOF/HSMS/MSE,” Journal of Pharmaceutical and Biomedical Analysis, vol. 81-82, pp. 202–209, 2013.
- H. Chen, L. Chen, D. D. Tang et al., “Metabolomics reveals hyperlipidemic biomarkers and antihyperlipidemic effect of poria cocos,” Current Metabolomics, vol. 4, no. 2, pp. 104–115, 2016.
- H. Miao, “The antihyperlipidemic effect of Fu-Ling-Pi is associated with abnormal fatty acid metabolism as assessed by UPLC-HDMS-based lipidomics,” RSC Advances, vol. 5, no. 79, pp. 64208–64219, 2015.
- T. Yokozawa, P. D. Zheng, H. Oura, and F. Koizumi, “Animal model of adenine-induced chronic renal failure in rats,” Nephron, vol. 44, no. 3, pp. 230–234, 1986.
- D. Claramunt, H. Gil-Peña, R. Fuente et al., “Chronic kidney disease induced by adenine: a suitable model of growth retardation in uremia,” American Journal of Physiology—Renal Physiology, vol. 309, no. 1, pp. F57–F62, 2015.
- C. Wang, C.-M. Liu, L.-L. Wei et al., “A group of novel serum diagnostic biomarkers for multidrug-resistant tuberculosis by iTRAQ-2D LC-MS/MS and solexa sequencing,” International Journal of Biological Sciences, vol. 12, no. 2, pp. 246–256, 2016.
- N. A. Karp, W. Huber, P. G. Sadowski, P. D. Charles, S. V. Hester, and K. S. Lilley, “Addressing accuracy and precision issues in iTRAQ quantitation,” Molecular and Cellular Proteomics, vol. 9, no. 9, pp. 1885–1897, 2010.
- D.-D. Xu, D.-F. Deng, X. Li et al., “Discovery and identification of serum potential biomarkers for pulmonary tuberculosis using iTRAQ-coupled two-dimensional LC-MS/MS,” Proteomics, vol. 14, no. 2-3, pp. 322–331, 2014.
- C. Wang, L. L. Wei, L. Y. Shi et al., “Screening and identification of five serum proteins as novel potential biomarkers for cured pulmonary tuberculosis,” Scientific Reports, vol. 5, Article ID 15615, 2015.
- S. Wang, L. Lin, J. Zhou, S. Xiong, and D. Zhou, “Effects of yiqi chutan tang on the proteome in Lewis lung cancer in mice,” Asian Pacific Journal of Cancer Prevention: APJCP, vol. 12, no. 7, pp. 1665–1669, 2011.
- L. Wang, X. K. Huang, L. Wan, and Y. Yang, “Effect of fushengong decoction on the expression of nephrin mRNA in kidney of rats with chronic renal failure,” Journal of Sichuan University (Medical Science Edition), vol. 47, no. 3, pp. 342–346, 2016.
- L. Wan, X. K. Huang, L. Wang, and Y. Yang, “The effects of fushengong formula on renal function and shh signal pathway of rats with chronic renal failure,” Journal of Traditional Chinese Medicine, vol. 56, pp. 1771–1774, 2015.
- X.-M. Wu, Y.-B. Gao, F.-Q. Cui, and N. Zhang, “Exosomes from high glucose-treated glomerular endothelial cells activate mesangial cells to promote renal fibrosis,” Biology Open, vol. 5, no. 4, pp. 484–491, 2016.
- C. Xin, Z. Xia, C. Jiang, M. Lin, and G. Li, “Xiaokeping mixture inhibits diabetic nephropathy in streptozotocin-induced rats through blocking TGF-β1/Smad7 signaling,” Drug Design, Development and Therapy, vol. 9, pp. 6269–6274, 2015.
- Q.-Z. Wang, H.-Q. Gao, Y. Liang, J. Zhang, J. Wang, and J. Qiu, “Cofilin1 is involved in hypertension-induced renal damage via the regulation of NF-κB in renal tubular epithelial cells,” Journal of Translational Medicine, vol. 13, no. 1, 2015.
- T. Kato, N. Mizuguchi, and A. Ito, “Blood pressure, renal biochemical parameters and histopathology in an original rat model of essential hypertension (SHRSP/Kpo strain),” Biomedical Research (Japan), vol. 36, no. 3, pp. 169–177, 2015.
- L. C. Plantinga, C. Drenkard, S. O. Pastan, and S. S. Lim, “Attribution of cause of end-stage renal disease among patients with systemic lupus erythematosus: the georgia lupus registry,” Lupus Science & Medicine, vol. 3, no. 1, Article ID e000132, 2016.
- R. Inagi, “RAGE and glyoxalase in kidney disease,” Glycoconjugate Journal, vol. 33, no. 4, pp. 619–626, 2016.
- Y. B. Y. Sun, X. Qu, G. Caruana, and J. Li, “The origin of renal fibroblasts/myofibroblasts and the signals that trigger fibrosis,” Differentiation, vol. 92, no. 3, pp. 102–107, 2016.
- M. Zeisberg and E. G. Neilson, “Mechanisms of tubulointerstitial fibrosis,” Journal of the American Society of Nephrology, vol. 21, no. 11, pp. 1819–1834, 2010.
- L. S. Keir and C. B. Langman, “Complement and the kidney in the setting of Shiga-toxin hemolytic uremic syndrome, organ transplantation, and C3 glomerulonephritis,” Transfusion and Apheresis Science, vol. 54, no. 2, pp. 203–211, 2016.
- S. Morimura, M. Sugaya, and S. Sato, “Interaction between CX3CL1 and CX3CR1 regulates vasculitis induced by immune complex deposition,” The American Journal of Pathology, vol. 182, no. 5, pp. 1640–1647, 2013.
- S. C. Fagerholm, H. S. Lek, and V. L. Morrison, “Kindlin-3 in the immune system,” American Journal of Clinical & Experimental Immunology, vol. 3, no. 1, pp. 37–42, 2014.
- K. M. Huntoon, Y. Wang, C. A. Eppolito et al., “The acute phase protein haptoglobin regulates host immunity,” Journal of Leukocyte Biology, vol. 84, no. 1, pp. 170–181, 2008.
- J. Wassell, “Haptoglobin: function and polymorphism,” Clinical Laboratory, vol. 46, no. 11-12, pp. 547–552, 2000.
- B. Ghafouri, A. Carlsson, S. Holmberg, A. Thelin, and C. Tagesson, “Biomarkers of systemic inflammation in farmers with musculoskeletal disorders; a plasma proteomic study,” BMC Musculoskeletal Disorders, vol. 17, no. 1, 2016.
- A. Shah, H. Singh, V. Sachdev et al., “Differential serum level of specific haptoglobin isoforms in small cell lung cancer,” Current Proteomics, vol. 7, no. 1, pp. 49–56, 2010.
- S. Weiz, M. Wieczorek, C. Schwedler et al., “Acute-phase glycoprotein N-glycome of ovarian cancer patients analyzed by CE-LIF,” Electrophoresis, 2016.
- L. Sun, S. Hu, L. Yu et al., “Serum haptoglobin as a novel molecular biomarker predicting colorectal cancer hepatic metastasis,” International Journal of Cancer, vol. 138, no. 11, pp. 2724–2731, 2016.
- K. L. Graves and D. J. Vigerust, “Hp: an inflammatory indicator in cardiovascular disease,” Future Cardiology, vol. 12, no. 4, pp. 471–481, 2016.
- T. Costacou and T. J. Orchard, “The Haptoglobin genotype predicts cardio-renal mortality in type 1 diabetes,” Journal of Diabetes and Its Complications, vol. 30, no. 2, pp. 221–226, 2016.
- V. Sandim, D. D. A. Pereira, D. E. Kalume et al., “Proteomic analysis reveals differentially secreted proteins in the urine from patients with clear cell renal cell carcinoma,” Urologic Oncology: Seminars and Original Investigations, vol. 34, no. 1, pp. 5.e11–5.e25, 2016.
- S. L. Saraf, X. Zhang, B. Shah et al., “Genetic variants and cell-free hemoglobin processing in sickle cell nephropathy,” Haematologica, vol. 100, no. 10, pp. 1275–1284, 2015.
- T. J. Orchard, W. Sun, P. A. Cleary et al., “Haptoglobin genotype and the rate of renal function decline in the diabetes control and complications trial/epidemiology of diabetes interventions and complications study,” Diabetes, vol. 62, no. 9, pp. 3218–3223, 2013.
- U. Wormser, J. Mandrioli, M. Vinceti et al., “Reduced levels of alpha-1-antitrypsin in cerebrospinal fluid of amyotrophic lateral sclerosis patients: a novel approach for a potential treatment,” Journal of Neuroinflammation, vol. 13, no. 1, 2016.
- H. Ma, Y. Lu, H. Li et al., “Intradermal αl-antitrypsin therapy avoids fatal anaphylaxis, prevents type 1 diabetes and reverses hyperglycaemia in the NOD mouse model of the disease,” Diabetologia, vol. 53, no. 10, pp. 2198–2204, 2010.
- C. Grimstein, Y.-K. Choi, C. H. Wasserfall et al., “Alpha-1 antitrypsin protein and gene therapies decrease autoimmunity and delay arthritis development in mouse model,” Journal of Translational Medicine, vol. 9, article 21, 2011.
- A. S. Elshikha, Y. Lu, M.-J. Chen et al., “Alpha 1 antitrypsin inhibits dendritic cell activation and attenuates nephritis in a mouse model of lupus,” PLoS ONE, vol. 11, no. 5, Article ID e0156583, 2016.
- N.-J. Kwak, E.-H. Wang, I.-Y. Heo et al., “Proteomic analysis of alpha-1-antitrypsin in immunoglobulin A nephropathy,” Proteomics—Clinical Applications, vol. 1, no. 4, pp. 420–428, 2007.
- Z. Guo, X. Liu, M. Li et al., “Differential urinary glycoproteome analysis of type 2 diabetic nephropathy using 2D-LC-MS/MS and iTRAQ quantification,” Journal of Translational Medicine, vol. 13, no. 1, 2015.
- A. Smith, V. L'Imperio, G. De Sio et al., “α-1-Antitrypsin detected by MALDI imaging in the study of glomerulonephritis: its relevance in chronic kidney disease progression,” Proteomics, vol. 16, no. 11-12, pp. 1759–1766, 2016.
- M. Navarro-Muñoz, M. Ibernon, J. Bonet et al., “Uromodulin and α1-antitrypsin urinary peptide analysis to differentiate glomerular kidney diseases,” Kidney and Blood Pressure Research, vol. 35, no. 5, pp. 314–325, 2012.
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