Metabolomics Strategies For Discovering Chronic Kidney Disease-Mineral And Bone Disorder Related Biomarkers And Investigating The Mechanism Of Bone Metabolism Toxicity

Chronic kidney disease-mineral and bone disorder(CKD-MBD)characterized by systemic disorders,is a common complication for chronic renal failure,which is associated with decreased quality of life and increased cardiovascular morbidity and mortality in CKD.However,even as the gold standard for CKD-MBD diagnosis,the application of bone biopsy is failed to popularize,and the current applied biochemical indicators for CKD-MBD diagnosis are challenged due to limited specificity and sensitivity.Therefore,more reliable biomarkers for CKD-MBD diagnostic are particularly urgent to be found.Protein bound uremic toxins(PBUTs)can be accumulated in vivo due to renal dysfunction and can’t be removed effectively by conventional dialysis,furthermore,many reports have suggested that PBUTS are associated with the onset and progression of CKD-MBD,and PBUTs could be the potential biomarkers for CKD-MBD.However,the evaluation standard for PBUTs level in kidney failure is relatively lack,and the real toxicity mechanism of PBUTs in CKD-MBD is not clear,which also limits the discovery of therapeutic targets and new drugs.Therefore,by combining metabolic profile analysis and metabolic targeted analysis to identify CKD-MBD related biomarkers,and develop the quantitative method for the representative PBUTs.Furthermore we took pcresol sulfate(PCS)as an example,and integrated metabolomics and transcriptomics to uncover the potential toxicity mechanism of PBUTs on bone metabolism.The main contents are as follows:1.Firstly,we applied UHPLC-QTOF/MS based serum metabolic profile analysis to three typical clinical manifestations of CKD-MBD,including: two cross-sectional studies:(1)A serum metabolic profiling method based on UHPLC-QTOF/MS analysis was applied to investigate the metabolic changes related with secondary hyperparathyroidism(SHPT),32 potential serum biomarkers that were primarily involved in protein synthesis and metabolism,amino acid metabolism,energy metabolism and steroid hormones metabolism,were screened and identified,and the performance of the prediction model constructed by these biomarkers for the diagnosis of SHPT were evaluated,where 71.1% accuracy,73.7% sensitivity and 68.4% specificity were achieved respectively,and the prediction results suggested that these potential biomarkers identified appeared to have excellent diagnostic ability for CKD-MBD,and need to be further investigated;(2)A serum metabolic profiling method based on UHPLC-QTOF/MS analysis was applied to investigate the metabolic changes related with uremic pruritus(UP),combing multivariate and univariate analysis,22 potential biomarkers related with UP were identified.Nine of the 22 metabolites in combination were characterized by a maximum area-under-receiver operating characteristic curve(AUC= 0.899)with a sensitivity of 85.1% and a specificity of 83.0% for the diagnosis of UP.Our results indicate that serum metabolic profiling might serve as a promising approach for the diagnosis of uremic pruritus and that the identified biomarkers may improve the understanding of pathophysiology of this disorder;one intervention study:(3)CKD-MBD can cause blood system dysfunction,and renal anemia is its clinical manifestation.In order to identify patients who will respond to IV iron therapy and who will not respond to,a metabolic profiling method based on UHPLCQTOF/MS analysis was applied to investigate the serum from 41 renal anemia patients with complete,partial or non-response to IV iron therapy and multivariate analysis methods were used to identify the potential biomarkers that could predict the response to IV iron therapy in renal anemia patients.Oleamide and ascorbate 2-sulfate(AS)were identified and verified as potential biomarkers for renal anemia.A prediction model constructed with oleamide and AS correctly identified approximately 83.3% of patients who were non-responsive to IV iron therapy and 87.5% of patients who had a complete response to IV iron therapy.The model has excellent discriminant performance,with an AUC of 0.901.These results show promise for larger studies that could advance more personalized treatment protocols for renal anemia patients.In general,the three LC-MS based metabolic profiling for CKD-MBD related bone disorder,blood dysfunction and skin abnormalities have identified the potential serum biomarkers for CKD-MBD,and provided some new evaluation index for the diagnosis,intervention and treatment for this systemic disorder.2.Next,based on differences between the results from three serum metabolic profile analysis and the observation for the significant correlation of PBUTs with CKD-MBD reported previously,and the fact that lack of evaluation system for the real levels of PBUTs in renal failure.We took the fifteen representative PBUTs(Nδ-(Carboxymethyl)lysine,Pentosidine,Kynurenine,Quinolinic acid,Kynurenic acid,pHydroxyhippuric acid,p-Cresyl sulfate,Hippuric acid,4-Ethylphenyl sulfate,Melatonin,Phenylacetic acid,CMPF)as the analysis targets,and different protein precipitation reagents and different types of solid phase extraction pellets were systematically compared for the extraction efficiency of PBUTS in serum,and developed a high performance liquid chromatography-electrospray ionization-tandem quadruple mass spectrometry(HPLC-ESI-MS/MS)for simultaneous determination fifteen PBUTs in serum by using carbamazepine and ketoprofen as internal standards in positive and negative mode respectively.Then we used the developed method for determination of PBUTs in serum of uremic patients and healthy control after passing the methodology validation.The results of serum preparation showed that precipitation with methanol in 96 well non-drip filtration plate for lipid and protein depletion can increase the absolute response of PBUTs in serum and can be an alternative option for extract PBUTs.The results of methodology showed that the established method is suitable for the analysis and detection of PBUTs levels in actual human samples,and the quantitative measurement results show that the fifteen PBUTs levels were significantly increased in uremic patients when compared with the healthy controls.This study not only provides a new sample treatment for MS-based metabolomics of CKD-MBD,but also expand our understanding of the true level of PBUTs in uremic population with the developed quantitative methods.Moreover,the quantitative results indicated that some other PBUTS with abnormal levels and potential toxic effects,except for PCS and IS.In total,this research has focused on PBUTs group,combing optimization of sample pretreatment and development quantitative methods for PBUTs,which provides an platform for revealing the true level of PBUTs in uremic patients.3.Based on the results from metabolic targeted analysis,we found there is a close relationship between increased PUBTs levels with the development of CKD-MBD,therefore,we took PCS as an example and the MC3T3-E1 osteoblast cell as the study target,a cell metabolic profiling method based on UHPLC-QTOF/MS analysis was applied to investigate the metabolic changes in PCS treated groups and identified 74 potential metabolic biomarkers in cell extraction,with ingenuity pathway analysis(IPA),15 unique pathways were enriched by the identified metabolites variables.Simultaneously,a cell transcriptomics method based on RNA-sequence analysis was applied to investigate the differentially expressed genes in PCS treated groups and identified 3679 potential gene biomarkers,with signal transduction pathways significant analysis,58 unique pathways were enriched by the identified gene variables.Furthermore,glutathione metabolism and glycerophospholipid metabolism were enriched and significantly altered at both metabolic level and gene level when compared with the pathways enriched by metabolic variables and gene variables.In addition,based on the metabolites variables and differentially expressed genes data imported in IPA,the biological functions changed in PCS treated groups were also enriched,including upregulated cell apoptosis and down-regulated cell cycle,DNA replication,recombination and repair function,and these mentioned biological dysfunction were consistent with the results of cell activity,cell apoptosis and cell cycle tests before.This study has uncovered the possible toxic effect mechanism of PBUTs in bone metabolism by integrating transcriptomics and metabolomics,which provides an alternative attempt for pathogenesis study of PBUTs.In summary,through combing metabolic profiling analysis,metabolic target analysis,transcriptomics and various bioinformatics strategies,our study has screened and identified the potential biomarkers for early diagnosis and response evaluation of CKDMBD,established the evaluation platform for real levels of PBUTs in vivo,and further integrated transcriptomics and metabolomics to uncover the possible toxic effect mechanism of PBUTs in bone metabolism,which provide an alternative attempt for pathogenesis study of PBUTs.