Proteomic-based Research Of Urinary Biomarker Of Glomerular Disease

Glomerular disease is still the most common kidney disease and the leading cause of end stage renal disease(ESRD).Exploring new biomarker of glomerular disease to improve its diagnosis and treatment is important.Proteomics method is increasingly being used in clinical research,involved in finding markers about diagnosis,prognosis,drug efficacy and adverse reactions.Duo to non-invasive,easy collection,and carrying information directly from kidney,urine is preferred choice for searching biomarkers of kidney disease.Urine proteome of glomerular disease is special.Because of disrupt of infiltration membranous,a large number plasma protein leaked into the urine,which exacerbate inhibition of high abundance and increase dynamic range of protein concentration.In this situation,it is hard to get a full picture of urine proteome of glomerular disease and finding useful biomarker of low abundance.We first used albumin/IgG antibody depletion combined with two-dimensional liquid chromatography tandem Mass spectrometry(2D-LC/MS/MS)to overcome the large dynamic range as seen in urine of patients with severe proteinuria to mine the lower abundance protein and obtained a comprehensive urine proteomic data of these patients and a number of differential protein between FSGS and MN.We use these information to further explore the biomarker of glomerular disease.Part Ⅰ Establishment of comparative urine proteomics research strategy in patients with glomerular diseaseObjective:To achieve comprehensively urine proteomics profile and screen differentially expressed urine protein in membranous nephropathy(MN)and focal segmental glomerulosclerosis(FSGS)patients with nephrotic syndromeMethods:15 MN patients and 15 FSGS patients with nephrotic syndrome were selected.Their urine samples were analyzed by Albumin/IgG antibody depletion combined with iTRAQ(isobaric tags for relative and absolute quantitation)labeled 2D-LC MS/MS(Two dimensional liquid chromatography-tandem mass spectrometry)for profiling urine proteomics and screening differentially expressed urine proteins.GO(Gene ontology)analysis was used to categorize identified protein and according to molecular function、biological process and cellular component.Results:A total of 809 urine proteins were identified in MN and FSGS patients with nephrotic syndrome,of which the 10 most abundance proteins are:serotransferrin,alpha-1-acid glycoprotein 1,alpha-1-acid glycoprotein 2,ceruloplasmin,zinc-alpha-2-glycoprotein,alpha-1-antitrypsin,haptoglobin,alpha-1B-glycoprotein,serum albumin,protein AMBP.GO analysis revealed that the molecular function of identified protein mainly included catalytic activity、binding and receptor activity,the biological process mainly involved metabolic process、cellular process and development process,the cellular component mainly contains extracellular region、cell part and extracellular matrix.90 proteins which fulfilled the criteria was choose as differentiated proteins between FSGS and MN.Conclusion:Our proteinuria proteomics profile is the largest for now and provides a basis for future study.Our differentially expressed protein need to be validated as biomarkers for diagnosis、therapy and prognosis.Part Ⅱ Proteomics-based screening of disease-associated proteins in the urine of patients with kidney diseaseObjective:To search for disease-associated proteins in the urine of patients with kidney disease by proteomics-based screening.Methods:Proteomic data of the urine of kidney disease patients was obtained from profile of pooled sample of patients with nephrotic syndrome.Proteomic data of the plasma,urine,and kidney of normal individuals were obtained from the 2013 PeptideAtlas database.Proteins in the urine proteome of kidney disease patients were compared to those of normal individual with protein abundance enrichment analyze.The achieved proteins were defined as disease-associated proteins.Of these,that were present in the proteome of the urine of kidney disease patients and kidney of normal individuals,but were not present in the proteome of the urine and plasma of normal individuals were considered as injured kidney-derived components.disease-associated proteins were validated in independent cohort including 50 FSGS,50MN,50 normal control.Results:Forty disease-associated proteins were identified,one of these protein,Mannose-binding lectin(MBL),was successfully validated in an independent cohort.Four injured kidney-derived components in disease-associated proteins were identified:ubiquitin-60S ribosomal protein L40,S-phase kinase-associated protein 1,transforming growth factor beta 2 receptor,and glutathione S-transferase A3.Conclusions:Our study identified 40 potential biomarkers for kidney disease.These biomarkers need to be further validated and explored for clinical meaning.Part Ⅲ Discovery of a new biomarker differentiating FSGS and Minimal change diseaseObjective:To search for biomarker differentiating FSGS and minmal change disease(MCD)by validating differentiated proteins indentified in discovery phase of proteomics.Methods:urinary fibrinogen as differentiated protein identified in previous study was validated by ELISA in independent cohorts containing patients with FSGS (n=50),and MCD(n=40),and normal controls(n=50).Fibrinogen was detected by immunofluorescence in kidney biopsy sample of patients with FSGS and MCD.renal interstitial fibrosis of patients with FSGS was quantitative analysis by Aperio ScanScope system.Results:It was found that although urinary fibrinogen level was higher both in FSGS and MCD as compared with normal controls(4425,1529-13153 ng/mg creatinine versus 2.18,1.63-2.85 ng/mg creatinine,P<0.001;169.9,76.89-391.6 ng/mg creatinine versus 2.18,1.63-2.85 ng/mg creatinine,P<0.001).Urinary fibrinogen levels in FSGS patients(4425,1529-13153 ng/mg creatinine)were significantly higher than that of MCD(169.9,76.89-391.6 ng/mg creatinine,P<0.001).Urinary fibrinogen allowed distinguishing between FSGS and MCD with an AUC of 0.957(95%confidence interval 0.920-0.994,P<0.001),and a sensitivity of 88%and specificity of 91.5%with an optimized cutoff value of 800 ng/mg creatinine.Further study showed that urinary fibrinogen levels were positively correlated with 24 hour urine protein(r=0.359,P=0.01),and two tubular injury markers:urinary N-acetyl-β-D-glucosaminidase(NAG)(r=0.447,P=0.001)and retinol-binding protein(RBP)(r=0.558,P<0.001).We also identified an increased fibrinogen immunostaining in tubulointerstitial space of FSGS patients,compared to MCD patients.Conclusions:These results suggested that urinary fibrinogen is a potential biomarker differentiating FSGS from MCD,and higher level of urinary fibrinogen may also indicate the presence of marked tubulointerstitial lesions in these patients.