Screening New Urine Biomarkers Of IgA Nephropathy With High Rsolution Mass Spectrometry And Clinical Validation

Background and objective:IgA nephropathy(IgAN) is one of the most common primary glomerular diseases and the main cause of chronic kidney disease(CKD) and end-stage renal disease in the world. Currently, renal biopsy is still the gold standard of diagnosis of IgAN. As an invasive examination, renal biopsy is risky and can't be done repeatedly. Thus, finging out noninvasive biomarkers of IgAN is of great significance. This study aims at screening potential urine biomarkers of IgA nephropathy with high rsolution mass spectrometry and clinical validation, eveluating the application value the biomarkers and discussing the potential pathogenesis of IgAN.Methods:(1) Choosing 24 patients between 20 to 50 years old who were diagnosed with IgA nephropathy by renal biopsy from August 2014 to January 2015 in the PL A general hospital(Lee grade IgAN III, IgAN III-IV, IgAN IV, IgAN IV-V and IgAN V for 13 cases,4 cases,5 cases,1 case and 1 case in each group), and 30 healthy people between 20 to 50 years old without common diseases whose routine blood, blood biochemistry, routine urine examination were all negative. Collecting urine of the second time since morning of the IgAN patients and healthy people, detecting the urine samples with Lable free LS/MS and screening out differentially expressed proteins between them. Finally, Analysing results with GO(Gene Ontology) analysis and pathway enrichment.(2) According to the results of mass spectrometry combined with the potential pathogenesis of IgAN, we choose complement C3, complement C4, epidermal growth factor (EGF), iron metabolism related proteins including transferrin (TF), ceruloplasmin(CP), lactoferrin(LTF) and to verify. The above markers were validated in 10 IgAN,10 MCD,10 IMN and 10 healthy people by enzyme linked immunosorbent assay(ELISA). Analyzing correlation between these protein with common clinical and pathological markers such as serum creatinine(SCr), urea nitrogen(UN),24-hour urinary protein quantity, estimated glomerular filtration rate(eGFR) and the degree of complement C3 deposition in renal tissue.Results:(1) High resolution mass spectrometry dection found a total of 394 urinary proteins between IgAN and healthy people, in which 77 urinary proteins were only found in IgAN group. Besides,317 urinary proteins were found differentally expressed in IgAN patients and healthy people.152 urinary proteins were higher and 165 proteins were lower in IgAN group than in healthy people. GO analysis and pathway enrichment found meaningful differential urine proteins mainly involved in such biological processes or signal pathways as lipid metabolism, blood coagulation and thrombosis, iron metabollism, activation of complement system, call adhesion, cell motility, calcium signal pathway and MAPK signal pathway.(2) We used the ELISA method to validate these protein and found that urine complement C3, complement C4, TF and CP were all increased significantly in IgAN group, IMN group and MCD group compared with NC group. The urine TF increased more than 100 times in disease groups. Urine EGF were decreased significantly in IgAN group, IMN group and MCD group compared with NC group.But there were no differences among disease groups. Urine LTF decreased significantly in MCD group compared with NC group, IgAN group and IMN group. But there were no differences among NC group, IgAN group and IMN group. In addition, the level of urine complement C3 and complement C4 was positively related to the 24-hour urinary protein quantity and urine EGF was negatively related to the 24-hour urinary protein quantity.Conclusions:In the study, the unique urine expression profile was difined by high resolution mass spectrometry. Some urinary protein were validated by the ELISA method. The urinary transferrin might be a biomarker of early diagnosis of kidney diseases. The urinary complement C3, EGF, TF, CP and LTF might be a group of biomarkers which could be used to distinguish MCD and IgAN, IMN. In addition, urinary complement C3, complement C4 and EGF might reflect the activity of IgAN together with 24-hour urinary protein quantity.