Screening And Verifying The Protein Biomarkers From Urine In Patients With IgA Nephropathy

Background and objectives:IgA nephropathy (IgAN) is the most common primary kidney disease and the leading cause of end-stage renal disease in China and worldwide. Currently, the diagnosis of IgAN is still dependent on renal biopsy. Most of IgAN is progressive, however, renal biopsy cannot be done frequently and repeatedly for its invasive property. Therefore, non-invasive biomarkers of IgAN are needed. The aim of this study is to detect specific protein biomarkers from urine supernatant and determine its application value in IgAN.Methods:1.Urine supernatant is added to Raybiotech protein array. Then, all differential expression protein biomarkers between 15 IgAN patients (Lee grade IgAN-Ⅰ-Ⅱ, IgAN-Ⅲ and IgAN-Ⅳ-Ⅴ,5 in each group),12 glomerular disease control (DC) patients (MCD, MN and FSGS,4 in each group) and 5 normal control (NC) persons are screened out.2.Differential expressed protein biomarkers are further validated in a large urine samples from 49 IgAN patients (8 patients in Lee’s grade IgAN-Ⅰ-Ⅱ,31 in IgAN-Ⅲ and 10 in IgAN-Ⅳ-Ⅴ),28 DC patients (MCD 8 patients, MN 8 each and FSGS 12) and 14 NC persons by enzyme linked immune-sorbent assay (ELISA) method.Results:1.The results by Raybiotech protein array:8 differential expressed protein biomarkers were screened out between IgAN group and MCD group; 20 between IgAN group and MN group. Compared among the subgroups,6 differential expressed protein between IgAN-I-II group and MCD group,20 between IgAN-I-II and MN group,9 between IgAN-Ⅲ group and MN group; differential expressed protein between IgAN-Ⅳ-Ⅴ group and NC group, IgAN-Ⅳ-Ⅴ group and MN group in 3 each. Differential expressed protein between IgAN group and NC group, IgAN-Ⅰ-Ⅱ and IgAN-Ⅳ-Ⅴ group, IgAN-Ⅰ-Ⅱ and NC group, IgAN-Ⅲ and IgAN-Ⅳ-Ⅴ group, IgAN-Ⅲ and NC group were screened out, and each with 1 differential protein.2.Verifing research by ELISA:The expression level of E-Cadherin is significantly lower in IgAN group than in DC group (7.11±4.46 vs 13.92±7.65 ng/ml and 121.84±125.95 vs 749.00±684.09 pg/ml, p<0.01). Compared with MN (18.02±8.40ng/ml) and FSGS (14.51±8.10 ng/ml) groups, the expression level of E-Cadherin in IgAN groups is significantly decreased (7.11±4.46 ng/ml). The expression level of HER2/ErbB2 is significantly lower in IgAN group than in DC group. The level of HER2/ErbB2 is significantly decreased in IgAN group compared with MN (121.84±125.95 vs 990.22±406.18 pg/ml, p<0.01) and MCD groups (121.84±125.95 vs 1051.70±928.43 pg/ml, p<0.01). Lyve-1 in urine supernatantis is significantly higher in IgAN group than in NC group (684.98±223.35 vs 62.12±76.43 pg/ml, p<0.01).Angiostatin is significantly higher in IgAN group than in NC group (255.15±288.43 vs 3.31±5.96 ng/ml, p<0.05) but significantly lower than in DC group (255.15±288.43 vs 499.98±314.24 ng/ml, p<0.05). Further analysis revealed that angiostatin is also significantly decreased in IgAN group (255.15±288.43 ng/ml) compared with MN (579.47±211.69 ng/ml) and MCD (664.56±375.95 ng/ml) groups.3.The correlation analysis between protein biomarkers and clinicopathological factors:angiostatin showed a positive correlation with SCr (r=0.939,p<0.001), blood urea nitrogen(BUN, r=0.931,p<0.001), Cys C(r=0.923,p<0.001), U-Prot(r=0.784, p<0.001), global sclerosis (r=0.718, p<0.001) and crescent (r=0.588,p<0.001) There is a negative correlation between angiostatin and eGFR (r=-0.721, p<0.001) Angiostatin levels in severe IgAN group (Lee’s grade IgAN-Ⅳ-Ⅴ group) is significantly higher than in IgAN-Ⅰ-Ⅱ (620.43±271.47 vs 107.38±83.37 ng/ml, p<0.01) and IgAN-Ⅲ group (620.43±271.47 vs 117.89±143.84 ng/ml,p<0.01).In IgAN with endothelial cell proliferation group, the level of angiostatin is significantly higher than in IgAN without endothelial cell proliferation group (406.95±336.56 vs 189.41±234.70, p<0.01). Along with the increase of the severity in interstitial inflammatory, interstitial fibrosis and inflammatory cells infiltration, the level of angiostatin is increased.Conclusions:In this study, protein array technology was used for screening out more protein biomarkers from urine supernatant of IgAN. Some protein biomarkers were turn out to be good biomarkers in the non-invasive diagnosis and condition assessment of IgAN. The level of angiostatin is significantly differential expressed between IgAN group and control group. It has a good correlation with clinical and pathological indicators. Angiostatin may be a specific biomarker in IgAN and play a therapeutic role in the progression of the disease. However, its mechanism and clinical application needs further research.