Urinary complement proteins in IgA nephropathy progression from a relative quantitative proteomic analysis

Abstract

Aim: IgA nephropathy (IgAN) is one of the leading causes of end-stage renal disease (ESRD). Urine testing is a non-invasive way to track the biomarkers used for measuring renal injury. This study aimed to analyse urinary complement proteins during IgAN progression using quantitative proteomics.

Methods: In the discovery phase, we analysed 22 IgAN patients who were divided into three groups (IgAN 1-3) according to their estimated glomerular filtration rate (eGFR). Eight patients with primary membranous nephropathy (pMN) were used as controls. Isobaric tags for relative and absolute quantitation (iTRAQ) labelling, coupled with liquid chromatography-tandem mass spectrometry, was used to analyse global urinary protein expression. In the validation phase, western blotting and parallel reaction monitoring (PRM) were used to verify the iTRAQ results in an independent cohort (N = 64).

Results: In the discovery phase, 747 proteins were identified in the urine of IgAN and pMN patients. There were different urine protein profiles in IgAN and pMN patients, and the bioinformatics analysis revealed that the complement and coagulation pathways were most activated. We identified a total of 27 urinary complement proteins related to IgAN. The relative abundance of C3, the membrane attack complex (MAC), the complement regulatory proteins of the alternative pathway (AP), and MBL (mannose-binding lectin) and MASP1 (MBL associated serine protease 2) in the lectin pathway (LP) increased during IgAN progression. This was especially true for MAC, which was found to be involved prominently in disease progression. Alpha-N-acetylglucosaminidase (NAGLU) and α-galactosidase A (GLA) were validated by western blot and the results were consistent with the iTRAQ results. Ten proteins were validated in a PRM analysis, and these results were also consistent with the iTRAQ results. Complement factor B (CFB) and complement component C8 alpha chain (C8A) both increased with the progression of IgAN. The combination of CFB and mucosal addressin cell adhesion molecule-1 (MAdCAM-1) also showed potential as a urinary biomarker for monitoring IgAN development.

Conclusion: There were abundant complement components in the urine of IgAN patients, indicating that the activation of AP and LP is involved in IgAN progression. Urinary complement proteins may be used as biomarkers for evaluating IgAN progression in the future.

Keywords: Complement proteins; IgA nephropathy; Proteomics; Urine; α-N-acetylglucosaminidase.

Figure 1. The workflow of differential urinary proteome analysis, western blotting and parallel reaction monitoring validation.

Figure 2. Hierarchical clustering analysis of 747 urinary proteins from four pooled samples.

(A) Heatmap of urinary proteins in IgAN subgroups and pMN group (two technical replicates). The color bar from orange to blue represented the fold change from increasing to decreasing of the proteins in four groups. (B) Mfuzz correlation analysis of urinary proteins among IgAN subgroups. Proteins in cluster 1 were increased with IgAN progression; Proteins in cluster 2 were decreased with IgAN progression; Proteins cluster 3 (n = 142) and Cluster 4 (n = 291) showed no gradual changes with IgAN progression. Color bar represented Z-score change from −2 to 2.

Figure 3. Functional analysis of urinary proteins in cluster 1 and cluster 2 of IgAN patients.

(A) KEGG pathway enrichment analysis of proteins in cluster 1; (B) protein-protein interaction (PPI) networks were created for cluster 1 proteins; (C) KEGG pathway enrichment analysis of proteins in cluster 2; (D) PPI networks were created for cluster 2 proteins. In the KEGG enrichment analysis, the abscissa represented the percent of corresponding genes under each pathway classification (p value was noted in the bracket). The ordinate represented enrichment pathway. In the protein interaction networks, circle nodes represented for genes/proteins, rectangle for KEGG pathways. Blue solid lines represented inhibition; red solid lines represented activation; blue dotted lines represented KEGG pathways. The significance of the pathways represented by −log (p value) was shown by color bar with dark blue as the most significant. The color of circle nodes from red to blue represented the fold change from increasing to decreasing of the proteins.

Figure 4. Comparison of urinary complement proteins in IgAN 1-3 and pMN patients.

CP, classical pathway; LP, lectin pathway; AP, alternative pathway; MAC, membrane attack complex.

Figure 5. Western blotting validation of NAGLU and GLA in another patient cohort.

In the WB experiments, equal amount of urine protein from each individual sample was loaded. The validation set included IgAN-1 (n = 6), IgAN-2 (n = 8), IgAN-3 (n = 3) and pMN (n = 5) patients. Means and standard deviations were represented in the figure, and a nonparametric test were used to analysis the data. * Indicates a p value < 0.05.

Figure 6. The PRM results of validated differential proteins.

IgAN-1: eGFR >90 ml/min/1.73 m², IgAN-2: eGFR: 60–90 ml/min/1.73 m², IgAN-3: eGFR: 30–60 ml/min/1.73 m². * Indicates a p value < 0.05, ** indicates a p value < 0.01, *** indicates a p value < 0.001, **** indicates a p value < 0.0001, ns indicates a p value > 0.05. CFB, complement factor B; C8A, complement component C8 alpha chain; SERPINA5, plasma serine protease inhibitor; CD58, lymphocyte function-associated antigen 3; MADCAM1, mucosal addressin cell adhesion molecule 1; AXL, tyrosine-protein kinase receptor UFO; ZG16B, zymogen granule protein 16 homolog B; EGF, pro-epidermal growth factor; DNASE2, deoxyribonuclease-2-alpha; LRG1, leucine-rich alpha-2-glycoprotein.

Figure 7. The analysis of PRM results.

(A) Receiver operating characteristic (ROC) analysis of IgAN-2 and IgAN-1; (B) ROC curve analysis of IgAN-3 and IgAN-1; (C) heatmap of correlations between PRM results and clinical tests. Color bar represented the correlations from −0.6 to 0.6. eGFR, estimated glomerular filtration rate; C3, complement component 3; C4, complement component 4; 24hUP, 24-h urinary protein; BUN, blood urine nitrogen; MAP, mean arterial pressure; M, mesangial hypercellularity; E, endocapillary cellularity; S, segmental sclerosis; T, interstitial fibrosis/tubular atrophy; C, crescents.