IgA nephropathy (IgAN) is one of the most common glomerular diseases, and activation of the complement pathways plays an important role in its pathogenesis (1–6). Immune complexes containing IgA1 and autoantibodies are deposited in the kidney, activating complements and causing immune inflammation, finally leading to mesangial cell and tissue injury (1–3). Several early studies have confirmed that multiple components of complement system are positive in biopsy kidney tissues of IgAN, including C3, properdin, C4b Binding Protein (C4BP), etc. (3, 4). Renal biopsies of patients with IgAN showed glomerular deposition of mannose-binding lectin (MBL), L-ficolin, and MBL-associated serine proteases (MASP) (7). Collectin 11, an initiator of the complement lectin pathway, was involved in both acute kidney injury and chronic tubulointerstitial fibrosis (8). It suggested the involvement of the alternative and lectin pathways in the pathogenesis of IgAN (2–6, 9).

Increasing evidence has shown that the activated serum or glomerular complements were related to the clinical characteristics of IgAN patients (proteinuria, hematuria, renal function) and renal pathological changes (2–6). Urinary C3a and C5a levels correlated with crescents (10). Furthermore, glomerular deposition of FHR5, C3, C3dg, C4d, C5b9, and collectin 11 was associated with IgAN progression (8, 11–13). The arteriolar C4d in IgAN kidney biopsy tissue was associated with mean arterial pressure, chronic microangiopathy, and disease progression (14). Complement factor H (CFH) and complement factor H-related protein genes (CFHRs) were also investigated. CFHR-5 was associated with kidney histological severity (15, 16) and serum CFHR-5 was independently associated with the progression of IgAN (12, 15).

Previous studies have often focused on one or several complement components. The different positive rates of different complement proteins in serum, glomeruli, or mesangial areas of IgAN patients might be influenced by the disease status or treatment (13). There is a lack of data about complement activation profile of IgAN patients. The progression in liquid chromatography-tandem mass spectrometry (LC–MS/MS)-based proteomic studies provides an important opportunity for systematic complement analysis. Our previous studies demonstrated that it could reveal the characteristics of proteome and protein glycosylation (17, 18), and provide noninvasive measures for differential diagnosis of kidney diseases (19). The high-quality proteomic assay based on mass spectrometry can reveal a complete profile of the complement activation in IgAN patients.

Therefore, we used proteomic analysis to detect the complements in the urine and analyze their correlation with the clinical and pathological manifestations of IgAN patients. In this study, we first performed liquid chromatography-tandem mass spectrometry analysis of urine proteins from patients with different renal diseases as well as healthy controls, to obtain urine complement profile in patients with IgAN. The correlation between complement activation and clinical or pathological parameters in IgAN patients was analyzed. Then, an enzyme-linked immunosorbent assay (ELISA) was used to validate the complement components in an isolated IgAN group. It is expected to provide information for the monitoring and treatment of IgAN patients, especially for complement-targeted therapy.

This is a concise version. Please check the Supplementary Materials for the complete version.

The complement system consists of nearly 60 proteins, which include proteins in three classical, lectin and alternative activation pathways (22), proteases interacting in a cascade-like fashion, multiple regulatory factors, and receptors (23). In this study, we used untargeted proteomic studies that first reported the urinary complement profile in IgAN. We detected 44 urinary complement proteins by LC–MS/MS and selected 7 complement proteins for validation by ELISA.

The complement system serves as part of the innate immune system. In recent years, the role of the complement system in IgAN has attracted more attention (14, 16, 24, 25). It has been reported that glomerular MASP1/3 and MASP2 deposition was associated with crescent formation (26) and deposition of arteriolar C4d of the kidney was associated with progressive kidney disease in IgAN (14). Furthermore, the role of CFH and CFHRs in IgAN has attracted extensive attention from researchers (12, 16, 27, 28) since genome-wide association studies showed that deletion of the CFHR1-3 gene is highly protective in patients with IgAN (29, 30). Higher plasma CFHR5 levels were associated with severe histological features (15), proteinuria, hypertension, and reduced eGFR (12) in IgAN. The previous findings were mostly derived from serum or renal tissue, and only a few data from urine samples of IgAN patients. Changes in urine complement proteins could reflect complement overproduction, secretion, and/or deposition in the damaged kidney. In our study, we identified multiple up-regulated and down-regulated complement proteins in the urine of patients with IgAN ( Figure 4 ).

Our results reveal that the urinary complement proteins of abnormal abundance are mainly enriched in the alternative and terminal pathways. A growing body of evidence implies that the activation of alternative pathways especially contributes to severe chronic lesions of kidney in IgAN patients (31). High abundance of urinary CFH and CFHR2, two members of the alternative pathway, is closely associated with glomerulosclerosis and TIF. It indicates that the activation of CFH and CFHR2 is a clue of the chronicity of IgAN. Our study agrees with previous studies (32, 33) and re-emphasizes the role of CFH and CFHRs in renal chronic damage in IgAN patients and their potential value as biomarkers of chronic injury. CFH is one of the most important circulating regulators of the alternative pathway and can accelerate the decay of the alternative pathway C3 convertase. Our results demonstrate that the CFH was positively correlated with 24h proteinuria and negatively correlated with eGFR. It is inconsistent with the results of CFH in serum. We speculate that the increase in proteinuria might be accompanied by an increase in urine CFH excretion (molecular weight of 155KDa), resulting in a decrease in circulatory CFH in IgAN patients. It has also been shown in previously published study (33). CFHR5 was an independent risk factor for IgAN progression (12, 15). CFHR genes are mainly transcribed in the liver and the proteins are distributed in plasma. CFHR proteins are emerging complement modulators, activators, and immune regulators (34). Each CFHR protein binds to C3b, iC3b, C3dg, and C3d. Several CFHR proteins interact with ligands such as glycosaminoglycans, monomeric CRP, pentraxin-3, malondialdehyde, and laminins of the kidney, and bind to the surfaces of apoptotic and necrotic cells (34). For the first time, we found a high abundance of CFHR2 in the urine of IgAN patients. It suggests that the liver might contribute to the overproduction of CFHR2, which is filtered from the glomeruli to the urine and could play an important role in the progression of IgAN. The evidence from previous and our studies indicates that complement activation is associated with the development of glomerular and tubulointerstitial injury.

The urinary CFB and C3 are correlated with hematuria, which suggests that CFB and C3 are associated with the activity of IgAN. In particular, C6 and CD55, which are associated with the cellular/fibrocellular crescents, indicate that IgAN is in pathological activity. Notably, our proteomic analysis showed a negative correlation between urinary CD55 and proteinuria, which is consistent with the study of Zhao et al. (35), suggesting a protective effect of CD55 on IgAN progression. Moreover, an interesting finding was that the difference of C1r and C1s were statistically significant between IgAN and healthy controls. Previous studies have shown that the lectin and alternative pathways were important in complement activation in patients with IgAN, but there was a lack of evidence for activation of the classical pathway. Our results from urine analysis imply the activation of C1r and C1s of the classic complement pathway in IgAN. It implied that the activation of classical pathway also participated in the pathogenesis of IgAN and need further study.

Collectin12, a member of the collectin family, is a pattern recognition molecule that initiates complement activation via the alternative pathway (36, 37). Collectin12 is coded by the collagen lectin gene COLEC12 (38). Previous studies have shown that colec12 correlates with the severity of diabetic retinopathy (39), involving in myelin internalization (40) and promotes IL-23 expression by dendritic cells (41). Our study finds that urinary colec12 correlated with fibrous crescents and glomerulosclerosis in IgAN. It is the first report which demonstrates the renal chronic lesion of IgAN was related to urinary colec12.

Therapeutic complement regulation has attracted the attention of physicians. Several ongoing clinical trials targeting complements have included IgAN patients. The interventions targeting factor B in IgAN included small molecule LNP023 (NCT03373461), an antisense inhibitor of CFB mRNA IONIS-FB-LRx (NCT04014335). Moreover, a humanized monoclonal antibody to MASP-2, narsoplimab, is used to treat IgAN patients with persistent proteinuria (NCT03608033). There is an emerging need for complement-targeted therapy monitoring, and thus our study provides a highlight for future complement-targeted therapy and a potential basis for effective monitoring of complement interventions, particularly in IgAN.

An advantage of our study was the comprehensive quantification of urinary complement proteins using a targeted mass spectrometric method. It presents a profile of urinary complement protein in IgAN patients. The correlation between urinary complement proteins and clinical as well as pathological characteristics was analyzed. However, this study has several limitations. The origins of the complement proteins detected in urine were unclear. Moreover, we did not monitor the dynamic changes of the complements after the treatment of the IgAN by different agents.

In conclusion, multiple abnormally increased components of complement system were detected in the urine of IgAN patients, suggesting that the abnormal activation of complement system in IgAN patients is obvious. A high abundance of urinary complement proteins was significantly associated with clinical or pathological parameters. These components could be potential biomarkers for monitoring IgAN and provide clues for therapeutic choice targeting complement system of IgAN patients.

The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the iProX partner repository with the dataset identifier PDX038451.

The studies involving human participants were reviewed and approved by the Medical Ethics Committees at the Sichuan Provincial People’s Hospital. The patients/participants provided their written informed consent to participate in this study.

DW designed and performed experiments, collected and analyzed data, interpreted experimental results and wrote the manuscript. CW collected the samples and clinical data. CW and YZ critiqued the experimental design. YZ performed liquid chromatography-tandem mass spectrometry (LC–MS/MS) analyses. DW performed ELISA experiments and conducted most of the experiments with help from SC, YL, and LW. GL designed the project, led the research team, analyzed data, and critiqued the manuscript. All authors have reviewed the manuscript. All authors contributed to the article and approved the submitted version.