Background and objectives: Increased circulating galactose-deficient IgA1 and subsequently complement activation both play important roles in the pathophysiology of IgA nephropathy. However, their relationship to disease severity and progression remains unclear. Design, setting, participants, & measurements: We assessed 1210 participants in a cohort study of biopsy-proven IgA nephropathy at Peking University First Hospital. Plasma concentrations of galactose-deficient IgA1 and complement component C3 were measured at the time of biopsy. We tested associations of galactose-deficient IgA1 and galactose-deficient IgA1/C3 ratio with CKD progression event, defined as ESKD or 50% decline in eGFR, using Cox proportional hazards models and restricted cubic splines. Results: After a median follow-up of 43 months (interquartile range, 24–76 months), 172 (14%) participants reached the CKD progression event. The association of galactose-deficient IgA1 levels and CKD progression event showed a nonlinear relationship. The risk of CKD progression events was greater with higher plasma galactose-deficient IgA1 levels but reached a plateau when galactose-deficient IgA1>325 U/ml, whereas the risk of CKD progression events monotonically increased with higher galactose-deficient IgA1/C3 ratio. After adjustment for traditional risk factors (demographics, eGFR, proteinuria, hypertension, Oxford pathologic score, and corticosteroids/immunosuppressive therapy), higher levels of galactose-deficient IgA1/C3 ratio were independently associated with CKD progression event (per natural log-transformed [galactose-deficient IgA1/C3], hazard ratio, 2.03; 95% confidence interval [95% CI], 1.25 to 3.29; P=0.004). In reference to the first quartile of the galactose-deficient IgA1/C3 ratio, hazard ratios were 1.71 (95% CI, 1.01 to 2.89) for the second quartile, 1.55 (95% CI, 0.91 to 2.63) for the third quartile, and 2.17 (95% CI, 1.33 to 3.56) for the fourth quartile. Conclusions: In IgA nephropathy, plasma galactose-deficient IgA1/C3 ratio was associated with CKD progression event independent of clinical and biopsy characteristics.

Monoclonal immunoglobulin produced by clonal plasma cells is the main cause in multiple myeloma and monoclonal gammopathy of renal significance. Because of the complicated purification method and the low stoichiometry of purified protein and glycans, site-specific N-glycosylation characterization for monoclonal immunoglobulin is still challenging. To profile the site-specific N-glycosylation of monoclonal immunoglobulins is of great interest. Therefore, in this study, we presented an integrated workflow for micro monoclonal IgA and IgG purification from patients with multiple myeloma in the HYDRASYS system, in-agarose-gel digestion, LC-MS/MS analysis without intact N-glycopeptide enrichment, and compared the identification performance of different mass spectrometry dissociation methods (EThcD-sceHCD, sceHCD, EThcD and sceHCD-pd-ETD). The results showed that EThcD-sceHCD was a better choice for site-specific N-glycosylation characterization of micro in-agarose-gel immunoglobulins (~2 μg) because it can cover more unique intact N-glycopeptides (37 and 50 intact N-glycopeptides from IgA1 and IgG2, respectively) and provide more high-quality spectra than sceHCD, EThcD and sceHCD-pd-ETD. We demonstrated the benefits of the alternative strategy in site-specific N-glycosylation characterizing micro monoclonal immunoglobulins obtained from bands separated by electrophoresis. This work could promote the development of clinical N-glycoproteomics and related immunology.

Recent studies discovered the prominent presence of anti-nephrin autoantibodies in minimal change disease (MCD) and focal segmental glomerulosclerosis (FSGS). However, widely different and often unconventional autoantibody detection methods were used in these studies, making it challenging to standardize anti-nephrin antibody detection and quantification across different studies. Here, we compare methods of conventional ELISA, immunoprecipitation (IP)-based on-beads ELISA, immunoprecipitation-Western blotting (IP-WB), and cell- and tissue-based immunofluorescence staining with two cohorts totaling 169 patients and control individuals. Different assay methods and antigen preparations led to method-specific false-positive and false-negative results. In general, high-quality antigens produced in human cells, combined with IP-based assays, yielded the most robust and reliable results. Among 63 and 24 samples from patients with FSGS or MCD, respectively, two patients with FSGS showed strong antibody signals in both ELISA-based assays and IP-WB, while approximately half of patients with MCD had weak signals detectable only by IP-WB. These findings highlight the importance of standardizing antibody detection methods.

Background: IgA nephropathy is a common primary glomerulonephritis caused by mesangial deposition of poly-IgA complexes. The disease follows a variable course of clinical progression, with a high risk of kidney failure. Although no specific therapy is available, enzymatic strategies to clear IgA deposits are being considered for the treatment of rapidly progressive IgA nephropathy. Methods: We chose an IgA protease of commensal bacterium Clostridium ramosum, termed AK183, as the template for constructing a recombinant biologic. To extend the t _1/2 in blood, we fused AK183 to the Fc segment of human IgG1. Activities of this Fc-AK183 fusion protein toward the cleavage and subsequent clearance of IgA were tested in mouse models. Results: First, we discovered an autocleavage activity of AK183 that separates the N-terminal protease from its C-terminal autotransporter β domain. Therefore, we grafted Fc to the N terminus of AK183 and demonstrated its week-long enzymatic activity in mice. In addition, the proteolytic fragments of IgA generated in the reaction with Fc-AK183 were effectively removed from circulation via kidney filtration. The combined actions of Fc-AK183-mediated cleavage and subsequent renal clearance of IgA resulted in a lasting obliteration of blood IgA, as demonstrated in a human IgA-injection model and in a humanized α1KI transgenic model. Fc-AK183 was also able to remove chronic IgA and associated complement C3 deposits in the glomerulus. Conclusion: We constructed a chimeric fusion of IgA protease with Fc and demonstrated its long-lasting efficacy as a promising targeted therapy for IgA nephropathy in mouse models.

Background and objectives: Poly-IgA immune complex formation and glomerular deposition play a key role in IgA nephropathy. Our study sought to develop a new methodology for one-step serologic detection of poly-IgA levels. Design, setting, participants, & measurements: A novel ELISA method using recombinant CD89 as a “capturing” probe was established for detecting poly-IgA immune complex in plasma. We applied semiquantitative measurements of these poly-IgA indices in patients recruited at Peking University First Hospital who had IgA nephropathy or other kidney disease types, as compared with healthy controls. The longitudinal trend of the poly-IgA index and the association with pathologic parameters and treatment responses were evaluated. Finally, we analyzed the molecular composition of poly-IgA complexes in patients by mass spectrometry. Results: Recombinant CD89–mounted ELISA plates specifically captured plasma poly-IgA. The levels of poly-IgA immune complex (26.7 [interquartile range (IQR) 17.1–42.6] U/ml) in IgA nephropathy were significantly higher than those in healthy controls (15.5 [IQR 10.7–20.0] U/ml; P<0.001) or in controls with non-IgA nephropathy disease (14.8 [IQR 10.5–21.9] U/ml; P<0.001). Higher levels of poly-IgA immune complex were associated with lower eGFR and worse kidney outcome. Accuracy parameters and concordant statistics showed good discrimination between IgA nephropathy and healthy controls based on poly-IgA index levels (area under the curve [AUC], 0.78; 95% confidence interval [95% CI], 0.72 to 0.83; P<0.001), significantly outperforming galactose-deficient IgA1 levels (AUC, 0.70; P=0.05). Corticosteroid and immunosuppressant treatments lowered poly-IgA indices. After a recombinant CD89–directed workflow in conjunction with mass spectrometry, we also analyzed the molecular composition of IgA immune complex in patients with IgA nephropathy. Conclusions: Higher level of recombinant CD89–bound poly-IgA immune complex was associated with the severity of the disease and with treatment response to steroids and immunosuppressants.

Introduction: Galactose-deficient IgA1 (GdIgA1) is critical in the formation of immunodeposits in IgA nephropathy (IgAN), whereas the origin of GdIgA1 is unknown. We focused on the immune response to fecal microbiota in patients with IgAN. Methods: By running 16S ribosomal RNA gene sequencing, we compared IgAN samples to the control samples from household-matched or non-related individuals. Levels of plasma GdIgA1 and poly-IgA complexes were measured, and candidate microbes that can either incite IgA-directed antibody response or degrade IgA through specific IgA protease activities were identified.Results: The IgAN group showed a distinct composition of fecal microbiota as compared to healthy controls. Particularly, high abundance of Escherichia-Shigella was associated with the disease group based on analyses using receiver operating characteristic (area under curve, 0.837; 95% CI, 0.738–0.914), principle coordinates, and the linear discriminant analysis effect size algorithm (linear discriminant analysis score, 4.56; p < 0.001). Accordingly, the bacterial levels directly correlated with high titers of plasma GdIgA1(r = 0.36, p < 0.001), and patients had higher IgA1 against stx2(2.88 ± 0.46 IU/mL vs. 1.34 ± 0.35 IU/mL, p = 0.03), the main antigen of Escherichia-Shigella. Conversely, the healthy controls showed relatively higher abundance of the commensal bacteria that produce IgA-degrading proteases. Particularly, the abundance of some intestinal bacteria expressing IgA proteases showed an inverse correlation with the levels of plasma GdIgA1 in IgAN.Conclusion: Our data suggest that mucosal IgA production, including those of GdIgA1, is potentially linked to the humoral response to gut Escherichia-Shigella as one of the sources of plasma GdIgA1. Conversely, the IgA protease-producing microbiota in the gut are suppressed in patients with IgAN.