| BackgroundAberrant O-glycosylation of IgA1 plays an important role in IgA nephropathy pathogenesis.Previous proteomic studies analyzed O-glycans of the circulating IgA1 hinge region and found that the N-acetylgalactosamine(GalNAc)and galactose number in the hinge region of IgA1 of patients with IgA nephropathy was lower than that in healthy participants.However,the diagnostic performance of the O-glycosylation traits in the hinge region of plasma IgA1 for IgA nephropathy remains unelucidated.The present study aimed to determine the difference in plasma IgAl hinge region O-glycoforms among IgA nephropathy,non-IgA nephropathy disease controls(DCs),and healthy controls(HCs)via liquid chromatography tandem mass spectrometry(LC-MS/MS),and to further evaluate the diagnostic performance of plasma IgA1 glycosylation traits.MethodsSixty-two patients with biopsy-proven primary IgA nephropathy,30 age-and sex-matched non-IgA nephropathy disease controls(10 patients with membranous nephropathy,10 with focal segmental glomerulosclerosis,and 10 with minimal change disease),and 30 healthy participants were prospectively recruited.Plasma galactose deficient-IgAl levels were measured using a KM5 5 kit.Plasma IgA was extracted using IgA immunoaffinity beads.After de-N-glycosylation,reduction,alkylation,trypsin digestion,and O-glycopeptide enrichment via HILIC,LC-MS/MS and Byonic software were applied to analyze the IgA1 O-glycosylation patterns and we derived the plasma IgA1 O-glycosylation traits.We conducted quantitative analysis of IgAl glycosylation patterns and O-glycosylation traits among the three groups,and correlation analysis between IgAl O-glycosylation characteristics and clinicopathological characteristics in IgAN patients.The diagnostic performance of IgAl O-glycosylation characteristics for IgAN was further evaluated.Results①We identified 48 intact IgA1 O-glycopeptides from the 122 enrolled participants.Plasma IgAl O-glycosylation patterns were significantly changed in IgA nephropathy patients compared to those with non-IgA nephropathy disease controls and healthy participants.②The representative mass spectrum and corresponding relative abundance of main 12 glycopeptides in IgA1 HR were shown in Table 1.2.Compared with DC,the relative abundance of GalNAc3Gal3 in the IgAN group was significantly higher,while the relative abundance of GalNAc5Gal4 and GalNAc6Gal5 was significantly lower.Compared with HC,the relative abundance of GalNAc3Gal3 and GalNAc4Gal2 in the IgAN group was significantly higher,while the relative abundance of GalNAc5Gal5 and GalNAc6Gal4 was significantly lower.③Compared with DCs and HCs,the GalNAc number was lowest in IgA nephropathy patients(IgAN vs DC vs HC:4.358 ± 0.104 vs 4.471 ± 0.073 vs 4.488 ±0.135,p<0.001).In addition,a similar result was observed for the galactose number in the IgA1 hinge region(IgAN vs DC vs HC:3.591 ±0.126 vs 3.733 ±0.147 vs 3.712±0.134,P<0.001).④The analysis of the correlations between plasma Gd-IgAl levels and the numbers of GalNAc and Gal in IgAN patients showed that the number of Gal was significantly negatively correlated with the plasma Gd-IgAl level(r=-0.283,p=0.026).We firstly found that the number of Gal in the IgA1 HR was positively correlated with the level of serum C3(r=0.335,p=0.008).⑤Compared with DC and HC,the AUC values of Gal,GalNAc,and the combination of Gal and GalNAc in diagnosing IgAN were significantly higher than those of plasma Gd-IgAl levels(Gal,GalNAc,GalNAc-Gal panel for DC:0.793 vs.0.805 vs.0.852;Gal,GalNAc,GalNAc-Gal panel for HC:0.764 vs.0.809 vs.0.844).We further combined plasma IgA level with IgAl O-glycosylation traits(combined Gal and GalNAc)to form a panel for IgAN diagnosis.The GalNAc-Gal-IgA panel had the strongest diagnostic performance among all the above five markers for distinguishing IgAN from DC and HC participants,with AUC values of 0.911(95%CI:0.854-0.967)the DC group and 0.927(95%CI:0.866-0.988)when compared with the HC group.Conclusions①Compared with HCs and DCs,the GalNAc and Gal number of plasma IgAl HR in IgAN patients were significantly lower.②The Gal number of plasma IgA1 HR in IgAN patients correlated with plasma Gd-IgA1 and C3 level.③The panel containing GalNAc,Gal,and circulating IgA showed excellent diagnostic performance and is promising for application in clinical practice.BackgroundThe initiating factor of IgA nephropathy(IgAN)is the elevation of circulating galactose-deficient IgA1.The complement proteins play an important role in the pathogenesis of IgAN.In previous studies,urinary complement proteins(UCP)of IgAN patients were detected by ELISA and association was found between urinary UCP and disease severity.However,there was few studies to explore the UCP profiles in different pathways by targeted proteomics in IgAN patients.In this study,we analyzed UCP profiles and explored the correlation between UCP and clinicopathological parameters by parallel reaction monitoring(PRM).MethodsSixty-four patients with primary IgAN diagnosed by renal biopsy in our hospital were enrolled.The urine was collected from the patients in biopsy morning.The urinary proteins were extracted with acetone,and followed by reduction,alkylation,trypsin digestion and C18 extraction for desalting.A total of 27 complement proteins of interest were selected and screened by Skyline software in the pooled urine sample.Each urine sample was analyzed with a reverse-phase C18 capillary LC column.The raw MS data was acquired using Orbitrap Fusion Lumos Tribrid to further quantitatively analyze the screened complement proteins in each urine sample.The complement proteins were classified into five groups,including classical pathway(CP),lectin pathway(LP),alternative pathway(AP)complement regulatory proteins,membrane attack complex(MAC)and complement C3.Spearman correlation analysis was conducted between the complement protein quantification and clinicopathological parameters in IgAN patients.Results①Sixty-four patients with biopsy-proven primary IgA nephropathy were enrolled,including 37 males and 27 females,with an average age of 40.37±12.80 years,eGFR of 73.72±24.71 mL/kg/1.73m2 and 24h urinary protein excretion rate(UPER)of 1.86 ±1.06 g.②A total of 30 urinary complement peptides were identified in the pooled sample.The median CV value of the identified complement peptides was 0.48,and the CV value of the 80%complement peptide was less than 0.6,and most of the complement peptides were stable.③Nineteen complement proteins were identified in urine samples of all IgAN patients,and urinary C5 was negatively correlated with eGFR.④Urinary C3 was positively correlated with UPER.Ficolin-2 and mannose binding lectin(MBL)serine protease-1 of LP were negatively correlated with UPER,while C4a was positively correlated with UPER.The decay-accelerating factor(DAF)in AP was negatively correlated with UPER.⑤Urinary C3 was positively correlated with MEST-C total score,DAF was negatively correlated with MEST-C total score,and C9 in MAC was positively correlated with MEST-C total score.Conclusions① Urinary complement proteins,especially C3,C4a,MAC and DAF,were correlated with disease severity in IgAN patients.② The changes of urinary complement proteins suggested that AP and LP activation play an important role in the pathogenesis of IgAN. |
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