29th Annual Meeting of the Glomerular Disease Collaborative Network

29th Annual Meeting of the Glomerular Disease Collaborative Network Updates on the Pathogenesis IgA Nephropathy and IgA Vasculitis (HSP) J. Charles Jennette, M.D. Brinkhous Distinguished Professor and Chair of Pathology and Laboratory Medicine University of North Carolina at Chapel Hill

IgA NEPHROPATHY IgA nephropathy is glomerular disease with IgA dominant or codominant mesangial immunoglobulin (Predominantly IgA1)

700 600 500 Frequency of Renal Biopsy Diagnoses vs Age at UNC from 1985-2007 Causes of Nephritis Lupus IgA Nep ANCA GN TBM Lesion Fibrillary GN Post Infect GN Lupus GN Anti GBM GN Hereditary Nep 400 300 200 IgAN ANCA GN 100 0 Anti-GBM GN 1 9 10 19 20 29 30 39 40 49 50 59 60 69 70 79 >79 Jennette JC, Falk RJ. NKF Primer on Kidney Disease, 6th Ed, 2014, Chapter 16:152-163

IgA Nephropathy mesangial immune complex deposits

Mesangial dense deposits Focal thinning of the GBM

RBC penetrating the glomerular capillary wall in thin basement membrane lesion Collar et al. Kid Intern 59:2069-2072, 2001

IgA Nephropathy Focal or Diffuse Mesangioprolifertive Glomerulonephritis Histologically Normal Glomeruli Focal or Diffuse Proliferative or Necrotizing Glomerulonephritis Focal or Diffuse Sclerosing Glomerulonephritis Frequency in Biopsy Specimens Crescentic Glomerulonephritis

IgA Nephropathy Focal or Diffuse Mesangioprolifertive Glomerulonephritis Histologically Normal Glomeruli Focal or Diffuse Proliferative or Necrotizing Glomerulonephritis Focal or Diffuse Sclerosing Glomerulonephritis Crescentic Glomerulonephritis Asymptomatic Hem / Proteinuria Mild to Moderate Nephritis Severe to Rapidly progressive Nephritis Chronic Nephritis

Analysis of the Oxford Data Using the ISN/RPS Classification Approach J C Jennette, Y Hu, SL Hogan, S Troyanov, M Haas. Unpublished data

IgA NEPHROPATHY Mesangial IgA Mesangial and Capillary Wall IgA

% IgA Nephropathy Specimens with Capillary Wall Electron Dense Deposits Relative to Histologic Phenotype 50 45 40 35 30 25 20 15 10 5 0 n=447

Mesangial IgA Mesangial and Capillary Wall IgA The presence of capillary wall IgA deposits was associated with a higher mesangial cellularity score and endocapillary proliferation. The presence of IgG was associated with a higher mesangial cellularity score and endocapillary proliferation. There was no significant association between the location of IgA or the presence of IgG and rate of loss of renal function. There was a trend towards poorer renal survival in those patients with glomerular IgG. Shubha S. Bellur, et al. Nephrol Dial Transplant (2011) 26: 2533 2536

IgA nephropathy probably can be caused by multiple different etiologies and pathogenic processes: 1) abnormally glycosylated IgA 2) antibodies against abnormally glycosylated IgA 3) reduced clearance of circulating IgA complexes 4) increased affinity for or reduced clearance of IgA deposits from the glomerular mesangium 5) excessive IgA antibody production in response to mucosal antigen exposure 6) increased permeability of mucosa to antigen 7) combinations of these factors Aberrant glycosylation of IgA1 may be caused by genetically determined aberrant mucosal immune responses to infections.

IgA1 but not IgA2 has a hinge region with 0-linked glycan chains. Each O-glycan chain is based on a core N-acetyl galactosamine unit (GalNAc) O-linked to serine or threonine. This can exist alone but usually carries galactose and sialic acid. Each IgA1 molecule can carry a mixture of chain types. VL VH CL CH1 Hinge Region CH2 CH3 IgA1 Ser/Thr-O-GalNAc Ser/Thr-O-GalNAc--Galactose Ser/Thr-O-GalNAc--Galactose-Sialic Acid Ser/Thr-O-GalNAc----Galactose-Sialic Acid Sialic Acid - In IgA nephropathy and IgA vasculitis, serum IgA1 has reduced terminal sialation and galactosylation (X), resulting in increased exposure of terminal GalNAc.

Patients with IgA nephropathy have increased serum galactose-deficient IgA1 levels. Moldoveanu Z, Wyatt RJ, et al. Kidney Int. 2007;71:1148-54. A lectin that recognizes N-acetylgalactosamine was used in an ELISA to measure serum galactose-deficient IgA1. The diagnostic sensitivity 76.5%, with specificity 94%; the positive predictive value was 88.6% and the negative predictive value was 78.9%.

Abnormal IgA glycosylation in Henoch-Schonlein purpura restricted to patients with clinical nephritis. Allen AC, Willis FR, Beattie TJ, Feehally J. Nephrol Dial Transplant. 1998 Apr;13(4):930-4. Children with HSP nephritis showed significantly higher IgA1-VV binding than children with HSP lacking renal involvement (P=0.004), children with post-streptococcal GN (P=0.002), or control children (P=0.005). Adults with IgAN showed significantly higher IgA1-VV binding than adults with non-iga glomerulonephritis or adult controls. There was no difference in IgA1-VV binding between adults with HSPN and IgAN.

Aberrant sialylation of serum IgA1 was associated with prognosis of patients with IgA nephropathy Ding JX, et al. Clin Immunol. 2007;125:268-74.

IgA1 hinge glycopeptide characterization by quadrupole orthogonal acceleration time-of-flight MS & MS/MS tandem mass spectroscopy (Q-TOF LC-MS/MS) GalNAc Galactose Sialic acid 4 3 4 4 1 1 5 3 1 5 4 3 5 4 1 5 3 2 4 4 3 5 4 3 4 4 4 Healthy Control 5 3 3 5 4 4 5 5 4 m/z 1-2 sialic acids per glycopeptide 3-4 sialic acids per glycopeptide Glycopeptides in higher mass range contain more sialic acids Provided by Olivier Lardinois, PhD, UNC Kideny Center

Quadrupole orthogonal acceleration time-of-flight tandem mass spectroscopy in MS & MS/MS modes (Q TOF LC MS/MS) Healthy Control Active IgAN Healthy Control treated with neuraminidase (=sialidase) Provided by Olivier Lardinois, PhD, UNC Kideny Center

Comparison of glycosylation profiles between healthy controls and active IgAN Conclusion: Sialylation is lower in IgAN Profiles with 2 or more 1.200 sialic acids are decreased Relative abundance 1.000 0.800 0.600 0.400 0.200 0.000 NeuAc = Sialic acid; Gal = Galactose; GalNAc = N Acetylgalactosamine Average of: 5 Healthy Controls (n = 5) 6 Active IgAN(n = 6) Provided by Olivier Lardinois, PhD, UNC Kideny Center

Glycan-specific IgG antibodies may form immune complexes with aberrantly glycosylated IgA1 IgG IgA1

Glycan-specific IgG antibodies have 88% specificity and 95% sensitivity for IgA nephropathy Suzuki H, et al. Aberrantly glycosylated IgA1 in IgA nephropathy patients is recognized by IgG antibodies with restricted heterogeneity. J Clin Invest. 2009;119:1668-77

Correlation between severity of hematuria/proteinuria and serum Gd-IgA1 or IgA/IgG-IC Serum Abnormal IgA1 Glycosylation Hematuria 1+ to 4+ Proteinuria 1+ to 4+ Serum IgA-IgG Immune Complexes Hematuria 1+ to 4+ Proteinuria 1+ to 4+ Suzuki H, et al. Clin Exp Nephrol. 2014;18:770-7.

Hypothesis: Glycan-specific IgG antibodies binding to abnormally gylcosylated IgA cause IgA nephropathy Abnormally glycosylated IgA1 molecules ( ), even in the absence of antigen, self aggregate or bind to antibodies directed against the abnormal glycosylation ( ) and localize in the glomerular mesangium where they activate complement by the lectin and alternative pathways..

Discovery of new risk loci for IgA nephropathy implicates genes involved in immunity against intestinal pathogens. Kiryluk K, et al. Nat Genet. 2014;46:1187-96. Genome-wide association study (GWAS) of IgA nephropathy in 20,612 individuals of European and East Asian ancestry. Multiple risk alleles, most associated with maintenance of the intestinal epithelial barrier and response to mucosal pathogens, including risk of inflammatory bowel disease. The risk alleles suggest host-intestinal pathogen interactions in establishing genetic susceptibility to IgAN.