Karen Molyneux

An update on the pathogenesis and treatment of IgA nephropathy.

Over the past two decades significant progress has been made in unravelling the complex pathogenesis of immunoglobulin A nephropathy (IgAN). Excess amounts of poorly galactosylated immunoglobulin (Ig)A1 in the serum appear to be the trigger for generation of glycan-specific IgG and IgA autoantibodies, resulting in the formation of circulating IgA immune complexes, which are pivotal to the development of nephritis. It remains unclear why there is an increase in poorly galactosylated IgA1 molecules in the serum in IgAN. One intriguing possibility is that this IgA is derived from displaced mucosal B cells, which have mis-homed from their mucosal induction sites to systemic sites, where they secrete polymeric, poorly galactosylated IgA directly into the circulation rather than onto mucosal surfaces. Lack of a clear appreciation of the origins of poorly galactosylated IgA1 and an incomplete understanding of immune complex formation have hampered development of specific therapeutic strategies to prevent mesangial IgA deposition. Clinicians have therefore been left to manage patients with generic therapies, mainly by control of blood pressure and renin-angiotensin blockade. A paucity of high-quality clinical trials has meant that evaluation of additional therapies, particularly immunosuppressive regimens, has been difficult and there remains a great deal of confusion over the optimum treatment of patients at high risk of progressive chronic kidney disease.

O-Glycosylation of Serum IgA1 Antibodies against Mucosal and Systemic Antigens in IgA Nephropathy

In IgA nephropathy (IgAN), serum IgA1 with abnormal O-glycosylation deposits in the glomerular mesangium. The underlying mechanism of this IgA1 O-glycosylation abnormality is poorly understood, but recent evidence argues against a generic defect in B cell glycosyltransferases, suggesting that only a subpopulation of IgA1-committed B cells are affected. For investigation of whether the site of antigen encounter influences IgA1 O-glycosylation, the O-glycosylation of serum IgA1 antibodies against a systemic antigen, tetanus toxoid (TT), and a mucosal antigen, Helicobacter pylori (HP), was studied in patients with IgAN and control subjects. Serum IgA1 was purified from cohorts of patients with IgAN and control subjects with HP infection and after systemic TT immunization. The IgA1 samples were applied to HP- and TT-coated immunoplates to immobilize specific antibodies, and IgA1 O-glycosylation profiles were assessed by binding of the O-glycan-specific lectin Vicia villosa using a modified ELISA technique. Although total serum IgA1 had raised lectin binding in IgAN, the O-glycosylation of the specific IgA1 antibodies to TT and HP did not differ between patients and control subjects. In both groups, IgA1 anti-HP had higher lectin binding than IgA1 anti-TT. This study demonstrates that IgA1 O-glycosylation normally varies in different immune responses and that patients produce the full spectrum of IgA1 O-glycoforms. IgA1 with high lectin binding was produced in response to mucosal HP infection in all subjects. The raised circulating level of this type of IgA1 in IgAN is likely to be a consequence of abnormal systemic responses to mucosally encountered antigens rather than a fundamental defect in B cell O-glycosylation pathways.

O-Glycosylation of Serum IgD in IgA Nephropathy

In IgA nephropathy (IgAN), serum IgA1 with abnormal O-glycosylation preferentially deposits in the glomerular mesangium. The control of O-glycosylation is poorly understood. Among Ig isotypes, only IgD, produced early in B cell development, and IgA1, produced by mature B cells, are O-glycosylated. For investigation of the stage of B cell maturation at which the defect seen in IgAN arises, the O-glycosylation of serum IgA1 and IgD was studied in IgAN and controls. Serum was obtained from 20 patients with IgAN and 20 control subjects. The O-glycosylation profiles of native and desialylated IgA1 and IgD were measured in an ELISA-type system using the lectins Helix aspersa and peanut agglutinin, which bind to alternative forms of O-glycan moieties. The lectin-binding patterns of the two immunoglobulins differed in all participants, with that of IgD suggesting that it is more heavily galactosylated than IgA1. Defective O-glycosylation of IgA1, probably taking the form of reduced galactosylation, was confirmed in IgAN in this study. This undergalactosylation was not shared by IgD; in contrast, IgD carried more galactosylated O-glycans in IgAN than controls. The contrasting lectin-binding patterns of IgA1 and IgD shows that Ig O-glycosylation is differentially controlled during B cell maturation. Compared with controls, O-glycosylation in IgAN is incomplete in IgA1 but more complete in IgD. These observations show that abnormal IgA1 O-glycosylation in IgAN is not due to an inherent defect in glycosylation mechanisms but arises only at a later stage in B cell development and may be secondary to aberrant immunoregulation.

Immunopathogenesis of IgAN

The defining hallmark of IgA nephropathy (IgAN) is deposition of polymeric IgA1 in the glomerular mesangium accompanied by a mesangial proliferative glomerulonephritis. The mechanisms involved in mesangial polymeric IgA1 deposition and the initiation of inflammatory glomerular injury remain unclear. This lack of a complete understanding of the pathogenesis of IgAN has meant that there is still no treatment known to modify mesangial deposition of IgA. Increasing evidence, however, supports the importance of IgA-containing immune complex formation as a pivotal factor driving mesangial IgA deposition and triggering of glomerular injury. A number of potentially important changes to the IgA1 molecule have been identified in IgAN, which may contribute to immune complex formation. These changes suggest that the polymeric IgA1 that deposits in IgA nephropathy is derived from mucosally primed plasma cells. The presence of this IgA in the circulation reflects displacement of mucosal B lineage cells to systemic sites and may be the result of mishoming of lymphocytes trafficking along the mucosa–bone marrow axis.

Spleen Tyrosine Kinase Is Important in the Production of Proinflammatory Cytokines and Cell Proliferation in Human Mesangial Cells following Stimulation with IgA1 Isolated from IgA Nephropathy Patients

IgA immune complexes are capable of inducing human mesangial cell (HMC) activation, resulting in release of proinflammatory and profibrogenic mediators. The subsequent inflammation, cellular proliferation, and synthesis of extracellular matrix lead to the progression of IgA nephropathy (IgAN). Spleen tyrosine kinase (SYK) is an intracellular protein tyrosine kinase involved in cell signaling downstream of immunoreceptors. In this study, we determined whether SYK is involved in the downstream signaling of IgA1 stimulation in HMC, leading to production of proinflammatory cytokines/chemokines and cell proliferation. Incubation of HMC with IgA1 purified from IgAN patients significantly increased the synthesis of MCP-1 in a dose-dependent manner. There was also significantly increased production of IL-6, IL-8, IFN-γ–inducible protein-10, RANTES, and platelet-derived growth factor-BB. Stimulation of HMC with heat-aggregated IgA1 purified from IgAN patients induced significantly increased HMC proliferation. Both pharmacological inhibition of SYK and knockdown of SYK by small interfering RNA significantly reduced the synthesis of these mediators and inhibited HMC proliferation. Moreover, positive immunostaining for total and phospho-SYK in glomeruli of kidney biopsies from IgAN patients strongly suggests the involvement of SYK in the pathogenesis of IgAN. To our knowledge, we demonstrate, for the first time, the involvement of SYK in the downstream signaling of IgA1 stimulation in HMC and in the pathogenesis of IgAN. Hence, SYK represents a potential therapeutic target for IgAN.

B-cell O-galactosyltransferase activity, and expression of O-glycosylation genes in bone marrow in IgA nephropathy

In IgA nephropathy (IgAN), pathogenic IgA1 is likely derived from bone marrow (BM) cells and exhibits reduced O-galactosylation. Defective O-galactosylation may arise from the compromised expression or function of the enzyme beta-galactosyltransferase and/or its molecular chaperone (Cosmc). We measured B-cell O-galactosylation activity and the relative gene expression of beta-galactosyltransferase and Cosmc in peripheral blood and BM taken from patients with IgAN and controls. O-galactosylation activity was measured in peripheral and BM B cells by the incorporation of radiolabeled galactose into an asialo-mucin acceptor. Gene expression of beta-galactosyltransferase and Cosmc was measured by real-time PCR and related to that of the enzyme GalNAc-T2 (UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase-2), which synthesizes the core O-glycan. Neither the B-cell O-galactosylation activity nor the gene expression of the enzyme or chaperone was different between patients and controls. However, the relationships between the O-glycosylation of serum IgA1, galactosylation activity, and beta-galactosyltransferase gene expression showed different patterns in IgAN and controls. In IgAN, O-galactosylation activity correlated with beta-galactosyltransferase gene expression, but not with IgA1 O-glycosylation, suggesting that factors other than the availability of beta-galactosyltransferase or Cosmc are responsible for altered IgA1 O-glycosylation.

A single amino acid deletion in the α2(I) chain of type I collagen produces osteogenesis imperfecta type III

RNase A protection analysis was used in the search for the cause of a non-lethal osteogenesis imperfecta (OI) phenotype (Sillence type III). Cleavage of the hybrid formed between a normal α2(I) sequence and RNA isolated from the patient indicated the presence of a mismatch. The position of the mismatch was determined and the corresponding area of COL1A2 was amplified using the polymerase chain reaction. Sequencing of cloned amplified DNA revealed the deletion, which was not present in either parent, of the final three bases of exon 19 in one of the patients two COL1A2 alleles. The deletion results in the loss of amino acid 255 (a valine encoded by the last codon of exon 19) of the triple helical region of half of the α2(I) collagen chains but does not disrupt the splicing of the heterogeneous nuclear RNA (hnRNA). This provides further evidence that OI type III may result from autosomal dominant mutations rather than only from autosomal recessive mutations as had previously been believed.

Galactosylation of IgA1 Is Associated with Common Variation in C1GALT1.

IgA nephropathy (IgAN), an important cause of kidney failure, is characterized by glomerular IgA deposition and is associated with changes in O-glycosylation of the IgA1 molecule. Here, we sought to identify genetic factors contributing to levels of galactose-deficient IgA1 (Gd-IgA1) in white and Chinese populations. Gd-IgA1 levels were elevated in IgAN patients compared with ethnically matched healthy subjects and correlated with evidence of disease progression. White patients with IgAN exhibited significantly higher Gd-IgA1 levels than did Chinese patients. Among individuals without IgAN, Gd-IgA1 levels did not correlate with kidney function. Gd-IgA1 level heritability (h2), estimated by comparing midparental and offspring Gd-IgA1 levels, was 0.39. Genome-wide association analysis by linear regression identified alleles at a single locus spanning the C1GALT1 gene that strongly associated with Gd-IgA1 level (β=0.26; P=2.35×10-9). This association was replicated in a genome-wide association study of separate cohorts comprising 308 patients with membranous GN from the UK (P<1.00×10-6) and 622 controls with normal kidney function from the UK (P<1.00×10-10), and in a candidate gene study of 704 Chinese patients with IgAN (P<1.00×10-5). The same extended haplotype associated with elevated Gd-IgA1 levels in all cohorts studied. C1GALT1 encodes a galactosyltransferase enzyme that is important in O-galactosylation of glycoproteins. These findings demonstrate that common variation at C1GALT1 influences Gd-IgA1 level in the population, which independently associates with risk of progressive IgAN, and that the pathogenic importance of changes in IgA1 O-glycosylation may vary between white and Chinese patients with IgAN.

The solution structures of native and patient monomeric human IgA1 reveal asymmetric extended structures: implications for function and IgAN disease.

Detailed analytical ultracentrifugation and X-ray/neutron scattering data and a new atomistic modelling approach revealed asymmetric extended solution structures for human IgA1 that account for its receptor-binding function. IgA1 with different hinge O-galactosylation patterns showed similar structures.

Disease specific biomarkers of abdominal aortic aneurysms detected by surface enhanced laser desorption ionization time of flight mass spectrometry.

INTRODUCTION Biomarkers have the potential to improve the clinical management of patients with AAA. REPORT A prospective, proteomics discovery study was undertaken to compare patients with AAA (n = 20) to matched screened controls (n = 19) for plasma protein expression. Surface-Enhanced-Laser-Desorption-Ionization Time of Flight Mass Spectrometry (SELDI ToF MS) coupled with Artificial Neural Networks (ANN) analysis identified six protein related diagnostic biomarker ions with a combined AUC of 0.89. DISCUSSION This study discovered a signature plasma protein profile for patients with AAA and demonstrated that mass spectrometric based research for disease specific biomarker of AAA is feasible.