A study on the mechanism of IgA-deposition on renal glomerular mesangium in IgA nephropathy -the role of circulating complex of IgA-fibronectin fragment in the interaction between IgA and mesangial cell matrix-

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Introduction : It is generally believed that IgA nephropathy is a consecquence of mesangial trapping of circulating macromolecular IgA.We have demonstrated that fibronectin fragment was found in patient's serum and was capable of binding IgA in vitro (J Am Soc Nephrol, 10 : 256, 1999). Therefore, we studied here if serum IgA from patient exists in association with fibronectin fragment, thereby forming macromolecular IgA, and if it deposits on the extracellular matrix in a fibronectin-dependent manner. Methods : Serum IgA from 10 patients with IgA nephropathy and 10 normal controls were obtained by jacalin-immobilized affinity chromatography. They were reacted with the confluent cell layer of cultured human fibroblasts for 3 hr and were cultured with medium for an additional 24 hr. IgA was detected by immunofluorescence microscory. Each three samples from both groups were applied to native SDS-PAGE (3.5%), and IgA and fibronectin fragments were detected by western blotting. Results : In … More all patients, but not in controls, the coarse and punctate clusters of IgA staining were demonstrated on the cell layer. The deposition was abolished by pretreatment of samples with anti-human fibronectin monoclonal antibodies, and was augmented with an addition of cathepsin D-digested fibronectin. Western blot analysis showed that major reactivities of patient's IgA were at 380 kDa and 200 kDa, shifting to higher molecular mass than an expected size from multiples of monomeric IgA (160 kDa). In a parallel experiment, a panel of anit-human fibronectin monoclonal antibodies reacted mainly with 440 kDa molecule presumably representing intact fibronectin in both patients and controls. However, broad and intense reactivity from 480-to 330-kDa was demonstrated by a monoclonal antibody to C-terminal region of fibornectin only in patients. These reactivities were also confirmed by using ELISA experiment, in which fibronectin fragment was detected in serum IgA captured by anti-human IgA coated plate. Thus serum IgA co-existed with fibronectin (fragment) in patient. Conclusion : Serum IgA from patients with IgA nephropathy is associated with fibronectin fragments to form a macromolecular IgA, thereby contributing to IgA depostion on extracellular matrix of mesangium through the mechanism of assembly of exogenous fibronectin into extracellular matrix. Less