Analysis of nephritogenic antigens in human glomerular basement membrane by two-dimensional gel electrophoresis

Collagenase digests of GBM were partially purified by column chromatography and analyzed by 2-D gel electrophoresis. Silver staining of 2-D gels showed charge- and size-related heterogeneity of proteins in the 45 to 50 kDa and 25 to 27 kDa regions. These components were transferred to nitrocellulose sheets and reacted with 10 human anti-GBM autoantibodies. Detection of bound anti-GBM autoantibodies to blotted proteins was carried out with peroxidase-labeled goat anti-human IgG and revealed binding predominantly to the cationic (pI 8 to 9.0) 45 to 50 kDa and 25 to 27 kDa components. Positive-staining patterns of blotted proteins were similar with all anti-GBM autoantibodies except that three sera additionally identified neutral (pI 5.5 to 6.5) protein components. One anti-GBM autoantibody, which developed following renal transplantation, lacked reactivity with the most cationic components in the 25 to 27 kDa region. These findings suggest heterogeneity of nephritogenic GBM antigens. The cationic 45 to 50 kDa components were sensitive to reduction, while one neutral 45 to 50 kDa component was resistant; a complex array of 25 to 30 kDa proteins (pI 5.5 to 7.5) were observed by silver staining postreduction. None of the reduced protein components reacted with anti-GBM antibodies, suggesting that epitopes on nephritogenic GBM antigens may be related to disulfide-bonded regions. Although there is variable immunohistochemical reactivity of anti-GBM autoantibodies with the GBM of infant kidneys, 2-D gels of collagenase-digested human infant GBM blotted and reacted with anti-GBM autoantibodies and showed staining patterns similar to that of adult GBM. These studies demonstrate the presence of nephritogenic antigens in the GBM of immature human kidney which are not detectable by immunohistochemical analysis.