Quantitative Analysis of Glomerular Type IV Collagen Alpha3–5 Chain Expression in Children with Thin Basement Membrane Disease

Abstract

Thin basement membrane disease (TBMD) and Alport syndrome, two forms of childhood nephritis, have generally been considered to be hereditary diseases. In Alport syndrome, several reports have demonstrated pathogenic mutations of the genes encoding type IV collagen α3, 4 and/or 5 chain [α3, 4 and/or 5(IV)]. Previous immunohistochemical studies indicated that these antigens were absent from the glomerular basement membrane (GBM) in Alport syndrome, whilst a normal labeling pattern was maintained in TBMD. In order to understand the role of the α3, 4 and/or 5(IV) antigens in TBMD, we used confocal laser scanning microscopy (CLSM) to examine cryosections of renal biopsies from 12 children with TBMD and 11 control children with IgA nephropathy (IgAN) without proteinuria. All tissue sections were stained with a mixture of FITC-conjugated rat monoclonal antibodies directed against human α3(IV), α4(IV) or α5(IV) and a Texas red-conjugated rat monoclonal antibody raised against human α2(IV). CLSM was performed and quantitative analysis of the ratio of the staining signal for α3(IV), α4(IV) or α5(IV) to α2(IV) [α3(IV), α4(IV) or α5(IV)/α2(IV)] along the GBM was determined. The average number of pixels for α3(IV), α4(IV) or α5(IV)/α2(IV) was 3.52 ± 1.49, 3.54 ± 1.25 and 1.09 ± 0.49 in TBMD and 3.62 ± 1.46, 3.99 ± 1.53 and 1.77 ± 0.47 in control subjects, respectively. Statistical analysis indicated that α5(IV)/α2(IV) ratio was significantly lower (p < 0.01) in children with TBMD compared to controls. These findings raise the possibility that TBMD might be caused by an abnormality of the α5(IV) antigen along the GBM.