Background: Alport syndrome (AS) is an inherited nephropathy characterized by glomerular basement membrane (GBM) abnormalities due to mutations in the type IV collagen genes. Through immunofluorescence analysis, the absence of alpha3(IV), alpha4(IV) and alpha5(IV) chains within the GBM has been shown in the majority of AS cases. In some atypical AS cases, however, staining of the GBM with antibodies against the alpha3(IV), alpha4(IV) and alpha5(IV) chains appeared normal. In this study, we studied these atypical AS cases by quantitative analysis of the expression of type IV collagen subchains in GBM.
Methods: Twelve patients diagnosed with AS, yet having normal staining for alpha3(IV) and alpha5(IV) chains in the GBM, were recruited. Quantitative analysis of type IV collagen subchains in the GBM was performed using confocal microscopy and immunofluorescence double label techniques.
Results: The absolute amounts of alpha3(IV), alpha4(IV) and alpha5(IV) were significantly lower in AS patients than that in normal subjects, associated with up-regulated expression of type IV collagen in GBM. It was found that eight cases had decreased ratios of alpha3(IV)/IV, alpha4(IV)/IV and alpha5(IV)/IV in the GBM simultaneously; one had reduced levels of alpha3(IV)/IV and alpha5(IV)/IV but had a normal level of alpha4(IV)/IV, and one had reduced alpha3(IV)/IV with normal alpha4(IV)/IV and alpha5(IV)/IV levels. The remaining two patients had normal ratios of alpha3(IV)/IV, alpha4(IV)/IV and alpha5(IV)/IV.
Conclusions: Confocal analysis demonstrated for the first time that the ratios of alpha3(IV)/IV, alpha4(IV)/IV and alpha5(IV)/IV in the GBM decreased in patients with AS, even though routine immunofluorescence staining for alpha(IV) chains appeared normal. This result not only sheds light on the pathogenesis of AS, but also provides an alternative approach to diagnose atypical AS cases.