Phenotype analysis of perlecan mutated mice in which heparan sulfates are removed by the use of ES cell.

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In the process of glomerular filtration, glomerular basement membranes (GBM) serve as a charge barrier to retain negatively-charged macromolecules in the circulation. On the molecular basis, GBM heparan sulfate prteoglycans (HSPG) have been thought of as key components that constitute the barrier. This negative charge theory has long been generally accepted. However, there is no direct evidence at the molecular level which links the collapse of the barrier to the development of proteinuria. Perlecan is a major GBM HSPG. Perlecan encompasses five molecular domains. Exon 3 of the first domain encodes heparan sulfate attachment sites. The present study was designed to evaluate negative charge theory from a different perspective. By the use of embryonic stem (ES) cell technology, the exon 3 was removed without frame shift. As a result, homozygous mouse was produced having mutated perlecan molecule. Our biochemical analysis at the post-transcriptional level demonstrated fibroblast obtained from the homozygote produed perlecan without heparan sulfates having an intact size of the core molecule. Although homozygotes did not spontaneously show massive proteinuria, intra-peritoneal albumin overload resulted in significant increase in urinary protein excretion after 2 weeks. Our immunoelectron microscopic investigation disclosed PEI stained sites in the GBM of homozygotes was not different from those of wild-type controls. The results strongly indicated other GBM HSPG compensate the loss of perlecan heparan sulfate. Agrin, another GBM HSPS, was not a candidate.