Glomerular laminin isoform transitions: errors in metanephric culture are corrected by grafting

Glomerular basement membrane (GBM) assembly and maturation are marked by th e replacement of laminin-1 (containing alpha1-, beta1-, and gamma1-chains) with laminin-11 (consisting of alpha5-, beta2-, and gamma1-chains). Similar ly, the alpha1- and alpha2-chains of type IV collagen are replaced by colla gen alpha3-, alpha4-, and alpha5(IV)-chains. The cellular origins of these molecules and mechanisms for isoform removal and substitution are unknown. To explore glomerular laminin isoform transitions in vitro, we assessed met anephric organ cultures. Standard culture conditions do not support endothe lial cell differentiation, and glomerular structures that form in vitro are avascular. Nevertheless, extensive podocyte development occurs in these cu ltures, including the formation of foot processes and assembly of a GBM-lik e matrix. Here, we show that the podocyte-specific markers, glomerular epit helial protein 1 and nephrin, which are normally expressed in capillary loo p stage glomeruli in vivo, are also expressed by glomerular figures that fo rm in organ culture. However, the GBM-like segments that form in vitro do n ot undergo normal laminin isoform switching. Instead, both laminin alpha1- and alpha5- chains are present, as is the b1- chain, but not beta2. When av ascular organ-cultured kidneys are grafted into anterior eye chambers, howe ver, kidney-derived angioblasts establish extensive vasculature by 6 days, and glomeruli are lined by endothelial cells. We evaluated embryonic day 12 (E12) vascular endothelial growth factor receptor (Flk1)-lacZ kidneys that had first been grown in organ culture for 6-7 days and then grafted into w ild-type mice. Correct laminin isoform substitution occurred and correlated with the appearance of endothelial cells expressing Flk1. Our findings ind icate that endothelial cells, and/or factors present in the circulation, me diate normal GBM laminin isoform transitions in vivo.