Partial Rescue of Glomerular Laminin α5 Mutations by Wild-Type Endothelia Produce Hybrid Glomeruli

Abstract

Both endothelial cells and podocytes are sources for laminin ␣1 at the inception of glomerulogenesis and then for laminin ␣5 during glomerular maturation. Why glomerular basement membranes (GBM) undergo laminin transitions is unknown, but this may dictate glomerular morphogenesis. In mice that genetically lack laminin ␣5, laminin ␣5␤2␥1 is not assembled, vascularized glomeruli fail to form, and animals die at midgestation with neural tube closure and placental deficits. It was previously shown that renal cortices of newborn mice contain endothelial progenitors (angioblasts) and that when embryonic day 12 kidneys are transplanted into newborn kidney, hybrid glomeruli (host-derived endothelium and donor-derived podocytes) result. Reasoning that host endothelium may correct the glomerular phenotype that is seen in laminin ␣5 mutants, ␣5 null embryonic day 12 metanephroi were grafted into wild-type newborn kidney. Hybrid glomeruli were identified in grafts by expression of a host-specific LacZ lineage marker. Labeling of glomerular hybrid GBM with chain-specific antibodies showed a markedly stratified distribution of laminins: ␣5 was found only on the inner endothelial half of GBM, whereas ␣1 located to outer layers beneath mutant podocytes. For measurement of the contribution of host endothelium to hybrid GBM, immunofluorescent signals for laminin ␣5 were quantified: Hybrid GBM contained approximately 50% the normal ␣5 complement as wild-type GBM. Electron microscopy of glomerular hybrids showed vascularization, but podocyte foot processes were absent. It was concluded that (1) endothelial and podocyte-derived laminins remain tethered to their cellular origin, (2) developing endothelial cells contribute large amounts of GBM laminins, and (3) podocyte foot process differentiation may require direct exposure to laminin ␣5.

Cellular Origins of Type IV Collagen Networks in Developing Glomeruli

“…Within 5 d after transplantation, sections of the engrafted Col4a3 knockout kidneys contained numerous LacZ-positive, host-derived endothelial cells (Figure 4A). As shown previously, this indicated the formation of hybrid glomeruli within grafts, 6 which in this case contained wildtype endothelial cells and Col4a3 mutant podocytes. When serial sections were incubated with anti-collagen ␣1␣2␣1(IV), most basement membranes within the host kidney as well as within grafts labeled positively, including the immature GBMs found within the host kidney glomeruli and hybrid glomeruli within grafts ( Figure 4B).…”

“…Furthermore, quantitative confocal image analysis showed that GBMs that form in these wildtype/ Lama5 mutant glomerular hybrids can contain up to approximately one-half the full complement of laminin ␣5 immunolabel, indicating that substantial amounts of GBM laminin can stem from the endothelium. 6 By contrast, and as shown here, when Alport mouse metanephroi were grafted into wildtype hosts, there was no expression of collagen ␣3␣4␣5(IV) in hybrid glomeruli within grafts, despite the presence of abundant wildtype endothelial cells, indicating once again a strictly podocyte origin for this collagen IV heterotrimer.…”

“…4 Furthermore, when laminin ␣5 knockout metanephroi are grafted into renal cortices of wildtype newborn host mice, host-derived endothelial cells migrate into glomeruli developing within grafts and these cells express laminin ␣5. 6 This partially rescues the glomerular Lama5 null phenotype, which ordinarily results in completely avascular glomeruli. Furthermore, quantitative confocal image analysis showed that GBMs that form in these wildtype/ Lama5 mutant glomerular hybrids can contain up to approximately one-half the full complement of laminin ␣5 immunolabel, indicating that substantial amounts of GBM laminin can stem from the endothelium.…”

“…5 By transplanting embryonic day 12 (E12) metanephroi into kidney cortices of newborn wildtype mice, we partially rescued the kidney phenotype and observed well vascularized hybrid glomeruli within grafts containing wildtype endothelial cells and laminin ␣5 mutant podocytes. 6 However, the GBMs that form within these glomerular hybrids are stratified and contain laminin ␣5 on their inner, subendothelial surfaces and laminin ␣1 on their outer, podocyte halves. Intriguingly, the podocytes fail to form foot processes, suggesting that an absence of the laminin ␣1-␣5 isoform switch stunts podocyte differentiation.…”

“…Intriguingly, the podocytes fail to form foot processes, suggesting that an absence of the laminin ␣1-␣5 isoform switch stunts podocyte differentiation. 6 Unlike laminin ␣5, laminin ␤2 expression is not required for normal glomerular development. However, mice with genetic delection of the Lamb2 gene become proteinuric by 8 d after birth, display effaced foot processes, and suffer renal failure by 4 wk of age.…”