Molecular and genetic analyses of renal capillary development: Studying the angiopoietin/Tie axis

Abstract

We postulated that endothelial precursors present at the inception of nephrogenesis differentiate into capillaries when experimental conditions resemble those found in vivo [Loughna S, Hardman P, Landels E, et al : A molecular and genetic analysis of renal glomerular capillary development. Angiogenesis 1:84-101, 1997; Loughna S, Yuan HT, Woolf AS: Effects of oxygen on vascular patterning in Tie1/LacZ metanephric kidneys in vitro. Biochem Biophys Res Commun 247:361-366, 1998]. At the start of nephrogenesis, mesenchyme contains no capillaries, yet we detected vascular endothelial growth factor in uninduced mouse embryonic (E) day E11 metanephric mesenchyme; receptor tyrosine kinase markers characteristic of endothelial precursors [vascular endothelial cell growth factor receptor (VEGFR) and Tie] were also expressed. We explanted day E13 Tie-1/LacZ metanephroi when nephron precursors are surrounded by reporter gene-expressing cells but glomeruli are not yet formed. In serum-free culture in normoxia, avascular glomeruli developed but existing vessels regressed. In hypoxia, however, transgene-expressing cells between nascent tubules formed large masses with poorly developed lumens; these resembled blood islands in yolk sac which arise by vasculogenesis. Glomeruli differentiating in hypoxia were devoid of capillaries but, when we transplanted Tie-1/LacZ E11 metanephroi into nephrogenic cortex of wild-type mice, a milieu permissive for differentiation of metanephric rudiments, transgene-expressing capillary loops formed in glomeruli. These experiments support an in situ origin of renal endothelia. We speculate that metanephroi are hypoxic in vivo to permit vasculogenesis; however, other, yet to be defined, cues are required for normal microcirculation patterning. Angiopoietin-1 (Ang-1) stimulates vascular network differentiation through Tie-2, while Ang-2 modulates this activation. At day E14, the forming metanephric artery expresses Ang-2; in neonates, expression was maintained in renal artery branches and smaller cortical vessels; differentiating mesangial cells transiently expressed Ang-2 [Yuan H-T, Suri C, Landon DN, et al : Angiopoietin-2 is a site specific factor in differentiation of mouse renal vasculature. J Am Soc Nephrol 11:1055-1066, 2000]. In the first postnatal weeks there was a lineage-switch of Ang-2 expression from vessels to epithelia (especially thin descending limb of loops of Henle). Because Tie-2 is widely-expressed by differentiating renal endothelia, we hypothesized that Ang-2 deficiency would disrupt kidney vessel patterning [Pitera JE, Woolf AS, Gale NW, et al : Dysmorphogenesis of kidney cortical peritubular capillaries in angiopoietin-2 deficient mice. Am J Pathol 165:1895-1906, 2004]. The normal renal cortical peritubular space contains fenestrated capillaries with few pericytes; they receive water and solutes which proximal tubules reclaim from the glomerular filtrate. In Ang-2 null mutants, pericyte-markers [α-smooth muscle actin (α-SMA), neural/glial cell 2 chondroitin sulfate proteoglycan (NG2), platelet-derived growth factor receptor β (PDGFRβ), and desmin] were up-regulated in cortical peritubular locations near to CD31/Tie-2 expressing capillaries. Total and tyrosine-phosphorylated Tie-2 increased in null mutant kidneys, and electron microscopy confirmed disorganized capillaries. Hence, Ang-2 deficiency causes dysmorphogenesis of cortical peritubular capillaries, with adjacent cells expressing pericyte-like markers; the latter effect may be caused by disturbed paracrine signaling between endothelia and surrounding mesenchymal precursor cells.