Development of kidney glomerular endothelial cells and their role in basement membrane assembly

Abstract

Data showing that the embryonic day 12 (E12) mouse kidney contains its own pool of endothelial progenitor cells is presented. Mechanisms that regulate metanephric endothelial recruitment and differentiation, including the hypoxia-inducible transcription factors and vascular endothelial growth factor/vascular endothelial growth factor receptor signaling system, are also discussed. Finally, evidence that glomerular endothelial cells contribute importantly to assembly of the glomerular basement membrane (GBM), especially the laminin component, is reviewed. Together, this forum offers insights on blood vessel development in general, and formation of the glomerular capillary in particular, which inarguably is among the most unique vascular structures in the body.

Keywords: glomerular basement membrane; laminin; podocytes; type IV collagen; vascular endothelial growth factor.

Figure 1

Light micrograph of section of developing rat kidney showing comma-shaped nephric figure. A vascular cleft (large arrow) has formed and is the site of angioblast ingress. Visceral epithelial cells (VE) will develop into podocyes, and parietal epithelial cells (PE) will line Bowman's capsule. A small Bowman's space (BS) can be observed. Note mitotic figures (double arrows) in developing tubular segment of the forming nephron. UD: Ureteric duct. Reproduced with permission (ref. 36).

Figure 2

Electron micrograph of vascular cleft region of an S-shaped stage nephron from a newborn rat that had received an injection of anti-laminin IgG directly conjugated to horseradish peroxidase (HRP). An erythrocyte (E) is densely stained due to the peroxidatic activity of hemoglobin. HRP is present throughout the full thickness of the developing GBM separating the endothelium (En) and primitive glomerular epithelium (Ep). Some strands of basement membrane-like material (arrows) lie within the cleft near putative mesangioblasts (M). HRP is also present in developing tubular basement membranes (TBM) beneath developing tubular epithelium (TEp). Reproduced with permission (ref. 43).

Figure 3

Electron micrograph of developing glomerular capillary loop. The endothelial cell (En) is large and contains only a few fenestrations at this stage. Only a few, relatively broad foot processes (fp) are present in the podocyte (Po) cell layer. A double basement membrane between the endothelium and podocytes can be seen clearly (arrows). Note the loose mesangial matrix (arrowheads) in the mesangium (M). Reproduced with permission (ref. 36).

Figure 4

In maturing stage glomeruli, a well developed, thin and extensively fenestrated endothelium (En) is evident. Double basement membranes are generally absent, but several examples of subepithelial projections of basement membrane can be seen (*). In the podocyte layer (Po) foot process (fp) interdigitation is well underway, and the filtration slits are spanned by slit diaphragms (arrows). CL: capillary lumen; US: urinary space. Reproduced with permission (ref. 38).

Figure 5

Diagram illustrating the formation of a hybrid glomerulus containing host- and graft-derived angioblasts, and graft-derived podocytes. Reproduced with permission (ref. 101).

Figure 6

HIF-2α expression in kidney. (A) E13.5 metanephros from a HIF2α^+/− stained for β-galactosidase histochemistry shows a branching, vessel-like pattern. (B) E14 HIF2a^−/− kidney showing β-galactosidase reaction product in glomerular endothelial cells migrating into the vascular cleft (VC), and in capillary loop stage glomeruli (G). Also, small arrows denote individual cells in the metanephric mesenchyme also containing β-galactosidase. (C) The same slide in B was also labeled with the lectin, BsLB4, an endothelial marker. (D) Co-localization of β-galactosidase and BsLB4 shows complete overlap. (E) Comma-shaped nephric figure from newborn mouse showing HIF2α/LacZ expression by developing podocytes (*). (F) View of newborn mouse showing widespread expression of HIF2α/LacZ in vascular endothelial cells and glomeruli. (G) Boxed region from (F) is shown at higher power. Note HIF2α/LacZ expression in some (arrows) but not all podocytes (arrowheads). (H) Frozen section of a 4 week HIF2α^+/− mouse showing intense β-galactosidase product in an endothelial pattern, and in smooth muscle cells of arteries (arrowhead). (I) Frozen section of a 4 week HIF2α^+/− mouse showing a small artery. Reaction product is seen in both endothelial cells (arrow) and smooth muscle cells (arrowheads). (J) Vascular endothelial cells (arrow) are positive for HIF2α/LacZ and a few tubular epithelial cells (arrowhead) also express HIF2α/LacZ. Reproduced with permission (ref. 76).

Figure 7

Electron microscopy of lightly fixed sections from newborn rat kidney incubated in vitro with anti-laminin IgG-HRP. Note HRP is present within double basement membrane (white arrows) and within biosynthetic apparatus (arrows) of the endothelium (En) and epithelium (Ep). Reproduced with permission (ref. 43).

Figure 8

Sections showing hybrid glomeruli. Top panels are from separate samples processed for LacZ, lower panels show corresponding serial sections immunolabeled for laminin. (A and C) Host tissue is intensely blue and can easily be distinguished from graft (dashed black line demarcates margin between host and graft tissue). Note ingress of a number of host-derived cells into graft, and the formation of hybrid glomeruli (arrows) containing host (blue) endothelial cells. (B and D) Immunofluorescence images of serial sections doubly labeled for laminin α1 (green) and α5 (red) chains. Laminin α5 protein is present in GBMs of hybrid glomeruli. (* marks same tubule in serial sections). Reproduced with permission (ref. 100).

Figure 9

Separate confocal images of the same hybrid glomerulus, dually labeled for laminin α1 (green) and laminin α5 (red). Note laminin α5 presence in vascular stalk (VS) as well as GBM. A higher power view of merged image (C) is shown in (D). GBM in glomerular hybrid is stratified; laminin α5 is on endothelial surface, whereas laminin α1 (which ordinarily is not present in capillary loop stage glomeruli) occupies podocyte surface of GBM. Central areas of signal overlap appear yellow. Reproduced with permission (ref. 100).

Figure 10

Ultrastructural examination of hybrid glomeruli. Tissue was developed with Bluo-gal, an alternative substrate for β-galactosidase, to mark endothelial cells of host (ROSA26) origin. Host-derived, Bluo-gal positive endothelial cells (En; arrows) and Bluo-gal negative podocytes (Po). In general, GBMs are poorly organized than and podocyte foot processes are absent. RBC: erythrocyte, signifying perfusion. Reproduced with permission (ref. 100).

Figure 11

To evaluate the relative contribution of endothelial cells to developing GBMs, immunofluorescence signal strengths across GBMs were quantified from host glomeruli (A) and compared with those from hybrid glomeruli (B) in adjacent areas containing metanephric grafts. Histogram plots show peak intensity values for laminin α5 chain (red) at bisected regions of the GBM. Only background levels for laminin α1 chain (green) are observed at these same points in normal glomeruli (A), whereas hybrids show abnormally high levels of α1 in outer layer of GBMs (B). Reproduced with permission (ref. 100).