The origin of renal vascular
The thickness of a glomerulus is approximately 97.4 µm . To record the developmental process of glomerular capillaries more accurately and completely, we developed an immunofluorescence method using ultra-thick section. Sections were generated at 150 µm. Negative control was performed for each antibody (n = 3) used in the following studies, and no non-specific signal was detected (data not shown). Then, we used immunostaining to detect the expression of FLK1 and CD34 which were expessed in EPCs but also in mature blood vessels [7, 10]. Compared with 15 µm tissue sections (Fig. 1a), we found that no individual FLK1 expression (FLK1+) or CD34+ cells were found in the ultra-thick fetal kidney sections (n = 6 / 6), and all of these endothelial cells were interconnected to form blood vessels (Fig. 1b, c). To determine whether these blood vessels were mature, we detected two mature blood vessels markers, endomucin and CD31 at E 13 to E 16 [11, 12]. We observed that CD31 was localized between endothelial cells (Fig. 1d), which demonstrated a stable connection between endothelial cells (n = 5 / 5). Endomucin was widely expressed in renal vessels (n = 5) and increased with the development of embryos (Fig. 1e, f). These data indicate that blood vessels in the embryonic kidney exist in a continuous and mature manner.
In that case, what is the source of renal blood vessels in the fetal kidney? Blood vessel sprouting was observed (n = 6) around Six2+ cap mesenchyme (Fig. 1g; Additional file 2: Video S1). Sprouting requires endothelial cell proliferation . We detected cell proliferation with Ki-67 . A large number of Ki-67+ cells appeared in the renal cortex, of which 27.09% ± 4.32% were vascular endothelial cells (Fig. 1h). This suggests that the source of the vascular endothelial cells in the early stage of kidney development is sprouting and proliferation. These results further confirm a recently published study on renal vascular development , which demonstrate that polygonal networks of vessels form in cycles at the periphery of the kidney by angiogenesis. However, the fine development of glomerular capillary need further elucidation.
Detailed development processes of glomerular capillary
Subsequently, we tracked the development of glomerular capillary with two markers of early glomerulus before and after birth. WT1 can label developing podocytes. NCAM1 is a marker of renal epithelial cells. Nephron begins to appear on the E 13 and stop producing new nephrons within a few days after birth. During this period, new glomeruli appeared every day (n = 8). We found that glomerular capillary formed in the same way before and after birth (Additional file 1: Fig. S1, a-i). The nascent nephron epithelium progresses through four stages: renal vesicle, comma-shaped body, S-shaped body, maturation . At first, capillaries formed a network surrounding renal vesicle (n = 5) (Fig. 2a). In the comma and S-shaped stages, 2-4 capillaries budding from the capillary network began to extend into the cleft of the glomerulus (n = 6) (Fig. 2b, c; Additional file 3: Video S2). These capillaries intersected and fused in the cleft, forming a capillary bed with multiple holes (Fig. 2c-f). Sprouting was also found on the capillary bed (n = 8). The sprouting of these marginal vessels expanded the area of the capillary bed through extension and anastomosis (Fig. 2e, f; Additional file 4 and 5: Videos S3 and S4).
In the process of developing into the Bowman’s capsule, the front end of the S-shaped body extended and encapsulated the capillaries that extend into the glomerular cleft (n = 5) (Fig. 3a). Influenced by the change of glomerular cleft shape, the capillary bed also bended and tended to be bowl shaped (Fig. 3b; Additional file 6: Video S5). Then the capillary bed began to expand and became spherical. During this process, we did not find sprouting from glomerular capillaries (n = 8 / 8) (Fig. 3b, c). Previous studies have shown that glomerular capillaries are enlarged via intussusceptive angiogenesis [17, 18]. Intussusceptive angiogenesis is a type of new blood vessel formation in which a capillary is longitudinally split into two vascular channels due to the formation and merging of intraluminal tissue pillars . Ki-67 was used to detect the proliferation of endothelial cells during this period (Fig. 3c). We found that endothelial cells are highly proliferative (n = 7 / 7) during the maturation of the glomerulus.
At comma-shaped and S-shaped stages, 2-4 capillaries extended into cleft to form a capillary bed. But the mature glomerulus had only one afferent arteriole and one efferent arteriole . How were these two vessels formed? During the development of the capillary bed toward the mature glomerulus, pruning of superfluous vessels was found . As shown in Figure 3D and Video S6, a blood vessel disconnected and regressed from the glomerulus, while the afferent and efferent arterioles developed into larger vessels. Subsequently, the glomerulus began to further expand, and the largest glomerulus was approximately 70 µm in diameter on the first day after birth (Fig. 3e). During the process of glomerular maturation, the expression of CD34 began to increase gradually in the glomerular artery (n = 8), while the expression in the peritubular capillaries began to decrease or lost after glomerular maturation (Fig. 3f). Therefore, CD34 can be used as a mature marker of glomerular capillaries.
Molecular mechanism of glomerular capillary development
The molecular mechanism of glomerular capillary formation is another important research topic. Previous studies have shown that VEGFA is a key factor in development of development of glomerular capillary. Loss of the VEGFA gene from developing podocytes in mice results in reduced glomerular endothelium, but not a complete loss [21, 22]. This suggests that VEGFA has other sources. Using immunofluorescence performed with ultra-thick section, we found that VEGFA was highly expressed in all the epithelial cells (n = 6 / 6) at the comma-shaped and S-shaped stages (Fig. 4a, b), This indicates that, in addition to podocytes, VEGFA secreted by other epithelial cells may also responsible for the formation of capillary network around Bowman’s capsules and renal tubules. VEGFA is also the reason for multiple capillaries to enter the glomerular cleft to form the capillary bed (Fig. 4b). CXCR4 is another factor that plays an important role in the development of glomerular capillaries . We found that CXCR4 was mainly expressed in some glomerular endothelial cells during the development of the capillary bed into the mature glomerulus (n = 6) (Fig. 4c, d). Previous studies have shown that cxcr4 mutants exhibited glomeruli with aneurismal dilation of the capillaries . However, the reason for this structure is not clear. Immunofluorescence performed with ultra-thick section showed that FLK1 decreased significantly in the glomerular vessels of cxcr4 mutants (n = 6 / 6) (Fig. 4e). FLK1 plays a decisive role in the proliferation and differentiation of blood vessels . These results indicate that the glomerular endothelial cells of cxcr4 mutant did not differentiate correctly in the process of capillary bed expansion into glomerulus.