Glomerular endothelial cell maturation depends on ADAM10, a key regulator of Notch signaling

Abstract

The principal function of glomeruli is to filter blood through a highly specialized filtration barrier consisting of a fenestrated endothelium, the glomerular basement membrane and podocyte foot processes. Previous studies have uncovered a crucial role of endothelial a disintegrin and metalloprotease 10 (ADAM10) and Notch signaling in the development of glomeruli, yet the resulting defects have not been further characterized nor understood in the context of kidney development. Here, we used several different experimental approaches to analyze the kidneys and glomeruli from mice lacking ADAM10 in endothelial cells (A10ΔEC mice). Scanning electron microscopy of glomerular casts demonstrated enlarged vascular diameter and increased intussusceptive events in A10ΔEC glomeruli compared to controls. Consistent with these findings, genes known to regulate vessel caliber (Apln, AplnR and Vegfr3) are significantly upregulated in A10ΔEC glomeruli. Moreover, transmission electron microscopy revealed the persistence of diaphragms in the fenestrae of A10ΔEC glomerular endothelial cells, which was corroborated by the elevated expression of the protein PLVAP/PV-1, an integral component of fenestral diaphragms. Analysis of gross renal vasculature by light sheet microscopy showed no major alteration of the branching pattern, indicating a localized importance of ADAM10 in the glomerular endothelium. Since intussusceptions and fenestrae with diaphragms are normally found in developing, but not mature glomeruli, our results provide the first evidence for a crucial role of endothelial ADAM10, a key regulator of Notch signaling, in promoting the development and maturation of the glomerular vasculature.

Keywords: A disintegrin and metalloprotease 10 (ADAM10); Diaphragms; Endothelial cells; Fenestra; Glomeruli; Notch.

Fig. 1. Scanning electron microscopic analysis of glomerular vascular corrosion casts reveals an increase in intussusceptive events and in vascular diameter

A, B, C, D) Scanning EM images of a control (A, B) and A10ΔEC glomerulus (C, D red arrows indicate holes that were scored as likely intussusceptive events). E) Histogram of capillary vessel diameters. F) Relative number of glomeruli with zero to seven intussusceptive capillary loops visible on one hemisphere for control (n=108) and A10ΔEC animals (n=109). P-value <.0001

Fig. 2. Transmission electron microscopic analysis of A10ΔEC glomeruli shows persistence of electron dense material resembling diaphragms in the fenestra of glomerular endothelial cells

A, B) Low magnification images of normal and A10ΔEC glomeruli. Red asterisks in B) indicate dilated glomerular capillary loops. C, E) TEM of control glomerular endothelial fenestrae. Normal open fenestrae indicated by yellow arrows in E. D, F) TEM images of A10ΔEC glomerular endothelial fenestrae show the presence of electron dense material that is consistent with the presence of fenestral diaphragms in panels F (red arrows point to the apparent diaphragms spanning fenestrae in A10ΔEC glomerular endothelial cells). Scale bars A, B): 10 microns. C, D): 1 micron. E, F): 500 nm. Abbreviations: Pod, podocytes; GBM, glomerular basement membrane; GEC, glomerular endothelial cell; CL, capillary lumen; and RBC, red blood cell. G) Quantification of percent fenestrae with apparent diaphragms (p-value .0005). H) Quantification of the shortest diameter of fenestrae from tangential pore images (see Supplemental Figure 2 for example).

Fig. 3. Immunofluorescence analysis of glomeruli with markers for endothelial cells, podocytes and mesangial cells did not uncover major evident changes in the composition of glomerular cells

Staining for nuclei (DAPI), endothelial cells (anti-Endomucin), A) mesangial cells (PdgfrB), B) podocyte staining (Nephrin) and combined staining with these markers on control (left panels) and A10ΔEC glomeruli (right panels). Images were acquired using a 60x objective. White arrows in combined images point to representative areas were cell interactions appear normal when compared to control glomeruli. Scale bars: 11 μm.

Fig. 4. Comparable recruitment of endothelial cells into the vascular cleft at different stages of glomerular development

PECAM (CD31) staining of kidneys isolated from newborn controls or A10ΔEC mice show comparable staining patterns at different stages of maturation, the S-shaped body (top row), the maturing glomerulus (middle row) and a mature glomerulus (bottom row). Representative samples for sections from 3 different litters with mutant and control littermates are shown. Scale bars: 12.5 μm.

Fig. 5. Light sheet microscopy of the kidney vasculature of newborn (P0) mice reveals a normal arterial branching pattern in A10ΔEC animals

A) A representative image of a kidney from a newborn (P0) A10ΔEC mouse perfused with fluorescently tagged tomato-lectin. Kidneys were cleared using the iDisco clearing protocol and imaged using a light sheet microscope (see materials and methods for details). Scale bars: 200um. B) Pruned kidney vascular trees, in which the smaller vessels on the left of the dotted white line shown in A were digitally removed to highlight the branching pattern of the major arteries, scale bars: 227 μm. C) The main vascular branches were counted to assess possible changes in overall branching pattern of the large arteries and arterioles.

Fig. 6. qPCR analysis of glomeruli shows altered expression of genes involved in regulating vessel diameter and fenestral diaphragm formation

A) qPCR analysis of cDNA samples generated from glomeruli that had been enriched using the sieve method of glomerular isolation. Apelin and the apelin receptor (AplnR), Vegfr3, and plasmalemma vesicle associate protein (PLVAP, PV-1) were expressed at significantly higher levels in the mutant samples compared to controls. (p-values, respectively, .0004; .0327; .0108; and <.0001) B) Cxcr4 was expressed at a statistically lower level (p-value .0011). * indicates a p-value < 0.05. C) Representative images of glomeruli stained with anti-PLVAP antibody. The presence of anti-PLVAP is indicated by dark purple staining, and glomeruli are circled in blue (control) and red (A10ΔEC). Scale bar represents 100 microns.