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 (A10?EC mice). Scanning electron microscopy of glomerular casts demonstrated enlarged vascular diameter and increased intussusceptive events in A10?EC glomeruli compared to controls. Consistent with these findings, genes known to regulate vessel caliber (Apln, AplnR and Vegfr3) are significantly upregulated in A10?EC glomeruli. Moreover, transmission electron microscopy revealed the persistence of diaphragms in the fenestrae of A10?EC 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.
Project description:The coronary vasculature is crucial for normal heart function, yet much remains to be learned about its development, especially the maturation of coronary arterial endothelium. Here, we show that endothelial inactivation of ADAM10, a key regulator of Notch signaling, leads to defects in coronary arterial differentiation, as evidenced by dysregulated genes related to Notch signaling and arterial identity. Moreover, transcriptome analysis indicated reduced EGFR signaling in A10?EC coronary endothelium. Further analysis revealed that A10?EC mice have enlarged dysfunctional hearts with abnormal myocardial compaction, and increased expression of venous and immature endothelium markers. These findings provide the first evidence for a potential role for endothelial ADAM10 in cardioprotective homeostatic EGFR signaling and implicate ADAM10/Notch signaling in coronary arterial cell specification, which is vital for normal heart development and function. The ADAM10/Notch signaling pathway thus emerges as a potential therapeutic target for improving the regenerative capacity and maturation of the coronary vasculature.
Project description:During vertebrate angiogenesis, Notch regulates the cell-fate decision between vascular tip cells versus stalk cells. Canonical Notch signaling depends on sequential proteolytic events, whereby interaction of Notch with membrane-anchored ligands triggers proteolytic processing, first by Adam10 and then presenilins. This liberates the Notch intracellular domain, allowing it to enter the nucleus and activate Notch-dependent genes. Here we report that conditional inactivation of Adam10 in endothelial cells (A10?EC) recapitulates the increased branching and density of the retinal vasculature that is also caused by interfering with Notch signaling. Moreover, A10?EC mice have additional vascular abnormalities, including aberrant subcapsular hepatic veins, enlarged glomeruli, intestinal polyps containing endothelial cell masses, abnormal endochondral ossification, leading to stunted long bone growth and increased pathologic neovascularization following oxygen-induced retinopathy. Our findings support a model in which Adam10 is a crucial regulator of endothelial cell-fate decisions, most likely because of its essential role in canonical Notch signaling.
Project description:Fenestral and stomatal diaphragms are endothelial subcellular structures of unknown function that form on organelles implicated in vascular permeability: fenestrae, transendothelial channels, and caveolae. PV1 protein is required for diaphragm formation in vitro. Here, we report that deletion of the PV1-encoding Plvap gene in mice results in the absence of diaphragms and decreased survival. Loss of diaphragms did not affect the fenestrae and transendothelial channels formation but disrupted the barrier function of fenestrated capillaries, causing a major leak of plasma proteins. This disruption results in early death of animals due to severe noninflammatory protein-losing enteropathy. Deletion of PV1 in endothelium, but not in the hematopoietic compartment, recapitulates the phenotype of global PV1 deletion, whereas endothelial reconstitution of PV1 rescues the phenotype. Taken together, these data provide genetic evidence for the critical role of the diaphragms in fenestrated capillaries in the maintenance of blood composition.
Project description:Severe sepsis is often accompanied by acute kidney injury (AKI) and albuminuria. Here we studied whether the AKI and albuminuria associated with lipopolysaccharide (LPS) treatment in mice reflects impairment of the glomerular endothelium with its associated endothelial surface layer. LPS treatment decreased the abundance of endothelial surface layer heparan sulfate proteoglycans and sialic acid, and led to albuminuria likely reflecting altered glomerular filtration permselectivity. LPS treatment decreased the glomerular filtration rate (GFR), while also causing significant ultrastructural alterations in the glomerular endothelium. The density of glomerular endothelial cell fenestrae was 5-fold lower, whereas the average fenestrae diameter was 3-fold higher in LPS-treated than in control mice. The effects of LPS on the glomerular endothelial surface layer, endothelial cell fenestrae, GFR, and albuminuria were diminished in TNF receptor 1 (TNFR1) knockout mice, suggesting that these LPS effects are mediated by TNF-? activation of TNFR1. Indeed, intravenous administration of TNF decreased GFR and led to loss of glomerular endothelial cell fenestrae, increased fenestrae diameter, and damage to the glomerular endothelial surface layer. LPS treatment decreased kidney expression of vascular endothelial growth factor (VEGF). Thus, our findings confirm the important role of glomerular endothelial injury, possibly by a decreased VEGF level, in the development and progression of AKI and albuminuria in the LPS model of sepsis in the mouse.
Project description:Podocytes are the highly specialised cells within the glomeruli of the kidney that maintain the filtration barrier by forming interdigitating foot processes and slit-diaphragms. Disruption to these features result in proteinuria. Studies into podocyte biology and disease has been hampered by a paucity of in vitro models of this non-proliferative cell type. Here we characterise sieved glomeruli from kidney organoids derived from human pluripotent stem cells. Compared to conditionally immortalised podocytes, organoid-derived glomeruli show superior podocyte-specific gene and protein expression, morphology and functional properties. Using CRISPR-derived MAFB reporter iPSC lines, homozygous MAFB mutant organoids recapitulated the anticipated disease related transcriptional changes. Culture of kidney organoids on chicken chorioallantoic membrane resulted in glomerular vascularisation, glomerular filtration barrier assembly, formation of slit diaphragms and fenestrated endothelial cells. This definitively demonstrates that human iPSC kidney organoid-derived glomeruli can serve as an accurate model of human podocytopathies and glomerular disease in vitro. Overall design: Gene expression profiling of three immortalised podocyte line samples at 33 degrees celsius, three immortalised podocyte line samples at 37 degrees celsius and three day 25 kidney organoid derived glomeruli
Project description:The molecular signaling mechanisms between glomerular cell types during initiation/progression of diabetic kidney disease (DKD) remain poorly understood. We compared the early transcriptome profile between DKD-resistant C57BL/6J and DKD-susceptible DBA/2J (D2) glomeruli and demonstrated a significant downregulation of essential mitochondrial genes in glomeruli from diabetic D2 mice, but not in C57BL/6J, with comparable hyperglycemia. Diabetic D2 mice manifested increased mitochondrial DNA lesions (8-oxoguanine) exclusively localized to glomerular endothelial cells after 3 weeks of diabetes, and these accumulated over time in addition to increased urine secretion of 8-oxo-deoxyguanosine. Detailed assessment of glomerular capillaries from diabetic D2 mice demonstrated early signs of endothelial injury and loss of fenestrae. Glomerular endothelial mitochondrial dysfunction was associated with increased glomerular endothelin-1 receptor type A (Ednra) expression and increased circulating endothelin-1 (Edn1). Selective Ednra blockade or mitochondrial-targeted reactive oxygen species scavenging prevented mitochondrial oxidative stress of endothelial cells and ameliorated diabetes-induced endothelial injury, podocyte loss, albuminuria, and glomerulosclerosis. In human DKD, increased urine 8-oxo-deoxyguanosine was associated with rapid DKD progression, and biopsies from patients with DKD showed increased mitochondrial DNA damage associated with glomerular endothelial EDNRA expression. Our studies show that DKD susceptibility was linked to mitochondrial dysfunction, mediated largely by Edn1-Ednra in glomerular endothelial cells representing an early event in DKD progression, and suggest that cross talk between glomerular endothelial injury and podocytes leads to defects and depletion, albuminuria, and glomerulosclerosis.
Project description:It is known that endothelial cells in the kidney are also strongly VEGF-dependent. Whether intravitreal drugs can be detected within the glomeruli or affect VEGF in glomerular podocytes is not known. Therefore, the aim of this pilot study was to investigate the effects of a single intravitreal injection of aflibercept and ranibizumab on glomeruli of monkeys.The kidneys of eight cynomolgus monkeys, which were intravitreally injected either with 2 mg of aflibercept or with 0.5 mg of ranibizumab, were investigated one and seven days after injection. Two animals served as controls. The distribution of aflibercept, ranibizumab and VEGF was evaluated using anti-Fc- or anti-F(ab)-fragment and anti-VEGF antibodies respectively. The ratio of stained area/nuclei was calculated using a semi-quantitative computer assisted method. Glomerular endothelial cell fenestration was quantified in electron microscopy using a systematic uniform random sampling protocol and estimating the ratio of fenestrae per µm.Compared to the controls, the anti-VEGF stained area/nuclei ratio of the ranibizumab-treated animals showed no significant changes whereas the stained areas of the aflibercept-treated monkeys showed a significant decrease post-treatment. Immune reactivity (IR) against aflibercept or ranibizumab was detected in aflibercept- or ranibizumab treated animals respectively. The number of fenestrations of the glomerular endothelial cells has shown no significant differences except one day after aflibercept injection in which the number was increased.Surprisingly, both drugs could be detected within the capillaries of the glomeruli. After a single intravitreal injection of aflibercept, VEGF IR in the podocytes was significantly reduced compared to controls. Ranibizumab injection had no significant effect on the glomeruli's VEGF level. Whether this is caused by aflibercept's higher affinity to VEGF or because it is used in a higher stoichiometric concentration compared to ranibizumab remains to be investigated.
Project description:Endothelial-to-mesenchymal transition (EndMT) can cause loss of tight junctions, which in glomeruli are associated with albuminuria. Here we evaluated the role of EndMT in the development of albuminuria in diabetic nephropathy (DN). We demonstrated that EndMT occurs in the glomerular endothelium of patients with DN, showing by a decrease in CD31 but an increase in ?-SMA expression. In glomeruli of db/db mice, there was an increased ROCK1 expression in the endothelium plus a decreased CD31-positive cells. Cultured glomerular endothelial cells (GEnCs) underwent EndMT when stimulated by 30?mM glucose, and exhibited increased permeability. Meanwhile, they showed a higher ROCK1 expression and activation. Notably, inhibition of ROCK1 largely blocked EndMT and the increase in endothelial permeability under this high-glucose condition. In contrast, overexpression of ROCK1 induced these changes. Consistent alterations were observed in vivo that treating db/db mice with the ROCK1 inhibitor, fasudil, substantially suppressed the expression of ?-SMA in the glomerular endothelium, and reduced albuminuria. Thus we conclude that ROCK1 is induced by high glucose and it stimulates EndMT, resulting in increased endothelial permeability. Inhibition of ROCK1 could be a therapeutic strategy for preventing glomerular endothelial dysfunction and albuminuria in developing DN.
Project description:Endothelial cells contain several nanoscale domains such as caveolae, fenestrations and transendothelial channels, which regulate signaling and transendothelial permeability. These structures can be covered by filter-like diaphragms. A transmembrane PLVAP (plasmalemma vesicle associated protein) protein has been shown to be necessary for the formation of diaphragms. The expression, subcellular localization and fenestra-forming role of PLVAP in liver sinusoidal endothelial cells (LSEC) have remained controversial. Here we show that fenestrations in LSEC contain PLVAP-diaphragms during the fetal angiogenesis, but they lose the diaphragms at birth. Although it is thought that PLVAP only localizes to diaphragms, we found luminal localization of PLVAP in adult LSEC using several imaging techniques. Plvap-deficient mice revealed that the absence of PLVAP and diaphragms did not affect the morphology, the number of fenestrations or the overall vascular architecture in the liver sinusoids. Nevertheless, PLVAP in fetal LSEC (fenestrations with diaphragms) associated with LYVE-1 (lymphatic vessel endothelial hyaluronan receptor 1), neuropilin-1 and VEGFR2 (vascular endothelial growth factor receptor 2), whereas in the adult LSEC (fenestrations without diaphragms) these complexes disappeared. Collectively, our data show that PLVAP can be expressed on endothelial cells without diaphragms, contradict the prevailing concept that biogenesis of fenestrae would be PLVAP-dependent, and reveal previously unknown PLVAP-dependent molecular complexes in LSEC during angiogenesis.
Project description:Semaphorin3a (Sema3a), a chemorepellant guidance protein, plays crucial roles in neural, cardiac and peripheral vascular patterning. Sema3a is expressed in the developing nephron, mature podocytes and collecting tubules. Sema3a acts as a negative regulator of ureteric bud branching, but its function in glomerular development has not been examined. Here we tested the hypothesis that Sema3a regulates glomerular vascular development using loss- and gain-of-function mouse models. Sema3a deletion resulted in defects in renal vascular patterning, excess endothelial cells within glomerular capillaries, effaced podocytes with extremely wide foot processes and albuminuria. Podocyte Sema3a overexpression during organogenesis resulted in glomerular hypoplasia, characterized by glomerular endothelial cell apoptosis, delayed and abnormal podocyte foot process development, a complete absence of slit diaphragms and congenital proteinuria. Nephrin, WT1 and VEGFR2 were downregulated in Sema3a-overexpressing kidneys. We conclude that Sema3a is an essential negative regulator of endothelial cell survival in developing glomeruli and plays a crucial role in podocyte differentiation in vivo. Hence, a tight regulation of Sema3a dosage is required for the establishment of a normal glomerular filtration barrier.