RESUMO
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.
Assuntos
Diferenciação Celular/genética , Células Endoteliais/fisiologia , Glomérulos Renais/crescimento & desenvolvimento , Podócitos/fisiologia , Semaforina-3A/fisiologia , Animais , Apoptose/fisiologia , Contagem de Células , Núcleo Celular/metabolismo , Técnicas de Cocultura , Células Endoteliais/metabolismo , Células Endoteliais/ultraestrutura , Técnica Direta de Fluorescência para Anticorpo , Corantes Fluorescentes/metabolismo , Deleção de Genes , Regulação da Expressão Gênica no Desenvolvimento , Griffonia/química , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Indóis/metabolismo , Glomérulos Renais/ultraestrutura , Camundongos , Camundongos Knockout , Camundongos Mutantes , Camundongos Transgênicos , Lectinas de Plantas/metabolismo , Podócitos/citologia , Podócitos/metabolismo , Podócitos/ultraestrutura , Ratos , Proteínas Recombinantes/metabolismo , Semaforina-3A/genética , Semaforina-3A/metabolismoRESUMO
We sought to examine the pathogenic role of excessive VEGF-A expression in podocytes, since it has been reported that diabetic nephropathy and other glomerular diseases are associated with increased VEGF-A expression. The induction of podocyte-specific VEGF164 overexpression in adult transgenic mice led to proteinuria, glomerulomegaly, glomerular basement membrane thickening, mesangial expansion, loss of slit diaphragms, and podocyte effacement. When doxycycline-mediated VEGF164 was stopped, these abnormalities reversed. These findings were associated with reversible downregulation of metalloproteinase 9 and nephrin expression. Using transmission electron microscopy, we established that VEGF-A receptor-2 (VEGFR2) was expressed in podocytes and glomerular endothelial cells. We also found that VEGF164 induced VEGFR2 phosphorylation in podocytes. Further, we were able to co-immunoprecipitate VEGFR2 and nephrin using whole kidney lysates, confirming interaction in vivo. This implies that autocrine and paracrine VEGF-A signaling through VEGFR2 occurs in podocytes and may mediate the glomerular phenotype caused by VEGF164 overexpression. Thus, we suggest that podocyte VEGF164 overexpression in adult mice is sufficient to induce glomerular filtration barrier structural and functional abnormalities similar to those present in murine diabetic nephropathy.
Assuntos
Nefropatias Diabéticas/metabolismo , Nefropatias/metabolismo , Podócitos/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Fatores Etários , Animais , Comunicação Autócrina , Nefropatias Diabéticas/genética , Nefropatias Diabéticas/patologia , Genótipo , Membrana Basal Glomerular/patologia , Nefropatias/genética , Nefropatias/patologia , Metaloproteinase 9 da Matriz/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Transgênicos , Comunicação Parácrina , Fenótipo , Fosforilação , Podócitos/patologia , Ligação Proteica , Proteinúria/genética , Proteinúria/metabolismo , Transdução de Sinais , Regulação para Cima , Fator A de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
Class 3 semaphorins (sema 3) are secreted guidance proteins. Sema 3A expressed by endothelial cells controls vascular morphogenesis through integrin inhibition. Sema 3C is required for normal cardiovascular patterning. Here we examined the potential role of sema 3C as regulator of endothelial cell function in vitro using mouse glomerular endothelial cells (MGEC). We determined that MGEC express sema 3C mRNA and protein and its receptors mRNA. Recombinant sema 3C induced MGEC proliferation 18 +/- 2% above control, as assessed by bromodeoxyuridine (BrdU) incorporation, and reduced starvation-induced apoptosis by 46 +/- 3%, as indicated by an in situ marker of activated caspase 3. Sema 3C increased MGEC adhesion to fibronectin 79 +/- 13% and to collagen 55 +/- 12% as compared with control. Sema 3C-induced MGEC adhesion was prevented by integrin blocking antibodies and involved beta1 integrin serine phosphorylation. Sema 3C-induced MGEC adhesion and proliferation were similar to those induced by vascular endothelial growth factor (VEGF)-A. Sema 3C induced a 44 +/- 11% increase in MGEC directional migration and stimulated MGEC capillary-like network formation on collagen I gels. Collectively, our data indicate that sema 3C promotes glomerular endothelial cell proliferation, adhesion, directional migration, and tube formation in vitro by stimulating integrin phosphorylation and VEGF120 secretion, functions that are similar to VEGF-A and opposite to sema 3A.
Assuntos
Células Endoteliais/efeitos dos fármacos , Células Endoteliais/fisiologia , Integrina beta1/efeitos dos fármacos , Semaforinas/fisiologia , Animais , Adesão Celular , Movimento Celular , Proliferação de Células , Células Endoteliais/citologia , Endotélio Vascular/citologia , Integrina beta1/metabolismo , Integrinas/efeitos dos fármacos , Integrinas/metabolismo , Rim/citologia , Camundongos , Neovascularização Fisiológica , Fosforilação , Proteínas Recombinantes/farmacologia , Semaforinas/farmacologia , Fator A de Crescimento do Endotélio Vascular/farmacologiaRESUMO
Class 3 semaphorins are guidance proteins involved in axon pathfinding, vascular patterning and lung branching morphogenesis in the developing mouse embryo. Semaphorin3a (Sema3a) is expressed in renal epithelia throughout kidney development, including podocytes and ureteric bud cells. However, the role of Sema3a in ureteric bud branching is unknown. Here we demonstrate that Sema3a plays a role in patterning the ureteric bud tree in both metanephric organ cultures and Sema3a mutant mice. In vitro ureteric bud injection with Sema3a antisense morpholino resulted in increased branching, whereas recombinant SEMA3A inhibited ureteric bud branching and decreased the number of developing glomeruli. Additional studies revealed that SEMA3A effects on ureteric bud branching involve downregulation of glial cell-line derived neurotrophic factor (GDNF) signaling, competition with vascular endothelial growth factor A (VEGF-A) and decreased activity of Akt survival pathways. Deletion of Sema3a in mice is associated with increased ureteric bud branching, confirming its inhibitory role in vivo. Collectively, these data suggest that Sema3a is an endogenous antagonist of ureteric bud branching and hence, plays a role in patterning the renal collecting system as a negative regulator.
Assuntos
Semaforina-3A/genética , Semaforina-3A/fisiologia , Ureter/embriologia , Animais , Axônios/metabolismo , Ligação Competitiva , Padronização Corporal , Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Camundongos , Modelos Biológicos , Oligonucleotídeos Antissenso/química , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Recombinantes/metabolismo , Transdução de Sinais , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Vascular endothelial growth factor (VEGF-A) plays multiple roles in kidney development: stimulates cell proliferation, survival, tubulogenesis, and branching morphogenesis. However, the mechanism that mediates VEGF-A induced ureteric bud branching is unclear. Glial-derived neurotrophic factor (GDNF) signaling through tyrosine kinase c-RET is the major regulator of ureteric bud branching. Here we examined whether VEGF-A regulates RET signaling. We determined that ureteric bud-derived cells express the main VEGF-A signaling receptor, VEGFR2 and RET, by RT-PCR, immunoblotting, and immunocytochemistry. We show that the VEGF-A isoform VEGF(165) induces RET-tyr(1062) phosphorylation in addition to VEGFR2 autophosphorylation, that VEGF(165) and GDNF have additive effects on RET-tyr(1062) phosphorylation, and that VEGFR2 and RET co-immunoprecipitate. Functionally, VEGF(165) induces ureteric bud cell proliferation and branching morphogenesis. Similarly, in embryonic kidney explants VEGF(165) induces RET-tyr(1062) phosphorylation and upregulates GDNF. These findings provide evidence for a novel cooperative interaction between VEGFR2 and RET that mediates VEGF-A functions in ureteric bud cells.
Assuntos
Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Morfogênese/fisiologia , Proteínas Proto-Oncogênicas c-ret/metabolismo , Transdução de Sinais/fisiologia , Ureter/fisiologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Crescimento Celular , Linhagem Celular , Proliferação de Células , Humanos , Ureter/embriologiaRESUMO
Vascular endothelial growth factor (VEGF-A) signaling is required for endothelial cell differentiation, vasculogenesis, angiogenesis, and vascular patterning. During kidney morphogenesis, podocyte VEGF-A guides endothelial cells toward developing glomeruli. Podocyte VEGF-A expression continues throughout life but its function after completion of development remains unclear. Here, we examined the expression of VEGF-A and its receptors VEGFR1, VEGFR2, NP1, and NP2 in conditionally immortalized mouse podocytes cultured in undifferentiated and differentiated conditions using RT-PCR and Western analysis. VEGF-A secretion was assessed by ELISA and Western analysis. Upon podocyte differentiation, VEGF-A protein expression and secretion increased threefold. Differentiated podocytes expressed eightfold higher VEGFR2 mRNA levels than undifferentiated podocytes, whereas VEGFR1, sVEGFR1, NP1, and NP2 mRNA levels were similar. We examined the regulation and function of the VEGF-A system by exposing differentiated podocytes to recombinant VEGF(165) (20 ng/ml) or control media for 24 h. VEGF(165) induced a twofold increase in VEGFR2 mRNA and protein levels, whereas VEGFR1, sVEGFR1, NP1, and NP2 mRNA levels remained unchanged. VEGF(165) induced VEGFR2 phosphorylation. VEGF(165) reduced podocyte apoptosis approximately 40%, whereas anti-VEGFR2 neutralizing antibody enhanced it twofold. We determined that VEGF-A signaling regulates slit diaphragm proteins by inducing a dose-response podocin upregulation and increasing its interaction with CD2AP. The data indicate that podocytes in culture have a functional autocrine VEGF-A system that is regulated by differentiation and ligand availability. VEGF-A functions in podocytes include promoting survival through VEGFR2, inducing podocin upregulation and increasing podocin/CD2AP interaction.