Autocrine VEGF-A system in podocytes regulates podocin and its interaction with CD2AP.
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.
Guan
Fangxia
F
Dept. of Pediatrics/Nephrology, Albert Einstein College of Medicine, 1300 Morris Park Ave, Forchheimer Bldg., Rm. 708, Bronx, NY 10461, USA.
Villegas
Guillermo
G
Teichman
Jason
J
Mundel
Peter
P
Tufro
Alda
A
D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D002097:C-Reactive Protein / Q000032:analysis / Q000235:genetics / Q000502:physiology; D002454:Cell Differentiation / Q000502:physiology; D002460:Cell Line; D002470:Cell Survival / Q000502:physiology; D003598:Cytoskeletal Proteins; D005786:Gene Expression Regulation / Q000502:physiology; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins / Q000032:analysis / Q000235:genetics / Q000502:physiology*; D051379:Mice; D009419:Nerve Tissue Proteins / Q000032:analysis / Q000235:genetics / Q000502:physiology; D039943:Neuropilin-2 / Q000032:analysis / Q000235:genetics / Q000502:physiology; D050199:Podocytes / Q000737:chemistry / Q000166:cytology / Q000502:physiology*; D011506:Proteins / Q000032:analysis / Q000235:genetics / Q000502:physiology*; D051057:Proto-Oncogene Proteins c-akt / Q000032:analysis / Q000235:genetics / Q000502:physiology; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics; D040262:Receptors, Vascular Endothelial Growth Factor / Q000032:analysis / Q000235:genetics / Q000502:physiology; D015398:Signal Transduction / Q000502:physiology; D042461:Vascular Endothelial Growth Factor A / Q000032:analysis / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D040281:Vascular Endothelial Growth Factor Receptor-1 / Q000032:analysis / Q000235:genetics / Q000502:physiology; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000032:analysis / Q000235:genetics / Q000502:physiology; D000099153:Pentraxins
D048868:Adaptor Proteins, Signal Transducing; D002097:C-Reactive Protein; D003598:Cytoskeletal Proteins; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D039943:Neuropilin-2; D011506:Proteins; D051057:Proto-Oncogene Proteins c-akt; D012333:RNA, Messenger; D040262:Receptors, Vascular Endothelial Growth Factor; D042461:Vascular Endothelial Growth Factor A; D040281:Vascular Endothelial Growth Factor Receptor-1; D040301:Vascular Endothelial Growth Factor Receptor-2; D000099153:Pentraxins; C114545:CD2-associated protein; C407223:NPHS2 protein; C467480:vascular endothelial growth factor A, mouse