Acute podocyte vascular endothelial growth factor (VEGF-A) knockdown disrupts alphaVbeta3 integrin signaling in the glomerulus.
Podocyte or endothelial cell VEGF-A knockout causes thrombotic microangiopathy in adult mice. To study the mechanism involved in acute and local injury caused by low podocyte VEGF-A we developed an inducible, podocyte-specific VEGF-A knockdown mouse, and we generated an immortalized podocyte cell line (VEGF(KD)) that downregulates VEGF-A upon doxycycline exposure. Tet-O-siVEGF:podocin-rtTA mice express VEGF shRNA in podocytes in a doxycycline-regulated manner, decreasing VEGF-A mRNA and VEGF-A protein levels in isolated glomeruli to ~20% of non-induced controls and urine VEGF-A to ~30% of control values a week after doxycycline induction. Induced tet-O-siVEGF:podocin-rtTA mice developed acute renal failure and proteinuria, associated with mesangiolysis and microaneurisms. Glomerular ultrastructure revealed endothelial cell swelling, GBM lamination and podocyte effacement. VEGF knockdown decreased podocyte fibronectin and glomerular endothelial alpha(V)beta(3) integrin in vivo. VEGF receptor-2 (VEGFR2) interacts with beta(3) integrin and neuropilin-1 in the kidney in vivo and in VEGF(KD) podocytes. Podocyte VEGF knockdown disrupts alpha(V)beta(3) integrin activation in glomeruli, detected by WOW1-Fab. VEGF silencing in cultured VEGF(KD) podocytes downregulates fibronectin and disrupts alpha(V)beta(3) integrin activation cell-autonomously. Collectively, these studies indicate that podocyte VEGF-A regulates alpha(V)beta(3) integrin signaling in the glomerulus, and that podocyte VEGF knockdown disrupts alpha(V)beta(3) integrin activity via decreased VEGFR2 signaling, thereby damaging the three layers of the glomerular filtration barrier, causing proteinuria and acute renal failure.
Department of Pediatrics, Yale University School of Medicine, New Haven, Connecticut, United States of America.
Delma
D
Veron
Guillermo
G
Villegas
Pardeep Kumar
PK
Aggarwal
Claudia
C
Bertuccio
Juan
J
Jimenez
Heino
H
Velazquez
Kimberly
K
Reidy
Dale R
DR
Abrahamson
Gilbert
G
Moeckel
Michael
M
Kashgarian
Alda
A
Tufro
D000818:Animals; D001794:Blood Pressure / Q000187:drug effects; D002478:Cells, Cultured; D015536:Down-Regulation / Q000187:drug effects; D004318:Doxycycline / Q000494:pharmacology; D004727:Endothelium / Q000187:drug effects / Q000378:metabolism; D005353:Fibronectins / Q000378:metabolism; D055785:Gene Knockdown Techniques; D039302:Integrin alphaVbeta3 / Q000378:metabolism*; D051379:Mice; D023421:Models, Animal; D039942:Neuropilin-1 / Q000378:metabolism; D010641:Phenotype; D050199:Podocytes / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D011485:Protein Binding / Q000187:drug effects; D011507:Proteinuria / Q000150:complications / Q000378:metabolism / Q000473:pathology / Q000503:physiopathology; D034741:RNA, Small Interfering / Q000378:metabolism; D051437:Renal Insufficiency / Q000150:complications / Q000378:metabolism / Q000473:pathology / Q000503:physiopathology; D015398:Signal Transduction / Q000187:drug effects; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism*; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000378:metabolism
D005353:Fibronectins; D039302:Integrin alphaVbeta3; D034741:RNA, Small Interfering; D042461:Vascular Endothelial Growth Factor A; C467480:vascular endothelial growth factor A, mouse; D039942:Neuropilin-1; D040301:Vascular Endothelial Growth Factor Receptor-2; D004318:Doxycycline