VEGF-A is essential for the development and maintenance of the glomerular filtration barrier 
. Our studies uncover a specific molecular mechanism mediating VEGF-A requirement in the adult glomerulus. We show that in vivo
podocyte VEGF-A regulates fibronectin and alphaV
integrin signaling in the glomerulus, and that disruption of VEGFR2- alphaV
integrin crosstalk by acute podocyte VEGF knockdown damages the three layers of the glomerular filtration barrier, resulting in proteinuria and acute renal failure.
We generated a transgenic mouse model to knockdown VEGF in podocytes by doxycycline-regulated shRNA, enabling to study the pathogenic effects of local VEGF downregulation and repair processes in vivo
in a reversible manner, at difference from permanent loss of VEGF expression in knockout models. An immortalized podocyte cell line derived from these mice silences VEGF-A upon doxycycline exposure, decreasing both VEGF cell content and VEGF secretion. Differentiated VEGFKD
podocytes express prototypical podocyte proteins: nephrin, podocin, WT1, as well as VEGFR2 
. Accordingly, doxycycline-induced VEGF knockdown decreased VEGFR2 phosphorylation in VEGFKD
podocytes, resulting in cell shape and size change. Lee et al. reported a similar autocrine response in endothelial cells 
A surprising finding of this study was that in vivo
podocyte VEGF knockdown for only one week induced proteinuria and kidney failure (). Even though the circulating VEGF levels were normal (), short-term podocyte VEGF knockdown caused a glomerular phenotype characterized by low glomerular volume, mesangiolysis, microaneurisms and typical features of endotheliosis, including endothelial cell swelling, interdigitation into the GBM, widening and lamination of the lamina densa, and podocyte effacement. These abnormalities were not due to hypertension, microangiopathic anemia or decreased availability of circulating VEGF. Instead, they resulted from decreased VEGFR2 signaling in the glomerulus. The evidence for this conclusion is that mice with VEGF knockdown were normotensive, their hematocrit and VEGF circulating levels were normal, while VEGF mRNA and protein were significantly decreased in isolated glomeruli, and in the urine (). Accordingly, podocyte VEGF knockdown decreased VEGFR2 phosphorylation in cultured podocytes (). We previously reported that absence of VEGF-A signals promotes VEGFR2-nephrin interaction 
. This may have contributed to maintain normal nephrin expression in the setting of proteinuria. Alternatively, the latter could be due to the short duration of the experiment.
Podocyte VEGF knockdown glomerular phenotype is reminiscent of preeclampsia, an entity due, at least in part, to excess soluble VEGFR1 acting as a decoy receptor, effectively decreasing circulating VEGF availability 
. A previous report showed that long-term deletion of VEGF in podocytes, using a different genetic approach, induces thrombotic microangiopathy and hypertension 
. Our short-term podocyte VEGF knockdown did not develop such a severe phenotype, as determined by light microscopy, TEM examination and normal blood pressure. Instead, the glomerular phenotype induced by short-term podocyte VEGF knockdown was similar to milder cases of renal disease induced by anti-VEGF drugs 37
, supporting the concept that disruption of the tight regulation of glomerular VEGF causes acute and chronic renal disease.
An important conclusion drawn from our experiments is that decreased autocrine and paracrine VEGFR2 signaling induced by podocyte VEGF knockdown disrupts VEGFR2- alphaVbeta3 integrin crosstalk at the glomerular filtration barrier. The experimental evidence supporting this is as follows. First, podocyte VEGF knockdown downregulates alphaVbeta3 integrin in glomerular endothelial cells (). Second, VEGFR2 interacts with beta3 integrin and neuropilin-1 in the kidney in vivo and in cultured podocytes (). Third, podocyte VEGF knockdown disrupts alphaVbeta3 integrin activation in glomeruli (). Fourth, in cultured podocytes alphaVbeta3 integrin activation is negatively modulated cell-autonomously by VEGF knockdown, leading to reversible changes in podocyte shape and size ( and ).
In endothelial cells VEGF-A signaling activates integrin alphaV
via VEGFR2 
. VEGF-induced alphaV
integrin activation is dependent on affinity modulation and VEGFR2-beta3
integrin direct association 
. This inside-out mechanism of alphaV
integrin activation is consistent with our observation of decreased WOW1 and baseline AP5 labeling in VEGF knockdown glomeruli and podocytes. The interaction of VEGFR2- alphaV
integrin is a complex critical modulator of angiogenesis in vitro and in vivo 
. In cultured endothelial cells beta3
integrin silencing impairs cell adhesion, migration and capillary growth in response to VEGF 
. However, beta3
integrin null mice have increased VEGFR2 signaling 
integrin negatively regulates VEGF-mediated angiogenesis by limiting neuropilin-1 interaction with VEGFR2 
. Thus, we propose that low alphaV
integrin activity plays a compensatory role in the setting of decreased glomerular VEGFR2 signaling.
VEGF-A stimulates uPAR expression in endothelial cells 
. Excessive soluble uPAR, a biomarker of systemic inflammation 
, was shown to cause FSGS in mice and humans by binding and activating podocyte beta3
. Whether VEGF-A signaling regulates suPAR effects at the glomerular filtration barrier, remains to be determined.
Podocyte VEGF knockdown-induced disruption of VEGFR2 - alphaV
integrin signaling at the glomerular filtration barrier resulted in endothelial injury and GBM lamination. VEGF and alphaV
integrin normally provide endothelial cell survival signals, stimulate adhesion and fenestrae formation 
. Endotheliosis-associated GBM lamination appears to be due to defective alphaV
integrin-mediated endothelial adhesion or altered assembly of the GBM due to lower expression of fibronectin. Integrin alpha3
is crucial for podocyte development and GBM assembly 
. Deletion of alpha3
integrin caused disorganized GBM, associated with proteinuria and perinatal lethality 
. Podocyte-specific alpha3
integrin deletion resulted in massive proteinuria and GBM lamination 
. Podocyte beta1
integrin deletion resulted in effaced podocytes, multilaminated GBM, expansion of the lamina rara externa, and normal glomerular endothelium 
. Although podocyte VEGF knockdown induced GBM lamination in adult mice, the GBM splitting expanded the lamina rara interna adjacent to damaged endothelial cells. Consistent with this, podocyte VEGF knockdown did not alter beta1
integrin expression or phosphorylation.
In summary, our studies showed that acute podocyte VEGF knockdown in mice decreases autocrine and paracrine VEGFR2 signaling, induces fibronectin and alphaVbeta3 integrin downregulation and decreased activation in the glomerulus. Further, podocyte VEGF knockdown disrupted VEGFR2- alphaVbeta3 integrin functional interaction in the glomerulus, thereby damaging the three layers of the glomerular filtration barrier, resulting in proteinuria and acute renal failure. Collectively, our findings provide mechanistic insight on potential targets for intervention in pathological circumstances where kidney VEGF is disregulated.