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.

Summary Vascular endothelial growth factor-a (VEGF-A) is a protein secreted by podocytes that is necessary for survival of endothelial cells, podocytes and mesangial cells. VEGF-A regulates slit-diaphragm signaling and podocyte shape via VEGFR2-nephrin-nck-actin interactions. Chronic hyperglycemia-induced excess podocyte VEGF-A and low endothelial nitric oxide drive the development and the progression diabetic nephropathy. The abnormal cross talk between VEGF-A and NO pathways is fueled by the diabetic milieu increased oxidative stress. Recent findings on these pathogenic molecular mechanisms provide new potential targets for therapy for diabetic renal disease.

*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.

The onset of diabetic nephropathy (DN) is highlighted by glomerular filtration barrier abnormalities. Identifying pathogenic factors and targetable pathways driving DN is crucial to developing novel therapies and improving the disease outcome. Semaphorin3a (sema3a) is a guidance protein secreted by podocytes. Excess sema3a disrupts the glomerular filtration barrier. Here, using immunohistochemistry, we show increased podocyte SEMA3A in renal biopsies from patients with advanced DN. Using inducible, podocyte-specific Sema3a gain-of-function (Sema3a+) mice made diabetic with streptozotocin, we demonstrate that sema3a is pathogenic in DN. Diabetic Sema3a+ mice develop massive proteinuria, renal insufficiency, and extensive nodular glomerulosclerosis, mimicking advanced DN in humans. In diabetic mice, Sema3a+ exacerbates laminin and collagen IV accumulation in Kimmelstiel-Wilson-like glomerular nodules and causes diffuse podocyte foot process effacement and F-actin collapse via nephrin, αvβ3 integrin, and MICAL1 interactions with plexinA1. MICAL1 knockdown and sema3a inhibition render podocytes not susceptible to sema3a-induced shape changes, indicating that MICAL1 mediates sema3a-induced podocyte F-actin collapse. Moreover, sema3a binding inhibition or podocyte-specific plexinA1 deletion markedly ameliorates albuminuria and abrogates renal insufficiency and the diabetic nodular glomerulosclerosis phenotype of diabetic Sema3a+ mice. Collectively, these findings indicate that excess sema3a promotes severe diabetic nephropathy and identifies novel potential therapeutic targets for DN.

Background:Excess VEGF-A down-regulates nephrin causing glomerular disease. Nephrin interacts with VEGFR2 in vivo. Results: Nephrin-VEGFR2 interaction is direct, modulated by tyrosine phosphorylation, the VEGR2-nephrin complex involves Nck and actin, and VEGF-A signaling via this complex decreases cell size. Conclusion: This interaction links extracellular VEGF-A to slit diaphragms and the podocyte actin cytoskeleton. Significance: Our findings provide a molecular mechanism for VEGF-induced glomerular disease.

The tight regulation of vascular endothelial growth factor-A (VEGF-A) signaling is required for both the development and maintenance of the glomerular filtration barrier , but the pathogenic role of excessive amounts of VEGF-A detected in multiple renal diseases remains poorly defined. We generated inducible transgenic mice that overexpress podocyte VEGF 164 at any chosen stage of development. In this study, we report the phenotypes that result from podocyte VEGF 164 excess during organogenesis and after birth. On doxycycline induction, podocin-rtTA: tet-O-VEGF 164 mice express twofold higher kidney VEGF 164 levels than single transgenic mice, localized to podocytes. Podocyte VEGF 164 overexpression during organogenesis resulted in albuminuria at birth and was associated with glomerulomegaly, uniform podocyte effacement, very few and wide foot processes joined by occluding junctions, almost complete absence of slit diaphragms, and swollen endothelial cells with few fenestrae as revealed by transmission electron microscopy. Podocyte VEGF 164 overexpression after birth caused massive albuminuria in 70% of 2-week-old mice, glomerulomegaly, and minimal changes on light microscopy. Transmission electron microscopy showed podocyte effacement and fusion and morphologically normal endothelial cells. Podocyte VEGF 164 overexpression induced nephrin down-regulation without podocyte loss. VEGF 164 -induced abnormalities were reversible on removal of doxycycline and were unresponsive to methylprednisolone. Collectively, the data suggest that moderate podocyte VEGF 164 overexpression during organogenesis results in congenital nephrotic syndrome, whereas VEGF 164 overexpression after birth induces a steroid-resistant minimal change likedisease in mice.

Semaphorin-3A (Sema3a), a guidance protein secreted by podocytes, is essential for normal kidney patterning and glomerular filtration barrier development. Here, we report that podocyte-specific Sema3a gain-of-function in adult mice leads to proteinuric glomerular disease involving the three layers of the glomerular filtration barrier. Reversibility of the glomerular phenotype upon removal of the transgene induction provided proof-of-principle of the cause-and-effect relationship between podocyte Sema3a excess and glomerular disease. Mechanistically, excess Sema3a induces dysregulation of nephrin, matrix metalloproteinase 9, and αvβ3 integrin in vivo. Sema3a cell-autonomously disrupts podocyte shape. We identified a novel direct interaction between the Sema3a signaling receptor plexinA1 and nephrin, linking extracellular Sema3a signals to the slit-diaphragm signaling complex. We conclude that Sema3a functions as an extracellular negative regulator of the structure and function of the glomerular filtration barrier in the adult kidney. Our findings demonstrate a crosstalk between Sema3a and nephrin signaling pathways that is functionally relevant both in vivo and in vitro.

VEGF-A and nitric oxide are essential for glomerular filtration barrier homeostasis and are dysregulated in diabetic nephropathy. Here, we examined the effect of excess podocyte VEGF-A on the renal phenotype of endothelial nitric oxide synthase (eNOS) knockout mice. Podocyte-specific VEGF 164 gain of function in eNOS 2/2 mice resulted in nodular glomerulosclerosis, mesangiolysis, microaneurysms, and arteriolar hyalinosis associated with massive proteinuria and renal failure in the absence of diabetic milieu or hypertension. In contrast, podocyte-specific VEGF 164 gain of function in wild-type mice resulted in less pronounced albuminuria and increased creatinine clearance. Transmission electron microscopy revealed glomerular basement membrane thickening and podocyte effacement in eNOS 2/2 mice with podocytespecific VEGF 164 gain of function. Furthermore, glomerular nodules overexpressed collagen IV and laminin extensively. Biotin-switch and proximity ligation assays demonstrated that podocyte-specific VEGF 164 gain of function decreased glomerular S-nitrosylation of laminin in eNOS 2/2 mice. In addition, treatment with VEGF-A decreased S-nitrosylated laminin in cultured podocytes. Collectively, these data indicate that excess glomerular VEGF-A and eNOS deficiency is necessary and sufficient to induce Kimmelstiel-Wilsonlike nodular glomerulosclerosis in mice through a process that involves deposition of laminin and collagen IV and de-nitrosylation of laminin.