Acute Podocyte Vascular Endothelial Growth Factor (VEGF-A) Knockdown Disrupts alpha_Vbeta₃ Integrin Signaling in the Glomerulus

<div><p>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. <em>Tet-O-siVEGF:podocin-rtTA</em> mice express <em>VEGF</em> 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 <em>tet-O-siVEGF:podocin-rtTA</em> 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_Vbeta₃ integrin <em>in vivo</em>. VEGF receptor-2 (VEGFR2) interacts with beta₃ integrin and neuropilin-1 in the kidney <em>in vivo</em> and in VEGF^KD podocytes. Podocyte VEGF knockdown disrupts alpha_Vbeta₃ integrin activation in glomeruli, detected by WOW1-Fab. VEGF silencing in cultured VEGF^KD podocytes downregulates fibronectin and disrupts alpha_Vbeta₃ integrin activation cell-autonomously. Collectively, these studies indicate that podocyte VEGF-A regulates alpha_Vbeta₃ integrin signaling in the glomerulus, and that podocyte VEGF knockdown disrupts alpha_Vbeta₃ integrin activity via decreased VEGFR2 signaling, thereby damaging the three layers of the glomerular filtration barrier, causing proteinuria and acute renal failure.</p> </div

Blood Pressure in <i>siVEGF</i> mice.

<p>Systolic blood pressure and diastolic blood pressure is normal in VEGF knockdown mice. In control period and VEGF knockdown period, values are mean ±SE in peak of activity (dark period) and rest (light period).</p

General parameters from <i>siVEGF</i> mice.

<p>General parameters from <i>siVEGF</i> mice.</p

VEGF knockdown glomerular phenotype.

<p>(A) PAS stain shows mesangiolysis (arrowhead), microaneurisms (green asterisk) and decreased glomerular volume in VEGF-A knockdown glomeruli, scale bars  = 50 µm. (B) Quantitation of glomerular volume reveals that VEGF knockdown induces significant decrease in glomerular size. * indicates P<0.05 vs. control. (C-D) TEM: control glomeruli show normal ultrastructure; scale bars  = 200 nm (C) and 1 µm (D). (E-F) TEM: VEGF knockdown glomeruli show endothelial cell swelling, vacuolization and decreased fenestration; GBM lamination of the lamina densa, irregular thickening and interdigitations of the endothelium; podocyte foot process effacement; scale bars = 500′nm (E) and 1 µm (F). Cap = capillary, P = podocyte, *  = GBM.</p

VEGF knockdown downregulates fibronectin expression.

<p>(A) IHC: VEGF knockdown glomeruli show marked decrease in immunoreactive fibronectin, while nephrin expression is unchanged. Scale bar = 20 µm (B) Representative immunoblots show decreased fibronectin in VEGF knockdown kidney lysate, whereas nephrin and laminin expression levels are similar to controls. (C) ICC: Fibronectin expression decreases in doxycycline-induced VEGF^KD podocytes, while nephrin does not change. (D) Representative immunoblot shows fibronectin downregulation in induced VEGF^KD podocytes, scale bars = 10 µm. In (C) and (D) bar graphs show densitometric analysis, data are expressed as mean±SEM fold change in arbitrary units as compared to controls, n≥3, * indicate P<0.05 vs. control.</p

Podocyte VEGF knockdown downregulates endothelial alpha_Vbeta3 integrin.

<p>(A) Representative immunoblots show decreased alpha_Vbeta₃ integrin in VEGF knockdown kidney lysate, while beta₁ integrin and S⁷⁸⁵- beta₁ integrin (P-β1) remain at control levels. (B) Dual-immunostaining shows decreased alpha_Vbeta₃ integrin in VEGF knockdown glomeruli, with minimal co-localization with podocin, which is stable; negative controls shown. (C) Dual-immunostaining shows alpha_Vbeta₃ integrin and CD31 (endothelial marker) co-localization in control glomeruli, while in VEGF knockdown alpha_Vbeta₃ integrin decreases and CD31 does not. Note that immunoreactive alpha_Vbeta₃ signals appear higher than in (B) due to permeabilization required to detect CD31. (D) Representative immunoblots show alpha_Vbeta₃ integrin and beta₃ integrin levels in podocyte lysate unchanged upon VEGF knockdown. In (A) and (D) bar graphs show densitometric analysis, data are expressed as mean±SEM fold change as compared to controls, n≥3, * indicate P<0.05 vs. control. In (B) and (C) scale bars = 20 µm.</p

VEGF knockdown mouse model.

<p>(A) One week after doxycycline-induction, <i>podocin-rtTA:tet-O-siVEGF</i> adult mice decrease VEGF-A mRNA and VEGF-A protein levels to ∼20% of controls in isolated glomeruli. (B) Urine VEGF-A decreases in VEGF knockdown mice to ∼30% of control values. (C) Plasma VEGF-A is similar in VEGF knockdown and control mice. (D) VEGF immunohistochemistry shows normal expression in control (left), and decreased glomerular VEGF-A expression in VEGF knockdown mice (right), scale bar = 30 µm. (E) Low magnification VEGF immunohistochemistry shows absence of VEGF in some glomeruli in VEGF knockdown mice (right), (scale bar = 40 µm). (F) Glomerular VEGF-A (VEGF⁺ area/glomerular area x100) in VEGF knockdown mice (n = 135) decreases to ∼30% of controls (n = 118). In all bar graphs * indicates P<0.05 compared to control.</p

VEGF knockdown podocyte model.

<p>(A) Cell Tracker (green) and rhodamine phalloidin (red) labeling shows images of spindle-like undifferentiated VEGF^KD podocytes (top left) and rhomboidal/polygonal differentiated VEGF^KD podocytes (top right). Immunoblots show Sv40T antigen expressed only in undifferentiated VEGF^KD podocytes (bottom left). (B) Differentiated VEGF^KD podocytes exposed to doxycycline decreased VEGF-A cellular content and secreted VEGF-A. (C) Immunocytochemistry: differentiated VEGF^KD podocytes in control conditions express VEGFR2 (green) and nephrin (red), which co-localize (yellow, merge), cell nuclei labeled with DAPI (blue); scale bar = 10 µm. (D) Immunoblots show that VEGF-A knockdown did not change nephrin, podocin, WT-1 and VEGFR2 expression level in VEGF^KD podocytes; whereas VEGF-A knockdown decreased ^Y1175VEGFR2 phosphorylation as compared to control. Bar graphs show densitometric analysis of ≥3 immnublots/protein expressed as fold change mean ±SEM. (E) VEGF-A knockdown changed VEGF^KD podocyte shape and decreased their size, assessed by rhodamine phalloidin staining, which were reversible upon exposure to recombinant VEGF₁₆₅. (F) Quantitation of VEGF^KD podocyte area change induced by VEGF knockdown, reversibility by exposure to VEGF₁₆₅, expressed as fold change mean±SEM. Scale bar = 20 µm. In all bar graphs * indicate P<0.05 vs. control.</p

VEGF knockdown induces proteinuria and acute renal failure.

<p>(A) Immunoblot shows severe albuminuria in VEGF Knockdown mice. (B) ELISA: urinary albumin/creatinine ratio in VEGF knockdown mice is 10-fold higher than in controls. (C) Creatinine clearance is 62% lower in VEGF knockdown mice than in controls. (D) Plasma creatinine significantly increases in VEGF knockdown mice. * indicates P<0.05 vs. control.</p

VEGFR2-β3 integrin-neuropilin-1 interact <i>in vivo</i> and in cultured podocytes. VEGF knockdown decreases alpha_Vbeta₃ integrin activity.

<p>(A) VEGFR2 - beta₃ integrin - neuropilin1 (NRP1) co-immunoprecipitate <i>in vivo,</i> shown by reciprocal VEGFR2 and beta₃ integrin IP. Negative control is rabbit serum (RS). Immunoprecipitates were blotted with VEGFR2, beta₃ integrin, Y¹¹⁷⁵-VEGFR2, anti S⁷⁸⁵- beta₃ integrin and neuropilin-1 antibodies. (B) VEGFR2 - beta₃ integrin – NRP1 co-immunoprecipitate in cultured podocytes. IPs were performed as described in (A) using VEGF^KD podocyte lysates. (C) Dual-immunostaining shows decreased active alpha_vbeta₃ integrin (WOW-1) and total beta₃ integrin in glomeruli from VEGF knockdown mice. (D) Immunocytochemistry shows decreased active alpha_Vbeta₃ integrin (WOW-1) in VEGF knockdown as compared to control podocytes, blue nuclei (Hoechst 33342). Scale bars = 20 µm.</p