Vascular Endothelial Growth Factor A Signaling in the Podocyte-Endothelial Compartment Is Required for Mesangial Cell Migration and Survival

Clinical course of VEGF^hypo/loxP,Neph-Cre^+/− mice. (A) Representative SDS-PAGE blot shows frank nephrotic-range proteinuria (>5 g/L) in VEGF^hypo/loxP,Neph-Cre mice by 5 d of age. Molecular weight (MW) markers: lane 1, urine from a control (VEGF^hypo/loxP) mouse; lane 2, urine from a mutant (VEGF^hypo/loxP,Neph-Cre^+/−); and lanes 3 and 4, positive controls from mice with known albuminuria. The arrow points at the large amount of albumin (66.2 kD) present in a sick VEGF^hypo/loxP,Neph-Cre^+/− mouse. A total of 2 μl of urine was loaded in each lane. (B) The bar graph shows reduced hemoglobin (Hb) levels in VEGF^hypo/loxP,Neph-Cre^+/− mice compared with controls that carry the VEGF^hypo/loxP and VEGF^hypo/+,Neph-Cre^+/− genotypes. Values represent mean ± SD (n = 3 in each group); *P < 0.05. (C) Analysis of serum creatinine levels. Serum creatinine measurements were markedly increased in mutant mice by 10 d of age. Values represent mean ± SD (n = 3 in each group); **P < 0.01. 100% of VEGF^hypo/loxP,Neph-Cre^+/− mice (n = 18) were dead by 3 wk of age.

Mesangiolysis in VEGF^hypo/loxP,Neph-Cre^+/− mice. (A) hematoxylin and eosin (H&E) and Masson trichrome staining (MAS) demonstrate loss of mesangial cells within differentiated glomeruli as early as 1 to 3 d of age compared with a day 3 (early time point) control. The mesangial cell compartment was replaced by material that has a foamy appearance. (B) Vascular α-smooth muscle actin (VSMA-α) immunostaining confirms that some mesangial cells (brown) are still present in glomeruli at 3 d (early time point) but that they are completely lost by 10 d (late time point). Control glomeruli at P10 to P12 are stained positive for VSMA-α. Early time point includes days 1 to 3 of age (P1 to P3); late time point includes days 10 to 12 of age (P10 to P12).

Molecular marker analysis of cell compartments within the glomerulus of VEGF^hypo/loxP,Neph-Cre^+/− mice at birth and days 10 to 12 (late time point). (A) In situ hybridization: Podocyte-specific markers: Wilms tumor suppressor gene (WT-1) and nephrin are expressed by podocytes at levels similar to controls at day 1 (early time point). (B) PDGFR-β receptor marks mesangial cells in developing and mature glomeruli. At birth, PDGFR-β receptor staining is similar to controls but completely disappears by 10 to 12 d of age (late time point) compared with control glomeruli of the same stage. (C) Immunostaining for the podocyte cell marker WT-1 and the endothelial cell marker platelet endothelial cell adhesion molecule 1 (PECAM-1) versus WT-1 and the early mesangial cell marker desmin. At day 1 (early time point), dual immunofluorescent staining demonstrated the presence of both endothelial (green in the top row) and mesangial (green in the bottom row) cells. WT-1–positive cells are podocytes (red).

(A) The endothelial cell marker PECAM is present at birth and is not different between control and mutant VEGF^hypo/loxP,Neph-Cre^+/− glomeruli. (B) Double immunostaining demonstrates rapid injury in the endothelial compartment by P3 (early time point). Loss of endothelial cells demonstrated by the endothelial-specific marker PECAM (green in the top row) is apparent at this stage, whereas mesangial cells detected by the mesangial-specific marker desmin (green in the bottom row) are still present in the glomeruli of VEGF^hypo/loxNeph-Cre^+/− mice. Anti–laminin-α5 antibody (red) was used to outline the glomerular structure. (C) Immunostaining for the endothelial cell marker CD31 (PECAM) and the later mesangial cell marker VSMA-α. Double immunostaining confirms that endothelial cell loss precedes the loss of mesangial cells in VEGF^hypo/lox,Neph-Cre^+/− mice as PECAM-positive cells are lost before VSMA-α–positive cells. Magnification, ×63 in A and C; ×20 in B.

Endothelial cell loss precedes mesangiolysis in VEGF^hypo/loxP,Neph-Cre^+/− mice. Electron micrographs of glomeruli from control (a) and from VEGF^hypo/loxP,Neph-Cre^+/− (b) mice show the presence of both mesangial (big arrows) and endothelial (small arrows) cells at birth. By day 3 (c, *) endothelial capillaries are enlarged and lack endothelial cells, leaving dilated capillary “ghosts” (d, *). Mesangial cells are still found at P3 (c, arrow). (e) At higher magnification, the normal glomerular filtration barrier (GFB) can be seen to consist of podocytes, fenestrated endothelium, and intervening glomerular basement membrane (GBM). (f) In the GFB of VEGF^hypo/loxP,Neph-Cre^+/− mice, fenestrated endothelial cells are missing, whereas GBM (double arrows) and podocyte foot processes are well preserved; however, by P5, “coarsening” of foot processes becomes apparent (g, ***). (h) By day 5, both mesangial and endothelial cells are lost. Po, podocytes; en, endothelium; cap, capillary. Magnification, ×1500 in a, b, c, and h; ×25,500 in e, f, and g; ×3100 in d.

Effect of VEGF-A gene dose within podocytes on the mesangial cell compartment. Podocyte-selective deletion of both VEGF-A (approximately 100% loss) alleles in glomeruli results in perinatal lethality. No endothelial or mesangial cells are observed in the glomerulus. Mesangial cells can be observed in glomeruli from VEGF^hypo/loxP,Neph-Cre^+/− mice at birth, but these undergo dramatic mesangiolysis by 2 wk of age. Podocyte-selective loss of a single VEGF-A allele results in renal disease by 2.5 wk of age, characterized by proteinuria and swelling of endothelial cells, whereas mesangial cells are clearly present at this stage. Arrowheads, mesangial cells; ESKD, end-stage kidney disease.

Expression of major VEGF receptors in the glomerulus in vivo. (A) Paraffin sections of newborn mouse kidney from VEGFR-2,GFP neo-out mice were stained with GFP antibody (brown). Endothelial cells are seen to express the GFP protein during glomerulogenesis and in the adult glomerulus. (B) VSMA-α staining was performed in conjunction with GFP staining to mark the mesangial cells. No cells that expressed both VSMA-α and VEGFR-2 were observed. (C) To determine the expression of VEGFR-1 (Flt-1), kidneys from newborn VEGFR-1^lacZki/+ mice were harvested and stained for β-galactosidase activity. Endothelial cells clearly express lacZ, whereas other glomerular cell types do not. (D) Dual immunostaining on kidneys from newborn VEGFR-1^lacZki/+ mice demonstrates expression of β-galactosidase (lacZ) gene product in VEGFR-1–positive cells; PDGFR-β marks the mesangial cells. No cells clearly expressed both PDGFR-β and VEGFR-1.