VEGF(165) mediates glomerular endothelial repair

Abstract

VEGF(165), the most abundant isoform in man, is an angiogenic cytokine that also regulates vascular permeability. Its function in the renal glomerulus, where it is expressed in visceral epithelial and mesangial cells, is unknown. To assess the role of VEGF(165) in glomerular disease, we administered a novel antagonist - a high-affinity, nuclease-resistant RNA aptamer coupled to 40-kDa polyethylene glycol (PEG) - to normal rats and to rats with mesangioproliferative nephritis, passive Heymann nephritis (PHN), or puromycin aminonucleoside nephrosis (PAN). In normal rats, antagonism of VEGF(165) for 21 days failed to induce glomerular pathology or proteinuria. In rats with mesangioproliferative nephritis, the VEGF(165) aptamer (but not a sequence-scrambled control RNA or PEG alone) led to a reduction of glomerular endothelial regeneration and an increase in endothelial cell death, provoking an 8-fold increase in the frequency of glomerular microaneurysms by day 6. In contrast, early leukocyte influx and the proliferation, activation, and matrix accumulation of mesangial cells were not affected in these rats. In rats with PHN or PAN, administration of the VEGF(165) aptamer did not influence the course of proteinuria using various dosages and administration routes. These data identify VEGF(165) as a factor of central importance for endothelial cell survival and repair in glomerular disease, and point to a potentially novel way to influence the course of glomerular diseases characterized by endothelial cell damage, such as various glomerulonephritides, thrombotic microangiopathies, or renal transplant rejection.

Figures

Figure 1

Evaluation of VEGF165 aptamer attenuation of VEGF165-induced corneal angiogenesis. Hydron pellets with or without VEGF165 (3 pmol) were implanted in the corneal stroma of rats. Animals were treated intravenously twice daily with either PBS, aptamer, or the control aptamer (20 mg/kg per day) (a) or with the indicated doses of aptamer (b). Corneal neovascularization index was derived from estimates of the length and density of new vessels and the circumference of the neovascular response. Each group contained 8 eyes.

Figure 2

Glomerular cell proliferation at 48 hours after induction of anti–Thy 1.1 nephritis in rats receiving the VEGF165 aptamer (n = 6), PEG alone (n = 6), scrambled VEGF165 aptamer (n = 5), or PBS alone (n = 5). (a) BrdU incorporation in a rat receiving VEGF165 aptamer. Labeled cells are mostly localized to the mesangium (cluster of 3 cells in the center of the glomerulus); only 1 glomerular endothelial cell (top edge of the glomerulus) is labeled. ×1,000. (b) BrdU incorporation in a rat receiving PEG alone. Labeled cells are mostly endothelial cells. ×1,000. (c) PAS-stained renal section from a rat receiving VEGF165 aptamer. A mesangial cell mitosis is present (arrow). ×1,000. (d) PAS-stained renal section of a rat receiving PEG alone. An endothelial cell mitosis is present (arrow). ×1,000. (e) Quantitative evaluation of endothelial vs. nonendothelial glomerular cell proliferation (as defined by counts of mitotic figures or nuclei incorporating BrdU). NS, not significant.

Figure 3

Glomerular cell death and apoptosis detected by (a) TUNEL staining and (b) staining for nuclei containing single-stranded DNA (ssDNA) at 48 hours after induction of anti–Thy 1.1 nephritis in rats receiving the VEGF165 aptamer (n = 6) or PEG alone (n = 6). TUNEL-stained and ssDNA-positive cells were separated into those that localized to endothelial cell layers and others.

Figure 4

Mesangiolysis, glomerular microaneurysms, and proteinuria on days 2 and 6 after induction of anti–Thy 1.1 nephritis in rats receiving the VEGF165 aptamer (n = 6), PEG alone (n = 6), scrambled VEGF165 aptamer (n = 5), or PBS alone (n = 5). (a) Overview of histological changes in a rat receiving the VEGF165 aptamer on day 6 after disease induction. Four of 6 glomeruli show marked mesangiolysis, capillary dilation, and microaneurysm formation (arrowheads). A protein cast is present in 1 tubule (arrow). ×200. (b) Overview of histological changes in a rat receiving PEG alone on day 6 after disease induction. Marked mesangioproliferative changes are present in 2 glomeruli; the other 2 glomeruli exhibit milder degrees of mesangial expansion. No evidence of persistent mesangiolysis is present. ×200. (c) Semiquantitative evaluation of mesangiolysis, frequency of microaneurysms, and proteinuria.

Figure 5

Mesangial changes on days 2 and 6 after induction of anti–Thy 1.1 nephritis in rats receiving the VEGF165 aptamer (n = 6) or PEG alone (n = 6). Shown is a semiquantitative evaluation of the glomerular de novo expression of α-smooth muscle actin, PDGF B-chain, type IV collagen, and fibronectin.

Figure 6

Glomerular leukocyte influx on day 2 (48 hours) after induction of anti–Thy 1.1 nephritis in rats receiving the VEGF165 aptamer (n = 6) or PEG alone (n = 6). Shown is a quantitative evaluation of glomerular numbers of ED-1–positive monocytes/macrophages, proliferating ED-1–positive cells, polymorphonuclear neutrophils (PMNs), and hydrogen peroxide–producing leukocytes.

Figure 7

Glomerular ultrastructure in rats with PHN on day 7 after disease induction in a rat receiving the VEGF165 aptamer (a) and a rat receiving PEG alone (b). Both rats exhibit subepithelial immune deposits and foot-process fusion to an equal degree. There is no difference in endothelial cell structure or the number of endothelial fenestrations. ×12,000.