VEGF inhibition and renal thrombotic microangiopathy

Abstract

The glomerular microvasculature is particularly susceptible to injury in thrombotic microangiopathy, but the mechanisms by which this occurs are unclear. We report the cases of six patients who were treated with bevacizumab, a humanized monoclonal antibody against vascular endothelial growth factor (VEGF), in whom glomerular disease characteristic of thrombotic microangiopathy developed. To show that local reduction of VEGF within the kidney is sufficient to trigger the pathogenesis of thrombotic microangiopathy, we used conditional gene targeting to delete VEGF from renal podocytes in adult mice; this resulted in a profound thrombotic glomerular injury. These observations provide evidence that glomerular injury in patients who are treated with bevacizumab is probably due to direct targeting of VEGF by antiangiogenic therapy.

Figure 1. Microangiopathy in Patients Who Were Treated with Bevacizumab

Panel A shows the ultrastructure of a healthy glomerular filtration barrier, which is composed of three layers: the outermost podocyte layer, the fenestrated glomerular endothelial cells, and an intervening glomerular basement membrane. Urinary filtrate passes from the blood lumen to the urinary space. Podocytes produce vascular endothelial growth factor (VEGF). The tyrosine kinase receptors for VEGF (VEGFR-1 and VEGFR-2) are expressed by glomerular endothelial cells. A slit diaphragm is indicated by the arrow, and fenestrations are indicated by arrowheads. (Photomicrograph courtesy of Dr. Wilhelm Kriz, Mannheim, Germany.) Panel B shows silver staining of two representative glomeruli in biopsy specimens from patients. In a specimen from Patient 4 (left), mesangiolysis (single arrow), prominent endothelial swelling (arrowhead), red-cell fragments (double arrows), and thrombi are visible in some capillary loops. In a specimen from Patient 1 (right), the double contours of capillary basement membranes (arrows) can be seen. Panel C shows transmission electron micrographs of glomeruli from Patient 4 (left), revealing fibrillar material that is characteristic of fibrin (arrows), and from Patient 1 (right), revealing duplication of capillary basement membranes (arrow) and a marked widening of the subendothelial spaces by electron-lucent material (arrowheads).

Figure 2. Thrombotic Microangiopathy Caused by Genetic Deletion of VEGF from Glomeruli in a Murine Model

In Panel A, the successful excision of vascular endothelial growth factor (VEGF) from podocytes was confirmed by a lack of VEGF RNA expression after induction with doxycycline (+Dox), as compared with normal levels of Wilms’ tumor suppressor 1 (WT-1) RNA, another gene expressed by podocytes. Results from a control mouse (−Dox) are shown for comparison. Panel B shows the breeding strategy that was used to generate a time-specific and cell-specific knockout of VEGF in podocytes. In the absence of doxycycline (yellow circles), the reverse tetracycline transactivator protein (rtTA, red crescents) cannot bind to the tetracycline responsive element in the tetO-Cre transgene. In the presence of doxycycline, rtTA, under control of the podocyte-specific promoter (podocin), binds to initiate transcription of Cre recombinase specifically in podocytes. In Panel C, a kidney from a +Dox mouse 9 weeks after induction with doxycycline is pale, small, and sclerotic, findings that are consistent with end-stage kidney failure, as compared with a normal kidney from a −Dox mouse. As shown in Panel D, albuminuria was detected 4 weeks after induction in all mutant mice (arrow). All control (Con) and mutant (KO) mice received doxycycline. Molecular mass in kilodaltons is shown on the left. L denotes ladder, the molecular reference for protein size. In Panel E, in glomeruli that were stained with silver methenamine outlining basement membranes (subpanels a and b), the lumens of glomerular capillaries that are seen in control mice (subpanel a) are either obliterated or collapsed in mutant mice (subpanel b). Electron micrographs (EM) showing the ultrastructure of glomeruli (subpanels c, d, and f) reveal swollen endothelial cells (arrow, sub-panel d) and dense subendothelial deposits (white arrows, subpanel f) in capillary loops of VEGF mutants. Fenestrated endothelium (arrowheads, subpanel c) is shown in controls for comparison. Podocyte foot processes are relatively spared early in disease (white arrowheads, subpanel f). On light microscopy, Martius scarlet blue (MSB) staining shows an intracapillary thrombus in a mutant mouse (double arrows, subpanel e). In Panel F, schistocytes (arrows) were found in blood smears from 58% of mutant mice (subpanel a). Immunohistochemical staining for fibrin of glomeruli from VEGF mutants shows positive results (reddish color in subpanel b). There was no staining in glomeruli of control littermates (not shown).

Figure 3. Hypothetical Model of Disruption of VEGF Signaling in Renal Thrombotic Microangiopathy

The loss of function of vascular endothelial growth factor (VEGF) through genetic deletion (VEGF KO), pharmacologic inhibition, or an elevated level of circulating soluble fms-like tyrosine kinase 1 (sFlt-1) that binds VEGF is associated with damage to the glomerular endothelium characterized by swelling and thrombotic microangiopathy. VEGFR-2 denotes kinase insert domain receptor.