Table 1.
Details of the animals used in this study.
Figure 1.
Semi-quantitative computer assisted method used for the quantification and normalisation of the VEGF staining.
Glomeruli were defined as the area of interest (AOI), and then the AOI were isolated using the image j software. The nuclei in the AOI were then counted and finally the stained area of each AOI was determined.
Figure 2.
Example of a montage of transmission electron micrographs by using multiple image arrangements (MIAs) in order to provide the entire renal glomerulus.
These images were taken using the image analysis software iTEM at the magnification of ×3000. Here one day after aflibercept injection.
Figure 3.
Examples of representative transmission electron micrographs of (A) a fenestrated glomerular endothelium and of (B) peripheral versus mesangial portions of the glomerular endothelium (both one day after aflibercept injection).
(A) Blood lumen on the upper part, urinary space on the lower part of the image. The healthy glomerular filtration barrier consists of three layers [6]: the fenestrated glomerular endothelial cells, the intervening glomerular basement membrane and the podocyte processes and slit diaphragm. GBM = glomerular basement membrane, CL = capillary lumen, POD = podocyte. Arrows mark glomerular endothelial cells fenestrae (note the absence of diaphragm), asterisks mark podocyte foot processes, arrowheads mark podocyte slit diaphragm. (B) At this magnification, podocyte foot processes (asterisks) allow the clear identification of the capillary lumen (CL). In accordance with our definition, the peripheral portion begins where the endothelium and the glomerular endothelial basement membrane (GBM) run approximately parallel (marked by arrows). Arrows mark direction into which peripheral endothelium begins. In between the arrows the mesangium (Mes) and the mesangial portion of the capillary endothelium (MesE) is located. Note that in the mesangial portion there is no GBM adjacent to the fenestrated endothelium so that the described counting method is not applicable and the endothelium does not show the typical single- layered configuration. Magnification ×20000.
Figure 4.
Immune fluorescent photomicrographs of glomeruli from control (A), aflibercept-treated (B–D) and ranibizumab-treated (E–F) monkeys eyes.
In all figures, the asterisks label the spaces of the Bowman capsule. A) Kidney sections from the control animal did not show any specific staining with anti-human IgG-Fc antibody in the glomeruli. Only the erythrocytes (arrow) within the capillaries showed a weak fluorescence. B) One day after aflibercept injection, the endothelium cell layer and material within the capillaries of a glomerulus were highly fluorescent (white arrow) after labelling with an antibody against the Fc region of IgG. In an adjacent glomerulus, only the endothelium was stained (white arrowhead) whereas the lumina of the vessels did not contain IgG-Fc positive material (black arrow). C) Erythrocytes within the glomeruli (arrowhead) as well as the endothelium (arrow) were highly fluorescent. D) Seven days after aflibercept injection, the fluorescent material within the capillaries (arrowhead) and the fluorescence intensity of the endothelium became weaker. E) One day after ranibizumab injection, the endothelium cell layer (white arrow) and erythrocytes (arrowhead and black arrow in the inset) were fluorescent after staining with an antibody against human Fab of IgG. F) The specific fluorescence of the endothelium (arrow) and erythrocytes (arrowhead) was nearly lost seven days after injection of ranibizumab.
Figure 5.
Quantification and normalisation of aflibercept/ranibizumab staining.
Results of the analysis of aoi of glomeruli from kidneys of monkeys one and seven days after aflibercept (A) or ranibizumab (B) treatment and the corresponding controls, respectively after staining with the anti-Fc-fragment antibody for aflibercept and the anti-Fab fragment antibody for ranibizumab immune reactivity analysis; t-test against the corresponding control was performed: ** for p<0.001,*** for p<0.0001.
Figure 6.
Quantification and normalisation of the VEGF staining.
Results of the analysis of aoi of glomeruli from kidneys of monkeys one and seven days after ranibizumab and aflibercept treatment and the corresponding controls after anti-VEGF staining; t-test against control: * for p<0.05, *** for p<0.0001; t-test ranibizumab day 1 versus aflibercept day 1 and ranibizumab day 7 versus aflibercept day 7: # for p<0.05, ### for p<0,0001; t-test aflibercept day 1 versus aflibercept day7: +++ for p<0.0001.
Figure 7.
Examples of transmission electron micrographs used for the quantification of the glomerular endothelial cells fenestrations.
The red line drawn a long lamina rara interna, the length of line is in µm, the red crosses point out fenestrations. (A) after injection of the vehicle; (B) in the untreated control; (C) one day after injection of ranibizumab; (D) seven days after injection of ranibizumab; (E) one day after injection of aflibercept; (F) seven days after injection of aflibercept. Magnification ×20000.
Figure 8.
Box plot representation of the quantification of the fenestrations per µm depending on the treatment and its duration.
Application of the Student's test showed no significant differences in the number of fenestrations per µm except one day after aflibercept's injection where the number was increased compared to all other groups (*, p<0.05).