Figure 1.
Uptake of α-Gal A by human kidney ECs.
(A) Dual immunofluorescence staining showing α-Gal A in the glomerulus (G) in a renal biopsy from a Fabry patient who was infused with α-Gal A 2 h before the biopsy. Localization of α-Gal A is observed in the GECs as indicated by co-localization with CD34 (endothelial cell surface marker). Merged high power view demonstrates that α-Gal A is seen in GECs (white arrowheads) and in podocyte (blue arrowhead) in a human glomerulus from a Fabry disease patient. Labeling for α-Gal A is also seen in the proximal tubule (PT). Scale bar, 25 µm. (B) Peroxidase staining showing localization of α-Gal A (green arrowheads) in larger vessel ECs in a renal biopsy from a Fabry patient who was infused with α-Gal A 2 h before the biopsy. Scale bar, 25 µm. (C) Uptake of Alexa-Fluor 546-labeled α-Gal A (red) in cultured human GECs as a function of time at 37°C. At the indicated times, the cells were fixed and analyzed by confocal microscopy. Scale bar, 5 µm. (D) Co-localization (yellow) of α-Gal A (red) and lysosomes (green). A merged image is shown. Scale bar, 5 µm.
Figure 2.
M6PR and sortilin bind specifically to α-Gal A in GECs.
(A) Affinity chromatography indicate the migration of the different α-Gal A binding proteins (arrowheads). (B) Western blot analysis demonstrated that the two bands were M6PR and sortilin. Lysate from human GECs culture was used as a positive control.
Figure 3.
Immunofluorescent demonstration of sortilin, M6PR and PECAM-1 in cultured human GECs.
(A) Non-permeabilized GECs showing cell surface labeling of sortilin and M6PR. White arrowheads indicate potential cell surface labeling of the different receptors. (B) Permeabilized GECs showing intracellular labeling for both M6PR and sortilin. (C) Demonstration of the specific endothelial cell marker PECAM-1 in permeabilized GECs. (D) Endocytosed recombinant α-Gal A (red) co-localizes with both sortilin (green) and M6PR (green) in intracellular compartments after 60 min. A high-power view of the co-localization of M6PR with recombinant α-Gal A is shown, yellow color indicated with white arrowheads demonstrates co-localization. Controls were incubated with serum and detected with same secondary antibodies (data not shown). Nuclei (blue) were stained with DAPI. Scale bars, 5 µm.
Figure 4.
Uptake of 125I-labeled α-Gal A by human GECs.
(A and B) Human GECs were incubated with 125I-α-Gal A for different times at 37°C showing both total and cell-associated α-Gal A uptake. (C and D) Human GECs were incubated with different concentrations of 125I-α-Gal A for 12 h at 37°C showing both total and cell-associated α-Gal A uptake. (E) Uptake of 125I-α-Gal A and inhibition with M6P, RAP, a combination of both M6P and RAP, and α-Gal A. Uptake was assayed as described in the method section. Each point represents a mean of triplicates with standard deviations. Addition of inhibitors show significant (*) reductions (P<0.002) in the uptake of α-Gal A after 12 h.
Figure 5.
Binding of α-Gal A by M6PR and sortilin.
The purified ectodomains of M6PR and sortilin were immobilized on BIAcore chips. (A) SPR analysis of α-Gal A binding to purified human M6PR. (B) Binding of 50 nM α-Gal A to M6PR in the presence or absence of 50 µM M6P. (C) Inhibition of α-Gal A binding to sortilin by M6PR. Sortilin was saturated with M6PR prior to injection of α-Gal A. For comparison, sortilin was saturated with flow buffer prior to injection of α-Gal A.
Figure 6.
Expression of sortilin and M6PR in human GECs and in larger human renal vessel ECs.
Dual immunofluorescence shows co-localization of sortilin and M6PR with PECAM-1 in human GECs in the glomeruli (G) as seen in sections from a normal human kidney. The respective merge images are shown in both low and high magnifications. Sortilin and M6PR are localized in the GECs and co-localizes with PECAM-1 to some extent as seen by the merged images. High-power views demonstrate that the receptors are localized in the GECs as indicated with white arrowheads. Sortilin and M6PR labeling of podocytes is also observed as previously shown in podocytes [15]. Scale bars, 20 µm. (B) Dual immunofluorescence show co-localization of sortilin and M6PR with PECAM-1 and CD34 (EC cell surface markers), respectively. The receptors are localized in the ECs of the larger renal vessels as indicated with white arrowheads. Staining of sortilin and M6PR is also seen in SMCs (blue arrowheads). Scale bars, 25 µm.