Figure 1.
Uptake of recombinant α-Gal A by human podocytes.
(A) Peroxidase immunohistochemistry for α-Gal A in a biopsy from a male Fabry patient using anti-human α-Gal A antibody. The patient was intravenously infused with α-Gal A 2 h before the biopsy was taken. Labeling of a human glomerulus (G) showing α-Gal A localization in the podocytes (indicated with green arrowheads) and GL-3 inclusions seen as vacuoles (indicated with red arrowheads). Staining is also seen in parietal epithelial cells (indicated with yellow arrows). Scale bar, 25 µm. A high-power view of a portion of the glomerulus (top-right) demonstrates the localization of infused recombinant α-Gal in the podocyte. (B) For comparison, no α-Gal A labeling is seen in the podocytes in a biopsy from an untreated male Fabry patient. (C) The treated male Fabry patient shows no detectable labeling of endogenous α-Gal A in the collecting ducts (CD). Red arrowheads indicate heavy GL-3 inclusions. Scale bar, 25 µm. (D) A normal individual shows labeling of endogenous α-Gal A (green arrowheads) in both thick ascending limbs of Henle (TAL) and in CD. Scale bar, 25 µm. (E) Immunofluorescent demonstration of Alexa-Fluor 488-labeled α-Gal A uptake in human podocytes as a function of time at 37°C. At the indicated times, the cells were fixed and analyzed by confocal microscopy. Scale bar, 10 µm. (F) For colocalization of α-Gal A (green) and lysosomes (red) a merged image is shown. The yellow color illustrates colocalization. Scale bar, 5 µm.
Figure 2.
Megalin, M6PR and sortilin bind specifically to recombinant α-Gal A in podocytes.
(A) Affinity chromatography: the arrowheads indicate the migration of the different α-Gal A binding proteins. (B) Western blot analysis demonstrated that the three bands were megalin, M6PR, and sortilin as indicated with arrows. Lysate from human podocyte culture was used as a positive control.
Figure 3.
Uptake of 125I-labeled α-Gal A by human podocytes.
(A) Human podocytes were incubated with 125I-α-Gal A for different times. (B) Podocytes incubated with 125I-α-Gal A for 12 h in the presence or absence of RAP, sortilin propeptide, M6P, and with all three ligands combined at 37°C. Uptake was assayed as described in the method section of the paper. Values are means of triplicate experiments with standard deviations (SD). The addition of sortilin propeptide shows no significant inhibition, however the addition of M6P, RAP or a combination of all three inhibitors show significant reductions (* indicate P<0.001) in the α-Gal A uptake after 12 h.
Figure 4.
Binding and uptake of α-Gal A by sortilin.
(A) SPR analysis of α-Gal A binding to purified human sortilin and inhibition of binding by NT. The lower binding curve was corrected for the binding of NT itself to sortilin. (B) Uptake of Alexa-Fluor 488-labeled α-Gal A by wild-type and full-length sortilin transfected HEK293 cells in the presence or absence of M6P or M6P and NT for 60 min at 37°C. At the given time, cells were fixed, and analyzed by confocal microscopy. Scale bar, 10 µm.
Figure 5.
Megalin, sortilin, and M6PR are expressed in cultured podocytes.
(A) Immunofluorescent demonstration of megalin, sortilin, and M6PR in cultured human podocytes and (B) specific podocyte-markers podocin and nephrin. Cells were fixed, stained with the appropriate antibodies and detected with Alexa-Fluor-conjugated secondary antibodies. Controls were incubated with serum and detected with same secondary antibodies (data not shown). Nuclei (blue) were stained with DAPI. Scale bars, 5 µm. White arrows indicate potential cell surface labeling of the different receptors in the human podocytes.
Figure 6.
Expression of megalin, sortilin, and M6PR in human glomeruli.
(A) Expression of megalin, M6PR, sortilin, podocin, nephrin, APN mRNAs by RT-PCR (+). Negative controls (−) run without RT enzyme. The marker used was 100 bp ladder. (B) Immunoperoxidase labeling of paraffin sections from a normal human kidney incubated with antibodies to megalin, M6PR, and sortilin show that the brush borders of proximal tubules (PT) are heavily stained. The distal tubules (DT) are unstained for megalin, but stained for sortilin and M6PR. The glomeruli (G) show staining for megalin, sortilin and M6PR. Preabsorption of the antibodies with the respective antigens show loss of immunoreactivity, demonstrating antibody specificity. Tubular and podocyte staining was blocked by preincubation. Controls that included only the secondary antibody showed no labeling (data not shown). Scale bar, 50 µm. (C) Dual immunofluorescence and confocal microscopy with monoclonal anti-WT1 antibody (red) and polyclonal antibodies (green) against megalin, sortilin, and M6PR. The respective merge images are shown in both low and high magnifications. Megalin, sortilin, and M6PR are localized in the glomerular podocytes and co-localizes with WT-1 as seen by the merged images indicated with white arrow-heads. High-power views of a portion of a glomerulus show merged images of the receptors and WT1 demonstrating that megalin, sortilin, and M6PR localize primarily to the podocyte cell bodies. Scale bar, 50 µm.
Table 1.
Primers for RT-PCR.