Long-term enzyme replacement therapy is associated with reduced proteinuria and preserved proximal tubular function in women with Fabry disease

Background Fabry disease is an X-linked lysosomal storage disorder caused by mutations in the GLA gene. Deficiency of α-galactosidase A (α-Gal A) causes intracellular accumulations of globotriaosylceramide (GL-3) and related glycosphingolipids in all organs, including the kidney, often leading to end-stage renal failure. In women with Fabry disease, accumulation of GL-3 in the glomerular podocytes and other renal cells induces progressive, proteinuric nephropathy, but not as severe as in men. Enzyme replacement therapy (ERT) with recombinant α-Gal A reduces cellular GL-3 deposits in podocytes and tubular epithelial cells. We have previously shown that α-Gal A is delivered to these cells by different pathways involving different receptors. This study investigated the long-term changes in albuminuria, estimated glomerular filtration rate (eGFR) and urinary markers of both glomerular and tubular dysfunction in women with Fabry disease treated with ERT. Methods A retrospective, single centre, cohort study evaluated the long-term association between ERT, albuminuria and eGFR in 13 women with Fabry disease and mild renal involvement. In particular, we analysed the changes in the proteinuric profile, including the glomerular marker IgG, the tubular markers α1-microglobulin and retinol-binding protein (RBP), and the shared tubular and glomerular markers albumin and transferrin. Results ERT was associated with a significant reduction in albuminuria and a relatively stable eGFR. The decrease in albuminuria was paralleled by a decrease in both glomerular and tubular urine protein markers. Conclusions The data indicate that long-term ERT is associated with a reduction in albuminuria and glomerular and tubular urinary protein markers in women with Fabry disease and mild renal manifestation

Mannose 6-Phosphate Receptor and Sortilin Mediated Endocytosis of α-Galactosidase A in Kidney Endothelial Cells

Prominent vasculopathy in Fabry disease patients is caused by excessive intracellular accumulation of globotriaosylceramide (GL-3) throughout the vascular endothelial cells causing progressive cerebrovascular, cardiac and renal impairments. The vascular lesions lead to myocardial ischemia, atherogenesis, stroke, aneurysm, thrombosis, and nephropathy. Hence, injury to the endothelial cells in the kidney is a key mechanism in human glomerular disease and endothelial cell repair is an important therapeutic target. We investigated the mechanism of uptake of α-galactosidase A (α-Gal A) in renal endothelial cells, in order to clarify if the recombinant enzyme is targeted to the lysosomes via the universal mannose 6-phosphate receptor (M6PR) and possibly other receptors. Immunohistochemical localization of infused recombinant α-Gal A in a renal biopsy from a classic Fabry disease patient showed that recombinant protein localize in the endothelial cells of the kidney. Affinity purification studies using α-Gal A resins identified M6PR and sortilin as α-Gal A receptors in cultured glomerular endothelial cells. Immunohistochemical analyses of normal human kidney with anti-sortilin and anti-M6PR showed that sortilin and M6PR were expressed in the endothelium of smaller and larger vessels. Uptake studies in cultured glomerular endothelial cells of α-Gal A labeled with fluorescence and 125I showed by inhibition with RAP and M6P that sortilin and M6PR mediated uptake of α-Gal A. Biacore studies revealed that α-Gal A binds to human M6PR with very high affinity, but M6PR also binds to sortilin in a way that prevents α-Gal A binding to sortilin. Taken together, our data provide evidence that sortilin is a new α-Gal A receptor expressed in renal endothelial cells and that this receptor together with the M6PR is able to internalize circulating α-Gal A during enzyme replacement therapy in patients with Fabry disease

Receptor-Mediated Endocytosis of α-Galactosidase A in Human Podocytes in Fabry Disease

Injury to the glomerular podocyte is a key mechanism in human glomerular disease and podocyte repair is an important therapeutic target. In Fabry disease, podocyte injury is caused by the intracellular accumulation of globotriaosylceramide. This study identifies in the human podocyte three endocytic receptors, mannose 6-phosphate/insulin-like growth II receptor, megalin, and sortilin and demonstrates their drug delivery capabilities for enzyme replacement therapy. Sortilin, a novel α-galactosidase A binding protein, reveals a predominant intracellular expression but also surface expression in the podocyte. The present study provides the rationale for the renal effect of treatment with α-galactosidase A and identifies potential pathways for future non-carbohydrate based drug delivery to the kidney podocyte and other potential affected organs

Expression of sortilin and M6PR in human GECs and in larger human renal vessel ECs.

<p>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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039975#pone.0039975-Prabakaran1" target="_blank">[15]</a>. 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.</p

Binding of α-Gal A by M6PR and sortilin.

<p>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.</p

Uptake of ¹²⁵I-labeled α-Gal A by human GECs.

<p>(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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039975#s2" target="_blank">method</a> 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.</p

Uptake of α-Gal A by human kidney ECs.

<p>(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.</p

Uptake of ¹²⁵I-labeled α-Gal A by human podocytes.

<p>(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.</p