Distribution of α-Galactosidase A in Normal Human Kidney and Renal Accumulation and Distribution of Recombinant α-Galactosidase A in Fabry Mice

Preparation of Renal Tissue

Normal, uninvolved human renal tissue that was obtained from resected renal carcinoma kidneys and a from renal biopsy from a 62-yr-old female patient who had Fabry-Anderson disease and was not in treatment at the time for the biopsy was fixed in 4% formaldehyde in 0.1 M sodium cacodylate buffer (pH 7.2). The tissue was trimmed into small blocks, further fixed by immersion for 1 h in 1% formaldehyde, infiltrated with 2.3 M sucrose that contained 2% formaldehyde for 30 min, and frozen in liquid nitrogen. At the time for the biopsy, just before start of ERT, this patient had a plasma creatinine of 86 μM/L, GFR of 84 ml/min per 1.73 m² (⁵¹Cr-EDTA), and a urinary protein excretion of 2.0 g/24 h.

Human kidney sections were also obtained from paraffin-embedded blocks. α-Gal A–deficient mouse kidneys and normal mouse kidneys were fixed by perfusion retrograde through the aorta with 2% paraformaldehyde in 0.1 M sodium cacodylate buffer (pH 7.2). The tissue either was trimmed into small blocks, further fixed by immersion in 1% paraformaldehyde, infiltrated with 2.3 M sucrose for 30 min, and frozen in liquid nitrogen or was prepared for paraffin embedding. For morphologic and autoradiography studies, tissues were fixed in 1% glutaraldehyde, postfixed in 1% OsO₄, dehydrated, and embedded in Epon. All animal experiments were approved and carried out in accordance with provisions for the animal care license provided by the Danish National Animal Experiments Inspectorate and the Guide for the Care and Use of Laboratory Animals (U.S. Department of Health and Human Services, NIH Publication No. 86-23). Ethical approval for the human studies was granted by the Copenhagen Local Research Ethics Committee, and informed consent was obtained from each patient.

Antibodies and Proteins

Affinity-purified rabbit polyclonal anti (a)-α-Gal A was provided by Genzyme Corp. (Framingham, MA). Sheep a-rat megalin (11) and rabbit a-rat cubilin (12) were provided by Dr. Pierre Verroust (INSERM U538, Paris, France). Rabbit a-human vitamin D–binding protein (DBP), rabbit a-human cathepsin D, rabbit a-human retinol-binding protein (RBP), rabbit a-human albumin, and rabbit a-human transferrin were from DAKO A/S (Glostrup, Denmark). For double-labeling experiments to identify lysosomal structures, we used a polyclonal anti-rat cathepsin B (Upstate Biotechnology, Lake Placid, NY) and a-cathepsin D from DAKO. Peroxidase-conjugated secondary antibodies were purchased from DAKO. Fluorescence-conjugated secondary antibodies were purchased from Molecular Probes (Eugene, OR). Controls for unspecific binding were performed with nonspecific rabbit, goat, or sheep IgG from DAKO. Gold-conjugated goat anti-mouse and goat anti-rabbit IgG secondary antibodies were from British BioCell Int. (Cardiff, UK). Recombinant receptor-associated protein (RAP) was provided by Morten Nielsen (Department of Medical Biochemistry, University of Aarhus). Recombinant α-Gal A (Fabrazyme) was provided by Genzyme Corp.

Immunocytochemistry

For light microscopy, 0.8-μm cryosections were obtained at −80°C, and for electron microscopy, 70- to 90-nm sections were obtained at −100°C with an FCS Reichert Ultracut S cryoultramicrotome as described previously (13) and 2-μm paraffin sections were obtained with a Leica RM2165 microtome (Leica, Wetzlar, Germany). For immunolabeling, the sections were incubated with primary antibodies as indicated at room temperature for 1 h after preincubation in PBS that contained 0.05 M glycine and 1% BSA. The sections were subsequently incubated with peroxidase-conjugated secondary antibody (Dako), the peroxidase was visualized with diaminobenzidine, and the sections were subsequently counterstained with Maier’s stain for 2 min, or, for immunofluorescence, the sections were incubated with fluorescence-conjugated secondary antibodies as indicated. The sections were examined in a Leica DMR microscope that was equipped with a Leica DFC320 camera. Images were transferred by a Leica TFC Twain 6.1.0 program and processed using Adobe Photoshop 8.0. For EM immunolabeling the section were incubated with primary antibodies at 4 °C over night followed by incubation at room temperature for 1 h with 10 nm gold particles coupled to relevant immunoglobulins (BioCell, Cardiff, UK). The cryosections were embedded in methylcellulose containing 0.3% uranyl acetate and studied in a Philips CM100 electron microscope (Eindhoven, The Netherlands). Controls. Sections were incubated with secondary antibodies alone or with nonspecific IgG.

Immunofluorescence Microscopy of BN16 Cells

Rat yolk sac carcinoma (BN16) cells (14) were cultured as described previously (15,16). Briefly, BN16 cells were grown in 25-cm² plastic culture flasks (Corning Costar, Badhoevedrop, Holland) in Eagle’s minimal essential medium (Bio-Whittaker, Walkersville, MD) supplemented with 10% FCS (Biologic Industries, Fredensborg, Denmark), 2 mM l-glutamine, 50 U/ml penicillin, and 50 μg/ml streptomycin (Bio-Whittaker) in a humidified atmosphere of 5% CO₂-95% air at 37°C. The cells were subcultured every fourth day with a split ratio of 1:5 by using 0.02% EDTA and 0.05% trypsin (Bio-Whittaker). Experiments were carried out with confluent monolayers of BN16 cells cultured in 24-well plates (Nagle Nunc International, Hereford, UK) for uptake studies.

Immunofluorescence on BN16 cells was performed essentially as described previously (17). In brief, after incubation with α-Gal A at indicated times, the cells were fixed with 1% paraformaldehyde for 1 min, permeabilized with Triton X100, and preincubated with PBS (0.01 M [pH 7.4]) that contained 1% BSA for 15 min. The cells were then incubated with rabbit a-α-Gal A. Subsequently, the cells were incubated with FITC coupled to swine anti-rabbit (Dako) or Alexa fluor 488 (Molecular probes). Rabbit and sheep sera were used as controls.

Autoradiography

For autoradiographic studies, mice were administered an intravenous injection of 120 μl of ¹²⁵I-labeled α-Gal A (8.8 μg, approximately 18 × 10⁶ cpm, free iodine, 3.2%, iodinated by the chloramine-T method as described previously [18]). After 2, 4, or 24 h, the kidneys were fixed as described previously, and the tissue was postfixed, dehydrated, and embedded in Epon. The tissue was prepared for light microscope autoradiography on 1-μm-thick Epon sections using Ilford K2 emulsion (Essex, UK). The sections were exposed for 2 to 4 wk and developed in Kodak D19 developer (Balderup, Denmark). For electron microscope autoradiography, Ilford L4 emulsion was applied to 60-nm sections as described previously (19), and sections were exposed for 2 to 4 mo before development in Kodak D19 developer. The sections were studied in a Philips CM 100 electron microscope.

Tissues and Urine Samples

α-Gal A–deficient mice produced by gene targeting as described previously (5), and BALB/cA Bom mice were kept on a normal diet with free access to drinking water before infusion of recombinant α-Gal A (Fabrazyme) 1 mg/kg body wt. Urine was collected from the bladder immediately before perfusion of the mice and immediately frozen. Urine specimens were collected from patients with Fabry-Anderson disease (four male patients aged 15 to 37 yr and eight female patients aged 15 to 63 yr). In addition, urine samples were obtained from patients with Fabry-Anderson disease immediately before and 2 h after enzyme replacement infusions, 1 mg/kg body wt recombinant α-Gal A (Fabrazyme), and frozen until processed for immunoblotting. Mouse kidney cortical homogenates were obtained from the mice after perfusion of the kidneys through the abdominal aorta with saline. The kidney cortex was dissected from individual kidneys, minced, and homogenized in an ice-cold buffer (0.3 M sucrose, 25 mM imidazole, 8.5 μM leupeptin, 1 mM EDTA, 1 mM PMSF [pH 7.2]) for 30 s with an IKA Ultra-Turrax T8 homogenizer (IKA Labortechnik, Staufen, Germany). The homogenate was centrifuged at 4000 × g for 15 min and rehomogenized, and the supernatants were collected. All procedures were carried out on ice or at 4°C. BN16 cell homogenates were obtained similarly after incubation with α-Gal A at the indicated times, and the cells were scraped and processed.

Surface Plasmon Resonance Analysis

For the surface plasmon resonance (SPR) analyses, the BIAcore sensor chips (type CM5; Biosensor, Uppsala, Sweden) were activated with a 1:1 mixture of 0.2 M N-ethyl-N′-(3-dimethylaminopropyl) carbodiimide and 0.05 M N-hydroxysuccinimide in water according to the manufacturer. Rat cubilin and megalin purified by intrinsic factor-B₁₂ and RAP affinity chromatography, respectively, were immobilized as described previously (20). The SPR signal from immobilized cubilin generated BIAcore response units (RU) that were equivalent to 25 to 35 fmol/mm². The flow cells were regenerated with 20 μl of 1.5 M glycine-HCl (pH 3.0). The flow buffer was 10 mM Hepes, 150 mM NaCl, 1.5 mM CaCl₂, and 1 mM EGTA (pH 7.4). The binding data were analyzed using the BIA evaluation program. The number of ligands bound per immobilized receptor was estimated by dividing the ratio RU ligand/mass ligand with RU receptor/mass receptor. The Kd for binding was estimated using a BIA evaluation program.

ELISA and Enzyme Activity

α-Gal A ELISA assay was performed using a rabbit polyclonal antibody to human α-Gal A. A 96-well plate was coated with 0.2 μg of rabbit polyclonal antibody to human α-Gal A. Urine samples were diluted 1:10, 1:50, 1:500, and 1:1000 and incubated in the wells that contained the antibody. Bound α-Gal A was detected using biotinylated anti-human α-Gal A antibody and streptavidin–horseradish peroxidase with O-phenylenediamine as substitute (Sigma, St. Louis, MO). α-Gal A activity assay was performed using 4-methylumbelliferyl-α-D-galactoside (Sigma) as substrate. Urine samples were analyzed for activity with undiluted and at 1:10, 1:100, and 1:1000 dilutions. The samples were then incubated in 27 mM citric acid and 46 mM sodium phosphate dibasic buffer (pH 4.4). For inhibition of endogenous urine activity of α-Gal B or N-acetylgalactosaminidase, N-acetylgalactosamine was included in the assay at a concentration of 117 mM. The amount of α-Gal A was calculated against recombinant human α-Gal A standards, and the results are shown as average of different dilutions.

SDS-PAGE and Immunoblotting

Samples of renal cortical homogenates, BN16 cell homogenates, or urine were mixed with Laemmli sample buffer that contained 2.5% SDS, subjected to SDS-PAGE using polyacrylamide minigels (Biorad Mini Protean II, Hercules, CA), and transferred to nitrocellulose paper. All urine blots were loaded according to urine creatinine. Blots were blocked with 5% milk in PBS-T (80 mM Na₂HPO₄, 20 mM NaH₂PO₄, 100 mM NaCl, and 0.1% Tween 20 [pH 7.5]) for 1 h and incubated overnight at 4°C with primary antibody in PBS-T with 1% BSA. After washing in PBS-T, the blots were incubated for 1 h with horseradish peroxidase–conjugated secondary antibody diluted 1:3000. After a final wash, antibody binding was visualized using ECL enhanced chemiluminescence system (Amersham International, Bucks, UK). Controls involving incubation without primary antibody and incubation with nonspecific serum revealed no significant labeling.

Distribution of α-Gal A in Normal Human Kidney

The lysosomes of proximal convoluted tubules were intensively stained for α-Gal A (Figure 1, A and B). Labeling was also very pronounced in thick ascending limbs (TAL; Figure 1, B and C) and in interstitial cells (Figure 1B). Glomerular cells including podocytes and parietal epithelial cells of the Bowman’s capsule were unlabeled, and macula densa cells exhibited very little labeling (Figure 1B). In medullary rays TAL and pars recta of proximal tubules were intensively labeled (Figure 1C). In the medulla (Figure 1D), collecting ducts and thin limbs of Henle were strongly labeled. Throughout the kidney, no staining of vascular endothelial cells was observed, as opposed to the strong staining that was seen in endothelial cells of liver sinusoids (data not shown). That the tubular staining for α-Gal A was indeed lysosomal was confirmed by double immunofluorescence staining for cathepsin B (Figure 1E).

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Figure 1.

Horseradish peroxidase immunohistochemistry for α-galactosidase A (α-Gal A) in normal human kidney. (A) Proximal tubule labeling. (B) Labeling in proximal tubules (P) and distal tubules (D). Macula densa cells (MD) and glomerulus (G) show no labeling. Arrows indicate labeled interstitial cells. (C) Strongly labeled pars recta of proximal tubule (P) and thick ascending limbs of Henle (TAL) in medullary ray are seen. (D) Labeling of collecting ducts (CD) and thin limbs (TL) in renal inner medulla. (E) Immunofluorescence for α-Gal A (green) and cathepsin B (red) in renal proximal tubules. Yellow stain indicates co-localization. Bars as indicated.

Urinary Protein Excretion in Patients with Fabry-Anderson Disease

Figure 2 demonstrates the urinary excretion of four proteins in 12 patients with Fabry-Anderson disease. One male and one female patient have very significant excretion of albumin, transferrin, DBP, and RBP. In addition, two female and two male patients have intermediate excretions of the same proteins. It is notable that the excretion pattern is the same for all proteins investigated, including the 20-kD low molecular weight protein RBP and 50-kD DBP. For comparison, Western blots (data not shown) from a patient with minimal-change disease (urinary protein excretion 7 g/24 h) showed a very similar pattern of protein excretion.

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Figure 2.

Western blots of urine from 12 patients with Fabry-Anderson disease. The amounts of urine applied were normalized for creatinine concentration. One male and one female patient demonstrated very high excretion of all four proteins analyzed (albumin, transferrin, vitamin D–binding protein [DBP], and retinol-binding protein [RBP]). In addition, two female patients and two male patients had intermediate increased excretion of the proteins.

The urinary excretion of α-Gal A was investigated in patients in ERT before infusion of the enzyme and 2 h after. As illustrated (Figure 3) for the two male and two of the female patients, there was a highly significant increase in urinary excretion of the infused enzyme, indicating glomerular filtration of the enzyme in these patients.

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Figure 3.

Western blot analyses of urine that was collected from three female and two male patients with Fabry-Anderson disease (for one of the latter [the first four lanes], two different treatments of same patient before [B] and 2 h after [A] intravenous treatment with l mg/kg recombinant α-Gal A [Fabrazyme]). There is a high urinary excretion of the enzyme in two female patients and the male patients. One of the female patients and one of the male patients are the two patients with the heavy proteinuria shown in Figure 2. G, recombinant α-Gal A.

Biopsy of Female Patient with Fabry-Anderson Disease

A renal biopsy from the female patient with the intense proteinuria demonstrated in Figure 2, taken before ERT was instituted, revealed intensive accumulation of Gl3 in podocytes, parietal epithelial cells of Bowman’s capsule, distal tubule cells, vascular endothelial cells, and arteriolar wall cells (Figure 4, A through F). Although the accumulation was not observed in proximal tubule at the light microscopic level (Figure 4C), electron microscopy revealed a significant increase in lysosomal myelin figures (Figure 4F) but not as significant as in podocytes (Figure 4E). An analysis of the expression in this patient of the two endocytic receptors megalin and cubilin, the lysosomal enzyme cathepsin D, and reabsorbed vitamin D carrier protein DBP revealed no obvious changes (Figure 5).

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Figure 4.

Light and electron microscopy of kidney biopsy from the female patient with Fabry-Anderson disease and heavy proteinuria (see Figures 2 and 3). (A) Podocytes and glomerular parietal epithelial cells (arrow) show heavy accumulation of Gl3. (B) Endothelial cells (arrow) and cells of the wall of a small arteriole demonstrate globotriaosylceramide (Gl3) accumulation. (C) Proximal tubule epithelium appears normal at this magnification. (D) Lysosomal changes are observed in distal convoluted tubule (D). Part of the glomerulus (G) is seen at the right. (E) Electron micrograph of podocyte from the same patient demonstrating an extreme vacuolization. (F) Lysosomal lipid accumulation in proximal tubule cell. N, nucleus. Bars as indicated.

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Figure 5.

Expression of megalin (A), cubilin (B), cathepsin D (C), and reabsorbed DBP (D) as shown by immunocytochemistry on cryosections from biopsy from the same patient as in Figure 4. No obvious changes from normal are observed. Bar as indicated.

Uptake of Recombinant α-Gal A in Rat Yolk Sac Cells

Immortalized rat yolk sac cells that expressed high amounts of megalin and cubilin endocytosed avidly the α-Gal A (Figure 6A). The accumulated enzyme was found after 2 and 5 h but was not detectable after 24 h (Figure 6B). The uptake was highly reduced after addition of RAP, a ligand for megalin (19), inhibiting binding of virtually all ligands for megalin (10) (Figure 6C). RAP-inhibitive binding to megalin of α-Gal A was demonstrated by SPR analysis with a Kd of approximately 600 nM (Figure 6C). Cubilin did not bind α-Gal A (results not shown).

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Figure 6.

(A) Immunofluorescent demonstration of uptake of α-Gal A in BN16 cells after exposure to α-Gal A (15 μg/ml for 1 h). (B) Western blots for α-Gal A in BN16 cells after exposure for 1 h and chase incubation for 2, 5, and 24 h, left and right, inhibition by 1 μM receptor-associated protein (RAP). (C) Surface plasmon resonance analysis of α-Gal A binding to megalin and inhibition of binding by RAP. The binding curve at the right was corrected for the binding of RAP itself to megalin.

Renal Uptake of ¹²⁵I-Labeled α-Gal A in Mouse

After intravenous infusion of labeled α-Gal A, 1.1, 0.9, and 0.3% of the injected enzyme was found in the kidney after 2, 4, and 24 h, respectively, and 20, 20, and 12%, respectively, was found in the liver at the same time points. Autoradiography (Figure 7) revealed significant accumulation of the enzyme in lysosomes of proximal tubules by light and electron microscopic autoradiography.

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Figure 7.

Light (A) and electron (B) microscopic autoradiography of kidneys from mice that were administered an injection of ¹²⁵I-labeled α-Gal A 2 h before fixation. (A) Grains are located over the apical part of proximal tubules corresponding to the location of lysosomes in mouse proximal tubules. Distal tubules (D) show no labeling. (B) Autoradiographic grains are located over apical lysosomes. Bars as indicated.

Urinary Excretion and Renal Accumulation of Intravenously Infused Recombinant α-Gal A into Mice with Fabry-Anderson Disease

After infusion of recombinant enzyme into α-Gal A gene–deleted mice, the urinary bladder was emptied just before perfusion fixation of the kidney. Analyses of the urine by Western blotting and ELISA and by measurements of the enzyme activity revealed that significant amounts of enzymatically active enzyme were excreted in the urine. Relatively high enzyme activity per unit of α-Gal A protein was observed in earlier time points compared with that from later time points, indicating the loss of activity when α-Gal A was present longer in urine (Figure 8).

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Figure 8.

(A) Western blot of urine from α-Gal A knockout mice collected 2, 4, and 24 h after intravenous injection of recombinant α-Gal A. Controls, noninjected mice. (B) Content in urine analyzed by ELISA. Data are means ± SD of three to six mice. (C) Enzyme activity in urine, analyzed as described in Materials and Methods. Data are means ± SD of three to six mice except for 4 h, in which urine from one mouse only was analyzed.

The enzyme that accumulated in lysosomes of renal proximal tubules was visualized by immunoperoxidase, immunofluorescence, and immunogold techniques (Figure 9, A through C). Immunolabeling was found after 2, 4, and 24 h (Figure 9, A through H). In addition, labeling was seen in interstitial cells and in glomerular podocytes (Figure 9, D and G) but not in renal vascular endothelial cells as opposed to an intense uptake in hepatic sinusoidal endothelial cells in which accumulation was observed up to 24 h after infusion of α-Gal A (data not shown). No labeling was seen in noninjected mice with Fabry-Anderson disease or in absorption controls of injected mice.

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Figure 9.

Immunocytochemistry of kidneys from α-Gal A knockout mice that were administered an intravenous injection of α-Gal A. (A through E and G) Data from mice that were administered an injection 2 h before fixation. (F) Data from mice that were administered an injection 4 h before fixation. (H) Data from mice that were administered an injection 24 h before fixation. (A) Light microscopy of cryosection demonstrating avid uptake of enzyme in proximal tubules. (B) Electron microscopic immunogold labeling on cryosection showing proximal tubular lysosomal accumulation. (C) Double-labeling immunofluorescence (paraffin section) for α-Gal A (green) and cathepsin D (red). Yellow color indicates co-localization in lysosomes of the two enzymes. (D) Paraffin section demonstrating accumulation of α-Gal A in interstitial cells in outer stripe of outer medulla. (E) Initial part of proximal tubule (P) is labeled, and it seems that podocytes (arrows) are labeled. (F) At 4 h, labeling is seen at the initial part of proximal tubule. (G) At the electron microscopic level, lysosomal accumulation is seen in podocyte after 2 h (arrow). BM, glomerular basement membrane. (H) Even after 24 h, immunoreactive enzyme could still be found in proximal tubules. Bars as indicated.