Induced pluripotent stem cell-based disease modeling identifies ligand-induced decay of megalin as a cause of Donnai-Barrow syndrome

Abstract

Donnai-Barrow syndrome (DBS) is an autosomal-recessive disorder characterized by multiple pathologies including malformation of forebrain and eyes, as well as resorption defects of the kidney proximal tubule. The underlying cause of DBS are mutations in LRP2, encoding the multifunctional endocytic receptor megalin. Here, we identified a unique missense mutation R3192Q of LRP2 in an affected family that may provide novel insights into the molecular causes of receptor dysfunction in the kidney proximal tubule and other tissues affected in DBS. Using patient-derived induced pluripotent stem cell lines we generated neuroepithelial and kidney cell types as models of the disease. Using these cell models, we documented the inability of megalin R3192Q to properly discharge ligand and ligand-induced receptor decay in lysosomes. Thus, mutant receptors are aberrantly targeted to lysosomes for catabolism, essentially depleting megalin in the presence of ligand in this affected family.

Keywords: endocytosis; low-molecular-weight proteinuria; proximal tubule dysfunction; renal Fanconi syndrome.

Graphical abstract

Figure 1

Mutation c:G9575A in Donnai-Barrow syndrome disrupts renal megalin^R3192Qexpression. (a) Structural organization of the megalin polypeptide composed of complement-type repeats (CR), epidermal growth factor (EGF)–type repeats, and β-propellers. Mutation R3192Q (encoded by c:G9575A) in an EGF-type repeat is indicated by the red asterisk. (b) Immunohistological detection of megalin (arrowheads) in proximal tubule cells of kidney biopsies from a control subject but not from patient R3192Q_2. (c) Western blot analysis of spot urine documenting urinary loss of the megalin ligands vitamin D–binding protein (DBP), retinol-binding protein (RBP), α₁-microglobulin, and β₂-microglobulin in patients R3192Q_1 and R3192Q_2, but not in 3 control subjects. (d) Sequence analysis showing a homozygous mutation c:G9575A (red box) in LRP2 in induced pluripotent stem cell lines derived from patients R3192Q_1 and R3192Q_2 as compared with a control cell line. Sequences were aligned to the LRP2 reference sequence given above (NCBI reference sequence: NM_004525.2). To optimize viewing of this image, please see the online version of this article at www.kidney-international.org.

Figure 2

Cellular expression of megalin^R3192Qis impacted by a post-transcriptional mechanism. (a) Immunodetection of megalin (red) in induced pluripotent stem cell (iPSC) lines at the indicated time points of neuroectodermal differentiation. Cells were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (blue). Megalin expression was induced from day 5 onward in both genotypes. At day 9, receptor levels decreased in patient-derived cells as compared with control cells. Bar = 10 μm. (b) Transcript levels of LRP2 during differentiation into neuroectoderm cells in the control subject and patient R3192Q_1. Data are depicted as Δct normalized to day 0 of the control cells (ΔΔct) ± SD; n = 4 experiments with 2–3 biological replicates/experiment. Statistical analyses were performed by 2-way analysis of variance with the Bonferroni post-test. ∗P < 0.05; ∗∗∗P < 0.001. (c) Western blot analysis of megalin in R3192Q_1 and control iPSC lines at the indicated time points of neuroectodermal differentiation. The detection of α-tubulin served as loading control. (d) Megalin levels were quantified by densitometric scanning of replicate Western blots (exemplified in panel c) in control and R3192Q_1 neural precursor cells (NPCs) at days 5 and 9 of differentiation (n = 2–3 independent experiments, 2–4 biological replicates/experiment). Megalin levels in R3192Q_1 cells were comparable with control cells at day 5, but significantly decreased at day 9 of differentiation. Values are given as relative levels of expression compared with controls (set to 100% ± SD). Statistical significance was determined by Student’s t test (day 9) or 1-paired t test (day 5). ∗∗∗∗P < 0.0001. (e) Quantitative real-time polymerase chain reaction analysis of LRP2 transcript levels in a control and R3192Q_1 NPCs at days 5 and 9 of differentiation. (n = 3 independent experiments, 2–4 biological replicates/experiment). Levels are depicted as ct values normalized to transcript levels of GAPDH (Δct ± SD) used as an internal control. Transcript levels for LRP2 are unchanged at days 5 and 9 comparing genotypes (Student’s t test). To optimize viewing of this image, please see the online version of this article at www.kidney-international.org.

Figure 3

Mutation c:G9575A does not impact the endocytic activity of megalin^R3192Q. (a) Neural precursor cells (NPCs) from a control or from patient R3192Q_1 were treated for 2 hours in medium containing 10 μg/ml recombinant GST-SHH-N. Subsequently, cells were immunostained for GST-SHH-N (red) using anti-GST antisera and counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (blue). Bar = 25 μm. (b) NPCs from a control or from patient R3192Q_1 at day 9 of differentiation were treated overnight in medium with (+) or without (−) 10 μg/ml recombinant GST-SHH-N. Thereafter, levels of megalin and GST-SHH-N in cell extracts were determined by Western blotting. Detection of α-tubulin served as loading control. (c) Quantification of GST-SHH-N uptake in control and R3192Q_1 cells by densitometric scanning of replicate Western blots (exemplified in panel b). Levels are given relative to control cells (set to 100% ± SD). The amount of internalized GST-SHH-N is significantly lower in cells from patients R3192Q_1 as compared with control cells (n = 3 independent experiments, 2–4 biological replicates/experiment; Student’s t test). ∗P < 0.05. To optimize viewing of this image, please see the online version of this article at www.kidney-international.org.

Figure 4

The addition of SHH-N decreases stability of megalin^R3192Qin neural precursor cells (NPCs). (a) NPCs at day 5 of differentiation were treated with 10 μg/ml GST-SHH-N or blank medium overnight, and levels of megalin were determined in cell lysates by Western blotting thereafter. Detection of α-tubulin served as a loading control. (b) Quantification of megalin levels in control and R3192Q_1 NPC lines by densitometric scanning of replicate Western blots (exemplified in panel a). Levels are given as relative to the untreated condition (set to 100% ± SD). In the presence of GST-SHH-N, levels of megalin^R3192Q were significantly lower compared with that of the wild-type receptor (n = 4 independent experiments, 2–3 biological replicates/experiment). This difference was not seen in the control medium (blank) lacking the receptor ligand. Statistical significance was determined using Student’s t test. ∗P < 0.05; ∗∗P < 0.01. (c) Replicate layers of control and R3192Q_1 NPCs at day 5 of differentiation were treated with 10 μg/ml GST-SHH-N and 7.5 μg/ml cycloheximide. Cells were harvested at the indicated time points, and expression levels of megalin were determined by Western blotting. Detection of α-tubulin served as a loading control. (d) Megalin levels were quantified by densitometric scanning of replicate Western blots (exemplified in panel c) in control and R3192Q_1 NPCs after treatment with GST-SHH-N and cycloheximide (mean of 3 independent experiments). Receptor levels are given as a percentage of levels at time point 0 of treatment for each cell line. In the presence of a ligand, a significantly faster decay was observed for megalin^R3192Q as compared with the wild-type receptor (P < 0.0001 for genotype and time, unmatched 2-way analysis of variance).

Figure 5

Ligand-induced decay of megalin^R3192Qin induced pluripotent stem cell (iPSC)–derived renal proximal tubule epithelial-like cells (RPTECs). (a) Control and patient iPSC-derived RPTECs at day 8 of differentiation were treated with 10 μg/ml GST-SHH-N or blank (10 $\text{μ}$g/ml GST) medium overnight. Subsequently, megalin levels in cell lysates were determined by Western blotting. Detection of $\text{α}$-tubulin served as a loading control. (b) Megalin levels in a control and R3192Q_1 RPTECs were quantified by densitometric scanning of replicate Western blots (exemplified in panel a). Levels are given as relative to the untreated condition (set at 100% ± SD). In the presence of GST-SHH-N, levels of megalin^R3192Q were significantly lower as compared with the wild-type receptor (n = 3 independent experiments, 2–3 biological replicates/experiment). Statistical significance was determined using Student’s t test. ∗P < 0.05.

Figure 6

Binding of GST-SHH-N directs megalin^R3192Qto lysosomes. (a) Immunofluorescence detection of megalin (red) and GST-SHH-N (green) in control and patient neural precursor cells (NPCs) at day 7 of differentiation treated with 10 μg/ml GST-SHH-N for 2 hours. Cells were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (blue). Bar = 8 μm. (b) Extent of colocalization of megalin with GST-SHH-N as determined by Mander’s colocalization coefficient is increased for megalin^R3192Q as compared with wild-type megalin, suggesting the prolonged interaction of the mutant receptor with its ligand. One representative experiment is shown with data given as mean ± SD. This experiment was repeated 4 times with 25–40 cells/experiment analyzed. All 4 experiments showed statistical significance (Student’s t test). ∗∗∗P < 0.0001. (c) Immunofluorescence detection of megalin (red) and lysosomal marker LAMP1 (green) in control and patient NPCs at day 7 of differentiation treated with 10 μg/ml GST or GST-SHH-N for 2 hours. Cells were counterstained with DAPI. Bar = 8 μm. (d) Mander’s colocalization coefficient documents increased colocalization of megalin^R3192Q with LAMP1 in NPCs treated with GST-SHH-N (but not GST) as compared with wild-type megalin (n = mean of 4 experiments with 15–40 cells/experiment analyzed ± SD; Student’s t test). (e) Immunodetection of lysosomal marker LAMP1 (green), GST-SHH-N (red), and megalin (blue) in control and patient NPCs at day 7 of differentiation. Cells were treated with 10 $\text{μ}$g/ml GST-SHH-N for 2 hours and counterstained with DAPI (gray). Bar = 10 $\text{μ}$m. Higher magnifications of overview pictures are given as insets (bars = 4 $\text{μ}$m). Increased colocalization of megalin with GST-SHH-N in LAMP1+ lysosomal vesicles (white signals) was noted in patient as compared with control NPC lines. To optimize viewing of this image, please see the online version of this article at www.kidney-international.org.

Figure 7

Inhibition of lysosomal proteases prevents the ligand-induced decay of megalin^R3192Q. (a) Neural precursor cells (NPCs) at differentiation day 5 were treated overnight with 10 $\text{μ}$g/ml GST-SHH-N in the absence or presence of lysosomal protease inhibitors (+ lyso. inhibitor; see Supplementary Methods for details). Subsequently, megalin levels in cell lysates were determined by Western blotting. Detection of $\text{α}$-tubulin served as a loading control. (b) Quantification of megalin levels in control and R3192Q_1 NPCs by densitometric scanning of replicate Western blots (exemplified in panel a). Levels are given relative to GST-SHH-N without lysosomal inhibitors (set to 100% ± SD). Treatment with lysosomal inhibitors significantly increased levels of megalin^R3192Q as compared with nontreated cells (right panel). No effect of lysosomal inhibition was seen on levels of the wild-type receptor in control NPCs (left panel) (n = 4 independent experiments, 2–3 biological replicates/experiment). Statistical significance was determined using Student’s t test. ∗∗∗∗P < 0.0001.