Reduced ciliary polycystin-2 in induced pluripotent stem cells from polycystic kidney disease patients with PKD1 mutations

Abstract

Heterozygous mutations in PKD1 or PKD2, which encode polycystin-1 (PC1) and polycystin-2 (PC2), respectively, cause autosomal dominant PKD (ADPKD), whereas mutations in PKHD1, which encodes fibrocystin/polyductin (FPC), cause autosomal recessive PKD (ARPKD). However, the relationship between these proteins and the pathogenesis of PKD remains unclear. To model PKD in human cells, we established induced pluripotent stem (iPS) cell lines from fibroblasts of three ADPKD and two ARPKD patients. Genetic sequencing revealed unique heterozygous mutations in PKD1 of the parental ADPKD fibroblasts but no pathogenic mutations in PKD2. Undifferentiated PKD iPS cells, control iPS cells, and embryonic stem cells elaborated primary cilia and expressed PC1, PC2, and FPC at similar levels, and PKD and control iPS cells exhibited comparable rates of proliferation, apoptosis, and ciliogenesis. However, ADPKD iPS cells as well as somatic epithelial cells and hepatoblasts/biliary precursors differentiated from these cells expressed lower levels of PC2 at the cilium. Additional sequencing confirmed the retention of PKD1 heterozygous mutations in iPS cell lines from two patients but identified possible loss of heterozygosity in iPS cell lines from one patient. Furthermore, ectopic expression of wild-type PC1 in ADPKD iPS-derived hepatoblasts rescued ciliary PC2 protein expression levels, and overexpression of PC1 but not a carboxy-terminal truncation mutant increased ciliary PC2 expression levels in mouse kidney cells. Taken together, these results suggest that PC1 regulates ciliary PC2 protein expression levels and support the use of PKD iPS cells for investigating disease pathophysiology.

Figure 1.

iPS cell lines derived from patients with PKD are pluripotent and karyotypically normal. (A) Immunocytochemistry of pluripotency markers for representative lines from each patient on feeder layers. (B) Corresponding normal karyotypes of iPS lines. (C) Hematoxylin and eosin-stained teratoma sections showing pigmented epithelium (ectoderm), cartilage (mesoderm), and glandular epithelium (endoderm). Similar results were obtained for all control lines. (D) Directed differentiation of iPS lines into lineages expressing markers of the endoderm (SOX17), mesoderm (forkhead box protein F1 [FOXF1]), and ectoderm (NESTIN). Scale bars, 200 µm. AP, alkaline phosphatase; SSEA-3, stage-specific embryonic antigen 3; TRA-1-60, tumor rejection antigen 1–60.

Figure 2.

PKD iPS cells express PKD disease genes. Representative immunoblots of (A) PC1 and (B) PC2 with β-actin loading control in undifferentiated ES and iPS cell lines. A putative truncated PC1 band is observed for ADPKD patient 3 (arrowhead). (C) Immunoblots for PC1 and (D) PC2 with α-tubulin loading control for both in IMCD3 cells transfected with either empty vector negative control (−) or GFP-tagged full-length human PKD1 (+) compared with untransfected H9 human ES cells. (E) Representative immunoblot of FPC in ES and iPS lines. A small volume of lysate from a porcine renal tubular epithelial cell line stably expressing myc-tagged, human fibrocystin (hFPC-myc) was loaded three lanes to the right as a positive control. The extreme right lane is a mirrored, shorter exposure of the hFPC-myc lane that was superimposed onto the film. BJ, iPS cells derived from human foreskin fibroblasts; F5, hfib2-iPS5; HDF, iPS cells derived from adult human dermal fibroblasts.

Figure 3.

PKD iPS lines exhibit normal rates of replication, apoptosis, and ciliogenesis. (A) Representative immunocytochemistry and (B) quantification of cells positive for BrdU, phosphorylated histone H3 (pH3), and cleaved caspase-3 (c-casp3) in control (blue bars), ADPKD (red), or ARPKD (yellow) iPS cells. Scale bar, 50 µm. (C) Colocalization of the pluripotency marker OCT4 with acetylated α-tubulin (AcT) in representative stem cell lines after 7 days in feeder-free culture. Scale bar, 10 µm. (D) Percentage of ES or iPS cells with detectable cilia. One to two iPS lines were analyzed per patient, and iPS lines from the same patient had similar results; ∼1,000 cells were counted per condition per experiment (n≥3 experiments for each line). Error bars indicate SEM.

Figure 4.

PC2 is reduced at primary cilia in ADPKD iPS cells. (A) Colocalization of PC2 with acetylated α-tubulin (AcT) and the tight junction marker ZO-1 in representative ES or iPS lines. Insets show a close-up of a single cilium. (B) Percentage of cilia positive for PC2; ∼1,000 cells were counted per condition per experiment (n≥3 experiments). (C) Representative confocal x and y planes and corresponding z stacks of cells from these populations. Crosshairs in the merge illustrate the x and y planes shown in the z stack. (D) Ciliary lengths in these cell populations averaged from confocal z stacks. (E) Averaged intensity profiles of line scans through ∼45 individual cilia from these populations pooled from two to three different experiments. Data points represent averaged raw fluorescence intensity values from many images taken with identical exposures. Line scans were 16 µm in length. White dotted vertical line overlay in C illustrates how line scans were drawn. Scale bars, 10 µm. Error bars indicate SEM. P value, t test.

Figure 5.

Reduced ciliary PC2 is not due to a global loss of PC2 or PC1. Colocalization of PC2 with (A) acetylated α-tubulin and the tight junction marker ZO-1 or (B) the endoplasmic reticulum marker KDEL in iPS lines in a focal plane basal to the primary cilium. (C) Representative image showing lack of 7e12 antibody anti-PC1 immunofluorescence in H9 ES cells. Inset shows close-up view of one cilium. (D) Representative image of IMCD3 cells transfected with GFP-tagged human PC1. Transfected cells express variable levels of PC1. Whereas anti-GFP identifies PC1 in both the cell body and cilia, anti-PC1 only recognizes PC1 in the cell body. A close-up view of the red dotted rectangular region is shown below for each channel. The yellow dotted circle indicates a representative cilium. Untransfected cells (arrowhead) appear negative for PC1 staining. (E) PKD1 chromatograms representing wild-type, ADPKD fibroblasts, and derived PKD iPS colonies. Base pair positions are indicated relative to the start of the coding sequence, and amino acids are shown in blue shading. Red arrows highlight point mutations. (F) Chromatogram of a second iPS cell line (cell line 1B) derived from patient 1 showing loss of heterozygosity at the mutant locus. (G) Single nucleotide polymorphisms (SNPs; red arrows) in this iPS cell line. Exons and SNP identifiers are provided above the chromatograms. Scale bars, 10 µm.

Figure 6.

Ciliary PC2 is reduced in ADPKD somatic cells and hepatoblasts. Schematic of protocols for (A) stochastic differentation into embryoid bodies or (B) directed differentiation into hepatoblasts. Markers used to identify stages of differentiation are shown in red. (C) PC2 localization in EB outgrowth epithelial cells expressing ZO-1 at the membrane and AcT at the cilium. Insets show close-up of a representative cilium. (D) Representative immunofluorescence images of hepatoblast marker expression (column 1) and PC2 localization to cilia (columns 2–5). Average percentage of HNF4+ cells coexpressing AFP and CK19 is displayed at lower right of column 1 images. (E) Quantification of PC2 positive cilia in EB outgrowths or (F) hepatoblast monolayers (∼300 cells counted per condition per experiment; n≥3 experiments per line). (G) Corresponding ciliary length measurement for EB outgrowths or (H) hepatoblasts. (I) Averaged intensity profiles of line scans through ∼40 cell–cell junctions from somatic epithelial cells pooled from two to three different experiments. Data points represent averaged raw fluorescence intensity values from images taken with identical exposures. Line scans were 16 µm in length. Red dotted line overlay in C illustrates how line scans were drawn. (J) Average PC2/ZO-1 fluorescence ratios for the points at the apex of the curves shown in I. Patient 1 iPS lines were prone to fibroblastic differentiation under feeder-free conditions and, therefore, unavailable for hepatoblast differentiation. Scale bars, 10 µm. Error bars indicate SEM. P value, t test between the two pooled groups in brackets. BMP, bone morphogenetic protein.

Figure 7.

Wild-type PC1 promotes ciliary localization of endogenous PC2 in ADPKD hepatoblasts. (A) Average number of GFP-positive cells per confluent well of a 24-well plate in untransfected (control) or transfected (GFP-PKD1) populations. (B) Representative images of iPS-derived hepatoblasts from ADPKD patient 3 exogenously expressing GFP-tagged wild-type human PC1 full-length (GFP-PC1 positive) or neighboring untransfected control cells (GFP-PC1 negative) from the same cultures. GFP was detected in the green channel, PC2 was detected in the red channel, and AcT was detected in the far red channel. The merged image was pseudocolored without GFP to highlight colocalization of PC2 and acetylated tubulin. Scale bar, 5 µm. (C) Averaged intensity profiles of line scans through 25 individual cilia from GFP+ or untransfected neighboring cells pooled from four different experiments with hepatoblasts derived from ADPKD patients 2 or 3. Data points represent averaged raw fluorescence intensity values from images taken with identical exposures. Line scans were 16 µm in length. Red dotted line overlay in B illustrates how line scans were drawn. (D) Average PC2/AcT fluorescence ratios for the points at the apex of the curves shown in C. Error bars indicate SEM. P values, t test.

Figure 8.

Wild-type but not mutant PC1 promotes ciliary localization of endogenous PC2 in IMCD3 cells. (A) Transfection efficiencies (fraction of cells that expressed YFP) for YFP-tagged murine PC1 wild-type and mutant constructs. (B) Total number of cells with detectable PC2 at the cilium in untransfected (control) cultures or those cells transfected with murine PC1 constructs. (C) PC2 and AcT colocalization in IMCD3 cells expressing YFP-tagged wild-type murine PC1 full-length (mPC1), PC1 C-terminal truncation mutant (mPC1 mutant), or untransfected control cells from the same cultures. Merge is pseudocolored to highlight PC2/AcT colocalization. (D) Averaged intensity profiles of line scans through ∼75 individual cilia from these populations pooled from three different experiments. Data points represent averaged raw fluorescence intensity values from images taken with identical exposures. Line scans were 16 µm in length. Red dotted line overlay in C illustrates how line scans were drawn. (E) Average PC2/AcT fluorescence ratios for the points at the apex of the curves shown in D. (F) Ciliary lengths in these cell populations averaged from confocal z stacks. (G) Representative confocal x and y planes and corresponding z stacks of cells from these populations. Crosshairs in the merge illustrate the x and y plane shown in the z stack. (H) 4× and (I) 40× resolution bleed-through controls. Representative images of IMCD3 cells transfected with the wild-type construct of YFP-mPC1 incubated with primary antibodies against GFP and PC2 and subsequently incubated with both secondary antibodies (stained), no secondary antibodies (unstained), or only the secondary antibody to GFP (488 only) are shown. Corresponding fourth-channel images of AcT show cilia. Identical exposures are shown for all images. Scale bars, 5 µm in C, G, and I; 50 µm in H. Error bars in bar graphs and line scan time points indicate SEM. P values, t test.

Figure 9.

A model links loss-of-function mutations in PC1 to reduced ciliary PC2. In wild-type (WT) or ARPKD cells, pools of PC2 (green) associated with PC1 (pink) traffic from vesicles (Vs) to the cilium, whereas pools of unassociated PC2 traffic to the plasma membrane (PM) or endoplasmic reticulum (ER). In ADPKD cells, cellular concentrations of PC1 are diluted by mutant PC1 proteins (blue, PC1 mutant), resulting in diminished trafficking of PC2 to the cilium (thinner pink arrow). Reduced ciliary PC2 combined with reduced functional PC1 levels has a synergistic effect, leading to a pathogenic state.