Elucidating Mechanisms of Hypomorphic WDR19-Related Kidney Failure

Abstract

Introduction: Variants in the WDR19 gene, a crucial component of the intraflagellar transport (IFT) complex A, are associated with renal-cystic ciliopathies, a prevalent cause of renal failure of genetic origin. In the Arab Druze population, a WDR19 pathogenic missense variant (c.878G>A; p.Cys293Tyr, termed WDR19:C.878G>A) is the most common genetic cause of kidney failure manifesting as adult-onset, typically nonsyndromic chronic kidney disease (CKD). The underlying pathogenesis of this condition remains unclear.

Methods: We used CRISPR-Cas9 to induce patient-specific hypomorphic and loss-of-function (LoF) variants in human embryonic stem cells (hESCs), in addition to using patient-derived induced pluripotent stem cells (iPSCs) for differentiation into kidney organoids. Organoids were assessed by using immunofluorescence, electron microscopy, RNA-sequencing, and pathway analysis to elucidate the effects of these pathogenic variants on kidney development and ciliopathy characteristics.

Results: The WDR19 hypomorphic variant impairs nephron development, causing delayed kidney organoid differentiation from early stages, cystogenesis, and structural abnormalities in both tubular and glomerular structures. Mutant organoids displayed reduced ciliation and shortened cilia. Both mutated organoids exhibited Sonic hedgehog dysregulation, where the pathway was upregulated in the presence of severe LoF variant and significantly reduced ciliation. Elevated sonic hedgehog (Shh) signaling was associated with significant downregulation of fibroblast growth factor (FGF) 8 (FGF8) and transcriptomic alterations in associated pathways, suggesting an inverse pathways relationship during kidney organoid development.

Conclusion: Our study validates the pathogenic role of the WDR19 hypomorphic variant in adult-onset renal failure and highlights how hypomorphic pathogenic variants disrupt kidney development. These findings underscore the critical role of cilia in renal development, offering insight into the mechanisms of ciliopathies.

Keywords: WDR19; ciliopathies; kidney organoids.

Graphical abstract

Figure 1

(a) Generation of CSES7 embryonic stem cell line with the induced WDR19 c.878G>A pathogenic variant (WDR19:C.878G>A). Schematic representation of the plasmid-mediated CRISPR-Cas9 gene editing approach used in the current study. A plasmid containing guide RNA (gRNA) targeting the WDR19 locus and Cas9 was introduced into cells. For homology-directed repair (HDR), a donor template, comprising the c.878G>A variant, adjacent silent variants, and flanking homology arms, was delivered to facilitate precise genome editing. On the right is the Sanger sequencing confirmation of the WDR19:C.878G>A induced clone, compared with the isogenic WDR19-WT control. (b) Generation of the CSES7 embryonic WDR19 LoF embryonic stem cell line (WDR19-LoF). This CRISPR-Cas9 approach uses 2 gRNAs to create double-strand breaks in the intronic regions flanking Exon 8 of the WDR19 gene. The deletion of Exon 8 leads to a frameshift, introducing an early stop codon and producing a truncated, non-functional WDR19 protein. In the lower panel, the sequencing confirmation of the WDR19-ΔExon8 clone is shown, alongside electrophoresis results comparing WDR19-WT and the 432 bp shorter WDR19-ΔExon8 product. In addition, cDNA electrophoresis demonstrates no detectable product in the WDR19-ΔExon8 knockout line, confirming successful exon deletion and absence of full-length transcript. (c) WT kidney organoid generation with representative phase contrast images of the organoids on days 8, 12, 15, and 24 of differentiation; the latter is a mature kidney organoid. Scale bars: 500 μm. (d) Immunofluorescence confocal images of WDR19-WT line-derived kidney organoids on day 24 of differentiation. ECAD (red) marks the distal tubule, LTL (green) the proximal tubule, and PODXL (white) the glomeruli. Scale bars: 100 μm. WT, wild-type.

Figure 2

(a) Phase contrast images of the 3 human embryonic stem cell lines with identical genetic backgrounds—WDR19-WT, WDR19:C.878G>A, and WDR19-LoF—on day 4 of differentiation into kidney organoids at the monolayer stage. At this stage, the cells exhibit similar morphology. Scale bars: 500 μm. (b) Left panel: phase contrast representative images. of day 13, WDR19-WT and WDR19:C.878G>A organoids showing proper differentiation, reaching the renal vesicle stage, whereas no such structures are observed in WDR19-LoF organoids. Right panel: day 25 immunofluorescence images display nephron compartments. A noticeable scarcity of nephron structures is seen in WDR19-LoF organoids compared with WDR19-WT and hypomorphic WDR19:C.878G>A organoids. Scale bars: 500 μm. (c) Representative images of kidney organoids derived from WDR19-WT, WDR19:C.878G>A, and WDR19-iPSCs on days 13 and 15. On day 13, all 3 lines show similar rounded luminal structures positive for LHX1 (green), marking renal vesicles. By day 15, WDR19:C.878G>A and WDR19-iPSC organoids retain rounded “doughnut-like” renal vesicles, whereas WDR19-WT organoids progress to a more tubular and convoluted morphology, indicating advanced differentiation. (c’) Quantification of renal vesicles (RVs) per area in kidney organoids derived from WDR19-WT, WDR19:C.878G>A, and WDR19-iPSCs. WDR19:C.878G>A organoids display a significantly higher number of renal vesicles compared with WDR19-WT. Data points represent individual measurements, and error bars indicate the mean ± SEM. ∗∗∗P < 0.001. For renal vesicle quantification, WDR-G>A: n = 11, WDR-WT: n = 7, iPSC: n = 6. Scale bars: 100 μm (right) and 500 μm (left). For more information, see Materials and Methods section. (d) Phase contrast images showing differentiation timeline from days 11 to 18. WDR19:C.878G>A organoids show a markedly less organized appearance and early collapse starting from day 15 of differentiation. (e) Immunofluorescence images of mature kidney organoids (day 25) derived from WDR19-WT and WDR19:C.878G>A lines. WDR19:C.878G>A organoids exhibit abnormal tubular structures, including dilated LTL-positive proximal tubules (yellow arrowheads) and lack of structural continuity between different nephron compartments. Scale bars: 100 μm. (e’). Tubule-to-lumen area ratio analysis based on immunostaining images demonstrates significantly tubular dilatation in WDR19:C.878G>A organoids relative to WDR19-WT. Data is presented as mean ± SD, ∗∗∗∗P < 0.0001.(f) Electron microscopy images revealing disrupted tubular morphology, dilated lumens, and accumulation of cellular debris in WDR19:C.878G>A organoids, further indicating abnormal nephron architecture compared to WDR19-WT. Scale bars: 5–10 μm. (g) Electron microscopy images of kidney organoids revealing foot process–like structures resembling podocyte pedicels in WDR19-WT organoids (yellow arrowheads), which are absent in WDR19:C.878G>A organoids. Scale bars: 5 nm. Middle: immunofluorescence images of PODXL staining showing that WDR19:C.878G>A organoids exhibit smaller, less uniformly rounded glomerular structures compared to WDR19-WT. Scale bars: 100 μm. (h) Cystogenesis in kidney organoids treated with forskolin (25 μM) from days 14 to 25 of differentiation. Representative images show cyst-like dilations in WDR19:C.878G>A and WDR19-iPSC derived organoids, whereas WDR19-WT organoids do not form cysts. (h’). Quantification of cysts per area indicates a significant increase in cyst formation in WDR19:C.878G>A and iPSCs compared with WDR19-WT organoids. Data are presented as mean ± SEM, ∗∗∗∗P < 0.0001. WDR19-WT: n = 12; WDR19G>A: n = 7; iPSC: n = 5. Scale bars: 500 μm. LoF, loss-of function; WT, wild-type.

Figure 3

(a) Immunofluorescence staining of cilia using acetylated alpha-tubulin (red) and gamma-tubulin (green) as markers for axonemal structure and basal body, respectively. Both the WDR19:C.878G>A and WDR19-LoF cells exhibit significantly reduced cilia per unit area compared to wild-type cells (∗∗∗∗P < 0.0001), with WDR19-LoF showing the most severe reduction. Scale bars: 10 μm (upper panel) and 3 μm (lower panel). (b) Transmission electron microscopy images of primary cilia in WT and WDR19:C.878G>A cells. Cilia from WDR19-WT (left panel) appear normal, exhibiting typical structural features, whereas cilia from WDR19:C.878G>A mutant cells are shortened. Scale bars: 500 nm and 200 nm. The graph on the right quantifies cilia length based on n = 200 random measurements of acetylated alpha-tubulin positive cilia. Cells carrying the WDR19:C.878G>A variant exhibit significantly shorter cilia compared to WT cells (∗∗∗∗P < 0.0001). LoF, loss-of function; WT, wild-type.

Figure 4

(a) Western blot analysis of GLI3 processing in WDR19-WT, WDR19:C.878G>A, and WDR19-LoF lines on day 7 of differentiation. The GLI3F:GLI3S ratio quantifies Sonic Hedgehog (Shh) pathway activity, showing a positive correlation with pathway activation. (b) GLI1 relative expression during differentiation days 0, 7, and 18 in WDR19-WT, WDR19:C.878G>A, and WDR19-LoF lines. Due to severely disrupted kidney organoid differentiation, WDR19-LoF lines were not included beyond the monolayer stage (day 7). (c) Western blot analysis of GLI3 processing in WDR19-WT, WDR19:C.878G>A, and WDR19-LoF lines on day 18 of differentiation. WDR19-LoF line was excluded from this analysis due to severely disrupted kidney organoid differentiation. (d) FGF8 relative expression during differentiation days 0, 7, and 18 in WDR19-WT, WDR19:C.878G>A, and WDR19-LoF lines. Due to severely disrupted kidney organoid differentiation, WDR19-LoF lines were not included beyond the monolayer stage (day 7). (e) Hierarchical clustering of RNA-sequencing data showing the transcriptional differences between WDR19 c.878G>A-harboring organoids: WDR19:C.878G>A and the WDR19-iPSCs lines, and the WDR19-WT organoids. (f) Principal component analysis (PCA) plot of bulk RNA sequencing data. The plot illustrates the separation of samples across 3 biological groups: WDR19-WT (green squares), WDR19:C.878G>A (blue triangles), and WDR19-iPSC (red circles). The x-axis (PC1) explains 69% of the variance, whereas the y-axis (PC2) explains 29% of the variance. The clear separation between the WDR19 mutant lines and the wild-type samples highlights the significant transcriptional differences induced by the pathogenic variant. (g) Top 10 up- and downregulated GO biological pathways (green) and top 5 biological processes (blue) identified by RNA-seq analysis. The analysis is based on 960 genes that were differentially expressed in the same direction (either up- or downregulated) in both hypomorphic WDR19 mutant lines (WDR19:C.878G>A and WDR19-iPSC) compared to WDR19-WT. The numbers on the right indicate the number of altered genes out of the total genes in that GO term. GO, gene ontology; LoF, loss-of function; WT, wild-type.

Figure 5

(a) Differentiation of WDR19-WT, WDR19:C.878G>A, and WDR19-LoF cells into cerebral brain organoids. Immunofluorescence staining shows DAPI (blue), SOX2 (green), and TUJ2 (red), in the organoids. All 3 cell lines successfully differentiate into cerebral organoids. (b) Relative expression levels of neuronal markers TUJ1, PAX6, MAP2, and the signaling protein FGF8 on day 77 of differentiation.