Abstract: FR-PO0637
Development of a High-Throughput Three-Dimensional (3D) Tubulogenesis Assay to Study ADPKD Gene Function
Session Information
- Cystic Kidney Diseases: Basic and Translational Research
November 07, 2025 | Location: Exhibit Hall, Convention Center
Abstract Time: 10:00 AM - 12:00 PM
Category: Genetic Diseases of the Kidneys
- 1201 Genetic Diseases of the Kidneys: Monogenic Kidney Diseases
Authors
- Westermann, Lukas, Renal Division, Department of Medicine, Medical Center, Faculty of Medicine, University of Freiburg, Freiburg, Germany
- Rhein, Kilian, Renal Division, Department of Medicine, Medical Center, Faculty of Medicine, University of Freiburg, Freiburg, Germany
- Jahn, Johannes, Renal Division, Department of Medicine, Medical Center, Faculty of Medicine, University of Freiburg, Freiburg, Germany
- Schöler, Felix, Renal Division, Department of Medicine, Medical Center, Faculty of Medicine, University of Freiburg, Freiburg, Germany
- Reimund, Lea, Renal Division, Department of Medicine, Medical Center, Faculty of Medicine, University of Freiburg, Freiburg, Germany
- Mangel, Franziska, Renal Division, Department of Medicine, Medical Center, Faculty of Medicine, University of Freiburg, Freiburg, Germany
- Seyed Tarrah, Shanli, Renal Division, Department of Medicine, Medical Center, Faculty of Medicine, University of Freiburg, Freiburg, Germany
- Busch, Tilman, Renal Division, Department of Medicine, Medical Center, Faculty of Medicine, University of Freiburg, Freiburg, Germany
- Kottgen, Michael, Renal Division, Department of Medicine, Medical Center, Faculty of Medicine, University of Freiburg, Freiburg, Germany
Background
ADPKD is the most common monogenic kidney disease. Approximately 80% of ADPKD cases are caused by mutations in the PKD1 gene, which encodes the protein Polycystin-1 (PC1). Despite its central role in the disease, the function of PC1 remains poorly understood. Developing a reliable cellular model that reflects the disrupted tubular morphogenesis observed in ADPKD could provide valuable insights into PC1 function and help address current knowledge gaps.
Methods
Pkd1-deficient and isogenic wildtype mIMCD3 cells were seeded in a Matrigel-collagen-scaffold in 96-well plates. Within 7 days, cells organized into complex structures. Structures were imaged with a high-throughput confocal microscope. Data analysis was performed by a neural network, followed by automated segmentation for 3D structure detection enabling unbiased classification and quantification. Analysis of morphological alterations and longitudinal transcriptomic profiling of 3D structures aimed at identifying functional interaction partners of PC1.
Results
We have developed a high-throughput 3D tubulogenesis assay using mIMCD3 cells showing robust tubulogenesis in wildtype cells. Segmentation revealed a significant reduction of mean tubules per well in Pkd1 knockout (KO) vs isogenic control cells (71 vs 214 tubules, p<0.01) and a significant reduction in mean tubule area (1335 vs 2811 μm2; p<0.01). Differential gene expression analysis of genotype-dependent tubulogenesis identified potential signaling effectors of Pkd1.Transcriptomic analysis comparing early stages of tubulogenesis in wildtype and isogenic Pkd1 KO cells revealed 118 differentially expressed genes (53 genes up-, 65 downregulated). We characterized downregulated hit genes with cyst association in the 3D tubulogenesis assay. Inducible Tns-1 loss phenocopied the disturbed tubulogenesis observed in Pkd1 KO cells (number of tubules: 60 vs 113, p<0,05; tubule area: 2189 vs 3301 µm2, p<0,01). In a murine ADPKD model, we observed a reciprocal relationship between TNS-1 levels and cyst size.
Conclusion
We demonstrate the establishment of a high-throughput 3D tubulogenesis assay that allows for monitoring of PC1 function with an automated image analysis platform. This cell-based assay identifies TNS1 as a novel downstream target of PC1 which may play a role in cystogenesis.