Kidney Week

Abstract: FR-PO588

Analysis of Pkd1 Gene Function in a Three-Dimensional Tubulogenesis Assay

Session Information

• Cystic Kidney Diseases: Mechanisms and Models
  October 25, 2024 | 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: Cystic

Authors

• Westermann, Lukas, Renal Division, Department of Medicine, Medical Center, Faculty of Medicine, University of Freiburg, Freiburg, Germany

Lukas Westermann, MD

• Rhein, Kilian, Renal Division, Department of Medicine, Medical Center, Faculty of Medicine, University of Freiburg, Freiburg, Germany

Kilian Rhein

• Jahn, Johannes, Department of Radiology, Medical Center, Faculty of Medicine, University of Freiburg, Freiburg, Germany

Johannes Jahn

• Schöler, Felix, Renal Division, Department of Medicine, Medical Center, Faculty of Medicine, University of Freiburg, Freiburg, Germany

Felix Schöler

• Niedermoser, Matthias, Renal Division, Department of Medicine, Medical Center, Faculty of Medicine, University of Freiburg, Freiburg, Germany

Matthias Niedermoser

• Seidl, Elena, Renal Division, Department of Medicine, Medical Center, Faculty of Medicine, University of Freiburg, Freiburg, Germany

Elena Seidl

• Moser, Niklas, Renal Division, Department of Medicine, Medical Center, Faculty of Medicine, University of Freiburg, Freiburg, Germany

Niklas Moser

• Seyed Tarrah, Shanli, Renal Division, Department of Medicine, Medical Center, Faculty of Medicine, University of Freiburg, Freiburg, Germany

Shanli Seyed Tarrah, MS, MSc

• Reimund, Lea, Renal Division, Department of Medicine, Medical Center, Faculty of Medicine, University of Freiburg, Freiburg, Germany

Lea Reimund, MD

• Busch, Tilman, Renal Division, Department of Medicine, Medical Center, Faculty of Medicine, University of Freiburg, Freiburg, Germany

Tilman Busch

• Neubauer, Bjoern, Renal Division, Department of Medicine, Medical Center, Faculty of Medicine, University of Freiburg, Freiburg, Germany

Bjoern Neubauer, PhD

• Li, Yong, Institute of Genetic Epidemiology, Faculty of Medicine and Medical Center, University of Freiburg, Freiburg, Germany

Yong Li, PhD

• Kottgen, Anna, Institute of Genetic Epidemiology, Faculty of Medicine and Medical Center, University of Freiburg, Freiburg, Germany

Anna Kottgen, MD, MPH

• Kottgen, Michael, Renal Division, Department of Medicine, Medical Center, Faculty of Medicine, University of Freiburg, Freiburg, Germany

Michael Kottgen, MD

Background

Autosomal-dominant polycystic kidney disease (ADPKD) is the most common monogenic kidney disease. The majority of ADPKD patients (85%) harbors mutations in Pkd1 encoding for Polycystin-1 (PC1). PC1 function is still insufficiently understood. The establishment of a robust cellular readout for PC1 function mimicking impaired tubular morphogenesis observed in ADPKD could help to overcome these roadblocks.

Methods

Wildtype mIMCD-3 cells were seeded in a Matrigel-collagen-scaffold in the presence of hepatocyte growth factor in 96-well plates. Within 7 days, cells organized into complex tubular or spheroid structures. Structures were imaged with a confocal microscope. Data analysis was performed by training of a neural network (U-Net), followed by automated segmentation for 3D structure detection that enabled unbiased quantification and classification. Analysis of genotype-dependent morphological alterations and longitudinal transcriptomic profiling of 3D structures aimed at the identification of functional interaction partners of PC1.

Results

Automated segmentation revealed a significant reduction of mean tubules per well in Pkd1 knockout (KO) vs isogenic control cells (71 vs 214 tubules, respectively; p<0.0001) as well as a significant reduction in mean tubule area (1335 vs 2811 µm², respectively; p<0.0001). This phenotype was highly robust and confirmed in multiple Pkd1 KO and inducible Pkd1 knockdown (KD) cells. Longitudinal 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) with stringent cut-off-criteria (adjusted p<0.01, log₂ fold change ≤-1 and ≥1). We are currently investigating whether KD of the most significantly downregulated transcripts (GC, Scd1, C3, Chst15 and Tns1) phenocopies PC1 loss in the 3D tubulogenesis assay.

Conclusion

We demonstrate the establishment of a robust 3D tubulogenesis assay that allows for monitoring of PC1 function with an automated image analysis platform. This cell-based assay for PC1 function enables high-throughput screening for mechanistic studies and drug discovery.

Funding

• Government Support – Non-U.S.