ASN's Mission

To create a world without kidney diseases, the ASN Alliance for Kidney Health elevates care by educating and informing, driving breakthroughs and innovation, and advocating for policies that create transformative changes in kidney medicine throughout the world.

learn more

Contact ASN

1401 H St, NW, Ste 900, Washington, DC 20005

email@asn-online.org

202-640-4660

The Latest on X

Kidney Week

Please note that you are viewing an archived section from 2020 and some content may be unavailable. To unlock all content for 2020, please visit the archives.

Abstract: PO1514

Somatic Tubular Epithelial Cell Model of Type II Polycystic Kidney Disease Reveals Phenotypes of Altered Ciliary Length and Polycystin-Intraflagellar Complex Degradation

Session Information

Category: Genetic Diseases of the Kidneys

  • 1001 Genetic Diseases of the Kidneys: Cystic

Authors

  • Roy, Ankita, University of Washington, Seattle, Washington, United States
  • Vishy, Courtney E., University of Washington, Seattle, Washington, United States
  • Helms, Louisa, University of Washington, Seattle, Washington, United States
  • Gomez, Ivan G., University of Washington, Seattle, Washington, United States
  • Himmelfarb, Jonathan, Kidney Research Institute, Seattle, Washington, United States
  • Freedman, Benjamin S., University of Washington, Seattle, Washington, United States
Background

Autosomal dominant polycystic kidney disease (PKD) is a life-threatening monogenic disorder affecting 12 million people worldwide, commonly due to loss-of-function mutations in PKD1 or (type II) PKD2, encoding polycystin-1 (PC1) and polycystin-2 (PC2), respectively. These form a receptor-channel complex in the primary cilium whose molecular function remain uncertain, making it difficult to develop targeted therapeutics. Mice and organoids can express PKD phenotypes, but are slow and complex, posing challenges for mechanistic analysis.

Methods

To establish a somatic cell model of PKD, we generated five clones of porcine proximal tubule cell line (LLCPK1) completely lacking PC2 using the CRISPR-Cas system, alongside five isogenic control lines. Mutations were verified for each allele by TOPO cloning and clones were derived from a single subclone to minimize heterogeneity. Cells were evaluated for PC1 steady-state levels, ciliary defects, and cyst formation in low adhesion plates in the presence of cyst activators. Forskolin, 8-Br-cAMP and the myosin inhibitor blebbistatin promoted cystogenesis. Transcript analysis indicated that the effects of altered PC1 steady-state levels was post-transcriptional. Exogeneous myc-PC2 rescued PC1 in the PC2 null cells.

Results

In the absence of PC2, PC1 was thoroughly degraded, similar to findings in human and distinct from mouse. Notably, intraflagellar transport components including ARL13B and IFT88 were also reduced. Cilia in 2D cultures appeared short while in the cystic cells was lengthened. Pharmacologic agents that inhibit the proteasome and the lysosome stabilize PC1 in these cells and rescues the ciliary phenotype. Additionally, the drugs reduce cyst size in human PKD organoids lacking PKD2.

Conclusion

We have established a somatic tubular epithelial cell model for PKD, which can be rapidly assessed for molecular and cellular phenotypes. This reveals the necessity of PC2 for proper ciliary structure and to prevent the degradation of a protein complex containing PC1 and intraflagellar transport components. Overall the porcine lineage appears more similar to human cells compared to mouse. Targeting degradation pathways rescues this novel complex and may represent a useful therapeutic approach for PKD.

Funding

  • NIDDK Support