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Abstract: FR-PO576

Heterozygous PKD Organoids Show Increased Sensitivity to Forskolin-Stimulated Cystogenesis

Session Information

Category: Genetic Diseases of the Kidneys

  • 1201 Genetic Diseases of the Kidneys: Cystic

Authors

  • Thomas, Chardai J., University of Washington School of Medicine, Seattle, Washington, United States
  • Freedman, Benjamin S., University of Washington School of Medicine, Seattle, Washington, United States
  • Vishy, Courtney E., University of Washington School of Medicine, Seattle, Washington, United States
Background

Polycystic kidney disease (PKD) is inherited as a single heterozygous loss-of-function mutation, but theorized to require additional loss-of-function to cause cystogenesis. Activation of cAMP can stimulate cystogenesis, but whether this happens in heterozygotes is not well understood. We set out to test this in human kidney organoids.

Methods

We established human pluripotent stem cells in allelic series using CRISPR base editing to introduce nonsense mutations previously documented in ADPKD patients – PKD1 R2430X and Q3838X and PKD2 R186X and R872X. Mutations were confirmed by sequencing and protein changes by immunoblot. Homozygous mutant, heterozygous mutant, and isogenic control stem cells were differentiated into kidney organoids to determine if nonsense mutations conferred a cystic phenotype. Each genotype was treated with 0 uM or 30 uM forskolin, a stimulant of the cAMP pathway.

Results

Heterozygous cells expressed ~50% of PC1 or PC2 protein compared to controls. When differentiated into human kidney organoids, homozygous mutants spontaneously formed cysts whereas heterozygous mutants expressed no detectable phenotype, similar to non-mutant controls. Following forskolin treatment, heterozygous organoids showed a significant increase in the number of cysts formed which was not observed in homozygous mutant or non-mutant organoids (Figure 1).

Conclusion

Heterozygosity alone is insufficient to cause expression of PKD phenotypes in human kidney organoids. However, when treated with forskolin, heterozygosity sensitizes tubules to become cystic. Thus, even partial loss of function may enhance the PKD phenotype in the context of high levels of cAMP.

Control (+/+), heterozygous (+/-), and homozygous (-/-) organoids ± forskolin (FSK). Mean ± s.e.m. n = 4 independent experiments, ** p < 0.01

Funding

  • NIDDK Support