Kidney Week

Abstract: TH-PO672

Disruption of the EF-Hand Domain of Polycystin-2 Ameliorates Cystic Disease Caused by Polycystin-1 Deficiency

Session Information

• ADPKD: Genetic and Model Studies
  October 25, 2018 | Location: Exhibit Hall, San Diego Convention Center
  Abstract Time: 10:00 AM - 12:00 PM

Category: Genetic Diseases of the Kidney

• 1001 Genetic Diseases of the Kidney: Cystic

Authors

• Krappitz, Matteus, Yale School of Medicine, New Haven, Connecticut, United States

Matteus Krappitz,

• Hollmann, Till Amadeus, Yale School of Medicine, New Haven, Connecticut, United States

Till Amadeus Hollmann,

• Rümmele, David R., Friedrich-Alexander-Universität Erlangen-Nürnberg, Detmold, Germany

David R. Rümmele,

• Staudner, Tobias, Friedrich-Alexander-Universität Erlangen-Nürnberg, Detmold, Germany

Tobias Staudner,

• Dong, Ke, Yale School of Medicine, New Haven, Connecticut, United States

Ke Dong,

• Fedeles, Sorin V., Yale University School of Medicine, New Haven, Connecticut, United States

Sorin V. Fedeles,

• Gallagher, Rachel, Yale University School of Medicine, New Haven, Connecticut, United States

Rachel Gallagher,

• Cai, Yiqiang, Yale University School of Medicine, New Haven, Connecticut, United States

Yiqiang Cai,

• Zhang, Chao, Yale School of Medicine, New Haven, Connecticut, United States

Chao Zhang,

• Somlo, Stefan, Yale University , New Haven, Connecticut, United States

Stefan Somlo,

Background

Polycystin-2 (PC2/TRPP2), the product of one of the genes mutated in ADPKD, is expressed in the endoplasmic reticulum (ER) and ciliary membranes. The COOH-terminal tail of PC2 contains a potential Ca²⁺ binding EF-hand motif. A mouse model termed Pkd2^TEAA, in which critical residues at the EF-hand were substituted with alanine (T769A, E772A) to inactivate the Ca²⁺-binding properties of PC2-EF, was generated via CRISPR/Cas9 methodology [ASN2017, TH-PO597]. The Pkd2^TEAA mutant does not result in loss of PC2 function as evidenced by the lack of cysts in Pkd2^TEAA/TEAA animals. In this study we assessed the impact of the Pkd2^TEAA allele on Pkd1 derived cysts.

Methods

The Pkd1^R2220W human REJ mutant (Pkd1^RW) is a representative candidate of a PC1 hypomorphic missense mutation as it leads to a clear yet partial defect in PC1 biogenesis and cleavage. Mutant alleles were further modified by insertion of a V5 epitope tag in-frame at the C-terminus to detect the mutant proteins (Pkd1^RW-V5 and Pkd2^TEAA-V5). The Pkd2^TEAA mouse was crossed onto a Pkd1^RW/flox background under the control of Pkhd1-Cre (collecting duct-specific). The kidneys were examined by histological and morphological parameters.

Results

Compared to Pkd1^RW/flox; Pkhd1-Cre mice, the kidneys at P24 from Pkd1^RW/flox; Pkd2^TEAA/+; Pkhd1-Cre mice displayed a decreased KW/BW ratio (0.16 ± 0.02 vs 0.067 ± 0.04, ***p<0.001) and BUN (mg/dL: 93.6 ± 24.97 vs. 38.6 ± 19.54, **p<0.0013). We observed a marked increase in apoptosis specifically in cyst-lining epithelia expressing Pkd2^TEAA. No significant difference in the protein levels of PC1 or PC2 proteins could be detected between the different genotypes.

Conclusion

Inactivation of the Ca²⁺-binding properties of PC2 EF-hand on a partially PC1 deficient background results in improved cystic phenotype possibly via specific apoptosis of PC1 mutant cyst-lining epithelial cells. Modulators of PC2 EF-hand function may alter PC2 function in vivo to slow cyst progression in the setting of some PC1 missense mutations.

Funding

• NIDDK Support