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Abstract: PO1511

Role of Polycystin 2 in Endoplasmic Reticulum Calcium Release and Pathogenesis of Polycystic Kidney Disease

Session Information

Category: Genetic Diseases of the Kidneys

  • 1001 Genetic Diseases of the Kidneys: Cystic

Authors

  • Padhy, Biswajit, University of Iowa Carver College of Medicine, Iowa City, Iowa, United States
  • Xie, Jian, University of Iowa Carver College of Medicine, Iowa City, Iowa, United States
  • Huang, Chou-Long, University of Iowa Carver College of Medicine, Iowa City, Iowa, United States
Background

The prevailing view is that PKD is a ciliopathy. Yet, polycystin-2 contains an ER retention signal, is most abundantly expressed in ER. Early studies showed that PC2 is involved in agonist-induced ER Ca2+ release. Decrease in ER Ca2+ release in PC2-deficient cells is believed to contribute to cAMP overproduction and cystogenesis, the foundation for treatment by tolvaptan. Recent patch-clamp recordings reveal that PC2 conducts predominantly monovalent cation with ~40X more selective to K+ than Ca2+. The low selectivity for Ca2+raises the question regarding the role of PC2 in Ca2+ release in ER and thus the role of ER-localized PC2 in PKD pathogenesis. Cation exchange and/or parallel anion movement across ER membrane is imperative for Ca2+ release.TRIC-B (trimeric intracellular cation-B) is a known ER resident K+ channel mediates counterion exchange for IP3R and RyR-mediated Ca2+ release.

Methods

For in vitro studies PC2-deleted renal epithelial cell line and HEK cells with CRISPR-Cas9-mediated gene knockout of TRICB channel are used. ER Ca2+ release stimulated by carbachol or ATP is assayed by increases in the intracellular Ca2+ measured by using Fura-2 calcium imaging. Zebrafish is used as in vivo PKD model. Gene specific antisense morpholinos are used for targeted knockdown of PC2 and TRICB in zebrafish embryos. Dorsal curvature and pronephric cystic phenotypes are analyzed.

Results

Agonist-stimulated ER Ca2+ release is blunted in PC2-null epithelial cells. Expression of WT PC2 restores ER Ca2+ release. Expressing WT TRIC-B channel, but not LOF mutant, rescues ER Ca2+ release in PC2-null cells. Similarly, ER Ca2+ release is blunted in cells deleted of TRIC-B, and expression of WT PC2 partially rescues defective ER Ca2+ release in TRIC-B-null cells. Zebrafish embryos injected with PC2 antisense morpholino develops dorsal curvature and enlarged pronephric cyst, which can be rescued by co-injecting WT, but not LOF mutant, PC2 RNA. Co-injecting WT, but not LOF, TRIC-B RNA rescues defects in PC2-morphant fish.

Conclusion

PC2 is a monovalent cation channel facilitates ion exchange for IP3R-mediated Ca2+ release. Loss of this function of PC2 in ER plays a critical role in the cystogenesis of PKD.

Funding

  • NIDDK Support