Abstract: TH-PO581
Induced Inactivation of Pkd2 Results in Progressive Tubule Reduction and Loss in New, 3D Model of ADPKD Cystogenesis
Session Information
- Cystic Kidney Diseases - I
November 02, 2017 | Location: Hall H, Morial Convention Center
Abstract Time: 10:00 AM - 10:00 AM
Category: Genetic Diseases of the Kidney
- 801 Cystic Kidney Diseases
Authors
- Dixon, Eryn E., University of Maryland School of Medicine, Baltimore, Maryland, United States
- Woodward, Owen M., University of Maryland School of Medicine, Baltimore, Maryland, United States
Background
The role of PKD1/PKD2 loss in renal cytogenesis remains both unequivocal and undefined. Many disparate pathways are known to be altered in ADPKD without a definitive mechanistic pathway connecting back to the loss of either polycystin protein. A long standing roadblock has been the inability to model cystogenesis resulting from the spontaneous loss of PKD1/PKD2 as postulated by the two-hit ADPKD hypothesis.
Methods
To better understand the local effects of Pkd2 inactivation on cystogenesis, signaling, and organization of differentiated, E-cadherin positive, structures, a new, three-dimensional (3D) in vitro model has been developed that employs primary renal cells from an inducible Cre (Pkd2 Pax8 rtTA TetOCre+mTmG) mouse line. This 3D culture system combines a unique “sandwich” plating technique with a glial-derived neurotrophic factor (GDNF) growth factor cocktail to increase the yield of differentiated and complex epithelial structures, including spheroids and tubules. In addition, this new model system allows tracking of gross morphological changes of 3D structures and cellular components before and after the inactivation of Pkd2.
Results
Characterization of the differentiated tubule structures reveals that the cells of the organoids demonstrate typical apicobasolateral polarization and primary cilium as defined by basolateral Na+/K+-ATPase and lumenal acetylated tubulin, respectively. Interestingly, the differentiated organoids are positive for collecting duct markers, Dolichos biflorous agluttinin (DBA), and the apical water channel, aquaporin-2 (AQP2), while negative for proximal tubule markers, Lotus tetrogonobulus lectin (LTL) and ATP-binding cassette sub-family G member 2 (ABCG2). Addition of doxycycline induces Cre and mosaic inactivation of Pkd2 with an approximately 50% decrease in the abundance of total polycystin-2 (PC2). The loss of PC2 protein results in a progressive reduction and eventual loss of tracked tubule structures when compared to control organoids.
Conclusion
Changes in morphology of differentiated structures following inactivation of Pkd2 in this novel cystogenesis culture system suggest a defect in the 3D organization of epithelial structures that is dependent on PC2 and the study of cystogenesis in 3D organoid culture systems may provide novel insights into the pathogenesis of ADPKD.
Funding
- NIDDK Support