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Identification of ezrin in a novel 3D in vitro model for ADPKD as a new target in cystogenesis

The ciliopathy autosomal dominant polycystic kidney disease (ADPKD) is characterized by progressive cystogenesis mediated by two ciliary proteins, polcystin‐1 (PC1) and polycystin‐2 (PC2/TRPP2). While the mechanism of cystogenesis in ADPKD is undefined, loss of function mutations in PKD1 or PKD2, which encode for transmembrane proteins PC1 and PC2/TRPP2, have been identified as initiators in cyst formation. Polycystins have been implicated in apical signal transduction facilitated by the primary cilium. Therefore, it is critical for understanding the downstream mechanism of cyst formation, including effects of aberrant proliferation, mislocalization of growth signals, and alterations in fluid secretion machinery. Our novel three dimensional (3D) cystogenesis model cultures primary renal cells from Pkd2fl/fl Pax8rtTA TetOCre +mTmG mice in Matrigel to investigate the effects of Pkd2 inactivation on tubulogenesis. Tubule formation in this 3D culture method is promoted by seeding cells between two layers of Matrigel and the manipulation of glial derived neurotrophic factor (GDNF) exposure. Following the induction of Cre, we observe a strong correlation between the mosaic mTmG reporter, the reduction in PC2 protein abundance, and the increase of cystic structures, strongly supporting the successful inactivation of Pkd2. Using this model, we explored possible disruption of the apical compartment and inappropriate recruitment of secretory machinery by looking closely at the critical scaffold and ERM family protein, ezrin. We found there is a significant decrease in apically localized ezrin in cells with inactivated Pkd2, a finding consistent with a disruption in the structure of the apical compartment. In nephrectomized human ADPKD cystic epithelia, ezrin localization is altered along the apical compartment of small cysts, where total ezrin and the phospho‐ezrin/radixin/moesin (p‐ERM) complex colocalize with basolateral markers. Interestingly, appropriate apical localization of ezrin is present in larger cysts, indicating that ezrin may be a critical player in the initiation of cystogenesis. Our 3D cystogenesis model for ADPKD demonstrates characteristics consistent with human ADPKD cysts and identifies important defects in membrane sorting mechanisms.