The mechanism of cystogenesis in autosomal dominant polycystic kidney disease (ADPKD) remains ambiguous. While many altered signaling pathways have been identified as part of cyst formation, very little is known about the downstream effects of loss of function mutations in polycystin 1 (PC1) and polycystin 2 (PC2). These transmembrane proteins, encoded by PKD1 and PKD2 respectively, are suspected to form a signaling complex that has been localized to the membrane as well as the junctions. Preliminary immunocytochemistry data in human ADPKD epithelial cyst tissue reveals aberrant localization of aquaporin 2 (AQP2), as well as loss of zonula occludens 1 (ZO-1) in the tight junctions. These observations suggest a role for the polycystin proteins in the organization of the apical compartment of renal epithelial cells. This proposal introduces a novel, 3D ex- vivo organoid system developed to isolate the role of the pore forming subunit of the polycystin complex, PC2, in cyst formation, calcium (Ca2+) signaling, and epithelial organization. The model overcomes previous roadblocks to studying the role of PC2 by employing a genetically tractable, tetracycline responsive Cre recombinase to temporally control inactivation of Pkd2. Additionally, the culture system optimizes the development of complex, differentiated structures, including spheroids and tubules, that allow for tracking of morphological changes, as well as changes in localization and abundance of junction associated proteins following Pkd2 inactivation. Therefore, with the application of this new system, the role of PC2 in epithelial cell organization and how its inactivation contributes to cyst formation can be elucidated in relevant 3D structures. Preliminary data suggests that a mechanism of ADPKD cystogenesis is the disruption of apical compartment regulator, ezrin, an Ezrin-Radixin-Moesin (ERM) family protein. Ezrin is a regulator of apical transporters and membrane proteins, and stabilizes the F-actin cytoskeleton and apical junctional complex (AJC). In the 3D model system, following inactivation of Pkd2, total ezrin protein abundance is decreased, and ezrin expression is significantly decreased in cells that have inactivated Pkd2, compared to cells in control structures. This proposal investigates the hypothesis that PC2 is a regulator of Ca2+-dependent signaling pathways responsible for the activation and recruitment of ezrin to the apical membrane and maintenance of apical compartmentalization. Using my newly developed model system alongside gold standard biochemical and fluorescent techniques, data collected from proposed experiments will define the effect of Pkd2 inactivation on the localization and organization of the AJC in the mechanism of cystogenesis (Aim 1). Furthermore, it will define the role of PC2 mediated Ca2+ signaling on the regulation of ezrin function in renal epithelial cells (Aim 2).

Public Health Relevance

Autosomal dominant polycystic kidney disease (ADPKD) is one of the most common monogenic disorders worldwide, characterized by progressive cystogenesis and resulting in end stage renal disease (ESRD). A new model system identifies a potential role for ezrin in the disruption of apical compartmentalization in cystic renal epithelial cells. This proposal will define the downstream effect of polycystin-2 (PC2) signaling on the activation of ezrin; the data gathered from this proposal will elucidate a player in cystogenesis and potential therapeutic target for ADPKD.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Predoctoral Individual National Research Service Award (F31)
Project #
5F31DK117579-02
Application #
9647364
Study Section
Special Emphasis Panel (ZDK1)
Program Officer
Maric-Bilkan, Christine
Project Start
2018-07-01
Project End
2020-06-30
Budget Start
2019-07-01
Budget End
2020-06-30
Support Year
2
Fiscal Year
2019
Total Cost
Indirect Cost
Name
University of Maryland Baltimore
Department
Physiology
Type
Schools of Medicine
DUNS #
188435911
City
Baltimore
State
MD
Country
United States
Zip Code
21201