Document text
Principal Investigator: Eryn E. Dixon
Organization: UNIVERSITY OF MARYLAND BALTIMORE
Fiscal Year: 2019
Award: $37,082
Funding agency: National Institute of Diabetes and Digestive and Kidney Diseases
Project summary/abstract
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).
Terms: <3'5'-cyclic ester of AMP><3-D><3-D modeling><3-D structure><3-Dimensional><3-dimensional structure><3D><3D cell culture><3D culture><3D modeling><3D structure><A kinase anchoring protein><ADPKD><AKAP><AQP-CD><AQP2 protein><Adenosine Cyclic 3',5'-Monophosphate><Adenosine Cyclic Monophosphate><Adenosine Cyclic Monophosphate-Dependent Protein Kinases><Adenosine, cyclic 3',5'-(hydrogen phosphate)><Adult Polycystic Kidney Disease><Apical><Architecture><Autosomal Dominant Polycystic Kidney><Autosomal Dominant Polycystic Kidney Disease><Biochemical><Biologic Models><Biological Models><Body Tissues><CRE Recombinase><Calcium><Carrier Proteins><Cell Body><Cell Communication and Signaling><Cell Culture System><Cell Signaling><Cells><Cellular Matrix><Chemical Fractionation><Code><Coding System><Complex><Coon's Technic><Coon's Technique><Cyclic AMP><Cyclic AMP-Dependent Protein Kinases><Cyst><Cystic Kidney Diseases><Cystic Renal Diseases><Cytoskeletal System><Cytoskeleton><Data><Dependence><Development><Dimensions><Dominant Polycystic Kidney Disease><ESRD><End stage renal failure><End-Stage Kidney Disease><End-Stage Renal Disease><Engineering / Architecture><Enterobacteria phage P1 Cre recombinase><Epidermal Cyst><Epidermal Inclusion Cyst><Epidermoid Cyst><Epithelial><Epithelial Cells><Epithelial cyst><F-Actin><FRACN><Family><Family member><Filamentous Actin><Fluorescent Antibody Technic><Fluorescent Antibody Technique><Fluorescent Antinuclear Antibody Test><Fractionation><Fractionation Radiotherapy><GeneHomolog><Gold><Homolog><Homologous Gene><Homologue><Horn Cyst><Human><Immunofluorescence Technic><Immunofluorescence Technique><Inclusion Cyst><Integral Membrane Protein><Intracellular Communication and Signaling><Intrinsic Membrane Protein><Investigation><Ion Channel><Ionic Channels><Keratin Cyst><Keratinizing Cyst><Keratinous Cyst><Kidney><Kidney Urinary System><Kinases><L-Threonine><Lead><Liquid substance><Maintenance><Mediating><Membrane><Membrane Channels><Membrane Protein Gene><Membrane Proteins><Membrane-Associated Proteins><Mendelian disease><Mendelian disorder><Mendelian genetic disorder><Mice><Mice Mammals><Model System><Modeling><Modern Man><Monomeric G-Proteins><Monomeric GTP-Binding Proteins><Morphology><Murine><Mus><Occluding Junctions><Organoids><PKA><PKD1 protein><PKD2 protein><Pathway interactions><Pb element><Permeability><Phosphorylation><Phosphotransferase Gene><Phosphotransferases><Physiologic><Physiological><Pilar Cyst><Protein Family><Protein Kinase A><Protein Phosphorylation><Proteins><Receptor Protein><Regulation><Rho-associated kinase><Rho-kinase><Role><Sebaceous Cyst><Signal Pathway><Signal Transduction><Signal Transduction Systems><Signaling><Signaling Molecule><Small G-Proteins><Small GTPases><Structure><Surface Proteins><System><Techniques><Tetracycline Antibiotic><Tetracyclines><Threonine><Tight Junctions><Tissues><Transmembrane Protein><Transmembrane Protein Gene><Transphosphorylases><Transport Protein Gene><Transport Proteins><Transporter Protein><WCH-CD><Zonula Occludens><adenosine 3'5' monophosphate><apical membrane><aquaporin-2><aquaporin-CD><bacteriophage P1 recombinase Cre><basolateral membrane><biochemical tools><biochemistry tools><biological signal transduction><cAMP><cAMP-Dependent Protein Kinases><collecting duct water channel><combat><developmental><experiment><experimental research><experimental study><ezrin><fluid><fluorescent antibody><heavy metal Pb><heavy metal lead><immunocytochemistry><intracellular skeleton><liquid><loss of function mutation><membrane structure><membrane-organizing extension spike protein><moesin><monogenic disease><monogenic disorder><novel><pathway><pcy protein><phosphoprotein p81><planar cell polarity><polycystic breakpoint protein><polycystic kidney disease 1 protein><polycystic kidney disease 2 protein><polycystin 1><polycystin 2><postnatal><protein complex><radixin><radixin protein><receptor><recruit><renal><renal epithelium><rho><single-gene disease><single-gene disorder><small molecule inhibitor><social role><therapeutic target><three dimensional cell culture><three dimensional structure><three-dimensional modeling><trafficking>