Our finding that AQP2 constitutively recycles in the absence of the antidiuretic hormone, vasopressin (VP), has led to a rethinking of how VP causes AQP2 membrane accumulation and urinary concentration. Indeed, we recently found that an FDA approved drug, simvastatin, blocks AQP2 endocytosis and reduces urine output in VP- deficient Brattleboro rats. Our proposed studies continue the strategy of combining cell biological interrogation of AQP2 trafficking with functional studies in vivo to generate novel therapeutic approaches for disorders of fluid balance including NDI, as well as congestive heart failure and hyponatremia.
In Aim 1, we will explore the mechanism(s) underlying our recent discoveries that AQP2 itself is a catalyst for VP-induced actin depolymerization and that the actin-binding protein ezrin is an AQP2 associated protein involved in AQP2 endocytosis. Agents that target the ezrin binding domain on AQP2 would provide increased specificity of action for NDI treatment.
Aim 2 will first expand our promising work on phosphodiesterase (PDE5) inhibitors, calcitonin and statins, which promote AQP2 membrane accumulation and increase urine concentration. We will optimize treatment doses, times and delivery routes, and assess the effectiveness of drug combinations. Then, we will further develop our new chemical screening program to identify and characterize compounds that modulate exocytosis and endocytosis in AQP2- expressing cells using high throughput, fluorescence-based assays in cell cultures (we have already identified 40 chemicals that need further characterization). Compounds that modify (increase or decrease) AQP2 membrane accumulation in vitro will ultimately be tested in vivo. We will use a multidisciplinary approach ranging from in vitro protei association assays, novel cell culture models, cellular imaging and analysis (using the PMB Microscopy Core), high throughout chemical screening (via the Center for Systems Biology Chemical Biology COre) and whole animal studies. Our overall goal is to design novel therapies for water balance disorders, including NDI and hyponatremia, using a combination of directed and discovery approaches that bypass the VP/V2R signaling cascade to modulate cell surface expression of AQP2.

Public Health Relevance

The kidney controls how much of our daily water intake is reabsorbed back into the body, and how much is excreted in the urine. Sometimes this process is not balanced, resulting in diseases including hypertension (too much fluid and sodium reabsorption) or dehydration (too much urine production). Our work is aimed at finding the mechanisms by which the kidney controls these processes in order to cure kidney disease.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK096586-03
Application #
8731870
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Mullins, Christopher V
Project Start
2012-09-20
Project End
2016-08-31
Budget Start
2014-09-01
Budget End
2015-08-31
Support Year
3
Fiscal Year
2014
Total Cost
Indirect Cost
Name
Massachusetts General Hospital
Department
Type
DUNS #
City
Boston
State
MA
Country
United States
Zip Code
02199
Li, Wei; Jin, William W; Tsuji, Kenji et al. (2017) Ezrin directly interacts with AQP2 and promotes its endocytosis. J Cell Sci 130:2914-2925
Cheung, Pui W; Ueberdiek, Lars; Day, Jack et al. (2017) Protein phosphatase 2C is responsible for VP-induced dephosphorylation of AQP2 serine 261. Am J Physiol Renal Physiol 313:F404-F413
He, Qing; Bouley, Richard; Liu, Zun et al. (2017) Large G protein ?-subunit XL?s limits clathrin-mediated endocytosis and regulates tissue iron levels in vivo. Proc Natl Acad Sci U S A 114:E9559-E9568
Cheung, Pui W; Nomura, Naohiro; Nair, Anil V et al. (2016) EGF Receptor Inhibition by Erlotinib Increases Aquaporin 2-Mediated Renal Water Reabsorption. J Am Soc Nephrol :
Mamuya, Fahmy A; Cano-PeƱalver, Jose Luis; Li, Wei et al. (2016) ILK and cytoskeletal architecture: an important determinant of AQP2 recycling and subsequent entry into the exocytotic pathway. Am J Physiol Renal Physiol 311:F1346-F1357
Cheung, Pui W; Nomura, Naohiro; Nair, Anil V et al. (2016) EGF Receptor Inhibition by Erlotinib Increases Aquaporin 2-Mediated Renal Water Reabsorption. J Am Soc Nephrol 27:3105-3116
Nunes, Paula; Roth, Isabelle; Meda, Paolo et al. (2015) Ionic imbalance, in addition to molecular crowding, abates cytoskeletal dynamics and vesicle motility during hypertonic stress. Proc Natl Acad Sci U S A 112:E3104-13
Nair, Anil V; Keliher, Edmund J; Core, Amanda B et al. (2015) Characterizing the interactions of organic nanoparticles with renal epithelial cells in vivo. ACS Nano 9:3641-53
Herak-Kramberger, Carol M; Breljak, Davorka; Ljubojevi?, Marija et al. (2015) Sex-dependent expression of water channel AQP1 along the rat nephron. Am J Physiol Renal Physiol 308:F809-21
Arthur, Julian; Huang, Jianmin; Nomura, Naohiro et al. (2015) Characterization of the putative phosphorylation sites of the AQP2 C terminus and their role in AQP2 trafficking in LLC-PK1 cells. Am J Physiol Renal Physiol 309:F673-9

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