We believe that a combination of informed, targeted and unbiased drug screening is necessary to devise strategies to normalize water balance disorders, including nephrogenic diabetes insipidus and hyponatremia. The overall strategy proposed in this renewal application is designed to facilitate this goal. Over the past three years of funding, we have uncovered several important and previously unrecognized aspects of aquaporin 2 biology: 1) AQP2 catalyzes actin depolymerization in response to AVP: 2) AQP2 trafficking to the apical membrane involves transient basolateral insertion and redirection via transcytosis: 3) AQP2 recycles constitutively in the complete absence of any known phosphorylation events.
Aim 1 takes advantage of this new knowledge and interrogates the relationship between AQP2 phosphorylation, actin organization, and the polarity of AQP2 membrane delivery and accumulation in renal epithelial cells. This approach allows us to envisage more informed approaches to water balance disorders. Next, our use of high throughput chemical screening in the previous funding cycle led us to discover that the FDA approved cancer drug Erlotinib, an EGFR inhibitor, reduces urine output by 50% in lithium treated NDI mice.
Aim 2 will explore the mechanism by which EGFR inhibition alone, in the complete absence of VP, causes AQP2 phosphorylation and membrane accumulation in the absence of PKA stimulation. We need to identify which signaling pathway is responsible in order to fully understand how Erlotinib works in this setting, and to suggest alternative targeted approaches. Finally, our quest for additional new compounds that modulate AQP2 membrane accumulation will continue in Aim 3, in which a fluorescence assay will be used for unbiased screening of chemical libraries for inhibitors or stimulators of endocytosis. While endocytosis inhibitors are candidates for use in NDI, specific stimulators of AQP2 endocytosis could be useful in conditions of water overload that could lead to hyponatremia and even hypertension. We will also test exocytosis-inhibitor compounds that were identified in our previous screen for their ability to prevent AQP2 membrane accumulation, also a feature of drugs that would prevent water overloading. Our prior studies and the work proposed in this renewal application range from the in vitro characterization of protein interactions, through cell culture assays, to whole animal studies. We have developed new cell lines for high throughput chemical screens, a new AQP2-EGFP construct for live cell imaging, and we have a newly-established colony of conditional vasopressin receptor knockout mice in our facility for in vivo drug testing. Our work combines the need for a better understanding of basic mechanisms in order to drive translational medicine and clinical advances, with a more direct drug discovery approach using novel cell assays.

Public Health Relevance

The kidney controls how much of our daily salt and water intake is reabsorbed back into the body and how much is released as urine. Sometimes this process is not balanced, leading to diseases such as hypertension (too much fluid and sodium in the body) and dehydration (too much urine production). Our work is aimed at finding mechanisms that cause these diseases in order to develop drugs to cure them.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK096586-09
Application #
10005038
Study Section
Kidney Molecular Biology and Genitourinary Organ Development (KMBD)
Program Officer
Mullins, Christopher V
Project Start
2012-09-20
Project End
2021-08-31
Budget Start
2020-09-01
Budget End
2021-08-31
Support Year
9
Fiscal Year
2020
Total Cost
Indirect Cost
Name
Massachusetts General Hospital
Department
Type
DUNS #
073130411
City
Boston
State
MA
Country
United States
Zip Code
02114
Cheung, Pui W; Terlouw, Abby; Janssen, Sam Antoon et al. (2018) Inhibition of non-receptor tyrosine kinase Src induces phosphoserine 256-independent aquaporin-2 membrane accumulation. J Physiol :
Chen, Lihe; Lee, Jae Wook; Chou, Chung-Lin et al. (2017) Transcriptomes of major renal collecting duct cell types in mouse identified by single-cell RNA-seq. Proc Natl Acad Sci U S A 114:E9989-E9998
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
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 :
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

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