Urea concentration is maintained by tightly controlling urea transporter function. Recent advances have provided insight into the understanding of long-term regulation of urea transporters including: 1) cloning of two urea transporter genes, their promoters, and several cDNA is forms;2) creation of polyclonal antibodies;and 3) physiologic studies of urea transporter function and abundance. We have shown that the UT-A1 urea transporter is regulated by phosphorylation in rat inner medullary collecting duct (IMCD) suspensions and in a Madin-Darby canine kidney (MDCK) cell line (UT-A1-MDCK), which we created, that stably expresses UT-A1. We have also seen that vasopressin stimulates UT-A1 trafficking to the membrane of rat IMCD in a cAMP-dependent manner. Our hypothesis is that the vasopressin-mediated increase in intracellular cAMP stimulates a specific signaling complex of proteins that regulate UT-A1 function. Vasopressin increases intracellular cAMP via adenylyl cyclases. The nature of this stimulation over time is unknown. We will identify vasopressin-sensitive adenylyl cyclases in UT-A1-MDCK cells and monitor the synthesis of cAMP over time. Vasopressin-sensitive phosphodiesterases (PDEs) are located in the IMCD. We will identify the PDE(s) in the cells and determine if PDE activity is increased by vasopressin. Vasopressin stimulates phosphorylation of UT-A1. Treatment with the PKA inhibitor H-89 reduced phosphorylation indicating that PKA plays a role in UT-A1 phosphorylation. We have identified two PKA sites in UT-A1. It is unknown if phosphorylation at these sites is vasopressin-mediated. We will investigate whether vasopressin can stimulate phosphorylation with PKA-site deletion constructs of UT-A1.Aquaporin-2, another transporter in the IMCD, is inserted into the membrane after vasopressin treatment. Insertion has been shown to be regulated by a cAMP-signaling complex composed of an AKAP, PKA, and a PDE. UT-A1 also accumulates in the membrane after vasopressin stimulation. We will identify a cAMP signaling complex, identify key proteins in this complex, and monitor the resulting vasopressin-mediated compartmentalization of cAMP at the plasma membrane near UT-A1.

Public Health Relevance

The proposed studies will yield new information on the mechanisms underlying the disregulation of water homeostasis that occurs in common clinical disorders, such as congestive heart failure, cirrhosis, and nephrotic syndrome. We hope to clarify the mechanisms of urea movement to potentially provide insight into the treatment of disregulation of the body's water homeostasis.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Scientist Development Award - Research & Training (K01)
Project #
5K01DK082733-04
Application #
8136162
Study Section
Diabetes, Endocrinology and Metabolic Diseases B Subcommittee (DDK)
Program Officer
Rankin, Tracy L
Project Start
2008-09-30
Project End
2013-08-31
Budget Start
2011-09-01
Budget End
2012-08-31
Support Year
4
Fiscal Year
2011
Total Cost
$141,588
Indirect Cost
Name
Emory University
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
066469933
City
Atlanta
State
GA
Country
United States
Zip Code
30322
Blount, Mitsi A; Cipriani, Penelope; Redd, Sara K et al. (2015) Activation of protein kinase C? increases phosphorylation of the UT-A1 urea transporter at serine 494 in the inner medullary collecting duct. Am J Physiol Cell Physiol 309:C608-15
Hoban, Carol A; Black, Lauren N; Ordas, Ronald J et al. (2015) Vasopressin regulation of multisite phosphorylation of UT-A1 in the inner medullary collecting duct. Am J Physiol Renal Physiol 308:F49-55
Sim, Jae H; Himmel, Nathaniel J; Redd, Sara K et al. (2014) Absence of PKC-alpha attenuates lithium-induced nephrogenic diabetes insipidus. PLoS One 9:e101753
Sands, Jeff M; Blount, Mitsi A (2014) Genes and proteins of urea transporters. Subcell Biochem 73:45-63
Sands, Jeff M; Blount, Mitsi A (2014) Novel activators of aquaporin 2 membrane expression for the treatment of nephrogenic diabetes insipidus: less is more. Focus on ""High-throughput chemical screening identifies AG-490 as a stimulator of aquaporin 2 membrane expression and urine concentrati Am J Physiol Cell Physiol 307:C595-6
von Bergen, Tobias N; Blount, Mitsi A (2012) Chronic use of chloroquine disrupts the urine concentration mechanism by lowering cAMP levels in the inner medulla. Am J Physiol Renal Physiol 303:F900-5
Klein, Janet D; Blount, Mitsi A; Sands, Jeff M (2012) Molecular mechanisms of urea transport in health and disease. Pflugers Arch 464:561-72
Francis, Sharron H; Blount, Mitsi A; Corbin, Jackie D (2011) Mammalian cyclic nucleotide phosphodiesterases: molecular mechanisms and physiological functions. Physiol Rev 91:651-90
Klein, Janet D; Blount, Mitsi A; Sands, Jeff M (2011) Urea transport in the kidney. Compr Physiol 1:699-729
Klein, Janet D; Blount, Mitsi A; Frohlich, Otto et al. (2010) Phosphorylation of UT-A1 on serine 486 correlates with membrane accumulation and urea transport activity in both rat IMCDs and cultured cells. Am J Physiol Renal Physiol 298:F935-40

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