The long term goals of this proposed study are to delineate the mechanisms and regulation of aquaporin-2 (AQP2) trafficking in kidney collecting duct at normal and pathophysiological conditions. The antidiuretic hormone (arginine vasopressin, AVP) regulates osmotic water permeability (Pf) of kidney collecting duct, conferring the precise control of renal excretion of water. AVP stimulates Pf in collecting duct by triggering the translocation and fusion of cytoplasmic AQP2 containing vesicles to apical membrane of principal cells. By developing confocal imaging techniques using styryl dye FM1-43 as a plasma membrane marker to monitor exocytosis, fluorescien sulfonate as volume marker to monitor Pf and fluo-4 as intracellular Ca 2+ concentration ([Ca2+]i) marker in perfused rat inner medullary collecting duct (IMCD), it was found that AVP induces [Ca2+]i oscillations in individual IMCD cells, and that AVP induced apical exocytosis and increase of Pf are Ca 2+ dependent. AVP induced Ca 2+ mobilization is cAMP-dependent but is not sensitive to protein kinase A inhibitor at a dose known to inhibit AQP-2 phosphorylation. Physiological dose of atrial natriuretic factor (ANF) inhibits AVP stimulated Pf and [Ca2+]i oscillations.
The aims of this proposal are (1) to test whether AQP2 exocytosis is coupled to intracellular Ca2+ mobilization, (2) to test whether cAMP directly involved in Ca2+ mobilization and AQP2 exocytosis via cAMP-guanine-nucleotide-exchange factors (3) to elucidate the subcellular mechanisms and the physiological significance of [Ca2+]i oscillations in AVP induced exocytosis, (4) to determine the interactions between vasopressin and atrial natriuretic peptide in regulating [Ca2+]i and AQP2 exocytosis. All proposed studies will be conducted in live cells of perfused IMCD. Apical exocytosis is monitored by FM1-43 fluorescence. Pf is monitored by the emission of fluorescien sulfonate included in the luminal perfused. An UV pulse laser is used to manipulate [Ca2+]i via flash photolysis of photoactivatable intracellular Ca2+ chelators or caged signaling molecules without interrupting image acquisition. A reversible permeabilization procedure is used to delivery antibodies, inhibitory peptides, and impermeant caged compounds into IMCD cells. Complementary immunolocalization of AQP2 with fluorescence microscopy will be employed. This proposed study will provide new information on the mechanisms and regulation of the insertion and trafficking of AQP2 in intact cell of perfused IMCD. These may lead a better understand of the role of kidney collecting duct in water balance disorders.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK060501-03
Application #
6915015
Study Section
General Medicine B Study Section (GMB)
Program Officer
Mullins, Christopher V
Project Start
2003-09-20
Project End
2007-06-30
Budget Start
2005-07-01
Budget End
2006-06-30
Support Year
3
Fiscal Year
2005
Total Cost
$166,933
Indirect Cost
Name
University of South Florida
Department
Physiology
Type
Schools of Medicine
DUNS #
069687242
City
Tampa
State
FL
Country
United States
Zip Code
33612
Yip, Kay-Pong; Sham, James S K (2011) Mechanisms of vasopressin-induced intracellular Ca2+ oscillations in rat inner medullary collecting duct. Am J Physiol Renal Physiol 300:F540-8
Balasubramanian, Lavanya; Sham, James S K; Yip, Kay-Pong (2008) Calcium signaling in vasopressin-induced aquaporin-2 trafficking. Pflugers Arch 456:747-54
Balasubramanian, Lavanya; Yip, Kay-Pong; Hsu, Tai-Hsin et al. (2008) Impedance analysis of renal vascular smooth muscle cells. Am J Physiol Cell Physiol 295:C954-65
Ditlevsen, Susanne; Yip, Kay-Pong; Marsh, Donald J et al. (2007) Parameter estimation of feedback gain in a stochastic model of renal hemodynamics: differences between spontaneously hypertensive and Sprague-Dawley rats. Am J Physiol Renal Physiol 292:F607-16
Balasubramanian, Lavanya; Ahmed, Abu; Lo, Chun-Min et al. (2007) Integrin-mediated mechanotransduction in renal vascular smooth muscle cells: activation of calcium sparks. Am J Physiol Regul Integr Comp Physiol 293:R1586-94
Leong, Patrick K K; Devillez, Angela; Sandberg, Monica B et al. (2006) Effects of ACE inhibition on proximal tubule sodium transport. Am J Physiol Renal Physiol 290:F854-63
Yip, Kay-Pong (2006) Epac-mediated Ca(2+) mobilization and exocytosis in inner medullary collecting duct. Am J Physiol Renal Physiol 291:F882-90
Leong, Patrick K K; Yang, Li E; Landon, Carol S et al. (2006) Phenol injury-induced hypertension stimulates proximal tubule Na+/H+ exchanger activity. Am J Physiol Renal Physiol 290:F1543-50
Chon, Ki H; Raghavan, Ramakrishna; Chen, Yu-Ming et al. (2005) Interactions of TGF-dependent and myogenic oscillations in tubular pressure. Am J Physiol Renal Physiol 288:F298-307
Yip, Kay-Pong (2005) Flash photolysis of caged nitric oxide inhibits proximal tubular fluid reabsorption in free-flow nephron. Am J Physiol Regul Integr Comp Physiol 289:R620-R626