Access Type

Open Access Dissertation

Date of Award

January 2014

Degree Type

Dissertation

Degree Name

Ph.D.

Department

Physiology

First Advisor

Pablo A. Ortiz

Abstract

The thick ascending limb (TAL) in the kidney regulates extracellular fluid volume and blood pressure. The Na/K/2Cl cotransporter NKCC2 plays a central role in NaCl absorption by the TAL and blood pressure. NKCC2 trafficking to the apical membrane is a major mechanism to control NKCC2 activity. However, little is known about the proteins that mediate NKCC2 trafficking. Inhibition of the vesicle fusion proteins VAMP2 and VAMP3 blunts the increase in surface NKCC2 expression and NaCl absorption in response to stimulation by cAMP. In other cells, VAMPs mediate fusion of exocytic vesicles with the plasma membrane. Whether VAMP2 and VAMP3 mediate different pathways for NKCC2 exocytic delivery to the plasma membrane is not known. We hypothesized that VAMP2 and VAMP3 interact with NKCC2 in the TAL and differentially mediate constitutive and cAMP-stimulated exocytic delivery of NKCC2 to the plasma membrane. We silenced VAMP2 and VAMP3 in vivo via shRNAs in rat TALs and measured NKCC2 exocytic delivery and steady-state surface NKCC2 by surface biotinylation before and after cAMP stimulation. We observed that silencing VAMP2 decreased cAMP-stimulated exocytic delivery of NKCC2 and steady-state surface NKCC2 expression, whereas it did not affect constitutive NKCC2 trafficking in the absence of cAMP stimulation. Silencing VAMP3 decreased total NKCC2 expression and blunted constitutive NKCC2 exocytic delivery and steady-state surface NKCC2 expression, but had no effect on cAMP-stimulated NKCC2 trafficking. We also found by co-immunoprecipitation that VAMP2 and VAMP3 interact with NKCC2 and observed NKCC2 co-localization with VAMP2 and VAMP3 at the apical surface of TAL primary cultures. Interestingly, they co-localized at discrete microdomains at the apical surface. cAMP enhanced NKCC2-VAMP2 interaction, VAMP2 exocytic delivery and co-localization with NKCC2. Finally, to correlate NKCC2 trafficking with a renal phenotype, we studied renal function and blood pressure in VAMP3 knockout mice (VAMP2 knockouts die in utero). Urine volume was increased, urine osmolality decreased and Ca excretion was enhanced in VAMP3 knockout mice. This phenotype is similar to NKCC2 knockouts as described previously. When challenged to 24-hours water deprivation, VAMP3 knockouts experienced enhanced Na, Cl and K loss in the urine. Consistent with this renal phenotype, blood pressure was decreased in VAMP3 knockouts. We conclude that VAMP2 and VAMP3 interact and co-localize with NKCC2 in the TAL. VAMP2 mediates cAMP-stimulated NKCC2 trafficking while VAMP3 mediates constitutive trafficking. Also, VAMP3 is required for normal NKCC2 expression, renal function and blood pressure.

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