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Am J Physiol Renal Physiol (October 7, 2003). doi:10.1152/ajprenal.00307.2003
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Submitted on August 26, 2003
Accepted on September 30, 2003

Proteomic Analysis of Long-Term Vasopressin Action in Inner Medullary Collecting Duct of Brattleboro Rat

Bas W.M. van Balkom1, Jason D. Hoffert1, Chung-Lin Chou1, and Mark A. Knepper1*

1 Laboratory of Kidney and Electrolyte Metabolism, National Institutes of Health, National Heart, Lung and Blood Institute, Bethesda, MD, USA

* To whom correspondence should be addressed. E-mail: knep{at}helix.nih.gov.

Vasopressin regulates water and solute transport in the renal collecting duct. In addition to short-term regulation of aquaporin-2 trafficking, vasopressin also has long-term effects to regulate the abundances of aquaporins-2 and -3, and {beta}- and {gamma}-ENaC in collecting duct principal cells. To investigate further the direct and indirect long-term regulatory actions of vasopressin in the inner medullary collecting duct (IMCD), we used a proteomic approach, viz. difference gel electrophoresis (DIGE) coupled with MALDI-TOF identification of differentially expressed protein spots. dDAVP or vehicle was infused subcutaneously into Brattleboro rats for 3 days, and IMCD cells were purified from the inner medullas for proteomic analysis. 43 proteins were found to be regulated in response to vasopressin infusion, including 18 that were increased in abundance, 22 that were decreased, and 3 that were shifted in the gel, presumably due to post-translational modification. Immunocytochemistry confirmed collecting duct expression of several of the proteins that were identified. Immunoblot analysis of 9 of the proteins confirmed the changes seen by the DIGE method. Of these 9 proteins, 6 were increased in response to dDAVP infusion: NOS2, GRP78, HSP70, annexin II, glutaminase, and cathepsin D. The remaining 3 were decreased in response to dDAVP: aldehyde reductase I, adenylyl cyclase VI, and carbonic anhydrase II. The findings point to a role for vasopressin in the coordinate regulation of several determinants of nitric oxide levels (NOS2, arginase II, NADPH oxidase) and of proteins potentially involved in vasopressin escape (adenylyl cyclase VI and G-protein-coupled receptor kinase 4).




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