Na/H exchange activity is ubiquitous and mediates a variety of mammalian cell functions. In the renal proximal tubule, one specific isoform NHE-3 is the principal protein responsible for transepithelial NaCl and NaHCO3 absorption. Regulation of NHE-3 function is pivotal for acid-base and volume homeostasis. A number of hormones that regulate proximal tubule NHE-3 activity also modulate cellular cAMP levels and cAMP-dependent protein kinase activity. A fundamental unresolved question is the mechanism of regulation of NHE-3 by PKA. The present proposal tests the hypothesis that PKA directly phosphorylate NHE-3 leading to inhibition of its transport activity. This study will aim to establish that NHE-3 is a substrate for PKA and perform phosphopeptide mapping using bacterially expressed recombinant NHE-3 proteins in cell-free conditions as well as in mammalian cells transfected with the NHE-3 gene. The exact sites of PKA phosphorylation will then be mapped on NHE-3 and each of these sites will be systematically mutated to non-phosphorylatable residues. Using the wild type and the mutant NHE-3's, the relationship between PKA-induced phosphorylation and transport activity of NHE-3 will be established. This study will establish whether: 1) PKA inhibit NHE-3 function by direct phosphorylation, 2) PKA inhibits NHE-3 function by indirect means through intermediate regulators, or 3) both. This knowledge is of fundamental significance to the understanding of the kidney's role in acid-base and volume homeostasis.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
First Independent Research Support & Transition (FIRST) Awards (R29)
Project #
1R29DK048482-01
Application #
2148806
Study Section
General Medicine B Study Section (GMB)
Project Start
1995-02-01
Project End
2000-01-31
Budget Start
1995-02-01
Budget End
1996-01-31
Support Year
1
Fiscal Year
1995
Total Cost
Indirect Cost
Name
University of Texas Sw Medical Center Dallas
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
City
Dallas
State
TX
Country
United States
Zip Code
75390
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Di Sole, F; Hu, Ming-Chang; Zhang, Jianning et al. (2011) The reduction of Na/H exchanger-3 protein and transcript expression in acute ischemia-reperfusion injury is mediated by extractable tissue factor(s). Kidney Int 80:822-831
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Goetz, Regina; Nakada, Yuji; Hu, Ming Chang et al. (2010) Isolated C-terminal tail of FGF23 alleviates hypophosphatemia by inhibiting FGF23-FGFR-Klotho complex formation. Proc Natl Acad Sci U S A 107:407-12
Hu, Ming-Chang; Shi, Mingjun; Zhang, Jianning et al. (2010) Klotho deficiency is an early biomarker of renal ischemia-reperfusion injury and its replacement is protective. Kidney Int 78:1240-51
Hu, Ming Chang; Shi, Mingjun; Zhang, Jianning et al. (2010) Klotho: a novel phosphaturic substance acting as an autocrine enzyme in the renal proximal tubule. FASEB J 24:3438-50
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Geng, Weidong; Hill, Kathy; Zerwekh, Joseph E et al. (2009) Inhibition of osteoclast formation and function by bicarbonate: role of soluble adenylyl cyclase. J Cell Physiol 220:332-40
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