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1 The School of Medicine, Medical University of Ohio, Toledo, OH, USA
2 Department of Pediatrics, Steele Children's Research Center, University of Arizona, Tucson, AZ, USA
3 The School of Pharmacology, Medical University of Ohio, Toledo, OH, USA; The School of Medicine, Medical University of Ohio, Toledo, OH, USA
4 The School of Medicine, Medical University of Ohio, Toledo, OH, USA; The School of Pharmacology, Medical University of Ohio, Toledo, OH, USA
* To whom correspondence should be addressed. E-mail: jiliu{at}meduohio.edu.
Ouabain, a cardiotonic steroid and a specific inhibitor of the Na+/K+-ATPase, has been shown to significantly inhibit transcellular Na+ transport without altering the intracellular Na+ concentration ([Na+]i) in the epithelial cells derived from the renal proximal tubules. We therefore studied whether ouabain affects the activity and expression of Na+/H+ exchanger isoform 3 (NHE3) representing the major route of apical Na+ reabsorption in LLC-PK1 cells. Chronic basolateral, but not apical exposure to low concentration ouabain (50nM and 100nM) did not change [Na+]i, but significantly reduced NHE3 activity, NHE3 protein and mRNA expression. Inhibition of c-Src or phosphoinositide-3-kinase (PI3K) with PP2 or wortmannin, respectively, abolished ouabain-induced downregulation of NHE3 activity and mRNA expression. In caveolin-1 knock-down LLC-PK1 cells, ouabain failed to downregulate NHE3 mRNA expression and NHE3 promoter activity. Ouabain-response elements were mapped to a region between -450 and -1194 nt, where decreased binding of thyroid hormone receptor (TR) and Sp1 to their cognate cis-elements were documented in vitro and in vivo by protein/DNA array analysis, electrophoretic mobility shift assays (EMSA), supershift, and chromatin immunoprecipitation. These data suggest that, in LLC-PK1 cells, ouabain-induced signaling through the Na+/K+-ATPase-Src pathway results in decreased Sp1 and TR DNA binding activity and consequently in decreased expression and activity of NHE3. These novel findings may represent the underlying mechanism of cardiotonic steroid-mediated renal compensatory response to volume expansion and/or hypertension.
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