Cardiac glycoside downregulates NHE3 activity and expression in LLC-PK 1 cells

Shadi Oweis, Liang Wu, Pawel R Kiela, Hui Zhao, Deepak Malhotra, Fayez K Ghishan, Zijian Xie, Joseph I. Shapiro, Jiang Liu

Research output: Contribution to journalArticle

35 Citations (Scopus)

Abstract

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 (50 and 100 nM) 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 knockdown 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 -1,194 nt, where decreased binding of thyroid hormone receptor (TR) and Sp1 to their cognate cis-elements was documented in vitro and in vivo by protein/DNA array analysis, 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.

Original languageEnglish (US)
JournalAmerican Journal of Physiology - Renal Physiology
Volume290
Issue number5
DOIs
StatePublished - May 2006

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Cardiac Glycosides
Sodium-Hydrogen Antiporter
Ouabain
Protein Isoforms
Down-Regulation
LLC-PK1 Cells
RNA Isoforms
Thyroid Hormone Receptors
Transcytosis
Caveolin 1
Protein Array Analysis
Proximal Kidney Tubule
1-Phosphatidylinositol 4-Kinase
Chromatin Immunoprecipitation
Response Elements
Oligonucleotide Array Sequence Analysis
Epithelial Cells
Hypertension
Kidney
Messenger RNA

Keywords

  • c-Src
  • Caveolin-1
  • Kidney
  • Slc9a3
  • Sodium

ASJC Scopus subject areas

  • Physiology

Cite this

Cardiac glycoside downregulates NHE3 activity and expression in LLC-PK 1 cells. / Oweis, Shadi; Wu, Liang; Kiela, Pawel R; Zhao, Hui; Malhotra, Deepak; Ghishan, Fayez K; Xie, Zijian; Shapiro, Joseph I.; Liu, Jiang.

In: American Journal of Physiology - Renal Physiology, Vol. 290, No. 5, 05.2006.

Research output: Contribution to journalArticle

Oweis, Shadi ; Wu, Liang ; Kiela, Pawel R ; Zhao, Hui ; Malhotra, Deepak ; Ghishan, Fayez K ; Xie, Zijian ; Shapiro, Joseph I. ; Liu, Jiang. / Cardiac glycoside downregulates NHE3 activity and expression in LLC-PK 1 cells. In: American Journal of Physiology - Renal Physiology. 2006 ; Vol. 290, No. 5.
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