Cardiotonic steroids down-regulate sodium hydrogen exchanger expression in the proximal tubule cells

Our laboratory previously reported that ouabain decreases the transcellular sodium transport in LLCPK1 cell line by mechanisms other than affecting intracellular sodium concentration (Liu J 2002). We utilized for our experiments LLCPK1 cell line, which is an epithelial cells derived from pig renal proximal tubules. When cell were treated for 12 hours with low dose of ouabain (50nM and 100 nM), the intracellular sodium concentration was not affected. Moreover, NHE3, the major apical sodium transporter in proximal tubules, was significantly downregulated on mRNA level, as measured by StaRT-PCR. NHE3 protein was also downregulated when measured by westernblot. Total NHE activity was also decreased when measured by intracellular PH recovery after acidification. Ouabain-induced downregulation of NHE3 was blocked after inhibiting c-src or PI3K with PP2 or wortmannin respectively. Both c-src and PI3K are essential part of Na/K ATpase signaling cascade. We utilized transiently transfected cells with NHE3 promoter constructs in luceferase reporter vector to map the response elements. We mapped the ouabain response elements on NHE3 promoter to be (-450 to -1194). Furthermore, Ouabain-induced downregulation of NHE3 promoter activity was blocked in C2-9 cell line (siRNA-mediated caveolin-1 depleted cell line). We used C2-9 cell line to confirm the role of pump endocytosis in the ouabian signaling cascade to regulate NHE3 expression. Our laboratory identified that ouabain decreases the binding of transcription factors TR and Sp1 to their cognate cis-elements on the promoter. Our group utilized protein/DNA array analysis, chromatin immunoprecipitation and electrophoretic mobility shift assay (EMSA) to confirm the decrease of the transcription factors binding induced by ouabain.;We concluded that ouabain chronically downregulates NHE3 expression and acitivty in proximal tubule cells by endocytosis-mediated signaling cascade from Na/K ATPase. The signaling pathway involves c-src and PI3K to atenuate TR and Sp1 binding to NHE3 promoter.