In vitro characterization of aldosterone and cAMP effects in mouse distal convoluted tubule cells

Abstract

The distal nephron plays a capital role in the fine regulation of sodium reabsorption. Compared with the cortical collecting duct, much less information is available on the hormonal regulation of sodium transporter genes in the distal convoluted tubule (DCT), where the thiazide-sensitive Na(+)-Cl(-) cotransporter (NCC) is the major entry pathway for Na(+). The purpose of this study was to characterize the in vitro effects of aldosterone (Aldo; 1 microM) and cAMP (8-BrcAMP; 0.5 mM) on mouse DCT (mDCT) by using an immortalized mDCT cell line. Western blot analysis and semiquantitative RT-PCR were performed to analyze the expression of genes involved in sodium transport. The mDCTcell line expressed the 11 beta-hydroxysteroid dehydrogenase type 2 gene and both the mineralocorticoid and glucocorticoid receptor genes, suggesting Aldo responsiveness. In this sense, we found that mDCT cells expressed the amiloride-sensitive Na(+) channel (ENaC) and responded to Aldo by upregulating the alpha-subunit protein. Similarly, alpha(1) Na(+)-K(+)-ATPase protein was upregulated by Aldo and 8-BrcAMP. In addition, the Aldo intermediate gene sgk1 mRNA was increased in response to both Aldo and 8-BrcAMP, and the transcription factor HNF-3 alpha mRNA was induced by 8-BrcAMP. With respect to NCC regulation, although Aldo induced NCC protein levels in mice in vivo, neither Aldo nor 8-BrcAMP significantly induced the NCC mRNA or protein levels in mDCT cells. These results suggest that in mDCT, Aldo and cAMP modulate some downstream mediators and effectors in vitro but do not influence the expression of NCC in this cell model.