In vitro characterization of aldosterone and cAMP effects in mouse distal convoluted tubule cells.
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.
González-Núñez
Daniel
D
Servicio de Nefrología, and Labrotorio de Hormonología, Institut d'Investigacions Biomèdiques August Pi i Sunyer, Hospital Clinic, Universidad de Barcelona, 08036 Barcelona, Spain.
Morales-Ruiz
Manuel
M
Leivas
Alberto
A
Hebert
Steven C
SC
Poch
Esteban
E
D043209:11-beta-Hydroxysteroid Dehydrogenase Type 2 / Q000235:genetics / Q000378:metabolism; D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology*; D000450:Aldosterone / Q000494:pharmacology*; D000818:Animals; D002461:Cell Line, Transformed; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects / Q000502:physiology; D006360:Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism; D051541:Hepatocyte Nuclear Factor 3-alpha; D017874:Immediate-Early Proteins; D066298:In Vitro Techniques; D007535:Isomerases / Q000235:genetics / Q000378:metabolism; D007686:Kidney Tubules, Distal / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D019704:Protein Disulfide-Isomerases; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism; D011965:Receptors, Glucocorticoid / Q000235:genetics / Q000378:metabolism; D018161:Receptors, Mineralocorticoid / Q000235:genetics / Q000378:metabolism; D012964:Sodium / Q000378:metabolism*; D028021:Sodium-Potassium-Chloride Symporters / Q000235:genetics / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors; D000099184:Serum-Glucocorticoid Regulated Kinases
D043209:11-beta-Hydroxysteroid Dehydrogenase Type 2; D015124:8-Bromo Cyclic Adenosine Monophosphate; D000450:Aldosterone; D004268:DNA-Binding Proteins; D006360:Heat-Shock Proteins; D051541:Hepatocyte Nuclear Factor 3-alpha; D017874:Immediate-Early Proteins; D007535:Isomerases; D009687:Nuclear Proteins; D019704:Protein Disulfide-Isomerases; D017346:Protein Serine-Threonine Kinases; D011965:Receptors, Glucocorticoid; D018161:Receptors, Mineralocorticoid; D012964:Sodium; D028021:Sodium-Potassium-Chloride Symporters; D000254:Sodium-Potassium-Exchanging ATPase; D014157:Transcription Factors; D000099184:Serum-Glucocorticoid Regulated Kinases; C496500:Foxa1 protein, mouse; C513195:Pdia3 protein, mouse