Renin mRNA quantification using polymerase chain reaction in cultured juxtaglomerular cells. Short-term effects of cAMP on renin mRNA and secretion.
The aim of the present study was to set up a method to quantify renin mRNA levels in mouse renal juxtaglomerular cells, the main physiological site of renin synthesis. Because of the scarcity of the cells, a quantitative polymerase chain reaction had to be developed to measure renin mRNA. Juxtaglomerular cells were isolated and cultured for 2 days under various conditions, and renin mRNA was measured directly from the cytoplasm of the cultured cells without prior RNA purification. An internal standard consisting of a mutated renin mRNA with an insertion of 60 bp was designed to quantify the reaction, ensuring an identical detection and amplification efficiency to the target RNA. Renin mRNA could be precisely quantified between 0.6 and 20 pg, thus allowing its detection in approximately 5000 juxtaglomerular cells. Forskolin, an activator of adenylate cyclase, led to a concentration-dependent maximal threefold increase in renin mRNA in the cultures after 20 hours of incubation. The half-maximal effective dose was 3 x 10(-7) mol/L. The effect of forskolin was mimicked by 10(-5) mol/L isoproterenol, a beta-receptor agonist, and by 10(-5) mol/L isobutylmethylxanthine. A time-course study showed a rapid increase in renin mRNA within 3 hours after forskolin and isoproterenol addition. Renin secretion in the culture medium was measured in parallel and found to be stimulated by both agents. These results show that quantitative polymerase chain reaction is a suitable tool for studying renin gene expression in cultured juxtaglomerular cells. Our findings indicate that cAMP is a potent and fast activator of renin gene transcription and renin secretion in renal juxtaglomerular cells.
Della Bruna
R
R
INSERM Unit 36, Collège de France, Paris.
Kurtz
A
A
Corvol
P
P
Pinet
F
F
D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000502:physiology*; D007606:Juxtaglomerular Apparatus / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis* / Q000235:genetics; D012083:Renin / Q000235:genetics* / Q000378:metabolism
D012333:RNA, Messenger; D005576:Colforsin; D000242:Cyclic AMP; D012083:Renin; D015056:1-Methyl-3-isobutylxanthine