RARE RENAL TRANSCRIPTION OF THE ATRIAL-NATRIURETIC-FACTOR GENE IN RATS - DEMONSTRATION BY POLYMERASE CHAIN-REACTION

Renal synthesis of a peptide homologous to atrial natriuretic factor ( ANF) has been demonstrated. The aim of the present study was to determ ine if transcription of the ANF gene occurs in the kidney. Rat renal R NA was extracted from whole kidneys, and, separately, from the cortex and outer and inner medulla of rat kidneys. Probing with rat ANF-cDNA did not reveal a detectable message in Northern blot analysis, even wh en large quantities of RNA were used at low stringency hybridization c onditions. Therefore, reverse transcription (RT) followed by 35 cycles of polymerase chain reaction (PCR) was used to search for the renal m essage for ANF. Two 21-mer primers encompassing the 450 base pairs (bp ) of the coding region of the gene were used. Each cycle consisted of annealing at 56-degrees-C, extension at 72-degrees-C, and denaturation at 94-degrees-C. The PCR product was proven to be identical to the AN F gene by high stringency hybridization, which revealed the expected 4 50-bp hybrid band. Furthermore, the sequence of this product was ident ical to that of the coding region of the ANF gene. We used an RNA-spec ific PCR to obtain this band as a single reaction product. We conclude that the transcript of the ANF gene exists in the kidney, at extremel y low levels. The low abundance of the RNA message raises major concer ns about its physiologic relevance. Direct evidence for the translatio n of this transcript, and its quantification and localization, is stil l required to determine its significance.