Developmental expression of ROMK mRNA in rabbit cortical collecting duct.
The cortical collecting duct (CCD) is a major site of regulation of K+ homeostasis in the fully differentiated mammalian kidney. CCDs isolated from adult rabbits and microperfused in vitro secrete K+ into the tubular fluid at high rates. However, CCDs dissected from newborn animals show no significant net K+ secretion until the 3rd wk of life, at least in part because of a paucity of conducting apical secretory K+ (SK) channels. To determine whether the abundance of genes encoding the SK channel is developmentally regulated, we used reverse transcriptase-polymerase chain reaction (RT-PCR) and Northern blot analysis to test for the presence of mRNA encoding rat outer medullary K+ channel (ROMK), considered to be a major subunit of the SK channel, in kidney and single CCDs isolated from maturing rabbits. Using rat ROMK-specific primers, RT-PCR of rabbit kidney yielded an amplification product of expected size and sequence. Northern blot analysis identified a single band at approximately 2.9 kb in kidney at all ages. Densitometric analysis revealed a progressive increase in steady state expression of ROMK message in kidney after birth. RT-PCR of individual CCDs yielded a single band of predicted size for ROMK in all segments isolated from animals > or =3 wk old. In contrast, transcripts were not detected in any CCD samples obtained from 1-wk-old animals and were identified in only 30% of CCD samples isolated from 2-wk-old rabbits. In all of the latter tubular samples, a specific PCR product of correct size for beta-actin mRNA was detected. These results suggest that an increase in steady state expression of ROMK mRNA contributes to the developmental appearance of conducting secretory K+ channels in the CCD.
Benchimol
C
C
Department of Pediatrics, Mount Sinai School of Medicine, New York, NY 10029-6574, USA.
Zavilowitz
B
B
Satlin
L M
LM
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D004247:DNA; D018507:Gene Expression Regulation, Developmental; D007672:Kidney Cortex / Q000254:growth & development / Q000378:metabolism*; D007685:Kidney Tubules, Collecting / Q000254:growth & development / Q000378:metabolism*; D008969:Molecular Sequence Data; D015221:Potassium Channels / Q000235:genetics*; D024661:Potassium Channels, Inwardly Rectifying; D012333:RNA, Messenger / Q000235:genetics*; D011817:Rabbits; D020133:Reverse Transcriptase Polymerase Chain Reaction
C510692:Kcnj1 protein, rat; D015221:Potassium Channels; D024661:Potassium Channels, Inwardly Rectifying; D012333:RNA, Messenger; D004247:DNA