RT-PCR analysis of Na+/H+ exchanger mRNAs in rat medullary thick ascending limb.
The thick ascending limb (TAL) of rat kidney absorbs bicarbonate secondary to proton secretion, but displays both basolateral and luminal Na+/H+ exchange (NHE) activity. Several NHE genes, including NHE-1, NHE-2, NHE-3, and NHE-4, are expressed in the kidney. To identify the NHE isoforms expressed in the rat medullary TAL (MTAL), we used the reverse transcription-polymerase chain reaction (RT-PCR) to detect the mRNAs for NHE in microdissected MTAL. RT-PCR amplification from total RNA was performed between two specific primers for each NHE isoform. In rat kidney homogenate, the four NHE isoform mRNAs were detected, and the identity of the PCR products was demonstrated by the sizes of the fragments, digestion with restriction enzymes, and Southern blot analysis. In microdissected rat MTAL, NHE-3 was strongly expressed and NHE-1 mRNA was also detected, whereas NHE-2 and NHE-4 mRNAs were not detected. Therefore, NHE-3 could be the apical Na+/H+ exchanger, and NHE-1 could be the basolateral isoform in the MTAL.
Borensztein
P
P
Département de Physiologie, Université Pierre et Marie Curie, Paris, France.
Froissart
M
M
Laghmani
K
K
Bichara
M
M
Paillard
M
M
D000818:Animals; D001483:Base Sequence; D015139:Blotting, Southern; D017931:DNA Primers; D007679:Kidney Medulla / Q000378:metabolism*; D007684:Kidney Tubules / Q000378:metabolism*; D008297:Male; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000032:analysis / Q000096:biosynthesis* / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D015183:Restriction Mapping; D000074083:Sodium-Hydrogen Exchanger 3; D017923:Sodium-Hydrogen Exchangers / Q000096:biosynthesis*
D017931:DNA Primers; D012333:RNA, Messenger; C513110:Slc9a2 protein, rat; C000613387:Slc9a3 protein, rat; C081518:Slc9a4 protein, rat; D000074083:Sodium-Hydrogen Exchanger 3; D017923:Sodium-Hydrogen Exchangers; C069139:growth factor-activatable Na-H exchanger NHE-1