RT-PCR ANALYSIS OF NA+ H+ EXCHANGER MESSENGER-RNAS IN RAT MEDULLARY THICK ASCENDING LIMB/

The thick ascending limb (TAL) of rat kidney absorbs bicarbonate secon dary 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 revers e transcription-polymerase chain reaction (RT-PCR) to detect the mRNAs for NHE in microdissected MTAL. RT-PCR amplification from total RNA w as 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 frag ments, digestion with restriction enzymes, and Southern blot analysis. In microdissected rat MTAL, NHE-3 was strongly expressed and NHE-1 mR NA was also detected, whereas NHE-2 and NHE-4 mRNAs were not detected. Therefore, NHE-3 could be the apical Na-4/H+ exchanger, and NHE-1 cou ld be the basolateral isoform in the MTAL.