LOCALIZATION OF BUMETANIDE-SENSITIVE AND THIAZIDE-SENSITIVE NA-K-CL COTRANSPORTERS ALONG THE RAT NEPHRON

The present study was undertaken to investigate the mRNA localization of the two major kidney-specific Na-K-Cl transport proteins, the bumet anide-sensitive cotransporter (NKCC2 in rabbit and BSC1 in rat) and th e thiazide-sensitive cotransporter (TSC). NKCC2 from rabbit and mouse has been shown to exist in three isoforms (designated A, B, and F) tha t differ only in a 96-bp region. The divergent region of each of the t hree NKCC2 isoforms was cloned from rat kidney by a polymerase chain r eaction (PCR)-based strategy, and isoform-specific primers were chosen . RNA and cDNA were prepared from renal cortex and medulla and from mi crodissected nephron segments. Using reverse transcription (RT)-PCR, t he B isoform was detected only in cortex and the F isoform only in med ulla, whereas the A form was found in both. In dissected nephron segme nts, the B form was found exclusively in cortical thick ascending limb (CTAL) and macula densa-containing segment (MDCS), the F form only in medullary thick ascending limb (MTAL) and outer medullary collecting duct, and the A form in CTAL, MDCS, and MTAL. An additional isoform in cluding both A and F exons was identified by direct sequencing of a 59 2-bp product from medulla. The AF product was found only in the medull a and was localized exclusively in MTAL. TSC mRNA was detected exclusi vely in the distal convoluted tubule. Differential nephron localizatio n of NKCC2 isoforms suggests that Na-K-Cl cotransporters may differ in their transport characteristics to explain regulation of salt transpo rt along the nephron.