Intrarenal distribution of the colonic H,K-ATPase mRNA in rabbit

Background. Evidence suggests that the colonic H,K-ATPase isoform is expres sed in the kidney and that a mRNA species highly homologous to the rat and guinea pig HK alpha 2 is expressed in the cortical collecting duct (CCD) of the rabbit. The goals of this study were to determine if this mRNA is the rabbit homologue of HK alpha 2 or a novel isoform and to determine intraren al distribution of the HK alpha 2 mRNA in rabbit. Methods. 5'-RACE and Dye Deoxy Terminator chemistry were used to determine the full-length sequence of the rabbit HK alpha 2 mRNA. The intrarenal dist ribution of HK alpha 2 mRNA was determined in microdissected nephron segmen ts, connecting tubule (CNT), and CCD cells isolated by immunodissection, as well as in the three cell types of the CCD. Principal cells and alpha- and beta-intercalated cells were isolated by fluorescence-activated cell sorti ng. HK alpha 2 mRNA levels were determined by quantitative reverse transcri ption-polymerase chain reaction (RT-PCR) or single-nephron RT-PCR (SN-RTPCR ). Results. The full-length sequence of the rabbit kidney HK alpha 2 mRNA was determined. This transcript is identical to the one expressed in rabbit dis tal colon. In microdissected nephron segments, strong HK alpha 2 amplicons were present in the CNT, CCD, and outer medullary collecting duct (OMCD), w hereas no signal was detected in the proximal tubule, distal convoluted tub ule, think ascending limb, and inner medullary collecting duct. Roughly com parable levels of HK alpha 2 mRNA were present in all three CCD cell types, and the highest levels were observed in a subpopulation most likely corres ponding to CNT cells. Conclusions. These results suggest that the HK alpha 2 mRNA is expressed in rabbit collecting duct is identical in size and sequence to the one expres sed in rabbit distal colon. HK alpha 2 mRNA in the rabbit kidney is selecti vely expressed in the CNT, CCD, and OMCD, and all three collecting duct sub types express its mRNA.