CFTR EXPRESSION IN CORTICAL COLLECTING DUCT CELLS

The cystic fibrosis transmembrane conductance regulator (CFTR) is a ad enosine 3',5'-cyclic monophosphate-activated chloride channel located in the apical membrane of many epithelial cells, and it may play a sig nificant role in the kidney. Recent functional evidence from our labor atory suggests that CFTR may be expressed by the cortical collecting d uct (CCD). Therefore, in the present study, the reverse transcription- polymerase chain reaction (RT-PCR) technique was utilized to detect CF TR mRNA in the M-1 mouse CCD cell line and in immunoselected rabbit CC D cells. Primers were constructed to amplify the cDNA sequence encodin g the first nucleotide binding domain of CFTR. CFTR PCR products were obtained from both M-1 and rabbit CCD cDNA preparations. The identity of the product amplified from M-1 cell cDNA was confirmed by restricti on digestion analysis. The rabbit CCD PCR product was sequenced, and i ts deduced amino acid sequence was found to be 97% homologous to the c orresponding regions of human CFTR. The level of CFTR cDNA detected af ter 30 cycles of amplification of CCD cDNA was only 49 +/- 8 (n = 9) t imes lower than the level of beta-actin PCR product obtained from the same sample, suggesting that the levels of CFTR mRNA present in the CC D are physiologically relevant. Northern analysis, using a cRNA probe corresponding to the amplified region on the mRNA from CCD cells, reve aled a single hybridizing species with a size of similar to 6.5 kb. Fi nally, CFTR PCR was performed with cDNA preparations originating from principal cells (PC), beta-intercalated cells (beta-ICC), and alpha-IC C obtained by fluorescence-activated cell sorting of rabbit CCD. CFTR PCR products were obtained from all three cell types, with the most ab undant levels found in beta-ICC. beta-ICC expressed 25-fold (n = 4, P < 0.001) and 4.5-fold (n = 7, P < 9.001) higher levels than PC and alp ha-ICC, respectively. This distribution pattern suggests that, within the CCD, CFTR plays a role primarily in beta-ICC function.