THE CYSTIC-FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR MEDIATES TRANSEPITHELIAL FLUID SECRETION BY HUMAN AUTOSOMAL-DOMINANT POLYCYSTIC KIDNEY-DISEASE EPITHELIUM IN-VITRO

Transepithelial fluid secretion promotes the progressive enlargement o f cysts in autosomal dominant polycystic kidney disease (ADPKD). Recen t indirect evidence indicated that active chloride transport may drive net fluid secretion in cultures of epithelia derived from ADPKD cysts . We now report that forskolin, which stimulates adenylate cyclase, in creased the efflux rate constant for Cl-36 in monolayers of ADPKD cell s in vitro from 0.23 +/- 0.02 min(-1) to 0.44 +/- 0.05 min(-1) (N = 4) and that diphenylamine 2-carboxylate (DPC), which blocks chloride cha nnels, eliminated the forskolin-stimulated chloride efflux from these cells. To establish whether the cAMP-regulated chloride transporter, c ystic fibrosis transmembrane conductance regulator (CFTR), may potenti ally be involved in the chloride transport and fluid secretion of ADPK D epithelia, we examined CFTR mRNA and protein in these cultures. Nort hern blot hybridization using a human (h) CFTR cDNA probe demonstrated the presence of an similar to 6.5 kb transcript in total RNA from pol arized cultures of ADPKD, normal human kidney cortex (HKC), and T84 ce lls. Utilizing several antibodies to hCFTR, immunocytochemistry and co nfocal fluorescence microscopy localized an immunoreactive protein pri marily in the apical region of forskolin-stimulated ADPKD cells grown on permeable supports. This immunoreactivity could be eliminated by pr eincubation of antibody with immunizing peptide. To determine the effe ct of CFTR abundance on the magnitude of net fluid secretion, polarize d ADPKD cultures were treated with deoxyoligonucleotides that were eit her complementary (antisense), homologous (sense), or partially comple mentary (misantisense) to a sequence near the translation initiation s ite in hCFTR mRNA. Treatment with 5.0 mu M antisense oligonucleotide r esulted in a 73% reduction in forskolin-stimulated fluid secretion and a comparable reduction in the abundance of CFTR as detected by immuno cytochemistry. By contrast, treatment with 5.0 mu M sense oligonucleot ide reduced fluid secretion by only 34% and had less of an effect on C FTR abundance, while the effects of 5.0 mu M misantisense oligonucleot ide on both fluid secretion and CFTR abundance were insignificant. On the basis of these results we suggest that CFTR is a major mediator of forskolin-stimulated chloride and fluid secretion by epithelial cells of human polycystic kidneys in vitro.