Transepithelial fluid secretion promotes the progressive enlargement of
cysts in
autosomal dominant polycystic kidney disease (
ADPKD). Recent 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, increased the efflux rate constant for 36Cl in monolayers of
ADPKD cells 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 channels, eliminated the
forskolin-stimulated
chloride efflux from these cells. To establish whether the cAMP-regulated
chloride transporter,
cystic fibrosis transmembrane conductance regulator (CFTR), may potentially be involved in the
chloride transport and fluid secretion of
ADPKD epithelia, we examined CFTR
mRNA and
protein in these cultures. Northern blot hybridization using a human (h) CFTR
cDNA probe demonstrated the presence of an approximately 6.5 kb transcript in total
RNA from polarized cultures of
ADPKD, normal human kidney cortex (HKC), and T84 cells. Utilizing several
antibodies to hCFTR, immunocytochemistry and confocal fluorescence microscopy localized an immunoreactive
protein primarily in the apical region of
forskolin-stimulated
ADPKD cells grown on permeable supports. This immunoreactivity could be eliminated by preincubation of antibody with immunizing
peptide. To determine the effect of CFTR abundance on the magnitude of net fluid secretion, polarized
ADPKD cultures were treated with deoxyoligonucleotides that were either complementary (antisense), homologous (sense), or partially complementary (misantisense) to a sequence near the translation initiation site in hCFTR
mRNA. Treatment with 5.0 microM
antisense oligonucleotide resulted in a 73% reduction in
forskolin-stimulated fluid secretion and a comparable reduction in the abundance of CFTR as detected by immunocytochemistry. By contrast, treatment with 5.0 microM sense
oligonucleotide reduced fluid secretion by only 34% and had less of an effect on CFTR abundance, while the effects of 5.0 microM misantisense
oligonucleotide 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.