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| The cystic fibrosis transmembrane conductance regulator mediates transepithelial fluid secretion by human autosomal dominant polycystic kidney disease epithelium in vitro |
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| Author(s): Davidow CJ, Maser RL, Rome LA, Calvet JP, Grantham JJ |
| Source: KIDNEY INTERNATIONAL Volume: 50 Issue: 1 Pages: 208-218 Published: JUL 1996 |
| Times Cited: 57 References: 39 |
| Abstract: 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 Cl-36 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 similar to 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 mu M 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 mu M sense oligonucleotide reduced fluid secretion by only 34% and had less of an effect on CFTR abundance, while the effects of 5.0 mu M 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. |
| Document Type: Article |
| Language: English |
Addresses:
1. UNIV KANSAS, MED CTR, DEPT PHARMACOL, DIV NEPHROL, KANSAS CITY, KS 66160 USA
2. UNIV KANSAS, MED CTR, DEPT BIOCHEM & MOLEC BIOL, KANSAS CITY, KS 66160 USA
3. UNIV KANSAS, MED CTR, DEPT THERAPEUT, KANSAS CITY, KS 66160 USA
4. UNIV KANSAS, MED CTR, DEPT MED, KANSAS CITY, KS 66160 USA |
| Publisher: BLACKWELL SCIENCE INC, 238 MAIN ST, CAMBRIDGE, MA 02142 |
| Subject Category: Urology & Nephrology |
| IDS Number: UT645 |
| ISSN: 0085-2538 |
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| | | | | | | | | Record from Web of Science® | | | | | | | | | |