The cystic fibrosis transmembrane conductance regulator mediates transepithelial fluid secretion by human autosomal dominant polycystic kidney disease epithelium in vitro

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 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.

Small-Molecule CFTR Inhibitors Slow Cyst Growth in Polycystic Kidney Disease

“…29,30 As described at the beginning of this article, in vitro data implicate epithelial chloride secretion in the generation and maintenance of fluid-filled cysts. [11][12][13][14] As in other secretory epithelia, fluid secretion into the cyst lumen occurs by primary chloride exit across the cell apical mem- Figure 4. Structures of glycine hydrazide and malonic acid hydrazide CFTR inhibitors with their CFTR inhibition activity.…”

“…[11][12][13][14] The cystic fibrosis transmembrane conductance regulator protein (CFTR), a cAMP-regulated chloride channel, is believed to provide the principal route for chloride entry into expanding cysts. CFTR is expressed in the apical membrane of cyst-lining epithelial cells in PKD kidneys.…”

Strategies Targeting cAMP Signaling in the Treatment of Polycystic Kidney Disease

“…The role of CFTR in PKD is supported by whole-cell patch-clamp studies of cyst-derived epithelial cells, inhibition of fluid secretion by CFTR antisense oligonucleotides, inhibition of cyst growth in in vitro and in vivo models of cystogenesis, [86][87][88][89][90] and the milder cystic phenotype in patients affected by both ADPKD and cystic fibrosis. [91][92][93][94] However, an additional patient with coexisting ADPKD and cystic fibrosis progressed to ESRD after a lung transplant, possibly because of cyclosporin toxicity, and the disease severity of bpk CFTR double mutant mice was not less than the disease severity of bpk mice, a rapidly progressive model of PKD. 95,96 …”

Strategies to Inhibit Cyst Formation in ADPKD

“…It is currently believed that chloride is transported through cells from the basal to the lumenal side of the growing cyst and that the role of cAMP in this process is to activate the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel on the apical membrane of cyst-lining epithelial cells, thereby allowing net chloride secretion and ultimately fluid transport into the cyst (15,(17)(18)(19).…”