The cystic fibrosis transmembrane conductance regulator mediates transepithelial fluid secretion by human autosomal dominant polycystic kidney disease epithelium in vitro
The cystic fibrosis transmembrane conductance regulator mediates transepithelial fluid secretion by human autosomal dominant polycystic kidney disease epithelium in vitro
复制标题
DOI：
10.1038/ki.1996.304
复制
发表时间：
1996-07-01
影响因子：
19.6
通讯作者：
Grantham, JJ
中科院分区：
文献类型：
--
作者：
Davidow, CJ;Maser, RL;Grantham, JJ
经上皮液体分泌促进常染色体显性多囊肾病（ADPKD）囊肿的进行性扩大。最近的间接证据表明，积极的氯离子转运可能会驱动净液体分泌的上皮细胞来源于ADPKD囊肿的文化。我们现在报告，刺激腺苷酸环化酶的毛喉素，增加了体外ADPKD细胞单层中Cl-36的外排速率常数，从0.23 +/- 0.02 min（-1）增加到0.44 +/- 0.05 min（-1）（N = 4），而阻断氯离子通道的二苯胺2-羧酸盐（DPC）消除了这些细胞中毛喉素刺激的氯离子外排。为了确定cAMP调节的氯离子转运蛋白，囊性纤维化跨膜传导调节因子（CFTR），是否可能参与ADPKD上皮细胞的氯离子转运和液体分泌，我们检测了这些培养物中的CFTR mRNA和蛋白。使用人（h）CFTR cDNA探针的北方印迹杂交证明在来自ADPKD、正常人肾皮质（HKC）和T84细胞的极化培养物的总RNA中存在类似于6.5kb的转录物。利用几种抗体hCFTR，免疫细胞化学和共聚焦荧光显微镜定位的免疫反应蛋白主要在毛喉素刺激的ADPKD细胞生长在可渗透的支持物的顶端区域。这种免疫反应性可以通过抗体与免疫肽的预孵育来消除。为了确定CFTR丰度对净液体分泌量的影响，用与hCFTR mRNA中翻译起始位点附近的序列互补（反义）、同源（有义）或部分互补（错反义）的脱氧寡核苷酸处理极化ADPKD培养物。用5.0 μ M反义寡核苷酸处理导致毛喉素刺激的液体分泌减少73%，并且通过免疫细胞化学检测到CFTR丰度的减少相当。相比之下，用5.0 μ M正义寡核苷酸处理仅使液体分泌减少34%，并且对CFTR丰度的影响较小，而5.0 μ M错反义寡核苷酸对液体分泌和CFTR丰度的影响不显著。在这些结果的基础上，我们认为CFTR是一个主要的介质毛喉素刺激的氯化物和液体分泌的人多囊肾上皮细胞在体外。
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.