RESUMO
The localization of the multidrug resistance gene (mdr-1b) messenger ribonucleic acid (mRNA) along the rat nephron and its regulation was investigated under two different experimental situations: dehydration and high-Na+ diet. The mdr-1b mRNA was detected in glomeruli, proximal tubule segments, cortical and medullary thick ascending limbs, inner medullary collecting ducts and thin limbs of Henle's loop. Using the ribonuclease (RNase) protection assay (RPA), the abundance of mdr-1b mRNA was shown to be 35% less in renal cortex than in medulla. The mdr-1b mRNA expression in dehydrated rats in cortex or medulla did not differ from control. However, after 5 or 14 days on a high-Na+ diet, mdr-1b expression had decreased significantly in both cortex and medulla. There was no change in protein expression in dehydrated rats but a significant decrease occurred in rats fed the high-salt diet, confirming the results obtained with RPA. Our results suggest that the mdr-1b product is involved in extracellular volume regulation in rats.
Assuntos
Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Desidratação/metabolismo , Rim/metabolismo , Sódio na Dieta/farmacologia , Adrenalectomia , Aldosterona/farmacologia , Animais , Southern Blotting , Western Blotting , Dieta , Eletrólitos/metabolismo , Taxa de Filtração Glomerular/efeitos dos fármacos , Taxa de Filtração Glomerular/fisiologia , Rim/efeitos dos fármacos , Masculino , Néfrons/efeitos dos fármacos , Néfrons/metabolismo , Concentração Osmolar , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , RNA Mensageiro/isolamento & purificação , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ribonucleases/metabolismo , Fatores de Tempo , Membro 4 da Subfamília B de Transportadores de Cassetes de Ligação de ATPRESUMO
The expression of cystic fibrosis transmembrane conductance regulator (CFTR) in the thyroid has not been documented to date, although a role for CFTR in the thyroid follicular epithelium is suggested both clinically, by the occurrence of subclinical hypothyroidism in patients with cystic fibrosis (CF), and physiologically, by the presence of low-conductance, adenosine 3',5'-cyclic monophosphate-activated Cl channels in the follicular cells. Using reverse transcriptase-polymerase chain reaction with nested primers derived from exons 13 and 14 of the human CF gene, we have now documented the presence of CFTR mRNA in the human thyroid. Western blot analyses using six antibodies directed against different domains of human CFTR showed that a 165-kDa band was present in membrane extracts from bovine and human thyroid. This protein has the predicted size of mature CFTR and was not detected with preimmune serum or preadsorbed antiserum. By immunofluorescence and immunoperoxidase, CFTR was located in the follicular cells, with a diffuse, intracellular labeling pattern. Quantitative analysis revealed that 64% of the follicles were CFTR positive, but only 16% of the follicular cells were stained per follicle. The number of CFTR-positive cells was inversely proportional to the size of the follicle. These results 1) demonstrate the expression of CFTR at the mRNA and protein levels in human and bovine thyroid follicular cells and 2) suggest that CFTR expression could be instrumental in follicular enlargement.