The cystic fibrosis transmembrane conductance regulator (CFTR) is a adenosine 3',5'-cyclic monophosphate-activated chloride channel located in the apical membrane of many epithelial cells, and it may play a significant role in the kidney. Recent functional evidence from our laboratory suggests that CFTR may be expressed by the cortical collecting duct (CCD). Therefore, in the present study, the reverse transcription-polymerase chain reaction (RT-PCR) technique was utilized to detect CFTR mRNA in the M-1 mouse CCD cell line and in immunoselected rabbit CCD cells. Primers were constructed to amplify the cDNA sequence encoding the first nucleotide binding domain of CFTR. CFTR PCR products were obtained from both M-1 and rabbit CCD cDNA preparations. The identify of the product amplified from M-1 cell cDNA was confirmed by restriction digestion analysis. The rabbit CCD PCR product was sequenced, and its deduced amino acid sequence was found to be 97% homologous to the corresponding regions of human CFTR. The level of CFTR cDNA detected after 30 cycles of amplification of CCD cDNA was only 49 +/- 8 (n = 9) times lower than the level of beta-actin PCR product obtained from the same sample, suggesting that the levels of CFTR mRNA present in the CCD are physiologically relevant. Northern analysis, using a cRNA probe corresponding to the amplified region on the mRNA from CCD cells, revealed a single hybridizing species with a size of approximately 6.5 kb. Finally, CFTR PCR was performed with cDNA preparations originating from principal cells (PC), beta-intercalated cells (beta-ICC), and alpha-ICC obtained by fluorescence-activated cell sorting of rabbit CCD. CFTR PCR products were obtained from all three cell types, with the most abundant levels found in beta-ICC. beta-ICC expressed 25-fold (n = 4, P < 0.001) and 4.5-fold (n = 7, P < 0.001) higher levels than PC and alpha-ICC, respectively. This distribution pattern suggests that, within the CCD, CFTR plays a role primarily in beta-ICC function.
Aldosterone exerts complex effects on the cortical collecting duct (CCD): it increases Na+ and K+ transport, and it also influences H+ and HCO3 transport. Whether these latter effects represent direct action of aldosterone on intercalated cells (ICC) or are secondary to changes in the transport of other electrolytes is unclear. Because the presence of specific receptors is the prerequisite of a direct steroid action, and mineralocorticoid receptors (MR) have not yet been demonstrated in ICC, in this study we determined the density of MR directly in isolated principal cells (PC) and beta-ICC. Purified populations of these two cell types were obtained from rabbit renal cortex by immunodissection and fluorescence-activated cell sorting. We found that both PC and beta-ICC contained a significant number of MR, although receptor density was higher in PC than in beta-ICC (6,704 +/- 912 vs. 2,181 +/- 388 MR sites/cell; P < 0.001). 11 beta-Hydroxysteroid dehydrogenase (11 beta-OHSD), an enzyme that is present predominantly in mineralocorticoid target cells, exhibited a distribution similar to that of MR in the two cell types. 11 beta-OHSD activity, determined by measuring the rate of conversion of [3H]corticosterone to 11-dehydrocorticosterone, was 1.08 +/- 0.14 and 0.34 +/- 0.08 fmol.min-1 x 1,000 cells-1 (P < 0.001) in intact PC and beta-ICC, respectively. 11 beta-OHSD in both cell types utilized NAD as cofactor. These results suggest that beta-ICC are potential direct targets of aldosterone and that MR in both PC and beta-ICC are protected by 11 beta-OHSD.
In addition to the gastric isoform of H-K-ATPase, the colonic isoform is also expressed in the kidney, but its intrarenal localization and exact function are not known. The goal of this study was to determine whether the colonic H-K-ATPase is expressed in the rabbit cortical collecting duct (CCD) and whether it is regulated by changes in acid/base balance. With quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) with RNA isolated from immunodissected rabbit CCD cells and degenerate oligonucleotide primers, a PCR product of the predicted size (approximately 430 bp) was amplified. The amplified DNA was further characterized by nested PCR and sequencing. Direct sequencing of the 434-bp PCR product revealed 83% identity at the nucleotide level and an 80.4% identity at the deduced amino acid level to the rat colonic H-K-ATPase. With the same primers and cDNA originating from rabbit distal colon, a DNA fragment with a size and nucleotide sequence identical to that originating from CCD cells was amplified. Furthermore, using PCR screening, we isolated and sequenced a 1.5-kb cDNA clone from a rabbit CCD library. The predicted amino acid sequence of the protein encoded by this cDNA is 85 and 82% identical to the corresponding regions of the guinea pig and rat colonic H-K-ATPase, respectively, and 70% identical to the H-K-ATPase recently cloned from Bufo marinus, whereas it shows only 45 and 42% homology to the rat Na-K-ATPase alpha 1-subunit and the rat gastric H-K-ATPase, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
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