The distal convoluted tubule of rabbit kidney does not express a functional sodium channel.
We sought to assess whether the distal convoluted tubule (DCT) segment of the rabbit nephron expresses a functional epithelial sodium channel. First, the transepithelial voltage (V(te), lumen vs. bath) was measured in isolated perfused DCT segments (assessed separately in the upstream half and the downstream half of the DCT). V(te) was zero and not affected by amiloride or barium in the upstream DCT. V(te) was sometimes negative in the downstream DCT and depolarized by amiloride and hyperpolarized by barium, suggesting inclusion of connecting tubule (CNT) cells. To determine expression of epithelial sodium channel (ENaC) mRNA subunits by the upstream DCT, rabbit alpha-, beta-, and gamma-ENaC cDNA fragments were cloned and primers were selected for single-nephron RT-PCR analysis. Although alpha-ENaC was expressed by the DCT, beta- and gamma-ENaC were not detected in the DCT. In contrast, the CNT, CCD, and outer medullary collecting duct (OMCD) expressed all three subunits. Nedd4 was also not detected in the DCT but was expressed by the CNT, CCD, and OMCD. When upstream DCT fragments were grown to confluent monolayers in primary culture, the epithelia exhibited negative voltages and high transepithelial resistances and expressed mRNA for all three ENaC subunits as well as for Nedd4. The absence of a negative voltage and failure to detect transcript for beta- and gamma-ENaC and Nedd4 in the native rabbit DCT suggest that the sodium channel is not a significant pathway for sodium absorption by this segment. The phenotype conversion observed when DCT cells are grown in culture does not rule out the possibility that there may be conditions in which the DCT in the intact kidney expresses sodium channel activity. The results are consistent with the notion that DCT sodium transport is predominantly, if not exclusively, electroneutral.
Velázquez
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H
Renal Research, Veterans Affairs Connecticut Healthcare System, Research Office 151, West Haven, CT 06516, USA. heino.velazquez@yale.edu
Silva
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T
Andújar
E
E
Desir
G V
GV
Ellison
D H
DH
Greger
R
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D000818:Animals; D015152:Blotting, Northern; D002135:Calcium-Binding Proteins / Q000235:genetics; D003001:Cloning, Molecular; D056827:Endosomal Sorting Complexes Required for Transport; D053503:Epithelial Sodium Channels; D066298:In Vitro Techniques; D007668:Kidney / Q000378:metabolism; D007686:Kidney Tubules, Distal / Q000378:metabolism*; D008025:Ligases / Q000235:genetics; D008969:Molecular Sequence Data; D000075702:Nedd4 Ubiquitin Protein Ligases; D009399:Nephrons; D010477:Perfusion; D012333:RNA, Messenger / Q000378:metabolism; D011817:Rabbits; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015222:Sodium Channels / Q000235:genetics / Q000378:metabolism*; D014018:Tissue Distribution; D044767:Ubiquitin-Protein Ligases
D002135:Calcium-Binding Proteins; D056827:Endosomal Sorting Complexes Required for Transport; D053503:Epithelial Sodium Channels; D012333:RNA, Messenger; D015222:Sodium Channels; D000075702:Nedd4 Ubiquitin Protein Ligases; D044767:Ubiquitin-Protein Ligases; D008025:Ligases