Specific inhibition of epithelial Na+ channels by antisense oligonucleotides for the treatment of Na+ hyperabsorption in cystic fibrosis.
BACKGROUND
Cystic fibrosis (CF) respiratory epithelia are characterized by a defect Cl(-) secretion and an increased Na(+) absorption through epithelial Na(+) channels (ENaC). The present study aimed to find an effective inhibitor of human ENaC with respect to replacing amiloride therapy for CF patients. Therefore, we developed specific antisense oligonucleotides (AON) that efficiently suppress Na(+) hyperabsorption by inhibiting the expression of the alpha-ENaC subunit.


METHODS
We heterologously expressed ENaC in oocytes of Xenopus laevis for mass screening of AON. Additionally, primary cultures of human nasal epithelia were transfected with AON and were used for Ussing chamber experiments, as well as biochemical and fluorescence optical analyses.


RESULTS
Screening of several AON by co-injection or sequential microinjection of AON and ENaC mRNA in X. laevis oocytes led to a sustained decrease in amiloride-sensitive current and conductance. Using primary cultures of human nasal epithelia, we show that AON effectively suppress amiloride-sensitive Na(+) absorption mediated by ENaC in CF and non-CF tissues. In western blot experiments, it could be shown that the amount of ENaC protein is effectively reduced after AON transfection.


CONCLUSIONS
Our data comprise an initial step towards a preclinical test with AON to reduce Na(+) hyperabsorption in CF epithelia.
Sobczak
Katja
K
Institute of Animal Physiology, Westphalian Wilhelms-University Muenster, Muenster, Germany.
Segal
Andrei
A
Bangel-Ruland
Nadine
N
Semmler
Judith
J
Van Driessche
Willy
W
Lindemann
Hermann
H
Heermann
Ralf
R
Weber
Wolf-Michael
WM
D000818:Animals; D001483:Base Sequence; D003550:Cystic Fibrosis / Q000378:metabolism*; D017931:DNA Primers; D062686:Epithelial Sodium Channel Blockers; D053503:Epithelial Sodium Channels / Q000235:genetics; D006801:Humans; D008839:Microelectrodes; D008856:Microscopy, Fluorescence; D009297:Nasal Mucosa / Q000378:metabolism; D016376:Oligonucleotides, Antisense / Q000494:pharmacology*; D012333:RNA, Messenger / Q000235:genetics; D012964:Sodium / Q000378:metabolism*; D014982:Xenopus laevis
D017931:DNA Primers; D062686:Epithelial Sodium Channel Blockers; D053503:Epithelial Sodium Channels; D016376:Oligonucleotides, Antisense; D012333:RNA, Messenger; D012964:Sodium