Establishment of a mouse macula densa cell line with an nNOS promoter driving EGFP expression.
We describe a unique method for establishing a functionally intact macula densa cell line from immortalized renal cells in culture. The macula densa is involved in the tubuloglomerular feedback (TGF) system in the kidney and specifically expresses neuronal nitric oxide synthase (nNOS). A 347 bp portion of the nNOS promoter was used to drive the expression of enhanced green fluorescence protein (EGFP). An immortalized distal tubule (DT) cell line was derived from distal tubules microdissected from the kidneys of SV40 large T antigen transgenic mice. Immunofluorescence labeling using an antibody against nNOS revealed no specific EGFP expression in immunofluorescence-negative DT cells. The established cell line (NE-MD) showed a time-dependent increase in signals of the nNOS protein when they were incubated with 12 microM furosemide (an inhibitor of Na(+)-K(+)-2Cl(-) symporter) for 5 h. In conclusion, this newly developed macula densa cell line will be useful in studies of the TGF stem.
Yasuoka
Yukiko
Y
Department of Physiology, Kitasato University School of Medicine, Sagamihara 228-8555, Japan.
Kawada
Hideaki
H
Suzuki
Yoshiro
Y
Sato
Masahiro
M
Endou
Hitoshi
H
Obinata
Masuo
M
Kawahara
Katsumasa
K
D000818:Animals; D002460:Cell Line; D005091:Exons / Q000235:genetics; D005455:Fluorescent Antibody Technique; D005665:Furosemide / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects / Q000235:genetics* / Q000502:physiology; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics*; D015151:Immunoblotting; D007686:Kidney Tubules, Distal / Q000737:chemistry / Q000166:cytology* / Q000502:physiology; D051379:Mice; D008822:Mice, Transgenic; D052248:Nitric Oxide Synthase Type I / Q000032:analysis / Q000235:genetics* / Q000502:physiology; D011401:Promoter Regions, Genetic / Q000235:genetics* / Q000502:physiology; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors; D014162:Transfection; D019076:Transgenes / Q000235:genetics
D012333:RNA, Messenger; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D005665:Furosemide; D052248:Nitric Oxide Synthase Type I; C496321:Nos1 protein, mouse