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.