Low chloride stimulation of prostaglandin E₂ release and cyclooxygenase-2 expression in a mouse macula densa cell line

Reducing luminal NaCl concentration in the macula densa region of the nephron stimulates renin secretion, and this response is blocked by a specific inhibitor of cyclooxygenase-2 (COX-2) (Traynor, T. R., Smart, A., Briggs, J. P., and Schnermann, J. (1999) Am. J. Physiol. Renal Physiol. 277, F706-710). To study whether low NaCl activates COX-2 activity or expression we clonally derived a macula densa cell line (MMDD1 cells) from SV-40 transgenic mice using fluorescence-activated cell sorting of renal tubular cells labeled with segment-specific fluorescent lectins. MMDD1 cells express COX-2, bNOS, NKCC2, and ROMK, but not Tamm-Horsfall protein, and showed rapid ⁸⁶Rb⁺ uptake that was inhibited by a reduction in NaCl concentration and by bumetanide or furosemide. Isosmotic exposure of MMDD1 cells to low NaCl (80 mM) caused a prompt and time-dependent stimulation of prostaglandin E2 (PGE₂) release that was prevented by the COX-2 specific inhibitor NS-398 (10 μM). Reducing NaCl to 60 and 6 mM for 16 h increased COX-2 expression in a chloride-dependent fashion. Low NaCl phosphorylated p38 kinase within 30 rain and ERK1/2 kinases within 15 min without changing total MAP kinase levels. Low NaCl-stimulated PGE₂ release and COX-2 expression was inhibited by SB 203580 and PD 98059 (10 μM), inhibitors of p38 and ERK kinase pathways. We conclude that low chloride stimulates PGE₂ release and COX-2 expression in MMDD1 cells through activation of MAP kinases.