Role of Neuropathy Target Esterase (NTE) in high NaCl‐induced accumulation of glycerophosphocholine (GPC) by mouse Inner Medullary Collecting Duct 3 (mIMCD3) cells

Abstract

GPC is an osmoprotective compatible and counteracting organic osmolyte that accumulates in renal inner medullary cells in response to high NaCl and urea. We previously found that high NaCl increases GPC in MDCK cells. The GPC is derived from phosphatidylcholine (PC), catalyzed by increased activity of a phospholipase that was not identified at that time. NTE was recently shown to be a phospholipase that catalyzes production of GPC from PC (Zaccheo et al, JBC 279: 24024, 2004). The purpose of the present studies was to test whether NTE is activated by high NaCl in mIMCD3 cells, resulting in increased GPC. We find that high NaCl increases NTE mRNA 3-fold within 8 hours, and NTE protein by 4-fold at 16 hours and 8-fold at 48 hours. GPC (measured with a sensitive chemiluminescent assay) increases 2.5-fold. Di-isopropyl FluoroPhosphate (DFP) inhibits NTE esterase activity. It reduces GPC accumulation by 30% at 300 mosmol/kg and by 40% at 500 mosmol/kg (NaCl added). Further, a specific siRNA, which reduces NTE protein abundance by 50% at both 300 and 500 mosmol/kg, reduces GPC to the same extent as DFP at both osmolalities. We conclude that high NaCl increases NTE expression and phospholipase B activity, contributing to increased production and accumulation of GPC in mIMCD3 cells.