Glycerophosphocholine (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 and/or urea increases GPC in renal (Madin-Darby canine kidney, MDCK) cells and that the GPC is derived from phosphatidylcholine, catalyzed by a phospholipase that was not identified at that time. When neuropathy target esterase (NTE) was shown to be a phospholipase B that catalyzes production of GPC from phosphatidylcholine, we tested whether NTE contributes to the high NaCl-induced increase of GPC synthesis in renal cells, finding that it does. In mouse inner medullary collecting duct (mIMCD3) cells, high NaCl increases NTE mRNA and protein. Diisopropyl fluorophosphate, which inhibits NTE esterase activity, reduces GPC accumulation, as does an siRNA that specifically reduces NTE protein abundance. The 20-h half-life of NTE mRNA is unaffected by high NaCl, but knockdown of NFAT5/TonEBP by a specific siRNA inhibits the high NaCl-induced increase of NTE mRNA. Further, the lower renal inner medullary interstitial NaCl concentration that occurs chronically in ClCK1-/- mice and acutely in normal mice given furosemide is associated with lower NTE mRNA and protein. Thus, high NaCl increases transcription of NTE, mediated by NFAT5/TonEBP, and the resultant increase of NTE expression contributes to increased production and accumulation of GPC in mammalian renal cells in tissue culture and in vivo. We previously also found that high urea and/or NaCl inhibit the activity of a phosphodiesterase (GPC-PDE) that catalyzes breakdown of GPC to choline and glycerol phosphate, and that this contributes to osmotic induction of GPC. We identified the phosphodiesterase as Gdpd5. Recombinant Gdpd5 immunoprecipitated from mIMCD3 cells has GPC-PDE activity and the specific activity is lower if the cells have been exposed to high NaCl or urea indicating that high NaCl and high urea inhibit GDPD5 by post translational modification (PTM). We are currently identifying the amino acids involved in the PTMs. We identify two in HEK293 cells, namely cysteine 25 (C25) and threonine 587 (T587). Reactive oxygen species (ROS) are involved in the role of C25. High NaCl and urea increase ROS. When this increase is prevented by the antioxidant, N-acetyl cysteine, inhibition of GDPD5 is much less. Also, adding ROS in the form of H2O2, inhibits GDE-PDE activity of GDPD5 in which C25 is mutated to serine much less than it does of wild type GDPD5, and the remaining activity is further reduced by high NaCl and urea. T587 of GDPD5 is phosphorylated when NaCl and urea are not elevated, and high NaCl and urea decrease the phosphorylation. Mutation of T587 to alanine, which cannot be phosphorylated, decreases GPC-PDE activity of GDPD5. Finally, inhibition of CDK1 protein kinase reduces GDE-PDE activity of GDPD5 without altering phosphorylation at T587, which suggests that there may be additional PTMs. In order to understand better the cellular response to osmotic stress, like that that exists in the renal medulla, we are using protein mass spectrometry to study high NaCl-induced changes in protein phosphorylation and subcellular localization in HEK293 cells. We used Stable Isotopic Amino acids in Cell culture (SILAC) coupled to mass spectrometry to identify phosphorylation based signaling pathways in HEK293 cells. We identified more than 30,000 phosphopeptides in four biological replicate samples with 1% FDR. More than 7,000 unique phosphopeptides were quantified. 80% have a single phosphate group and 20% two or more phosphate groups. High NaCl significantly changes the abundance of 300 phosphopeptides. We identified functional category enrichment for these significantly changed phosphopeptides, and cellular pathways affected by hypertonicity. Network analysis of these results suggested that p38 MAPK might activate STAT1 and HSSP27 by phosphorylating them in response to high NaCl. We confirmed this by Western analysis with phosphospecific antibodies which showed that inhibition of p38 reduces high-NaCl-induced phosphorylation of STAT1 and HSP27. We used iTRAQ to quantify proteins in nuclear and cytoplasmic extracts from HEK293 cells exposed to high NaCl for one or eight hours or adapted to high NaCl for several passages. The abundance of 165 proteins changed in the nucleus or cytoplasm at at least one of the times. We are currently analyzing the patterns of change.

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National Heart, Lung, and Blood Institute
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