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Abstract: FR-PO1144

Diacylglycerol Kinase Epsilon (DGKE) Deficiency in Endothelial Cell Results in Marked Abnormalities in Phosphoinositides

Session Information

  • Pediatric Nephrology - I
    October 26, 2018 | Location: Exhibit Hall, San Diego Convention Center
    Abstract Time: 10:00 AM - 12:00 PM

Category: Pediatric Nephrology

  • 1600 Pediatric Nephrology


  • So, Vincent, University of Toronto, Hospital for Sick Children, Markham, Ontario, Canada
  • Choy, Christopher H., Ryerson University, Toronto, Ontario, Canada
  • Grimmer, Joanne, Children's Hospital of Western Ontario, London, Ontario, Canada
  • Botelho, Roberto, Ryerson University, Toronto, Ontario, Canada
  • Traynor-Kaplan, Alexis, University of Washington, North Bend, Washington, United States
  • Lemaire, Mathieu, Hospital for Sick Children, Toronto, Ontario, Canada

Loss-of-function mutations in DGKE cause a rare form of atypical hemolytic-uremic syndrome (aHUS) that does not implicate complement hyperactivation. Patients are typically diagnosed before age 1, have disease relapses until age 5 and develop end-stage kidney disease before adulthood. There is currently no treatment, but renal transplantation is safe.
Diacylglycerol (DAG) is produced when phosphatidylinositol 4,5-bisphosphate (PIP2) is cleaved by phospholipase C. DGKE is a lipid kinase that phosphorylates DAG to phosphatidic acid (PA). When produced, PA is shuttled to the endoplasmic reticulum where it is used to reconstitute the pool of PIP2 (as part of the PI cycle). The consequences of DGKE deficiency in human endothelial cells are unknown. A better understanding of DGKE disease pathophysiology is critical to developing new therapies for these patients.
Our objective is to quantify the impact of DGKE deficiency in endothelia on membrane lipids that play key roles in DGKE biology, including diacylglycerol, phosphatidic acid and phosphoinositides.


We used orthogonal methods to compare the lipid levels in wild-type and DGKE-deficient endothelial cells derived from human vein endothelial cells (HUVEC) with a CRISPR/Cas9-engineered deletion in the DGKE gene. These methods included mass spectrometry lipidomics, cellular labelling with 3H-inositol followed by high-performance liquid chromatography (HPLC), and live-tracking of transfected fluorescent-labelled PIP2 biosensor. The novel mass spectrometry lipidomics approach that we developed was also applied to an endothelial cell line derived from a patient with novel pathogenic DGKE mutations (c.A494G; p.D165G) and glomeruli extracted from the kidneys of a new Dgke-null mouse model (generated with CRISPR/Cas9).


Mass spectrometry lipidomics revealed significant PIP2 reduction in DGKE-null HUVEC. These results were corroborated using the same protocol applied to a patient-derived endothelial cell line and glomeruli from Dgke-null mice. 3H-inositol labelling and fluorescent lipid biosensor experiments confirmed a significant reduction in PIP2 in DGKE-deficient HUVEC.


Low endothelial PIP2 levels is likely to play a central role in the triggering the pro-thrombotic phenotype observed in patients with DGKE aHUS.


  • Other NIH Support