Kidney Week

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

Authors

• So, Vincent, University of Toronto, Hospital for Sick Children, Markham, Ontario, Canada

Vincent So,

• Choy, Christopher H., Ryerson University, Toronto, Ontario, Canada

Christopher H. Choy,

• Grimmer, Joanne, Children's Hospital of Western Ontario, London, Ontario, Canada

Joanne Grimmer,

• Botelho, Roberto, Ryerson University, Toronto, Ontario, Canada

Roberto Botelho,

• Traynor-Kaplan, Alexis, University of Washington, North Bend, Washington, United States

Alexis Traynor-Kaplan,

• Lemaire, Mathieu, Hospital for Sick Children, Toronto, Ontario, Canada

Mathieu Lemaire,

Background

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.

Methods

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).

Results

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.

Conclusion

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

Funding

• Other NIH Support