Phosphatidylinositol Cycle Disruption is Central to Atypical Hemolytic-Uremic Syndrome Caused by Diacylglycerol Kinase Epsilon Deficiency
Loss-of-function mutations in diacylglycerol kinase epsilon (DGKE) cause a rare form of atypical hemolytic-uremic syndrome (aHUS) for which there is no treatment besides kidney transplantation. Highly expressed in kidney endothelial cells, DGKE is a lipid kinase that phosphorylates diacylglycerol (DAG) to phosphatic acid (PA). Specifically, DGKE's preferred substrate is 38:4-DAG, that is DAG containing stearic acid (18:0) and arachidonic acid (20:4). DAG is produced when phosphatidylinositol
... phatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) is cleaved by phospholipase C (PLC). A better understanding of how DGKE deficiency impacts the endothelial lipid landscape is critical to developing a treatment for this condition. Methods: We used orthogonal methods to compare the lipid levels in two novel models of DGKE deficiency to their respective controls: an immortalized human umbilical vein endothelial cell (iHUVEC) engineered with CRISPR/Cas9 and a blood outgrowth endothelial cell (BOEC) from an affected patient. Methods included mass spectrometry lipidomics, radiolabeling of phosphoinositides with [3H]myo-inositol, and live-tracking of a transfected fluorescent PtdIns(4,5)2 biosensor. Results: Unexpectedly, mass spectrometry lipidomics data revealed that high 38:4-DAG was not observed in the two DGKE-deficient models. Instead, a reduction in 38:4-PtdIns(4,5)P₂ was the major abnormality.These results were confirmed with the other two methods in DGKE-deficient iHUVEC. Conclusion: Reduced 38:4-PtdIns(4,5)2 -but not increased 38:4-DAG- is likely to be key to the pro-thrombotic phenotype exhibited by patients with DGKE aHUS.