Loss of DGKε induces endothelial cell activation and death independently of complement activation

Abstract

Key Points• Loss of DGKe in endothelial cells induces cell death, impairs angiogenic responses, and leads to an activated and prothrombotic phenotype.• DGKE silencing in resting endothelial cells does not affect complement activation at their surface.Atypical hemolytic uremic syndrome (aHUS) is classically described to result from a dysregulation of the complement alternative pathway, leading to glomerular endothelial cell (EC) damage and thrombosis. However, recent findings in families with aHUS of mutations in the DGKE gene, which is not an integral component of the complement cascade, led us to consider other pathophysiologic mechanisms for this disease. Here, we demonstrate that loss of DGK« expression/activity in EC induces an increase in ICAM-1 and tissue factor expression through the upregulation of p38-MAPK-mediated signals, thus highlighting a proinflammatory and prothrombotic phenotype of DGK«-deficient ECs. More interestingly, DGKE silencing also increases EC apoptosis and impairs EC migration and angiogenesis in vitro, suggesting that DGKE loss-of-function mutations impair EC repair and angiogenesis in vivo. Conversely, DGKE knockdown moderately decreases the expression of the complement inhibitory protein MCP on quiescent EC, but does not induce complement deposition on their surface in vitro. Collectively, our data strongly suggest that in DGKE-associated aHUS patients, thrombotic microangiopathy results from impaired EC proliferation and angiogenesis rather than complement-mediated EC lesions. Our study expands the current knowledge of aHUS mechanisms and has implications for the treatment of patients with isolated DGKE mutations. (Blood. 2015;125(6):1038-1046 IntroductionAtypical hemolytic uremic syndrome (aHUS) is a severe form of thrombotic microangiopathy (TMA) that affects primarily the kidney. It is characterized by the occurrence of endothelial damage and fibrin/ platelet thrombi in the kidney microvasculature, leading to its typical triad of hemolytic microangiopathic anemia, thrombocytopenia, and acute renal injury.1 aHUS has a poor prognosis, with a 2% to 10% mortality rate, and about two-thirds of patients progress toward end-stage renal disease, and there is a high risk of recurrence of the disease after kidney transplantation.2 Over the past decade, many studies have highlighted the central role of complement alternative pathway dysregulations in the development of aHUS. [3][4][5] Several mutations in genes encoding complement regulatory proteins (factor H, 6 MCP, 7 factor I, 8 thrombomodulin 9 ) or components of the alternative C3 convertase (C3, 10 factor B 11 ), as well as the presence of circulating inhibitory anti-factor H antibodies, 12 have been shown to predispose to the development of aHUS. It is currently assumed that complement alternative pathway activation triggered mainly by infection or pregnancy 13 leads to endothelial cell (EC) damage and TMA. These observations led to the development of complement-targeted therapies for the treatment of aHUS, 14 and eculizumab, a mo...

High-Throughput Genetic Testing for Thrombotic Microangiopathies and C3 Glomerulopathies

“…An unexpected finding was the increased DGKE variant load in patients with C3G and IgAN and its possible association with hematuria, [26][27][28] which may reflect a role for DGKE in endothelial cell activation and damage. 29 CNVanalysis over the CFHR3-CFHR1 region confirmed the increased frequency of homozygosity for del(CFHR3-CFHR1) in aHUS patients (Table 3). We also noted an important limitation that the relatively small sample size reduces the statistical power of this study.…”

Modified Ham test for atypical hemolytic uremic syndrome

“…46 Because the relationship between these mutations and complement dysregulation is not fully clarified, diagnosis utilizing the present assay may not be feasible. 47 Thus, our results need to be confirmed by further prospective studies with larger TMA cohorts that include patients with these mutations. In conclusion, we have developed a modified Ham test with high reproducibility, sensitivity, and specificity in differentiating aHUS from other TMAs, based on complement-mediated apoptosis and death in GPI-AP-deficient cells.…”

Complement in hemolytic anemia

“…Rare cases of early onset (,12 months of age) aHUS harbor mutations in diacylglycerol kinase-e (DGKE); however, the mechanism of how DGKE mutations cause aHUS is unclear. 24 …”