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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 DGKε 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 DGKε-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.

Atypical hemolytic uremic syndrome (aHUS) is a rare renal thrombotic microangiopathy commonly associated with rare genetic variants in complement system genes, unique to each patient/family. Here, we report 14 sporadic aHUS patients carrying the same mutation, R139W, in the complement C3 gene. The clinical presentation was with a rapid progression to end-stage renal disease (6 of 14) and an unusually high frequency of cardiac (8 of 14) and/or neurologic (5 of 14) events. Although resting glomerular endothelial cells (GEnCs) remained unaffected by R139W-C3 sera, the incubation of those sera with GEnC preactivated with pro-inflammatory stimuli led to increased C3 deposition, C5a release, and procoagulant tissue-factor expression. This functional consequence of R139W-C3 resulted from the formation of a hyperactive C3 convertase. Mutant C3 showed an increased affinity for factor B and a reduced binding to membrane cofactor protein (MCP; CD46), but a normal regulation by factor H (FH). In addition, the frequency of at-risk FH and MCP haplotypes was significantly higher in the R139W-aHUS patients, compared with normal donors or to healthy carriers. These genetic background differences could explain the R139W-aHUS incomplete penetrance. These results demonstrate that this C3 mutation, especially when associated with an at-risk FH and/or MCP haplotypes, becomes pathogenic following an inflammatory endothelium-damaging event.

Atypical hemolytic uremic syndrome (aHUS) is characterized by genetic and acquired abnormalities of the complement system leading to alternative pathway (AP) overactivation and by glomerular endothelial damage, thrombosis, and mechanical hemolysis. Mutations per se are not sufficient to induce aHUS, and nonspecific primary triggers are required for disease manifestation. We investigated whether hemolysis-derived heme contributes to aHUS pathogenesis. We confirmed that heme activates complement AP in normal human serum, releasing C3a, C5a, and sC5b9. We demonstrated that heme-exposed endothelial cells also activate the AP, resulting in cell-bound C3 and C5b9. This was exacerbated in aHUS by genetic abnormalities associated with AP overactivation. Heme interacted with C3 close to the thioester bond, induced homophilic C3 complexes, and promoted formation of an overactive C3/C5 convertase. Heme induced decreased membrane cofactor protein (MCP) and decay-accelerating factor (DAF) expression on endothelial cells, giving Factor H (FH) a major role in complement regulation. Finally, heme promoted a rapid exocytosis of Weibel-Palade bodies, with membrane expression of P-selectin known to bind C3b and trigger the AP, and the release of the prothrombotic von Willebrand factor. These results strongly suggest that hemolysis-derived heme represents a common secondary hit amplifying endothelial damage and thrombosis in aHUS.