C3 glomerulopathy (C3G) is associated with dysregulation of the alternative pathway of complement activation, and treatment options for C3G remain limited. Complement factor H (FH) is a potent regulator of the alternative pathway and might offer a solution, but the mass and complexity of FH makes generation of full-length FH far from trivial. We previously generated a mini-FH construct, with FH short consensus repeats 1-5 linked to repeats 18-20 (FH), that was effective in experimental C3G. However, the serum of FH was significantly shorter than that of serum-purified FH. We introduced the oligomerization domain of human FH-related protein 1 (denoted by R1-2) at the carboxy or amino terminus of human FH to generate two homodimeric mini-FH constructs (FH and FH, respectively) in Chinese hamster ovary cells and tested these constructs using binding, fluid-phase, and erythrocyte lysis assays, followed by experiments in FH-deficient mice. FH and FH homodimerized in solution and displayed avid binding profiles on clustered C3b surfaces, particularly FH Each construct was >10-fold more effective than FH at inhibiting cell surface complement activity and restricted glomerular basement membrane C3 deposition significantly better than FH or FH FH had a C3 breakdown fragment binding profile similar to that of FH, a >5-fold increase in serum compared with that of FH and significantly better retention in the kidney than FH or FH FH may have utility as a treatment option for C3G or other complement-mediated diseases.

Atypical hemolytic uremic syndrome (aHUS) is frequently associated in humans with loss-of-function mutations in complement-regulating proteins or gain-of-function mutations in complement-activating proteins. Thus, aHUS provides an archetypal complement-mediated disease with which to model new therapeutic strategies and treatments. Herein, we show that, when transferred to mice, an aHUS-associated gain-of-function change (D1115N) to the complement-activation protein C3 results in aHUS. Homozygous C3 p.D1115N (C3KI) mice developed spontaneous chronic thrombotic microangiopathy together with hematuria, thrombocytopenia, elevated creatinine, and evidence of hemolysis. Mice with active disease had reduced plasma C3 with C3 fragment and C9 deposition within the kidney. Therapeutic blockade or genetic deletion of C5, a protein downstream of C3 in the complement cascade, protected homozygous C3KI mice from thrombotic microangiopathy and aHUS. Thus, our data provide in vivo modeling evidence that gain-of-function changes in complement C3 drive aHUS. They also show that long-term C5 deficiency is not accompanied by development of other renal complications (such as C3 glomerulopathy) despite sustained dysregulation of C3. Our results suggest that this preclinical model will allow testing of novel complement inhibitors with the aim of developing precisely targeted therapeutics that could have application in many complement-mediated diseases.

I n 2013 recessive mutations in DGKE, which encodes diacylglycerol kinase epsilon (DGKE), were first reported to cause atypical hemolytic uremic syndrome (aHUS) 1 and nephrotic syndrome, with glomerular microangiopathy said to resemble membranoproliferative (mesangiocapillary) glomerulonephritis (MPGN) 2 (Online Mendelian Inheritance in Man #615008), though the pathophysiological mechanisms remain poorly understood. aHUS is characterized by a clinical presentation with thrombocytopenia, microangiopathic hemolytic anemia, and organ injury. 3 aHUS is a broad term that has been used to refer to cases of thrombotic microangiopathy (TMA), in