BASIC RESEARCH www.jasn.org Blocking Properdin Prevents Complement-Mediated Hemolytic Uremic Syndrome and Systemic Thrombophilia Yoshiyasu Ueda,1 Takashi Miwa,1 Damodar Gullipalli,1 Sayaka Sato,1 Daisuke Ito,1 Hangsoo Kim,1 Matthew Palmer,2 and Wen-Chao Song1 Departments of 1Systems Pharmacology and Translational Therapeutics and 2Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania ABSTRACT Background Properdin (P) is a positive regulator of the alternative pathway of complement activation. Although P inhibition is expected and has been shown to ameliorate the alternative pathway of comple- ment-mediated tissue injury in several disease models, it unexpectedly exacerbated renal injury in a murine model of C3 glomerulopathy. The role of P in atypical hemolytic uremic syndrome (aHUS) is uncertain. Methods We blocked P function by genetic deletion or mAb-mediated inhibition in mice carrying a factor H (FH) point mutation, W1206R (FHR/R), that causes aHUS and systemic thrombophilia with high mortality. Results Pdeficiency completely rescued FHR/R mice from premature death and prevented thrombocyto- penia, hemolytic anemia, and renal disease. It also eliminated macrovessel thrombi that were prevalent in FHR/R mice. All mice that received a function-blocking anti-P mAb for 8 weeks survived the experimental period and appeared grossly healthy. Platelet counts and hemoglobin levels were significantly improved in FHR/R mice after 4 weeks of anti-P mAb treatment. One half of the FHR/R mice treated with an isotype control mAb but none of the anti-P mAb-treated mice developed stroke-related neurologic disease. Anti-P mAb-treated FHR/R mice showed largely normal renal histology, and residual liver thrombi were detected in only three of 15 treated mice. Conclusions These results contrast with the detrimental effect of P inhibition observed in a murine model of C3 glomerulopathy and suggest that P contributes critically to aHUS pathogenesis. Inhibition of P in aHUS may be of therapeutic benefit. J Am Soc Nephrol 29: ccc–ccc, 2018. doi: https://doi.org/10.1681/ASN.2017121244 Properdin (P) is a plasma glycoprotein and the only aneurysm, renal ischemia-reperfusion injury, in- known positive regulator of the alternative pathway flammatory arthritis, and allergic asthma.9–12 How- (AP) of complement activation.1–5 It is composed ever, it was observed in earlier studies that P defi- of seven type 1 thrombospondin repeats and present ciency or antibody neutralization unexpectedly in plasma at a concentration of about 5–15 mg/ml.6 P facilitates AP complement activation by binding to and stabilizing the C3 convertase C3bBb, and Received December 2, 2017. Accepted May 11, 2018. 7,8 thereby, it significantly extends its t1/2. There Published online ahead of print. Publication date available at is now considerable evidence to suggest that P www.jasn.org. plays a pathogenic role in AP complement–mediated fi Correspondence: Dr. Wen-Chao Song, University of Pennsylva- autologous tissue injury. For example, P de ciency nia School of Medicine, Room 1254 BRBII/III, 421 Curie Boule- has been shown to be protective in several murine vard, Philadelphia, PA 19104. Email: songwe@pennmedicine. models of complement-dependent diseases, includ- upenn.edu ing extravascular hemolysis, abdominal aortic Copyright © 2018 by the American Society of Nephrology J Am Soc Nephrol 29: ccc–ccc, 2018 ISSN : 1046-6673/2907-ccc 1 BASIC RESEARCH www.jasn.org exacerbated renal disease in murine models of C3 glomerulopathy Significance Statement (C3G) due to factor H (FH) knockout or C-terminal mutation.13,14 The latter finding highlighted the complex dynamics of comple- Dysregulation of the alternative pathway of complement activation ment activation and inhibition in the fluid phase and on target is associated with atypical hemolytic uremic syndrome (aHUS) and organs, and it raised intriguing questions regarding the mechanism C3 glomerulopathy (C3G). The role of properdin, a positive com- plement regulator, in aHUS and C3G remains uncertain. The authors of action of P and whether it is protective or pathogenic in atypical previously showed that properdin inhibition exacerbated a murine hemolyticuremicsyndrome(aHUS),anotherAPcomplement– model of C3G. In striking contrast, these studies show that blocking driven disease with renal injury as a hallmark phenotype. properdin in a murine model of aHUS prevented renal disease and Human aHUS is a form of thrombotic microangiopathy (TMA) systemic thrombophilia. These results suggest that properdin characterized by thrombocytopenia, hemolytic anemia, and renal contributes critically to renal disease in aHUS and that its inhibition in this disorder and other renal disorders may have therapeutic benefit. failure.15,16 Most aHUS cases are linked to abnormalities in the complement system, including loss of function mutations in genes encoding regulatory proteins, such as FH, or gain of functions Complete blood count was measured using an automated he- mutations in C3 and factor B.17–21 These complement defects pre- matology analyzer (XT-2000iV; Sysmex) at the Translational dispose patients to aHUS, which can be triggered by major health- Core Laboratory of the Children’s Hospital of Philadelphia.23 related events, such as infection, pregnancy, trauma, or surgery, whereby complement activation may be induced and inadequately Immunofluorescence Staining and Histology regulated, leading to microvascular injury.22 To better understand Kidneys were frozen in optimal cutting temperature compound the aHUS pathogenesis and facilitate development of novel thera- (Sakura), and 4-mm sections were cut. Sections were processed for peutic options, we previously generated a high-penetrance murine C3, C9, and fibrin deposition as described previously.23 For C9 model of aHUS by FH point mutation.23 Mice homozygous for the staining, a rabbit polyclonal anti-mouse C9 generated in house FH W1206R point mutation (FHR/R) developed characteristic was used. Details about generation and characterization of the aHUS symptoms, including low platelet counts, anemia, and renal anti-C9 antibody will be reported elsewhere. For costaining with disease, and they had high mortality.23 They also displayed a sys- C3 or C9, rat anti-mouse CD31 antibody (clone MEC13.3) (0.5 temic thrombophilia phenotype with formation of macrovessel mg/ml; BD Pharmingen) was used at 1:50 dilution to stain and thrombi in various organs, most prominently in the liver.23 In identify endothelial cells. Rat anti-mouse CD140b (PDGF this study, we have used the FHR/R mouse model and tested the receptor-b) antibody (clone ABP5) (0.5 mg/ml; eBioscience) role of P in aHUS using two separate approaches: by genetically was used at 1:25 to stain and identify mesangial cells. For these deleting the P gene9 and by mAb neutralization of P protein.11 Our two antibodies, Cy3-conjugated goat anti-rat IgG (0.2 mg/ml; results showed that P contributed critically to the pathogenesis of Invitrogen) was used at 1:100 dilution as the secondary anti- aHUS in FHR/R mice and that blocking P was sufficient to rescue body. A guinea pig polyclonal anti-nephrin antibody (Progen them from the disease. This outcome was in striking contrast with Biotechnik, Heidelberg, Germany) was used at 1:100 dilution to what had been observed previously in murine models of C3G, stain and identify podocytes followed by the use of Alexa Flour where blocking P exacerbated disease.13,14 Our data shed new light 555 goat anti-guinea pig IgG (2 mg/ml; Invitrogen) at 1:500 on the mechanism of action of P in different complement- dilution as the secondary antibody. Costained sections were mediated renal diseases and provide proof of concept in support examined on a Leica STED 33 super-resolution microscope. of therapeutic targeting of P in aHUS. To evaluate tissue pathology, isolated organs were fixed in 10% formalin-PBS–buffered saline and processed for hematoxylin and eosin and periodic acid–Schiff staining by AML Laboratories (www. METHODS amllabs.com). Kidney slides were graded by light microscopy in a blind fashion by a pathologist (M.P.) as described previously.23 Mice The source and generation of FH W1206R mutant mice (FHR/R) Electron Microscopy 2 2 fl fl and a P knockout mouse (P / ), generated by crossing P ox/ ox Electron microscopic analysis of kidney sections was per- and Ella-Cre mice, have been reported previously.9,23 All animal formed at the Electron Microscopy Resource Laboratory of experiments were approved by the Institutional Animal Care the University of Pennsylvania as described previously.13,23 and Use Committee of the University of Pennsylvania. AP and Lytic Complement Activity Assays Survival Curve LPS-induced AP complement activity assay and red blood cell The survival curve was analyzed using the GraphPad Prism lysis test for lytic complement activity in mouse plasma were program (La Jolla, CA) as described previously.13,23 performed as described.23 Both assays used lupiridin (Bivalir- udin;HospiraInc.,LakeForest, IL) anticoagulated mouse Measurement of BUN and Complete Blood Count plasma diluted to 10% and 50%, respectively. Nonsensitized BUN was measured using serum and urea nitrogen reagents sheep red blood cells (Complement Technology, Tyler, TX) (Sigma-Aldrich, St. Louis, MO) as described previously.23 were used in the lysis test. 2 Journal of the American Society of Nephrology J Am Soc Nephrol 29: ccc–ccc,2018 www.jasn.org BASIC RESEARCH (day 0) and then, at various time points after injection, and they were tested for to- tal AP complement activity. Treatment of FHR/R with Anti-P or Isotype Control mAb For therapeutic studies, 4-week-old FHR/R mice of mixed sexes were treated for 8 weeks with the anti-P mAb 14E111 or an isotype murine IgG1 control mAb (MOPC 31C)24 at 1 mg per mouse admin- istered intraperitoneally. Control mAb was administered weekly, and anti-P mAb was given either once weekly or twice weekly as specified in Results and figures.