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Acute kidney injury (AKI) remains a major reason for hospitalization with limited therapeutic options. Although complement activation is implicated in AKI, the role of C5a receptor 1 (C5aR1) in kidney tubular cells is unclear. Herein, aristolochic acid nephropathy (AAN) and folic acid nephropathy (FAN) models were used to establish the role of C5aR1 in kidney tubules during AKI in germline C5ar1 -/- , myeloid cell-specific, and kidney tubule-specific C5ar1 knockout mice. After aristolochic acid and folic acid injection, C5ar1 -/- mice had increased AKI severity and a higher degree of tubular injury. Macrophage depletion in C5ar1 -/- mice or myeloid cell-specific C5ar1 deletion did not affect the outcomes of aristolochic acid-induced AKI. RNA-sequencing data from renal tubular epithelial cells (RTECs) showed that C5ar1 deletion was associated with the down-regulation of mitochondrial metabolism and ATP production transcriptional pathways. Metabolic studies confirmed reduced mitochondrial membrane potential at baseline and increased mitochondrial oxidative stress after injury in C5ar1 -/- RTECs. Moreover, C5ar1 -/- RTECs had enhanced glycolysis, glucose uptake, and lactate production on injury, corroborated by metabolomics analysis of kidneys from AAN mice. Kidney tubule-specific C5ar1 knockout mice recapitulated exacerbated AKI observed in C5ar1 -/- mice in AAN and FAN. These data indicate that C5aR1 signaling in kidney tubules exerts renoprotective effects against toxin-induced AKI by limiting overt glycolysis and maintaining mitochondrial function, thereby revealing a novel link between the complement system and tubular cell metabolism., Competing Interests: Disclosure Statement None declared., (Copyright © 2025 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Schistosomiasis-induced pulmonary hypertension (PH) presents a significant global health burden, yet the underlying mechanisms remain poorly understood. Here, we investigate the involvement of platelets and the complement system in the initiation events leading to Schistosoma -induced PH. We demonstrate that Schistosoma exposure leads to thrombocytopenia, platelet accumulation in the lung, and platelet activation. In addition, we observed increased plasma complement anaphylatoxins C3a and C5a, indicative of complement system activation, and elevated platelet expression of C1q, C3, decay activating factor (DAF), and complement C3a and C5a receptors. Our findings suggest the active involvement of platelets in responding to complement system signals induced by Schistosoma exposure and form the basis for future mechanistic studies on how complement may regulate platelet activation and promote the development of Schistosoma -induced PH. NEW & NOTEWORTHY Schistosomiasis-induced pulmonary hypertension (PH) is a significant global health burden, yet the underlying mechanisms remain poorly understood. We demonstrate that Schistosoma exposure leads to platelet accumulation in the lung and platelet activation. We observed increased plasma levels of C3a and C5a, indicative of complement system activation, and elevated expression of platelet complement proteins and receptors. These findings underscore the role of platelets and complement in the inflammatory responses associated with Schistosoma -induced PH.

Uncontrolled complement activation can cause or contribute to glomerular injury in multiple kidney diseases. Although complement activation plays a causal role in atypical hemolytic uremic syndrome and C3 glomerulopathy, over the past decade, a rapidly accumulating body of evidence has shown a role for complement activation in multiple other kidney diseases, including diabetic nephropathy and several glomerulonephritides. The number of available complement inhibitor therapies has also increased during the same period. In 2022, Kidney Diseases: Improving Global Outcomes (KDIGO) convened a Controversies Conference, "The Role of Complement in Kidney Disease," to address the expanding role of complement dysregulation in the pathophysiology, diagnosis, and management of various glomerular diseases, diabetic nephropathy, and other forms of hemolytic uremic syndrome. Conference participants reviewed the evidence for complement playing a primary causal or secondary role in progression for several disease states and considered how evidence of complement involvement might inform management. Participating patients with various complement-mediated diseases and caregivers described concerns related to life planning, implications surrounding genetic testing, and the need for inclusive implementation of effective novel therapies into clinical practice. The value of biomarkers in monitoring disease course and the role of the glomerular microenvironment in complement response were examined, and key gaps in knowledge and research priorities were identified., (Copyright © 2024 Kidney Disease: Improving Global Outcomes (KDIGO). Published by Elsevier Inc. All rights reserved.)

Historically, the complement system (classical, lectin, alternative, and terminal pathways) is known to play a crucial role in the etiopathogenesis of many kidney diseases. Direct or indirect activation in these settings is revealed by consumption of complement proteins at the serum level and kidney tissue deposition seen by immunofluorescence and electron microscopy. The advent of eculizumab has shown that complement inhibitors may improve the natural history of certain kidney diseases. Since then, the number of available therapeutic molecules and experimental studies on complement inhibition has increased exponentially. In our narrative review, we give a summary of the main complement inhibitors that have completed phase II and phase III studies or are currently used in adult and pediatric nephrology. The relevant full-text works, abstracts, and ongoing trials (clinicaltrials.gov site) are discussed. Data and key clinical features are reported for eculizumab, ravulizumab, crovalimab, avacopan, danicopan, iptacopan, pegcetacoplan, and narsoplimab. Many of these molecules have been shown to be effective in reducing proteinuria and stabilizing kidney function in different complement-mediated kidney diseases. Thanks to their efficacy and target specificity, these novel drugs may radically improve the outcome of complement-mediated kidney diseases, contributing to an improvement in our understanding of their underlying pathophysiology., (© 2023. The Author(s), under exclusive licence to International Pediatric Nephrology Association.)

Complement-mediated diseases can be treated using systemic inhibitors. However, complement components are abundant in circulation, affecting systemic inhibitors' exposure and efficacy. Furthermore, because of complement's essential role in immunity, systemic treatments raise infection risk in patients. To address these challenges, we developed antibody fusion proteins combining the alternative-pathway complement inhibitor factor H (fH 1-5 ) with an anti-C3d monoclonal antibody (C3d-mAb-2fH). Because C3d is deposited at sites of complement activity, this molecule localizes to tissue complement while minimizing circulating complement engagement. These fusion proteins bind to deposited complement in diseased human skin sections and localize to activated complement in a primate skin injury model. We further explored the pharmacology of C3d-mAb-2fH proteins in rodent models with robust tissue complement activation. Doses of C3d-mAb-2fH >1 mg/kg achieved >75% tissue complement inhibition in mouse and rat injury models while avoiding circulating complement blockade. Glomerular-specific complement inhibition reduced proteinuria and preserved podocyte foot-process architecture in rat membranous nephropathy, indicating disease-modifying efficacy. These data indicate that targeting local tissue complement results in durable and efficacious complement blockade in skin and kidney while avoiding systemic inhibition, suggesting broad applicability of this approach in treating a range of complement-mediated diseases., Competing Interests: Declaration of interests This work was funded by Q32 Bio, Inc., which holds patents on ADX-097 and related molecules. Q32 Bio is currently conducting clinical trials using ADX-097. F.L., S.T.R., K.C.F., J.G.M., A.E.C., S.M.V., and S.W. are current employees and equity holders of Q32 Bio, Inc. M.J.S. and S.L.K. were employees of Q32 Bio, Inc. at the time of their contributions to the described work. J.M.T., and V.M.H. are co-founders of Q32 Bio, Inc. and retain equity in the company., (Copyright © 2024 Q32 Bio. Published by Elsevier Inc. All rights reserved.)

The complement cascade comprises soluble and cell surface proteins and is an important arm of the innate immune system. Once activated, the complement system rapidly generates large quantities of protein fragments that are potent mediators of inflammatory, vasoactive and metabolic responses. Although complement is crucial to host defence and homeostasis, its inappropriate or uncontrolled activation can also drive tissue injury. For example, the complement system has been known for more than 50 years to be activated by glomerular immune complexes and to contribute to autoimmune kidney disease. Notably, the latest research shows that complement is also activated in kidney diseases that are not traditionally thought of as immune-mediated, including haemolytic-uraemic syndrome, diabetic kidney disease and focal segmental glomerulosclerosis. Several complement-targeted drugs have been approved for the treatment of kidney disease, and additional anti-complement agents are being investigated in clinical trials. These drugs are categorically different from other immunosuppressive agents and target pathological processes that are not effectively inhibited by other classes of immunosuppressants. The development of these new drugs might therefore have considerable benefits in the treatment of kidney disease., (© 2023. Springer Nature Limited.)

B cell-targeted therapies are effective for treating multiple different kidney diseases in humans and also protect mice from Adriamycin nephropathy. Because glomerular IgM is frequently seen in both humans and mice with "nonimmune" forms of glomerular disease, we hypothesized that natural IgM binds to epitopes displayed in the injured glomerulus, exacerbating injury. To test this hypothesis, we induced Adriamycin nephropathy in BALB/C mice that cannot secrete soluble IgM (sIgM-/- mice) and compared them with BALB/C controls. Contrary to our prediction, we found that female sIgM-/- mice developed higher mortality and more severe kidney injury after injection of Adriamycin. The absence of soluble IgM did not reduce glomerular complement activation, and IgG was seen deposited within the injured glomeruli. Furthermore, we discovered that female sIgM-/- mice have higher levels of anti-cardiolipin IgG, and that IgG from these mice binds to epitopes in the injured kidney. These findings indicate that natural IgM may prevent generation of autoreactive IgG. Circulating levels of anti-cardiolipin IgG decreased after induction of kidney injury in female mice, consistent with deposition of the Abs in injured tissues. Better understanding of the mechanisms by which the immune system modulates and amplifies kidney injury may enable the development of targeted therapies to slow kidney disease progression., (Copyright © 2023 by The American Association of Immunologists, Inc.)

Significance Statement: Histologic quantification of complement C3 deposits in kidney biopsies provides prognostic information in patients with glomerulonephritis. Unfortunately, kidney biopsies are invasive procedures that cannot be performed regularly and only provide a snapshot of a small portion of one kidney at the time of sampling. We have developed a method to noninvasively detect specific C3 fragment deposition throughout both kidneys, using a monoclonal antibody targeting tissue-bound iC3b/C3d linked to a bioluminescent resonance energy transfer construct that emits near-infrared light. In a mouse model of glomerulonephritis, the probe detected iC3b/C3d in kidneys of live mice by bioluminescent imaging. This demonstrates that noninvasive imaging with an anti-iC3b/C3d probe can be used to monitor inflammation in the kidneys., (Copyright © 2023 by the American Society of Nephrology.)

Myelin oligodendrocyte glycoprotein (MOG) antibody-associated disease (MOGAD) is an inflammatory demyelinating CNS condition characterized by the presence of MOG autoantibodies. We sought to investigate whether human MOG autoantibodies are capable of mediating damage to MOG-expressing cells through multiple mechanisms. We developed high-throughput assays to measure complement activity (CA), complement-dependent cytotoxicity (CDC), antibody-dependent cellular phagocytosis (ADCP), and antibody-dependent cellular cytotoxicity (ADCC) of live MOG-expressing cells. MOGAD patient sera effectively mediate all of these effector functions. Our collective analyses reveal that (a) cytotoxicity is not incumbent on MOG autoantibody quantity alone; (b) engagement of effector functions by MOGAD patient serum is bimodal, with some sera exhibiting cytotoxic capacity while others did not; (c) the magnitude of CDC and ADCP is elevated closer to relapse, while MOG-IgG binding is not; and (d) all IgG subclasses can damage MOG-expressing cells. Histopathology from a representative MOGAD case revealed congruence between lesion histology and serum CDC and ADCP, and we identified NK cells, mediators of ADCC, in the cerebrospinal fluid of relapsing patients with MOGAD. Thus, MOGAD-derived autoantibodies are cytotoxic to MOG-expressing cells through multiple mechanisms, and assays quantifying CDC and ADCP may prove to be effective tools for predicting risk of future relapses.

The complement cascade is an important part of the innate immune system. In addition to helping the body to eliminate pathogens, however, complement activation also contributes to the pathogenesis of a wide range of kidney diseases. Recent work has revealed that uncontrolled complement activation is the key driver of several rare kidney diseases in children, including atypical hemolytic uremic syndrome and C3 glomerulopathy. In addition, a growing body of literature has implicated complement in the pathogenesis of more common kidney diseases, including acute kidney injury (AKI). Complement-targeted therapeutics are in use for a variety of diseases, and an increasing number of therapeutic agents are under development. With the implication of complement in the pathogenesis of AKI, complement-targeted therapeutics could be trialed to prevent or treat this condition. In this review, we discuss the evidence that the complement system is activated in pediatric patients with AKI, and we review the role of complement proteins as biomarkers and therapeutic targets in patients with AKI., (© 2022. The Author(s), under exclusive licence to International Pediatric Nephrology Association.)

The natural monoclonal antibody B4-IgM recognizes murine annexin 4 (mAn4) and exacerbates ischemia-reperfusion injury in many mouse models. During apoptosis, the intracellular mAn4 protein translocates to the membrane surface, remaining attached to the outer membrane leaflet where it is recognized by the anti-mAn4 B4-IgM antibody. B4-IgM does not recognize human annexin 4 (hAn4). However, the B4-IgM antibody epitope was detected by Western blot of unknown human proteins and by flow cytometry on all studied human cell lines undergoing apoptosis and on a minor subset of healthy cells. The B4-IgM antibody also recognizes the epitope on necrotic cells in cytoplasmic proteins, apparently entering through pores large enough to allow natural antibodies to penetrate the cells and bind to the epitope expressed on self-proteins. Using proteomics and site-directed mutagenesis, we found that B4-IgM binds to an epitope with post-translationally modified acetylated N-terminal methionine, followed by either glutamic or aspartic acid. The epitope is not induced by apoptosis or injury because this modification can also occur during protein translation. This finding reveals an additional novel mechanism whereby injured cells are detected by natural antibodies that initiate pathogenic complement activation through the recognition of epitopes that are shared across multiple proteins found in variable cell lines., (Copyright © 2023. Published by Elsevier Ltd.)

Introduction: In vivo , cancer cells respond to signals from the tumor microenvironment resulting in changes in expression of proteins that promote tumor progression and suppress anti-tumor immunity. This study employed an orthotopic immunocompetent model of lung cancer to define pathways that are altered in cancer cells recovered from tumors compared to cells grown in culture., Methods: Studies used four murine cell lines implanted into the lungs of syngeneic mice. Cancer cells were recovered using FACS, and transcriptional changes compared to cells grown in culture were determined by RNA-seq., Results: Changes in interferon response, antigen presentation and cytokine signaling were observed in all tumors. In addition, we observed induction of the complement pathway. We previously demonstrated that activation of complement is critical for tumor progression in this model. Complement can play both a pro-tumorigenic role through production of anaphylatoxins, and an anti-tumorigenic role by promoting complement-mediated cell killing of cancer cells. While complement proteins are produced by the liver, expression of complement proteins by cancer cells has been described. Silencing cancer cell-specific C3 inhibited tumor growth In vivo . We hypothesized that induction of complement regulatory proteins was critical for blocking the anti-tumor effects of complement activation. Silencing complement regulatory proteins also inhibited tumor growth, with different regulatory proteins acting in a cell-specific manner., Discussion: Based on these data we propose that localized induction of complement in cancer cells is a common feature of lung tumors that promotes tumor progression, with induction of complement regulatory proteins protecting cells from complement mediated-cell killing., Competing Interests: Author EP patent issued to Grid Therapeuticss for inhibitors of complement factor H; chief executive officer, founder, and board member of Grid Therapeutics; stock or stock options in Grid Therapeutics. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Kleczko, Poczobutt, Navarro, Laskowski, Johnson, Korpela, Gurule, Heasley, Hopp, Weiser-Evans, Gottlin, Bushey, Campa, Patz, Thurman and Nemenoff.)

The glomerulus is often the prime target of dysregulated alternative pathway (AP) activation. In particular, AP activation is the key driver of two severe kidney diseases: atypical hemolytic uremic syndrome and C3 glomerulopathy. Both conditions are associated with a variety of predisposing molecular defects in AP regulation, such as genetic variants in complement regulators, autoantibodies targeting AP proteins, or autoantibodies that stabilize the AP convertases (C3- and C5-activating enzymes). It is noteworthy that these are systemic AP defects, yet in both diseases pathologic complement activation primarily affects the kidneys. In particular, AP activation is often limited to the glomerular capillaries. This tropism of AP-mediated inflammation for the glomerulus points to a unique interaction between AP proteins in plasma and this particular anatomic structure. In this review, we discuss the pre-clinical and clinical data linking the molecular causes of aberrant control of the AP with activation in the glomerulus, and the possible causes of this tropism. Based on these data, we propose a model for why the kidney is so uniquely and frequently targeted in patients with AP defects. Finally, we discuss possible strategies for preventing pathologic AP activation in the kidney., (© 2022 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Uncontrolled alternative pathway activation is the primary driver of several diseases, and it contributes to the pathogenesis of many others. Consequently, diagnostic tests to monitor this arm of the complement system are increasingly important. Defects in alternative pathway regulation are strong risk factors for disease, and drugs that specifically block the alternative pathway are entering clinical use. A range of diagnostic tests have been developed to evaluate and monitor the alternative pathway, including assays to measure its function, expression of alternative pathway constituents, and activation fragments. Genetic studies have also revealed many disease-associated variants in alternative pathway genes that predict the risk of disease and prognosis. Newer imaging modalities offer the promise of non-invasively detecting and localizing pathologic complement activation. Together, these various tests help in the diagnosis of disease, provide important prognostic information, and can help guide therapy with complement inhibitory drugs., (© 2022 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Idiopathic nephrotic syndrome often responds to immunosuppressive treatment. Nevertheless, this syndrome-and the drugs used to treat it-remain important causes of patient morbidity. Idiopathic nephrotic syndrome is usually caused by minimal change disease or FSGS, diseases that primarily affect the podocytes. In spite of decades of research, the underlying causes of both diseases remain incompletely understood. There is, however, a large body of observational and experimental data linking the immune system with both minimal change disease and FSGS, including associations with systemic infections and hematologic malignancies. Perhaps most compellingly, many different immunomodulatory drugs are effective for treating idiopathic nephrotic syndrome, including biologic agents that have well-defined immune targets. In fact, the unexpected efficacy of targeted therapeutic agents has provided important new insights into the pathogenesis of these diseases. Given the large number of drugs that are available to deplete or block specific cells and molecules within the immune system, a better understanding of the immunologic causes of idiopathic nephrotic syndrome may lead to better diagnostic and therapeutic approaches., (Copyright © 2022 by the American Society of Nephrology.)

Complement activation at a particular location is determined by the balance of activating and inhibitory proteins. Factor H is a key regulator of the alternative pathway of complement, and genetic or acquired impairments in Factor H are associated with glomerular injury. The human Factor H-related proteins (FHRs) comprise a family of five proteins that are structurally related to Factor H. Variations in the genes or expression levels of the FHRs are also associated with glomerular disease, although the mechanisms of glomerular protection/injury are incompletely understood. To explore the role of the FHRs on complement regulation/dysregulation in the kidney, we expressed and purified recombinant murine FHRs (FHRs A, B, C and E). These four distinct FHRs contain binding regions with high amino acid sequence homology to binding regions within Factor H, but we observed different interactions of the FHRs with Factor H binding ligands, including heparin and C3d. There was differential binding of the FHRs to the resident kidney cell types (mesangial, glomerular endothelial, podocytes, and tubular epithelial). All four FHRs caused complement dysregulation on kidney cell surfaces in vitro, although the magnitude of the effect differed among the FHRs and also varied among the different kidney cells. However, only FHR E caused glomerular complement dysregulation when injected in vivo but did not exacerbate injury when injected into mice with ischemic acute kidney injury, an alternative pathway-mediated model. Thus, our experiments demonstrate that the FHRs have unique, and likely context-dependent, effects on the different cell types within the kidney., (Copyright © 2022 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

Extracellular vesicles (EV) are emerging mediators in several diseases. However, their role in the pathophysiology of antibody-mediated allograft rejection (AMR) has been poorly investigated. Here, we investigated the role of EV isolated from AMR patients in inducing tubular senescence and endothelial to mesenchymal transition (EndMT) and analyzed their miRNA expression profile. By multiplex bead flow cytometry, we characterized the immunophenotype of plasma AMR-derived EV and found a prevalent platelet and endothelial cell origin. In vitro, AMR-derived EV induced tubular senescence by upregulating SA-β Gal and CDKN1A mRNA. Furthermore, AMR-derived EV induced EndMT. The occurrence of tubular senescence and EndMT was confirmed by analysis of renal biopsies from the same AMR patients. Moreover, AMR-derived EV induced C3 gene upregulation and CFH downregulation in tubular epithelial cells, with C4d deposition on endothelial cells. Interestingly, RNase-mediated digestion of EV cargo completely abrogated tubular senescence and EndMT. By microarray analysis, miR-604, miR-515-3p, miR-let-7d-5p, and miR-590-3p were significantly upregulated in EV from AMR group compared with transplant controls, whereas miR-24-3p and miR-29a-3p were downregulated. Therefore, EV-associated miRNA could act as active player in AMR pathogenesis, unraveling potential mechanisms of accelerated graft senescence, complement activation and early fibrosis that might lead to new therapeutic intervention., (© 2022 The Authors. American Journal of Transplantation published by Wiley Periodicals LLC on behalf of The American Society of Transplantation and the American Society of Transplant Surgeons.)

Sustained complement activation is an underlying pathologic driver in many inflammatory and autoimmune diseases. Currently approved anti-complement therapies are directed at the systemic blockade of complement. Consequently, these therapies provide widespread inhibition of complement pathway activity, beyond the site of ongoing activation and the intended pharmacodynamic (PD) effects. Given the essential role for complement in both innate and adaptive immunity, there is a need for therapies that inhibit complement in diseased tissue while limiting systemic blockade. One potential approach focuses on the development of novel fusion proteins that enable tissue-targeted delivery of complement negative regulatory proteins. These therapies are expected to provide increased potency and prolonged tissue PD, decreased dosing frequency, and the potential for improved safety profiles. We created a library of bifunctional fusion proteins that direct a fragment of the complement negative regulator, complement receptor type 1 (CR1) to sites of tissue injury. Tissue targeting is accomplished through the binding of the fusion protein to complement C3 fragments that contain a surface-exposed C3d domain and which are covalently deposited on tissues where complement is being activated. To that end, we generated a fusion protein that contains an anti-C3d monoclonal antibody recombinantly linked to the first 10 consensus repeats of CR1 (CR1 1-10 ) with the intention of delivering high local concentrations of this complement negative regulatory domain to tissue-bound complement C3 fragments iC3b, C3dg and C3d. Biochemical and in vitro characterization identified several fusion proteins that inhibit complement while maintaining the C3d domain binding properties of the parent monoclonal antibody. Preclinical in vivo studies further demonstrate that anti-C3d fusion proteins effectively distribute to injured tissue and reduce C3 fragment deposition for periods beyond 14 days. The in vitro and in vivo profiles support the further evaluation of C3d mAb-CR1 1-10 as a novel approach to restore proper complement activation in diseased tissue in the absence of continuous systemic complement blockade., Competing Interests: KF, FL, JM, SR, SW, and SV are employees and shareholders of Q32 Bio Inc. SK and MS were previous employees of Q32 Bio Inc. and are shareholders of Q32 Bio Inc. SK is currently an employee of Compass Therapeutics. MS is currently an employee of Sanofi. ES is a paid consultant of Q32 Bio Inc. FT was previously a paid consultant of Q32 Bio Inc. JT is an employee of the University of Colorado School of Medicine and a consultant, founder, and shareholder of Q32 Bio Inc. VH is an employee of the University of Colorado School of Medicine; a founder, advisor, shareholder and previous consultant of Q32 Bio Inc. KF, FL, JM, SR, SW, SV, SK, MS, VH, and JT acknowledge a potential conflict of interest related to their relationship with Q32 Bio Inc. This work was funded by Q32 Bio Inc. (Waltham, MA, United States). The funder had the following involvement: study design, data collection, analysis and interpretation of data, preparation of this manuscript and the decision to submit it for publication. Medical writing and editorial support provided by Paul Littlebury, PhD, of Bioscript Stirling (Macclesfield, UK), was funded by Q32 Bio Inc., (Copyright © 2022 Fahnoe, Liu, Morgan, Ryan, Storek, Stark, Taylor, Holers, Thurman, Wawersik, Kalled and Violette.)

Studies have suggested imatinib mesylate (ImM) as a potential treatment for systemic lupus erythematosus nephritis (SLEN). However, ImM has limited renal excretion. The goal of the current research was to develop an ImM containing nanoformulation, conduct studies to evaluate pharmacokinetics, and determine whether kidney deposition can be enhanced in a mouse model of SLEN. A fish oil-based ImM oil-in-water nanoemulsion was developed and characterized for particle size, zeta potential, pH, and stability. MRL/MpJ-Faslrp (model of SLEN) and MRL/MpJ (control) mice (12-13 weeks) received one dose of ImM as either a nanoemulsion or naked drug. Pharmacokinetics and kidney deposition studies were performed. Statistics were conducted with a student's T-test. The nanoemulsion characteristics included particle size range of 60-80 nm, zeta potential of -6.6 to -7.8 mV, polydispersity index < 0.3, 3-day stability at 4 °C, and limited ImM leakage from the nanoemulsion in serum. Pharmacokinetics of the nanoformulation showed changes to pharmacokinetic parameters suggesting reduced systemic exposures (with reduced potential for toxicities) to ImM. Kidney deposition of ImM was threefold higher after 4 h in the MRL/MpJ-Faslrp mice that received the nanoformulation vs. naked drug. The current study showed encouraging results for development of a stable and well-characterized nanoemulsion for optimizing kidney deposition of ImM. Future strategies will define dose-efficacy and dose-toxicity relationships and evaluate approaches to further enhance kidney delivery and optimize deposition to the mesangial location of the kidney., (© 2021. Controlled Release Society.)

C3 glomerulopathy is a relatively new clinical entity that represents a challenge both to diagnose and to treat. As new therapeutic agents that act as complement inhibitors become available, many with an oral formulation, a better understanding of this disease and of the underlying complement dysregulation driving it has become increasingly useful to optimize patient care. Moreover, recent advances in research have clarified the role of complement in other glomerular diseases in which its role was less established, namely in immune-complex membranoproliferative glomerulonephritis (IC-MPGN), ANCA-vasculitis, IgA nephropathy, and idiopathic membranous nephropathy. Complement inhibitors are being studied in adult and adolescent clinical trials for these indications. This review summarizes current knowledge and future perspectives on every aspect of the diagnosis and management of C3 glomerulopathy and elucidates current understanding of the role of complement in this condition and in other glomerular diseases in children. An overview of ongoing trials involving therapeutic agents targeting complement in glomerular diseases is also provided., (© 2021. IPNA.)

Marginal zone (MZ) B cells produce broad-spectrum antibodies that protect against infection early in life. In some instances, antibody production requires MZ B cells to display pathogen antigens bound to major histocompatibility complex class II (MHC II) molecules to T cells. We describe the trogocytic acquisition of these molecules from conventional dendritic cells (cDCs). Complement component 3 (C3) binds to murine and human MHC II on cDCs. MZ B cells recognize C3 with complement receptor 2 (CR2) and trogocytose the MHC II-C3 complexes, which become exposed on their cell surface. The ubiquitin ligase MARCH1 limits the number of MHC II-C3 complexes displayed on cDCs to prevent their elimination through excessive trogocytosis. Capture of C3 by MHC II thus enables the transfer of cDC-like properties to MZ B cells.

About 5% of B cells in healthy mice and humans are allelically or isotypically included and hence co-express two different antibodies. In mice, dual antibody B cells (B 2R ) expand with systemic autoimmunity, co-express autoreactive and non-autoreactive antibodies, and participate in immune responses, but this phenomenon is strain dependent. This study was developed with two goals: 1) to establish the contribution of TLR and IFN receptor signaling to the development of germinal center B cells that express two antibodies in MRL/ lpr mice; and 2) to determine whether B 2R B cells are increased and particularly activated in a subset of adult patients diagnosed with systemic lupus erythematosus (SLE). Results from the MRL/ lpr studies indicate that the enhanced differentiation of dual-κ B cells into germinal center B cells is due to a heightened response to TLR7 and TLR9 signaling, further fueled by an increased response to type II IFN. To understand the clinical and translational implications of our observations in mouse B 2R B cells, cohorts of SLE patients and healthy controls were recruited and evaluated for expression of dual BCRs. Results from flow cytometry and microscopy revealed supraphysiological frequencies of κ + λ + B 2R cells in one fourth of the SLE patients. Abnormal numbers of κ + λ + B cells correlated with higher frequencies of activated naïve B cells and age-associated B cells, and a lower proportion of "B cells that are naïve IgD + " (BND). However, results from single cell V(D)J sequencing demonstrated that these high κ + λ + SLE patients harbored normal frequencies of κ + λ + and other B 2R B cells. and we further show that their B cells were instead decorated by κ and λ VH4-34 autoantibodies. Thus, our findings indicate that elevated flow cytometric detection of isotypically-included B cells can identify patients with high titers of B cell-reactive VH4-34 autoantibodies and abnormal distribution of B cell subsets relevant to autoimmunity., Competing Interests: JT receives royalties from Alexion Pharmaceuticals, Inc. and is a consultant for Q32 Bio, Inc., a company developing complement inhibitors. He also holds stock and will receive royalty income from Q32 Bio, Inc. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Peterson, Boackle, Taitano, Sang, Lang, Kelly, Rahkola, Miranda, Sheridan, Thurman, Rao, Torres and Pelanda.)

Introduction: Minimal change disease (MCD) is considered a podocyte disorder triggered by unknown circulating factors. Here, we hypothesized that the endothelial cell (EC) is also involved in MCD., Methods: We studied 45 children with idiopathic nephrotic syndrome (44 had steroid sensitive nephrotic syndrome [SSNS], and 12 had biopsy-proven MCD), 21 adults with MCD, and 38 healthy controls (30 children, 8 adults). In circulation, we measured products of endothelial glycocalyx (EG) degradation (syndecan-1, heparan sulfate [HS] fragments), HS proteoglycan cleaving enzymes (matrix metalloprotease-2 [MMP-2], heparanase activity), and markers of endothelial activation (von Willebrand factor [vWF], thrombomodulin) by enzyme-linked immunosorbent assay (ELISA) and mass spectrometry. In human kidney tissue, we assessed glomerular EC (GEnC) activation by immunofluorescence of caveolin-1 ( n = 11 MCD, n = 5 controls). In vitro , we cultured immortalized human GEnC with sera from control subjects and patients with MCD/SSNS sera in relapse ( n = 5 per group) and performed Western blotting of thrombomodulin of cell lysates as surrogate marker of endothelial activation., Results: In circulation, median concentrations of all endothelial markers were higher in patients with active disease compared with controls and remained high in some patients during remission. In the MCD glomerulus, caveolin-1 expression was higher, in an endothelial-specific pattern, compared with controls. In cultured human GEnC, sera from children with MCD/SSNS in relapse increased thrombomodulin expression compared with control sera., Conclusion: Our data show that alterations involving the systemic and glomerular endothelium are nearly universal in patients with MCD and SSNS, and that GEnC can be directly activated by circulating factors present in the MCD/SSNS sera during relapse., (© 2021 International Society of Nephrology. Published by Elsevier Inc.)

Background: Children who are critically ill with AKI suffer from high morbidity and mortality rates, and lack treatment options. Emerging evidence implicates the role of complement activation in AKI pathogenesis, which could potentially be treated with complement inhibitors. The purpose of this study is to evaluate the association between complement activation fragments and severity of AKI in children who are critically ill., Methods: A biorepository of samples from children who are critically ill from a prior multisite study was leveraged to identify children with stage 3 AKI and matched to patients without AKI on the basis of PELOD-2 (illness severity) scores. Specimens were analyzed for plasma and urine complement activation fragments of factor B, C3a, C4a, and sC5b-9. The primary outcomes were MAKE30 and severe AKI rates., Results: In total, 14 patients with stage 3 AKI (five requiring RRT) were matched to 14 patients without AKI. Urine factor Ba and plasma C4a levels increased stepwise as severity of AKI increased, from no AKI to stage 3 AKI, to stage 3 AKI with RRT need. Plasma C4a levels were independently associated with increased risk of MAKE30 outcomes (OR, 3.2; IQR, 1.1-8.9), and urine Ba (OR, 1.9; IQR, 1.1-3.1), plasma Bb (OR, 2.7; IQR, 1.1-6.8), C4a (OR, 13.0; IQR, 1.6-106.6), and C3a (OR, 3.3; IQR, 1.3-8.4) were independently associated with risk of severe stage 2-3 AKI on day 3 of admission., Conclusions: Multiple complement fragments increase as magnitude of AKI severity increases. Very high levels of urine Ba or plasma C4a may identify patients at risk for severe AKI, hemodialysis, and MAKE30 outcomes. The fragments may be useful as a functional biomarker of complement activation and may identify those patients to study complement inhibition to treat or prevent AKI in children who are critically ill. These findings suggest the need for further specific investigations of the role of complement activation in children who are critically ill and at risk of AKI., Competing Interests: A. Frazer-Abel reports having consultancy agreements with CSL Behring Pharmaceuticals and Ionis Pharmaceuticals; reports receiving honoraria from ECHO Just In Time Continuing Medical Education. B.P. Dixon reports having consultancy agreements with, and receiving honoraria from, Alexion Pharmaceuticals and Apellis Pharmaceuticals. J. Kendrick reports receiving research funding from Fresenius Medical Care Renal Therapies Group; and reports being a scientific advisor or member of the AstraZeneca Medical Advisory Committee, AMGEN Medical Advisory Board, Tricida Medical Advisory Board, and Velphoro Medical Advisory Board. J.M. Thurman reports having consultancy agreements with, having an ownership interest in, and receiving research funding from Q32 Bio, Inc.; and reports having patents and inventions with Alexion Pharmaceuticals and Q32 Bio, Inc. All remaining authors have nothing to disclose., (Copyright © 2021 by the American Society of Nephrology.)

Focal segmental glomerulosclerosis (FSGS) and minimal change disease (MCD) are common forms of idiopathic nephrotic syndrome. The causes of these diseases are incompletely understood, but the response of patients to immunosuppressive therapies suggests that their pathogenesis is at least in part immune mediated. Preclinical and clinical research indicates that activation of the classical pathway of complement contributes to glomerular injury in FSGS. Glomerular IgM deposits are also prominent in some patients, raising the possibility that IgM is a trigger of classical pathway activation. In the present study, we examined the pattern of complement activation in the glomeruli and plasma of patients with nephrotic syndrome. We also tested whether patients with FSGS and MCD have elevated levels of natural IgM reactive with epitopes on glomerular endothelial cells and cardiolipin. We found evidence of classical pathway activation in patients with idiopathic nephrotic syndrome compared with healthy control subjects. We also detected higher levels of self-reactive IgM to both targets. Based on these results, IgM and classical pathway activation may contribute to disease pathogenesis in some patients with FSGS and MCD. NEW & NOTEWORTHY IgM is detected in biopsies from some patients with nephrotic syndrome, although this has been attributed to passive trapping of the protein. We found, however, that IgM colocalizes with complement activation fragments in some glomeruli. We also found that affected patients had higher levels of IgM reactive to glomerular endothelial cell epitopes. Thus, IgM activates the complement system in the glomeruli of some patients with nephrotic syndrome and may contribute to injury.

Effects of cholecalciferol (VitD 3 ) and calcitriol (1,25-VitD 3 ), on the expression and function of major vitamin D metabolizing enzymes (cytochrome P450 [CYP]2R1, CYP24A1) and select drug transport pathways ( ABCB1/ P-gp, SLCO4C1 /OATP4C1) were evaluated in human kidney proximal tubule epithelial cells (hPTECs) under normal and uraemic serum conditions.hPTECs were incubated with 10% normal or uraemic serum for 24 h followed by treatment with 2% ethanol vehicle, or 100 and 240 nM doses of VitD 3 , or 1,25-VitD 3 for 6 days. The effects of treatment on mRNA and protein expression and functional activity of select CYP enzymes and transporters were assessedUnder uraemic serum, treatment with 1,25-VitD 3 resulted in increased mRNA but decreased protein expression of CYP2R1. Activity of CYP2R1 was not influenced by serum or VitD analogues. CYP24A1 expression was increased with 1,25-VitD 3 under normal as well as uraemic serum, although to a lesser extent. ABCB1 /P-gp mRNA expression increased under normal and uraemic serum, with exposure to 1,25-VitD 3 . SLCO4C1 /OATP4C1 exhibited increased mRNA but decreased protein expression, under uraemic serum + 1,25-VitD 3 . Functional assessments of transport showed no changes regardless of exposure to serum or 1,25-VitD 3 .Key findings indicate that uraemic serum and VitD treatment led to differential effects on the functional expression of CYPs and transporters in hPTECs.

The outbreak of COVID-19 disease, caused by SARS-CoV-2 beta-coronovirus, urges a focused search for the underlying mechanisms and treatment options. The lung is the major target organ of COVID-19, wherein the primary cause of mortality is hypoxic respiratory failure, resulting from acute respiratory distress syndrome, with severe hypoxemia, often requiring assisted ventilation. While similar in some ways to acute respiratory distress syndrome secondary to other causes, lungs of some patients dying with COVID-19 exhibit distinct features of vascular involvement, including severe endothelial injury and cell death via apoptosis and/or pyroptosis, widespread capillary inflammation, and thrombosis. Furthermore, the pulmonary pathology of COVID-19 is characterized by focal inflammatory cell infiltration, impeding alveolar gas exchange resulting in areas of local tissue hypoxia, consistent with potential amplification of COVID-19 pathogenicity by hypoxia. Vascular endothelial cells play essential roles in both innate and adaptive immune responses, and are considered to be "conditional innate immune cells" centrally participating in various inflammatory, immune pathologies. Activated endothelial cells produce cytokines/chemokines, dynamically recruit and activate inflammatory cells and platelets, and centrally participate in pro-thrombotic processes (thrombotic microangiopathies). Initial reports presented pathological findings of localized direct infection of vascular endothelial cells with SARS-CoV-2, yet emerging evidence does not support direct infection of endothelial or other vascular wall cell and thus widespread endothelial cell dysfunction and inflammation may be better explained as secondary responses to epithelial cell infection and inflammation. Endothelial cells are also actively engaged in a cross-talk with the complement system, the essential arm of innate immunity. Recent reports present evidence for complement deposition in SARS-CoV-2-damaged lung microcirculation, further strengthening the idea that, in severe cases of COVID-19, complement activation is an essential player, generating destructive hemorrhagic, capillaritis-like tissue damage, clotting, and hyperinflammation. Thus, complement-targeted therapies are actively in development. This review is intended to explore in detail these ideas., (© The Author(s) 2021.)

Cardiovascular disease (CVD) is the most common cause of death in patients with native and post-transplant chronic kidney disease (CKD). To identify new biomarkers of vascular injury and inflammation, we analyzed the proteome of plasma and circulating extracellular vesicles (EVs) in native and post-transplant CKD patients utilizing an aptamer-based assay. Proteins of angiogenesis were significantly higher in native and post-transplant CKD patients versus healthy controls. Ingenuity pathway analysis (IPA) indicated Ephrin receptor signaling, serine biosynthesis, and transforming growth factor-β as the top pathways activated in both CKD groups. Pro-inflammatory proteins were significantly higher only in the EVs of native CKD patients. IPA indicated acute phase response signaling, insulin-like growth factor-1, tumor necrosis factor-α, and interleukin-6 pathway activation. These data indicate that pathways of angiogenesis and inflammation are activated in CKD patients' plasma and EVs, respectively. The pathways common in both native and post-transplant CKD may signal similar mechanisms of CVD.

Rhabdomyolysis is frequently associated with kidney injury. Unfortunately, there are no specific treatments for this condition, and patient care primarily consists of supportive measures. In this edition of Kidney International, Boudhabhay et al. demonstrate that myoglobin released from injured skeletal muscle cells triggers tubulointerstitial complement activation. In a mouse model of the disease, interventions that scavenged free heme or that prevented complement activation ameliorated kidney injury, raising the possibility that these strategies may be effective treatments for the condition., (Copyright © 2020 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

Immunofluorescence staining of tissues has become a reliable and informative technique used in a diverse set of applications, ranging from simple detection of an antigen of interest in a specific location to the semiquantitative analysis of spatial relationships between multiple antigens and/or cell types. During complement activation, circulating complement proteins are covalently fixed to target tissues, providing a durable marker of complement activation in the tissue, and many of these proteins can be readily detected by immunofluorescence microscopy. In general, staining for complement fragments is much like staining for other noncomplement epitopes. However, one key difference is the diligence with which unfixed tissues must be handled when staining for complement fragment. Here we explain the process of dual staining frozen mouse kidney sections for the complement proteins C3 and C4. Throughout the protocol, we will emphasize important steps for preserving complement protein integrity as well as tips to improve the signal-to-noise ratio to improve overall image quality.

It has long been known that the complement cascade is activated in various forms of glomerulonephritis. In many of these diseases, immune-complexes deposit in the glomeruli and activate the classical pathway. Researchers have also identified additional mechanisms by which complement is activated in the kidney, including diseases in which the alternative and lectin pathways are activated. The kidney appears to be particularly susceptible to activation of the alternative pathway, and this pathway has been implicated as a primary driver of atypical hemolytic uremic syndrome, C3 glomerulopathy, anti-neutrophil cytoplasmic antibody-associated vasculitis, as well as some forms of immune-complex glomerulonephritis. In this paper we review the shared and distinct mechanisms by which complement is activated in these different diseases. We also review the opportunities for using therapeutic complement inhibitors to treat kidney diseases., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Although it was long believed that the complement system helps the body to identify and remove transformed cells, it is now clear that complement activation contributes to carcinogenesis and can also help tumors to escape immune-elimination. Complement is activated by several different mechanisms in various types of cancer, and complement activation fragments have multiple different downstream effects on cancer cells and throughout the tumor microenvironment. Thus, the role of complement activation in tumor biology may vary among different types of cancer and over time within a single tumor. In multiple different pre-clinical models, however, complement activation has been shown to recruit immunosuppressive myeloid cells into the tumor microenvironment. These cells, in turn, suppress anti-tumor T cell immunity, enabling the tumor to grow. Based on extensive pre-clinical work, therapeutic complement inhibitors hold great promise as a new class of immunotherapy. A greater understanding of the role of complement in tumor biology will improve our ability to identify those patients most likely to benefit from this treatment and to rationally combine complement inhibitors with other cancer therapies.

Kidney glomerulosclerosis commonly progresses to end-stage kidney failure, but pathogenic mechanisms are still poorly understood. Here, we show that podocyte expression of decay-accelerating factor (DAF/CD55), a complement C3 convertase regulator, crucially controls disease in murine models of adriamycin (ADR)-induced focal and segmental glomerulosclerosis (FSGS) and streptozotocin (STZ)-induced diabetic glomerulosclerosis. ADR induces enzymatic cleavage of DAF from podocyte surfaces, leading to complement activation. C3 deficiency or prevention of C3a receptor (C3aR) signaling abrogates disease despite DAF deficiency, confirming complement dependence. Mechanistic studies show that C3a/C3aR ligations on podocytes initiate an autocrine IL-1β/IL-1R1 signaling loop that reduces nephrin expression, causing actin cytoskeleton rearrangement. Uncoupling IL-1β/IL-1R1 signaling prevents disease, providing a causal link. Glomeruli of patients with FSGS lack DAF and stain positive for C3d, and urinary C3a positively correlates with the degree of proteinuria. Together, our data indicate that the development and progression of glomerulosclerosis involve loss of podocyte DAF, triggering local, complement-dependent, IL-1β-induced podocyte injury, potentially identifying new therapeutic targets., Competing Interests: Disclosures: J.M. Thurman reported a patent to US 7,999,082 issued "Alexion Pharmaceuticals, Inc." and a patent to US 8,703,140 B2 issued "Alexion Pharmaceuticals, Inc." J. Manrique reported personal fees from Alexion Pharmaceuticals, Inc. P.S. Heeger reported personal fees from Mallinckrodt Pharmaceuticals during the conduct of the study. No other disclosures were reported., (© 2020 Angeletti et al.)

Hepatocellular carcinoma (HCC) is difficult to detect, carries a poor prognosis, and is one of few cancers with an increasing yearly incidence. Molecular defects in complement factor H (CFH), a critical regulatory protein of the complement alternative pathway (AP), are typically associated with inflammatory diseases of the eye and kidney. Little is known regarding the role of CFH in controlling complement activation within the liver. While studying aging CFH-deficient (fH-/-) mice, we observed spontaneous hepatic tumor formation in more than 50% of aged fH-/- males. Examination of fH-/- livers (3-24 months) for evidence of complement-mediated inflammation revealed widespread deposition of complement-activation fragments throughout the sinusoids, elevated transaminase levels, increased hepatic CD8+ and F4/80+ cells, overexpression of hepatic mRNA associated with inflammatory signaling pathways, steatosis, and increased collagen deposition. Immunostaining of human HCC biopsies revealed extensive deposition of complement fragments within the tumors. Investigating the Cancer Genome Atlas also revealed that increased CFH mRNA expression is associated with improved survival in patients with HCC, whereas mutations are associated with worse survival. These results indicate that CFH is critical for controlling complement activation in the liver, and in its absence, AP activation leads to chronic inflammation and promotes hepatic carcinogenesis.

The complement system alternative pathway (AP) can be activated excessively in inflammatory diseases, particularly when there is defective complement regulation. For instance, deficiency in complement regulators CD55 and CD59, leads to paroxysmal nocturnal hemoglobinuria (PNH), whereas Factor H mutations predispose to atypical hemolytic uremic syndrome (aHUS), both causing severe thrombohemolysis. Despite eculizumab being the treatment for these diseases, benefits vary considerably among patients. Understanding the molecular mechanisms involved in complement regulation is essential for developing new treatments. Properdin, the positive AP regulator, is essential for complement amplification by stabilizing enzymatic convertases. In this study, the role of properdin in red blood cell (RBC) lysis and endothelial cell opsonization in these AP-mediated diseases was addressed by developing in vitro assays using PNH patient RBCs and human primary endothelial cells, where the effects of inhibiting properdin, using novel monoclonal antibodies (MoAbs) that we generated and characterized, were compared to other complement inhibitors. In in vitro models of PNH, properdin inhibition prevented hemolysis of patient PNH type II and III RBCs more than inhibition of Factor B, C3, and C5 (>17-fold, or >81-fold, or >12-fold lower molar IC 90 values, respectively). When tested in an in vitro aHUS hemolysis model, the anti-properdin MoAbs had 11-fold, and 86-fold lower molar IC 90 values than inhibition of Factor B, or C3, respectively ( P < 0.0001). When comparing target/inhibitor ratios in all hemolysis assays, inhibiting properdin was at least as efficient as the other complement inhibitors in most cases. In addition, using in vitro endothelial cell assays, the data indicate a critical novel role for properdin in promoting complement activation on human endothelial cells exposed to heme (a hemolysis by-product) and rH19-20 (to inhibit Factor H cell-surface protection), as occurs in aHUS. Inhibition of properdin or C3 in this system significantly reduced C3 fragment deposition by 75%. Altogether, the data indicate properdin is key in promoting RBC lysis and complement activation on human endothelial cells, contributing to the understanding of PNH and aHUS pathogenesis. Further studies to determine therapeutic values of inhibiting properdin in complement-mediated diseases, in particular those that are characterized by AP dysregulation, are warranted., (Copyright © 2020 Chen, Galwankar, Emch, Menon, Cortes, Thurman, Merrill, Brodsky and Ferreira.)

Pulmonary (arterial) hypertension (PH/PAH) is a life-threatening cardiopulmonary disorder. Experimental evidence suggests involvement of inflammatory and autoimmune processes in pathogenesis of PH/PAH, however the triggering and disease-promoting mechanisms remain unknown. The complement system is a key arm of innate immunity implicated in various pro-inflammatory and autoimmune diseases, yet, surprisingly little is known about the role of complement in PH/PAH pathogenesis. The preponderance of the existing data associates complement with PH/PAH via analysis of plasma and does not study the lung directly. Therefore, we aimed to resolve this by analyzing both the mechanisms of local lung-specific complement activation and the correlation of dysregulated plasma complement to clinical outcome in PAH patients. In our recent studies, reviewed herein, we show, for the first time, that  immunoglobulin-driven activation of the complement cascade, specifically its alternative pathway, in the pulmonary perivascular areas, is a key mechanism initiating pro-inflammatory processes in the early stage of experimental hypoxic PH (a form of "sterile inflammation"). In human patients with end-stage PAH, we have demonstrated that perivascular deposition of immunoglobulin G (IgG) and activation of the complement cascade are "longitudinally" persistent in the disease. We also showed, using unbiased network analysis, that plasma complement signaling, including again the Alternative pathway, is a prognostic factor of survival in patients with idiopathic PAH (IPAH). Based on these initial findings, we suggest that vascular-specific, immunoglobulin-driven dysregulated complement signaling triggers and maintains pulmonary vascular remodeling and PH. Future experiments in this area would facilitate discoveries on whether complement signaling can serve both as a biomarker and therapeutic target in PH/PAH., (Copyright ©2020 The Author(s).)

The complement cascade was first recognized as a downstream effector system of antibody-mediated cytotoxicity. Consistent with this view, it was discovered in the 1960s that complement is activated in the glomeruli of patients with immune complex glomerulonephritis. More recently, research has shown that complement system has many additional functions relating to regulation of the immune response, homeostasis, and metabolism. It has also become clear that the complement system is important to the pathogenesis of many non-immune complex mediated kidney diseases. In fact, in atypical hemolytic uremic syndrome and C3 glomerulopathy, uncontrolled complement activation is the primary driver of disease. Complement activation generates multiple pro-inflammatory fragments, and if not properly controlled it can cause fulminant tissue injury. Furthermore, the mechanisms of complement activation and complement-mediated injury vary from disease to disease. Many new drugs that target the complement cascade are in clinical development, so it is important to fully understand the biology of the complement system and its role in disease., (Copyright © 2019 National Kidney Foundation, Inc. Published by Elsevier Inc. All rights reserved.)

Antibody-mediated rejection (AbMR) adversely affects long-term graft survival in kidney transplantation. Currently, the diagnosis of AbMR requires a kidney biopsy, and detection of complement C4d deposition in the allograft is one of the diagnostic criteria. Complement activation also generates several soluble fragments which could potentially provide non-invasive biomarkers of the process. Furthermore, microvesicles released into the plasma from injured cells can serve as biomarkers of vascular injury. To explore whether soluble complement fragments or complement fragments bound to endothelial microvesicles can be used to non-invasively detect AbMR, we developed an in vitro model in which human endothelial cells were exposed to anti-HLA antibodies and complement sufficient serum. We found that complement fragments C4a and sC5b-9 were increased in the supernatants of cells exposed to complement-sufficient serum compared to cells treated complement-deficient serum. Furthermore, complement activation on the cell surface was associated with the release of microvesicles bearing C4 and C3 fragments. We next measured these analytes in plasma from kidney transplant recipients with biopsy-proven acute AbMR (n = 9) and compared the results with those from transplant recipients who also had impaired allograft function but who did not have AbMR (n = 30). Consistent with the in vitro results, complement fragments C4a and Ba were increased in plasma from patients with AbMR compared to control subjects (P < 0.001 and P < 0.01, respectively). Endothelial microvesicle counts were not increased in patients with AbMR, however, and the number of microvesicles with C4 and C3 bound to the surface was actually lower compared to control subjects (both P < 0.05). Our results suggest that plasma complement activation fragments may be useful as non-invasive biomarkers of antibody-mediated complement activation within the allograft. Complement-opsonized endothelial microvesicles are decreased in patients with AbMR, possibly due to enhanced clearance of microvesicles opsonized with C3 and C4 fragments., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Rationale: Pulmonary hypertension (PH) is a life-threatening cardiopulmonary disorder in which inflammation and immunity have emerged as critical early pathogenic elements. Although proinflammatory processes in PH and pulmonary arterial hypertension (PAH) are the focus of extensive investigation, the initiating mechanisms remain elusive. Objectives: We tested whether activation of the complement cascade is critical in regulating proinflammatory and pro-proliferative processes in the initiation of experimental hypoxic PH and can serve as a prognostic biomarker of outcome in human PAH. Methods: We used immunostaining of lung tissues from experimental PH models and patients with PAH, analyses of genetic murine models lacking specific complement components or circulating immunoglobulins, cultured human pulmonary adventitial fibroblasts, and network medicine analysis of a biomarker risk panel from plasma of patients with PAH. Measurements and Main Results: Pulmonary perivascular-specific activation of the complement cascade was identified as a consistent critical determinant of PH and PAH in experimental animal models and humans. In experimental hypoxic PH, proinflammatory and pro-proliferative responses were dependent on complement (alternative pathway and component 5), and immunoglobulins, particularly IgG, were critical for activation of the complement cascade. We identified Csf2/GM-CSF as a primary complement-dependent inflammatory mediator. Furthermore, using network medicine analysis of a biomarker risk panel from plasma of patients with PAH, we demonstrated that complement signaling can serve as a prognostic factor for clinical outcome in PAH. Conclusions: This study establishes immunoglobulin-driven dysregulated complement activation as a critical pathobiological mechanism regulating proinflammatory and pro-proliferative processes in the initiation of experimental hypoxic PH and demonstrates complement signaling as a critical determinant of clinical outcome in PAH.

Humoral autoimmunity is central to the development of systemic lupus erythematosus (SLE). Complement receptor type 2 (CR2)/CD21 plays a key role in the development of high-affinity Abs and long-lasting memory to foreign Ags. When CR2 is bound by its primary C3 activation fragment-derived ligand, designated C3d, it coassociates with CD19 on B cells to amplify BCR signaling. C3d and CR2 also mediate immune complex binding to follicular dendritic cells. As the development of SLE involves subversion of normal B cell tolerance checkpoints, one might expect that CR2 ligation by C3d-bound immune complexes would promote development of SLE. However, prior studies in murine models of SLE using gene-targeted Cr2 -/- mice, which lack both CR2 and complement receptor 1 (CR1), have demonstrated contradictory results. As a new approach, we developed a highly specific mouse anti-mouse C3d mAb that blocks its interaction with CR2. With this novel tool, we show that disruption of the critical C3d-CR2 ligand-receptor binding step alone substantially ameliorates autoimmunity and renal disease in the MRL/ lpr model of SLE., (Copyright © 2019 by The American Association of Immunologists, Inc.)