Systemic and local complement activation in peritoneal dialysis patients via conceivably distinct pathways.

BACKGROUND: Despite several advantages compared to haemodialysis (HD), peritoneal dialysis (PD) remains an underused dialysis technique due to its high technique failure rate related to membrane fibrosis and peritonitis events. Previous work has suggested a harmful role for the complement system in these processes, highlighting the need for a more comprehensive examination in PD. METHODS: Plasma levels of C1q, mannose-binding lectin (MBL), Properdin, Factor D, C3d/C3-ratio and soluble membrane attack complex (sC5b-9) were determined in PD patients (n = 55), HD patients (n = 41), non-dialysis chronic kidney disease (CKD) patients (n = 15) and healthy controls (n = 14). Additionally, C1q, MBL, Properdin, Factor D and sC5b-9 levels were assessed in the peritoneal dialysis fluid (PDF). In a subgroup, interleukin-6, matrix metalloproteinase-2 (MMP-2), myeloperoxidase (MPO) and elastase were measured in the PDF. RESULTS: PD patients had significantly higher systemic levels of sC5b-9 compared to healthy controls, CKD and HD patients (p < 0.001). Plasma levels of C1q and C3d/C3-ratios were significantly associated with systemic sC5b-9 levels (p < 0.001). Locally, sC5b-9 was detected in the PDF of all PD patients, and levels were approximately 33% of those in matched plasma, but they did not correlate. In the PDF, only Properdin levels remained significantly associated with PDF sC5b-9 levels in multivariate analysis (p < 0.001). Additionally, PDF levels of sC5b-9 positively correlated with elastase, MPO and MMP-2 levels in the PDF (p < 0.01). CONCLUSIONS: Our data reveal both systemic and local complement activation in PD patients. Furthermore, these two processes seem independent considering the involvement of different pathways and the lack of correlation.

Female but Not Male Mice Deficient in Soluble IgM Are Susceptible to Chemically Induced Glomerular Injury.

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.

A non-muscle myosin heavy chain 9 genetic variant is associated with graft failure following kidney transplantation.

BACKGROUND: Despite current matching efforts to identify optimal donor-recipient pairs for kidney transplantation, alloimmunity remains a major source of late transplant failure. Additional genetic parameters in donor-recipient matching could help improve longterm outcomes. Here, we studied the impact of a non-muscle myosin heavy chain 9 gene (MYH9) polymorphism on allograft failure. METHODS: We conducted an observational cohort study, analyzing the DNA of 1,271 kidney donor-recipient transplant pairs from a single academic hospital for the MYH9 rs11089788 C>A polymorphism. The associations of the MYH9 genotype with risk of graft failure, biopsy-proven acute rejection (BPAR), and delayed graft function (DGF) were estimated. RESULTS: A trend was seen in the association between the MYH9 polymorphism in the recipient and graft failure (recessive model, p = 0.056), but not for the MYH9 polymorphism in the donor. The AA-genotype MYH9 polymorphism in recipients was associated with higher risk of DGF (p = 0.03) and BPAR (p = 0.021), although significance was lost after adjusting for covariates (p = 0.15 and p = 0.10, respectively). The combined presence of the MYH9 polymorphism in donor-recipient pairs was associated with poor long-term kidney allograft survival (p = 0.04), in which recipients with an AA genotype receiving a graft with an AA genotype had the worst outcomes. After adjustment, this combined genotype remained significantly associated with 15-year death-censored kidney graft survival (hazard ratio, 1.68; 95% confidence interval, 1.05-2.70; p = 0.03). CONCLUSION: Our results reveal that recipients with an AA-genotype MYH9 polymorphism receiving a donor kidney with an AA genotype have significantly elevated risk of graft failure after kidney transplantation.

Noninvasive Detection of iC3b/C3d Deposits in the Kidney Using a Novel Bioluminescent Imaging Probe.

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.

Successful pharmacological intervention at different levels of the complement system in an in vitro complement fixation model for bullous pemphigoid.

Bullous pemphigoid (BP) is characterized by deposition of immunoglobulins and complement along the epidermal basement membrane (BM). In humans, there is a lack of functional studies targeting the complement system (CS). This study investigates activation of all complement pathways in BP skin biopsies. Moreover, pharmacological inhibition at different levels of the CS was investigated using anti-complement compounds in a complement fixation BP assay. In this retrospective study, 21 frozen biopsies from BP patients were stained by direct immunofluorescence for C1q, MBL, ficolin-2, C4d, properdin, C3c and C5b-9. Sera from 10 patients were analysed in a complement fixation assay in the presence of C1 inhibitor, anti-factor B monoclonal antibody (mAb), anti-C3 mAb and anti-C5 mAb and compared with dexamethasone. The two readouts were the quantity of complement deposited along the BM and the release of sC5b-9 in the supernatant. Our results show classical and alternative complement pathway activation in BP skin biopsies, but could not demonstrate significant lectin pathway activation. In contrast to dexamethasone, complement deposition along the BM could be selectively inhibited by anti-C1 and anti- factor B. More downstream, selective intervention at the level of C3 and C5 could effectively reduce complement deposition along the BM and the release of sC5b-9 in the supernatant. This study shows that selective intervention in either the classical, alternative or terminal pathway prevented deposition of complement along the BM in an in vitro BP model. The results of our study greatly encourage the clinical development of complement inhibitors for the treatment of BP.

An interleukin 6-based genetic risk score strengthened with interleukin 10 polymorphisms associated with long-term kidney allograft outcomes.

Of all kidney transplants, half are still lost in the first decade after transplantation. Here, using genetics, we probed whether interleukin 6 (IL-6) could be a target in kidney transplantation to improve graft survival. Additionally, we investigated if a genetic risk score (GRS) based on IL6 and IL10 variants could improve prognostication of graft loss. In a prospective cohort study, DNA of 1271 donor-recipient kidney transplant pairs was analyzed for the presence of IL6, IL6R, IL10, IL10RA, and IL10RB variants. These polymorphisms and their GRS were then associated with 15-year death-censored allograft survival. The C|C-genotype of the IL6 polymorphism in donor kidneys and the combined C|C-genotype in donor-recipient pairs were both associated with a reduced risk of graft loss (p = .043 and p = .042, respectively). Additionally, the GRS based on IL6, IL6R, IL10, IL10RA, and IL10RB variants was independently associated with the risk of graft loss (HR 1.53, 95%-CI [1.32-1.84]; p < .001). Notably, the GRS improved risk stratification and prediction of graft loss beyond the level of contemporary clinical markers. Our findings reveal the merits of a polygenic IL-6-based risk score strengthened with IL-10- polymorphisms for the prognostication and risk stratification of late graft failure in kidney transplantation.

Factor H related proteins modulate complement activation on kidney cells.

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.

Early-Career Complementologists (ECCO) - Past achievements and future directions.

The Early-Career Complementologists (ECCO) is a task force that was established, in close collaboration with the European Complement Network (ECN) and the International Complement Society (ICS), with the specific mission to support and connect early-career researchers (ECRs) in the complement field. ECRs are junior scientists at the early stages of their training which include undergraduate as well as graduate students, Ph.D. graduates, and post-doctoral fellows. This unique population within the scientific community represents the next generation of scientific leaders. However, ECRs are faced with key challenges and the COVID-19 pandemic has disproportionately impacted them. In this paper, we provide further insight into specific needs and challenges of ECRs in the complement field. We surveyed 52 ECRs in the complement field and assessed their perceptions of 1) mentor and peer support, 2) working conditions as well as 3) career interests and prospects. Furthermore, we review the various activities carried out by ECCO over the past years such as our social media presence, social events, and newly-created awards. We also discuss the future activities and events to be carried out by ECCO. Through these initiatives and activities, ECCO strives to boost collaborations between ECRs, provide recognition, and improve the visibility of their work. In addition, continuous joint efforts must also be made by the scientific community, research institutes, and funding organizations to nurture and invest in ECRs.

Evaluating the clinical utility of measuring levels of factor H and the related proteins.

After years of disappointing clinical results, the tide has finally changed and complement targeted-therapies have become a validated and accepted treatment option for several diseases. These accomplishments have revitalized the field and brought renewed attention to the prospects that complement therapeutics can offer. Streamlining diagnostics and therapeutics is imperative in this new era of clinical use of complement therapeutics. However, the incredible success in therapeutics has not been accompanied by the development of novel standardized tools for complement testing. Complement biomarkers can assist in the risk assessment and diagnosis of diseases as well as the prediction of disease progression and treatment response. Recently, a group of complement proteins has been suggested to be highly relevant in various complement-associated disorders, namely the human factor H (FH) protein family. This family of closely related proteins consists of FH, FH-like protein 1, and five factor H-related proteins, and they have been linked to eye, kidney, infectious, vascular, and autoimmune diseases as well as cancer. The goal of this review is to provide a comprehensive overview of the available data on circulating levels of FH and its related proteins in different pathologies. In addition, we examined the current literature to determine the clinical utility of measuring levels of the FH protein family in health and disease. Finally, we discuss future steps that are needed to make their clinical translation a reality.

Corrigendum: A Family Affair: Addressing the Challenges of Factor H and the Related Proteins.

[This corrects the article DOI: 10.3389/fimmu.2021.660194.].

Sex and Age-Related Differences in Complement Factors Among Patients With Intermediate Age-Related Macular Degeneration.

Purpose: Age-related macular degeneration (AMD) is an acquired degenerative disease of the retina classified into early, intermediate, and advanced AMD. A key factor in the pathogenesis of AMD is the complement system. The interaction of age and sex with the complement system may affect the risk of developing AMD. The purpose of this study was to determine if there were sex-specific differences in levels of complement factors among patients with the intermediate phenotype of AMD (iAMD) and explore the correlation between age and complement proteins. Methods: We studied complement factors in patients with iAMD and controls without AMD. Nonparametric, rank-based linear regressions including a sex by AMD interaction were used to compare levels for each analyte. Correlations between age and complement proteins were evaluated using the Spearman rank correlation coefficient. Results: We found significantly higher levels of factor B and factor I in females compared with males with iAMD, whereas no differences were seen in complement levels in male and female controls. The ratios of Ba/factor B, C3a/C3, C4b/C4, and C5a/C5 were not different in males and females with iAMD. Conclusions: We demonstrate disparities in a subset of systemic complement factors between females and males with iAMD, but apparent complement turnover as measured by ratios of activation fragments to intact molecules was not different between these groups. The results suggest that complement system levels, including complement regulator factor I, exhibits sex-related differences in patients with iAMD and highlights that stratification by sex might be helpful in the interpretation of clinical trials of anticomplement therapy.

Tumor Necrosis Factor-α Gene Polymorphism is Associated with Short- and Long-Term Kidney Allograft Outcomes.

Introduction: Kidney transplantation has excellent short-term results with current immunosuppression regimes, but long-term outcomes have barely improved over the past two decades. Hence, there is a need for new therapeutic options to increase long-term survival of kidney grafts. Drug development for kidney transplantation has slowly plateaued, limiting progress while making drug repurposing an attractive alternative. We, therefore, investigated the impact of tumor necrosis factor-alpha (TNF-α) gene (TNF) polymorphisms on kidney graft survival after transplantation. Methods: We performed a prospective cohort study to assess the association of TNF polymorphisms (rs1800629 G>A and rs3093662 A>G) with primary non-function and death-censored kidney allograft survival in 1271 kidney transplant pairs from the University Medical Center Groningen in The Netherlands. Results: The G-allele of the TNF rs3093662 polymorphism in donor kidneys was associated with a higher risk of immediate graft loss (odds ratio: 2.05; 95%-CI: 1.06-3.97; P = 0.032). Furthermore, the G-allele of this TNF rs3093662 polymorphism in the donor was also associated with worse 5-year, 10-year, and 15-year death-censored kidney graft survival (P < 0.05). The cumulative incidence of graft loss was 15.9% in the reference AA-genotype group and 25.2% in the AG/GG-genotype group, respectively. In multivariable analysis, the association between the TNF rs3093662 polymorphism in the donor and 15-year death-censored kidney graft survival remained significant (hazard ratio: 1.51; 95%-CI: 1.05-2.19, P = 0.028). Discussion: In conclusion, kidney allografts possessing a high-producing TNF polymorphism have a greater risk of immediate and late graft loss. Our study adds to a growing body of literature indicating the potential of TNF-α blockade in improving kidney transplantation outcomes.

A functional TGFB1 polymorphism in the donor associates with long-term graft survival after kidney transplantation.

BACKGROUND: Improvement of long-term outcomes in kidney transplantation remains one of the most pressing challenges, yet drug development is stagnating. Human genetics offers an opportunity for much-needed target validation in transplantation. Conflicting data exist about the effect of transforming growth factor-beta 1 (TGF-ß1) on kidney transplant survival, since TGF-ß1 has pro-fibrotic and protective effects. We investigated the impact of a recently discovered functional TGFB1 polymorphism on kidney graft survival. METHODS: We performed an observational cohort study analysing recipient and donor DNA in 1271 kidney transplant pairs from the University Medical Centre Groningen in The Netherlands, and associated a low-producing TGFB1 polymorphism (rs1800472-C > T) with 5-, 10- and 15-year death-censored kidney graft survival. RESULTS: Donor genotype frequencies of rs1800472 in TGFB1 differed significantly between patients with and without graft loss (P = 0.014). Additionally, the low-producing TGFB1 polymorphism in the donor was associated with an increased risk of graft loss following kidney transplantation (hazard ratio = 2.12 for the T-allele; 95% confidence interval 1.18-3.79; P = 0.012). The incidence of graft loss within 15 years of follow-up was 16.4% in the CC-genotype group and 31.6% in the CT-genotype group. After adjustment for transplant-related covariates, the association between the TGFB1 polymorphism in the donor and graft loss remained significant. In contrast, there was no association between the TGFB1 polymorphism in the recipient and graft loss. CONCLUSIONS: Kidney allografts possessing a low-producing TGFB1 polymorphism have a higher risk of late graft loss. Our study adds to a growing body of evidence that TGF-ß1 is beneficial, rather than harmful, for kidney transplant survival.

ECCO - A new initiative to support early-career researchers in the complement field.

Research on the complement system, like most areas of immunology, has seen tremendous progress over the last decades. Further advances in the complement field will rely on the next generation of scientific leaders, which are today's early-career researchers (ECRs). ECRs are emerging scientists who obtained their PhD degree within the past five years. They represent a distinct population within the scientific community, and accordingly have unique needs. Unfortunately, ECRs are faced with significant challenges that require customized solutions. The current paper provides a snapshot of the major obstacles ECRs face, such as an unhealthy work-life balance, lack of mentor and peer support, and uncertain career prospects. Efforts must consequently be taken to ensure stability and success of ECRs, not only to benefit these researchers in the early stages of their career, but the entire field of complement research. The Early-Career Complementologists (ECCO) was, therefore, launched as a new Task Force to support ECRs in the complement field. This new initiative aims to support and connect ECRs in the complement field worldwide. Furthermore, ECCO is supported by both the International Complement Society (ICS) and the European Complement Network (ECN); two professional societies led by scientists investigating the complement system.

The Contribution of Complement to the Pathogenesis of IgA Nephropathy: Are Complement-Targeted Therapies Moving from Rare Disorders to More Common Diseases?

Primary IgA nephropathy (IgAN) is a leading cause of chronic kidney disease and kidney failure for which there is no disease-specific treatment. However, this could change, since novel therapeutic approaches are currently being assessed in clinical trials, including complement-targeting therapies. An improved understanding of the role of the lectin and the alternative pathway of complement in the pathophysiology of IgAN has led to the development of these treatment strategies. Recently, in a phase 2 trial, treatment with a blocking antibody against mannose-binding protein-associated serine protease 2 (MASP-2, a crucial enzyme of the lectin pathway) was suggested to have a potential benefit for IgAN. Now in a phase 3 study, this MASP-2 inhibitor for the treatment of IgAN could mark the start of a new era of complement therapeutics where common diseases can be treated with these drugs. The clinical development of complement inhibitors requires a better understanding by physicians of the biology of complement, the pathogenic role of complement in IgAN, and complement-targeted therapies. The purpose of this review is to provide an overview of the role of complement in IgAN, including the recent discovery of new mechanisms of complement activation and opportunities for complement inhibitors as the treatment of IgAN.

Donor genetic variants in interleukin-6 and interleukin-6 receptor associate with biopsy-proven rejection following kidney transplantation.

Rejection after kidney transplantation remains an important cause of allograft failure that markedly impacts morbidity. Cytokines are a major player in rejection, and we, therefore, explored the impact of interleukin-6 (IL6) and IL-6 receptor (IL6R) gene polymorphisms on the occurrence of rejection after renal transplantation. We performed an observational cohort study analyzing both donor and recipient DNA in 1271 renal transplant-pairs from the University Medical Center Groningen in The Netherlands and associated single nucleotide polymorphisms (SNPs) with biopsy-proven rejection after kidney transplantation. The C-allele of the IL6R SNP (Asp358Ala; rs2228145 A > C, formerly rs8192284) in donor kidneys conferred a reduced risk of rejection following renal transplantation (HR 0.78 per C-allele; 95%-CI 0.67-0.90; P = 0.001). On the other hand, the C-allele of the IL6 SNP (at position-174 in the promoter; rs1800795 G > C) in donor kidneys was associated with an increased risk of rejection for male organ donors (HR per C-allele 1.31; 95%-CI 1.08-1.58; P = 0.0006), but not female organ donors (P = 0.33). In contrast, neither the IL6 nor IL6R SNP in the recipient showed an association with renal transplant rejection. In conclusion, donor IL6 and IL6R genotypes but not recipient genotypes represent an independent prognostic marker for biopsy-proven renal allograft rejection.

A Family Affair: Addressing the Challenges of Factor H and the Related Proteins.

Inflammation is a common denominator of diseases. The complement system, an intrinsic part of the innate immune system, is a key driver of inflammation in numerous disorders. Recently, a family of proteins has been suggested to be of vital importance in conditions characterized by complement dysregulation: the human Factor H (FH) family. This group of proteins consists of FH, Factor H-like protein 1 and five Factor H-related proteins. The FH family has been linked to infectious, vascular, eye, kidney and autoimmune diseases. In contrast to FH, the functions of the other highly homologous proteins are largely unknown and, hence, their role in the different disease-specific pathogenic mechanisms remains elusive. In this perspective review, we address the major challenges ahead in this emerging area, including 1) the controversies about the functional roles of the FH protein family, 2) the discrepancies in quantification of the FH protein family, 3) the unmet needs for validated tools and 4) limitations of animal models. Next, we also discuss the opportunities that exist for the immunology community. A strong multidisciplinary approach is required to solve these obstacles and is only possible through interdisciplinary collaboration between biologists, chemists, geneticists and physicians. We position this review in light of our own perspective, as principal investigators of the SciFiMed Consortium, a consortium aiming to create a comprehensive analytical system for the quantitative and functional assessment of the entire FH protein family.

Soluble CD59 in peritoneal dialysis: a potential biomarker for peritoneal membrane function.

INTRODUCTION: Various studies have reported the importance of complement regulators in preventing mesothelial damage during peritoneal dialysis (PD). Its assessment, however, is limited in clinical practice due to the lack of easy access to the peritoneal membrane. Recently, a soluble form of the complement regulatory protein CD59 (sCD59) has been described. We therefore aimed to investigate the role of sCD59 in PD. METHODS: Plasma sCD59 was measured in 48 PD patients, 41 hemodialysis patients, 15 non-dialysis patients with chronic kidney disease and 14 healthy controls by ELISA (Hycult; HK374-02). Additionally, sCD59 and sC5b-9 were assessed in the peritoneal dialysate. RESULTS: sCD59 and sC5b-9 were detectable in the peritoneal dialysate of all patients, and marginally correlated (r = 0.27, P = 0.06). Plasma sCD59 levels were significantly higher in PD patients than in patients with chronic kidney disease and healthy controls, but did not differ from hemodialysis patients. During follow-up, 19% of PD patients developed peritoneal membrane failure and 27% of PD patients developed loss of residual renal function. In adjusted models, increased sCD59 levels in the dialysate (HR 3.44, 95% CI 1.04-11.40, P = 0.04) and in plasma (HR 1.08, 95% CI 1.01-1.17, P = 0.04) were independently associated with the occurrence of peritoneal membrane failure. Higher plasma levels of sCD59 were also associated with loss of residual renal function (HR 1.10, 95% CI 1.04-1.17, P < 0.001). CONCLUSIONS: Our study suggests that sCD59 has potential as a biomarker to predict peritoneal membrane function and loss of residual renal function in PD, thereby offering a tool to improve patient management.

Complement-mediated kidney diseases.

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.

MASP-2 Is a Heparin-Binding Protease; Identification of Blocking Oligosaccharides.

It is well-known that heparin and other glycosaminoglycans (GAGs) inhibit complement activation. It is however not known whether fractionation and/or modification of GAGs might deliver pathway-specific inhibition of the complement system. Therefore, we evaluated a library of GAGs and their derivatives for their functional pathway specific complement inhibition, including the MASP-specific C4 deposition assay. Interaction of human MASP-2 with heparan sulfate/heparin was evaluated by surface plasmon resonance, ELISA and in renal tissue. In vitro pathway-specific complement assays showed that highly sulfated GAGs inhibited all three pathways of complement. Small heparin- and heparan sulfate-derived oligosaccharides were selective inhibitors of the lectin pathway (LP). These small oligosaccharides showed identical inhibition of the ficolin-3 mediated LP activation, failed to inhibit the binding of MBL to mannan, but inhibited C4 cleavage by MASPs. Hexa- and pentasulfated tetrasaccharides represent the smallest MASP inhibitors both in the functional LP assay as well in the MASP-mediated C4 assay. Surface plasmon resonance showed MASP-2 binding with heparin and heparan sulfate, revealing high Kon and Koff rates resulted in a Kd of ~2 µM and confirmed inhibition by heparin-derived tetrasaccharide. In renal tissue, MASP-2 partially colocalized with agrin and heparan sulfate, but not with activated C3, suggesting docking, storage, and potential inactivation of MASP-2 by heparan sulfate in basement membranes. Our data show that highly sulfated GAGs mediated inhibition of all three complement pathways, whereas short heparin- and heparan sulfate-derived oligosaccharides selectively blocked the lectin pathway via MASP-2 inhibition. Binding of MASP-2 to immobilized heparan sulfate/heparin and partial co-localization of agrin/heparan sulfate with MASP, but not C3b, might suggest that in vivo heparan sulfate proteoglycans act as a docking platform for MASP-2 and possibly prevent the lectin pathway from activation.