Felix Poppelaars

Distinct in vitro Complement Activation by Various Intravenous Iron Preparations

Background: Intravenous (IV) iron preparations are widely used in the treatment of anemia in patients undergoing hemodialysis (HD). All IV iron preparations carry a risk of causing hypersensitivity reactions. However, the pathophysiological mechanism is poorly understood. We hypothesize that a relevant number of these reactions are mediated by complement activation, resulting in a pseudo-anaphylactic clinical picture known as complement activation-related pseudo allergy (CARPA). Methods: First, the in-vitro complement-activating capacity was determined for 5 commonly used IV iron preparations using functional complement assays for the 3 pathways. Additionally, the preparations were tested in an ex-vivo model using the whole blood of healthy volunteers and HD patients. Lastly, in-vivo complement activation was tested for one preparation in HD patients. Results: In the in-vitro assays, iron dextran, and ferric carboxymaltose caused complement activation, which was only possible under alternative pathway conditions. Iron sucrose may interact with complement proteins, but did not activate complement in-vitro. In the ex-vivo assay, iron dextran significantly induced complement activation in the blood of healthy volunteers and HD patients. Furthermore, in the ex-vivo assay, ferric carboxymaltose and iron sucrose only caused significant complement activation in the blood of HD patients. No in-vitro or ex-vivo complement activation was found for ferumoxytol and iron isomaltoside. IV iron therapy with ferric carboxymaltose in HD patients did not lead to significant in-vivo complement activation. Conclusion: This study provides evidence that iron dextran and ferric carboxymaltose have complement-activating capacities in-vitro, and hypersensitivity reactions to these drugs could be CARPA-mediated.

Strong predictive value of mannose-binding lectin levels for cardiovascular risk of hemodialysis patients

Background Hemodialysis patients have higher rates of cardiovascular morbidity and mortality compared to the general population. Mannose-binding lectin (MBL) plays an important role in the development of cardiovascular disease. In addition, hemodialysis alters MBL concentration and functional activity. The present study determines the predictive value of MBL levels for future cardiac events (C-event), cardiovascular events (CV-event) and all-cause mortality in HD patients. Methods We conducted a prospective study of 107 patients on maintenance hemodialysis. Plasma MBL, properdin, C3d and sC5b-9 was measured before and after one dialysis session. The association with future C-events, CV-events, and all-cause mortality was evaluated using Cox regression models. Results During median follow-up of 27 months, 36 participants developed 21 C-events and 36 CV-events, whereas 37 patients died. The incidence of C-events and CV-events was significantly higher in patients with low MBL levels (<319 ng/mL, lower quartile). In fully adjusted models, low MBL level was independently associated with increased CV-events (hazard ratio 3.98; 95 % CI 1.88–8.24; P < 0.001) and C-events (hazard ratio 3.96; 95 % CI 1.49–10.54; P = 0.006). No association was found between low MBL levels and all-cause mortality. Furthermore, MBL substantially improved risk prediction for CV-events beyond currently used clinical markers. Conclusions Low MBL levels are associated with a higher risk for future C-events and CV-events. Therefore, MBL levels may help to identify hemodialysis patients who are at risk to develop cardiovascular disease. Electronic supplementary material The online version of this article (doi:10.1186/s12967-016-0995-5) contains supplementary material, which is available to authorized users.

Critical role for complement receptor C5aR2 in the pathogenesis of renal ischemia-reperfusion injury

The complement system, and specifically C5a, is involved in renal ischemia‐reperfusion (IR) injury. The 2 receptors for complement anaphylatoxin C5a (C5aR1 and C5aR2) are expressed on leukocytes as well as on renal epithelium. Extensive evidence shows that C5aR1 inhibition protects kidneys from IR injury; however, the role of C5aR2 in IR injury is less clear as initial studies proposed the hypothesis that C5aR2 functions as a decoy receptor. By Using wild‐type, C5aR1‐/‐, and C5aR2‐/‐ mice in a model of renal IR injury, we found that a deficiency of either of these receptors protected mice from renal IR injury. Surprisingly, C5aR2‐/‐ mice were most protected and had lower creatinine levels and reduced acute tubular necrosis. Next, an in vivo migration study demonstrated that leukocyte chemotaxis was unaffected in C5aR2‐/‐ mice, whereas neutrophil activation was reduced by C5aR2 deficiency. To further investigate the contribution of renal cell‐expressed C5aR2 vs. leukocyte‐expressed C5aR2 to renal IR injury, bone marrow chimeras were created. Our data show that both renal cell‐expressed C5aR2 and leukocyte‐expressed C5aR2 mediate IR‐induced renal dysfunction. These studies reveal the importance of C5aR2 in renal IR injury. They further show that C5aR2 is a functional receptor, rather than a decoy receptor, and may provide a new target for intervention.—Poppelaars, F., van Werkhoven, M. B., Kotimaa, J., Veldhuis, Z. J., Ausema, A., Broeren, S. G. M., Damman, J., Hempel, J. C., Leuvenink, H. G. D., Daha, M. R., van Son, W. J., van Kooten, C., van Os, R. P., Hillebrands, J.‐L., Seelen, M. A. Critical role for complement receptor C5aR2 in the pathogenesis of renal ischemia‐reperfusion injury. FASEB J. 31, 3193–3204 (2017). www.fasebj.org

New insight into the effects of heparinoids on complement inhibition by C1-inhibitor

Complement activation is of major importance in numerous pathological conditions. Therefore, targeted complement inhibition is a promising therapeutic strategy. C1‐esterase inhibitor (C1‐INH) controls activation of the classical pathway (CP) and the lectin pathway (LP). However, conflicting data exist on inhibition of the alternative pathway (AP) by C1‐INH. The inhibitory capacity of C1‐INH for the CP is potentiated by heparin and other glycosaminoglycans, but no data exist for the LP and AP. The current study investigates the effects of C1‐INH in the presence or absence of different clinically used heparinoids on the CP, LP and AP. Furthermore, the combined effects of heparinoids and C1‐INH on coagulation were investigated. C1‐INH, heparinoids or combinations were analysed in a dose‐dependent fashion in the presence of pooled serum. Functional complement activities were measured simultaneously using the Wielisa®‐kit. The activated partial thrombin time was determined using an automated coagulation analyser. The results showed that all three complement pathways were inhibited significantly by C1‐INH or heparinoids. Next to their individual effects on complement activation, heparinoids also enhanced the inhibitory capacity of C1‐INH significantly on the CP and LP. For the AP, significant potentiation of C1‐INH by heparinoids was found; however, this was restricted to certain concentration ranges. At low concentrations the effect on blood coagulation by combining heparinoids with C1‐INH was minimal. In conclusion, our study shows significant potentiating effects of heparinoids on the inhibition of all complement pathways by C1‐INH. Therefore, their combined use is a promising and a potentially cost‐effective treatment option for complement‐mediated diseases.

Renal expression of Toll-like receptor 2 and 4: Dynamics in human allograft injury and comparison to rodents

Activation of the innate immunity through Toll-like receptors (TLRs) has been postulated to play an important role in the pathophysiology of renal allograft dysfunction. TLR2 and TLR4 dynamics in different human post-transplant pathological entities has never been studied. Therefore, we evaluated pre- and post-transplantation protein expression of TLR2 and TLR4 in human kidney biopsies. Human kidney biopsies obtained from living kidney donors and patients with acute tubular necrosis, acute cellular and vascular rejection and interstitial fibrosis/tubular atrophy (IF/TA) were used. Translating results from animal studies to the clinical situation is highly important considering the upcoming clinical studies with TLR inhibitors in human renal transplantation. Hence, the TLR2 and TLR4 expression in healthy mouse and rat kidneys was analyzed and compared with human kidneys. In healthy human kidneys, TLR2 is expressed on the endothelium and Bowmans capsule, while TLR4 is expressed on the endothelium only. No tubular staining was found for both receptors in human kidneys. In contrast to human biopsies, TLR2 and TLR4 expression in rodents was observed on tubular epithelial cells. In all acute rejection human biopsies, increased infiltration of TLR4(+) leukocytes was observed. In conclusion, a discrepancy exists between human and rodent renal TLR expression, which suggests careful attention when translating results from rodent studies to the human situation. Additionally, this study revealed human TLR2 and TLR4 expression dynamics in human biopsies pre- and post-transplantation.

Complement-mediated inflammation and injury in brain dead organ donors

HighlightsDuring brain death, both local and systemic complement is activated in the donor.Complement activation in brain death is associated with allograft function and outcome after transplantation.Complement inhibition in the BD donor reduces inflammation and injury, thus improving graft function after transplantation. Abstract The importance of the complement system in renal ischemia‐reperfusion injury and acute rejection is widely recognized, however its contribution to the pathogenesis of tissue damage in the donor remains underexposed. Brain‐dead (BD) organ donors are still the primary source of organs for transplantation. Brain death is characterized by hemodynamic changes, hormonal dysregulation, and immunological activation. Recently, the complement system has been shown to be involved. In BD organ donors, complement is activated systemically and locally and is an important mediator of inflammation and graft injury. Furthermore, complement activation can be used as a clinical marker for the prediction of graft function after transplantation. Experimental models of BD have shown that inhibition of the complement cascade is a successful method to reduce inflammation and injury of donor grafts, thereby improving graft function and survival after transplantation. Consequently, complement‐targeted therapeutics in BD organ donors form a new opportunity to improve organ quality for transplantation. Future studies should further elucidate the mechanism responsible for complement activation in BD organ donors.

C1-Inhibitor Treatment Decreases Renal Injury in an Established Brain-Dead Rat Model

Background Kidneys derived from brain-dead (BD) donors have lower graft survival rates compared with kidneys from living donors. Complement activation plays an important role in brain death. The aim of our study was therefore to investigate the effect of C1-inhibitor (C1-INH) on BD-induced renal injury. Methods Brain death was induced in rats by inflating a subdurally placed balloon catheter. Thirty minutes after BD, rats were treated with saline, low-dose or high-dose C1-INH. Sham-operated rats served as controls. After 4 hours of brain death, renal function, injury, inflammation, and complement activation were assessed. Results High-dose C1-INH treatment of BD donors resulted in significantly lower renal gene expression and serum levels of IL-6. Treatment with C1-INH also improved renal function and reduced renal injury, reflected by the significantly lower kidney injury marker 1 gene expression and lower serum levels of lactate dehydrogenase and creatinine. Furthermore, C1-INH effectively reduced complement activation by brain death and significantly increased functional levels. However, C1-INH treatment did not prevent renal cellular influx. Conclusions Targeting complement activation after the induction of brain death reduced renal inflammation and improved renal function before transplantation. Therefore, strategies targeting complement activation in human BD donors might clinically improve donor organ viability and renal allograft survival.

Complement in renal transplantation : The road to translation

&NA; Renal transplantation is the treatment of choice for patients with end‐stage renal disease. The vital role of the complement system in renal transplantation is widely recognized. This review discusses the role of complement in the different phases of renal transplantation: in the donor, during preservation, in reperfusion and at the time of rejection. Here we examine the current literature to determine the importance of both local and systemic complement production and how complement activation contributes to the pathogenesis of renal transplant injury. In addition, we dissect the complement pathways involved in the different phases of renal transplantation. We also review the therapeutic strategies that have been tested to inhibit complement during the kidney transplantation. Several clinical trials are currently underway to evaluate the therapeutic potential of complement inhibition for the treatment of brain death‐induced renal injury, renal ischemia‐reperfusion injury and acute rejection. We conclude that it is expected that in the near future, complement‐targeted therapeutics will be used clinically in renal transplantation. This will hopefully result in improved renal graft function and increased graft survival.

The Complement System in Dialysis: A Forgotten Story?

Significant advances have lead to a greater understanding of the role of the complement system within nephrology. The success of the first clinically approved complement inhibitor has created renewed appreciation of complement-targeting therapeutics. Several clinical trials are currently underway to evaluate the therapeutic potential of complement inhibition in renal diseases and kidney transplantation. Although, complement has been known to be activated during dialysis for over four decades, this area of research has been neglected in recent years. Despite significant progress in biocompatibility of hemodialysis (HD) membranes and peritoneal dialysis (PD) fluids, complement activation remains an undesired effect and relevant issue. Short-term effects of complement activation include promoting inflammation and coagulation. In addition, long-term complications of dialysis, such as infection, fibrosis and cardiovascular events, are linked to the complement system. These results suggest that interventions targeting the complement system in dialysis could improve biocompatibility, dialysis efficacy, and long-term outcome. Combined with the clinical availability to safely target complement in patients, the question is not if we should inhibit complement in dialysis, but when and how. The purpose of this review is to summarize previous findings and provide a comprehensive overview of the role of the complement system in both HD and PD.

The lectin pathway in renal disease: old concept and new insights

The complement system is composed of a network of at least 40 proteins, which significantly contributes to health and disease. The lectin pathway (LP) is one of three pathways that can activate the complement system. Next to protection of the host against pathogens, the LP has been shown to play a crucial role in multiple renal diseases as well as during renal replacement therapy. Therefore, several complement-targeted drugs are currently being explored in clinical trials. Among these complement inhibitors, specific LP inhibitors are also being tested in renal abnormalities such as in immunoglobulin A nephropathy and lupus nephritis. Using various in vitro models, Yaseen et al. (Lectin pathway effector enzyme mannan-binding lectin-associated serine protease-2 can activate native complement component 3 (C3) in absence of C4 and/or C2. FASEB J 2017; 31: 2210-2219) showed that Mannan-associated serine protease2 can directly activate C3 thereby bypassing C2 and C4 in the activation of the LP. These new findings broaden our understanding of the mechanisms of complement activation and could potentially impact our strategies to inhibit the LP in renal diseases. In support of these findings, we present data of human renal biopsies, demonstrating the occurrence of the LP bypass mechanism in vivo. In conclusion, this review provides a detailed overview of the LP and clarifies the recently described bypass mechanism and its relevance. Finally, we speculate on the role of the C4 bypass mechanism in other renal diseases.