N.C.A.J. van de Kar

The fibrinolytic system in the hemolytic uremic syndrome : in vivo and in vitro studies

ABSTRACT: Fibrinolytic parameters and von Willebrand factor (vWF) antigen were measured in the plasma of 10 patients with hemolytic uremic syndrome (HUS). Samples were taken at presentation and again 2 wk later, before and after infusion of l-desamino-8-arginine vasopressin. Compared with the plasma values of healthy control children, levels of tissue-plasminogen activator (t-PA) antigen, plasminogen activator inhibitor type I (PAI-1) activity, and vWF as well as fibrin(ogen) degradation products were significantly elevated in the plasma of HUS patients on admission. No response of the fibrinolytic parameters and vWF were seen when l-desamino-8-arginine vasopressin infusion was given on admission. After 2 wk, t-PA antigen and vWF had partially returned to basal values, and t-PA antigen increased rapidly again after l-desamino-8-arginine vasopressin infusion. To investigate whether verocytotoxin contributes to the alteration of the fibrinolytic system found in HUS patients, purified verocytotoxin-1 (VT-1) was added to the media of cultured human endothelial cells. Addition of VT-1 alone did not change the production of t-PA, plasminogen activator inhibitor type I, and vWF antigen in these cells. However, when the endothelial cells were preincubated with tumor necrosis factor-a to increase the number of VT-1 receptors, VT-1 induced a marked decrease of the synthesis of t-PA, plasminogen activator inhibitor type I, and vWF. This was caused by a decrease in overall protein synthesis in the tumor necrosis factor-α-and VT-1-treated endothelial cells. We conclude from this study that the systemic fibrinolytic parameters measured in the plasma of HUS patients are probably not a direct effect of VT-1 on the endothelium but are sequelae of the disease in which the intestine and the kidney are predominantly affected.

Complement activation patterns in atypical haemolytic uraemic syndrome during acute phase and in remission.

Atypical haemolytic uraemic syndrome (aHUS) is associated with (genetic) alterations in alternative complement pathway. Nevertheless, comprehensive evidence that the complement system in aHUS patients is more prone to activation is still lacking. Therefore, we performed a thorough analysis of complement activation in acute phase and in remission of this disease. Complement activation patterns of the aHUS patients in acute phase and in remission were compared to those of healthy controls. Background levels of complement activation products C3b/c, C3bBbP and terminal complement complex (TCC) were measured using enzyme‐linked immunosorbent assay (ELISA) in ethylenediamine tetraacetic acid (EDTA) plasma. In vitro‐triggered complement activation in serum samples was studied using zymosan‐coating and pathway‐specific assay. Furthermore, efficiencies of the C3b/c, C3bBbP and TCC generation in fluid phase during spontaneous activation were analysed. Patients with acute aHUS showed elevated levels of C3b/c (P < 0·01), C3bBbP (P < 0·0001) and TCC (P < 0·0001) in EDTA plasma, while values of patients in remission were normal, compared to those of healthy controls. Using data from a single aHUS patient with complement factor B mutation we illustrated normalization of complement activation during aHUS recovery. Serum samples from patients in remission showed normal in vitro patterns of complement activation and demonstrated normal kinetics of complement activation in the fluid phase. Our data indicate that while aHUS patients have clearly activated complement in acute phase of the disease, this is not the case in remission of aHUS. This knowledge provides important insight into complement regulation in aHUS and may have an impact on monitoring of these patients, particularly when using complement inhibition therapy.

Eculizumab treatment efficiently prevents C5 cleavage without C5a generation in vivo.

To the editor: We read with interest the letter by Burwick et al in Blood [1][1] and were surprised by their conclusion that eculizumab failed to inhibit C5a generation in vivo. Eculizumab is a monoclonal antibody binding to human C5 preventing its cleavage to C5a and C5b. The authors investigated

Complement Factor H Serum Levels Determine Resistance to Pneumococcal Invasive Disease

Streptococcus pneumoniae is a major cause of life-threatening infections. Complement activation plays a vital role in opsonophagocytic killing of pneumococci in blood. Initial complement activation via the classical and lectin pathways is amplified through the alternative pathway amplification loop. Alternative pathway activity is inhibited by complement factor H (FH). Our study demonstrates the functional consequences of the variability in human serum FH levels on host defense. Using an in vivo mouse model combined with human in vitro assays, we show that the level of serum FH correlates with the efficacy of opsonophagocytic killing of pneumococci. In summary, we found that FH levels determine a delicate balance of alternative pathway activity, thus affecting the resistance to invasive pneumococcal disease. Our results suggest that variation in FH expression levels, naturally occurring in the human population, plays a thus far unrecognized role in the resistance to invasive pneumococcal disease.

Novel sequence variation affects GPIb α in post-diarrheal hemolytic uremic syndrome

Background: Hemolytic uremic syndrome (HUS) is one of the major causes of renal failure in children. In most cases the disease is caused by infection with Shiga toxin- producing Escherichia coli (STEC) and preceded by diarrhea. Only in 15% of cases STEC infection leads to HUS. Genetic predisposition of a patient to develop HUS after STEC infection might play a role, but very few reports on this subject are available. We describe a novel missense mutation in the GP1BA gene encoding platelet-receptor glycoprotein Ibα (GPIbα) in a severely affected HUS patient. Methods: GP1BA was screened by Sanger sequencing. Binding of recombinant GPIbα and von Willebrand factor (VWF) fragments was analyzed using surface plasmon resonance (SPR). The hematological studies using patient blood were performed. Results: The detected heterozygous mutation p.Pro46Leu is located in the proximity to one of the two GPIbα-VWF binding sites. SPR experiments show that the p.Pro46Leu leads to a small increase in GPIbα-VWF binding (p<0.001). The hemostatic parameters of patient blood after recovery from HUS show normal values. Conclusions: The described mutation affects GPIbα interaction with VWF in a mild gain-of-function manner and might have contributed to a prothrombotic state in the patient and to development of HUS.

KOUNCIL: Kidney-Oriented Understanding of Correcting Ciliopathies.

Objective Nephronophthisis is an autosomal recessive renal ciliopathy that constitutes the leading monogenic cause of end-stage renal disease in children. The KOUNCIL consortium is a collaboration between the UMC Utrecht, the Radboud UMC Nijmegen and UC London aimed at elucidating the genetic etiology and pathophysiological mechanisms underlying nephronophthisis and identifying drugs that prevent or delay renal insufficiency. Our goal is to improve genome diagnostics, genetic counselling and therapeutic options for nephronophthisis patients. Methods We employ next-generation sequencing to identify novel disease genes in 100 nephronophthisis patients included within the AGORA biobank project. The functional effect of novel mutations is assessed using in vitro and in vivo models. Genotypic and phenotypic patient characteristics are registered in a nephronophthisis database, facilitating correlation analyses and identification of early phenotypic markers. Newly identified nephronophthisis-genes are incorporated into diagnostic next-generation sequencing panels of ciliary genes. We use a systems-biology approach to identify and functionally characterize nephronophthisis-associated protein modules. Finally, we use high-throughput repurposing screens in zebrafish embryos to identify FDA-approved drugs that halt renal failure. Results With this approach, we expect to uncover the causal mutation in 60-90% of nephronophthisis patients. KOUNCIL members were involved in the recent identification of three novel genes (IFT172, WDR34 and WDR60) for nephronophthisis-related disorders. Clinical guidelines and new diagnostic tools for nephronophthisis are developed and implemented in diagnostics. We expect to identify drugs that can lead to novel therapies for nephronophthisis. Conclusion The KOUNCIL study is designed to advance understanding of renal ciliopathies and improve clinical care for nephronophthisis patients.