INFORMATION ABOUT PROJECT,
SUPPORTED BY RUSSIAN SCIENCE FOUNDATION
The information is prepared on the basis of data from the information-analytical system RSF, informative part is represented in the author's edition. All rights belong to the authors, the use or reprinting of materials is permitted only with the prior consent of the authors.
COMMON PART
Project Number22-15-00409
Project titleInvestigation of the mechanisms of complement membrane-attacking complex C5b-9 formation on the surface of endothelial cells in atypical hemolytic uremic syndrome. Search for prognostic signs to assess the severity and relapses of this disease.
Project LeadAvdonin Petr
AffiliationKoltzov Institute of Developmental Biology of Russian Academy of Sciences,
Implementation period | 2022 - 2024 |
Research area 05 - FUNDAMENTAL RESEARCH IN MEDICINE, 05-212 - Pediatrics and neonatology
KeywordsThrombotic microangiopathies, hemolytic uremic syndrome, complement, endothelial cells, microcirculation, kidneys, arterioles, von Willebrand factor multimers, macrophages-monocytes, hyaline thrombi, innate immunity, C3 glomerulopathy, next generation sequencing
PROJECT CONTENT
Annotation
HUS is a serious therapeutic problem in pediatrics and pediatric nephrology, being one of the leading causes of acute kidney injury (AKI) in children with potential transformation into end-stage chronic kidney disease (CKD) at various times from the onset of the disease. The diagnosis of HUS is based on a triad of symptoms: microangiopathic hemolytic anemia, thrombocytopenia, and AKI. These signs are components of the clinical and morphological syndrome - thrombotic microangiopathy (TMA), characterized by occlusive lesion of the vessels of the microvasculature resulting from damage to the endothelium. Most children (85-90%) are diagnosed with an infectious cyclic form of HUS (STEC-HUS / typical HUS), which develops as a result of damage to the vascular endothelium of the microvasculature with shiga toxin produced by strains of enterohemorrhagic E. coli or S.disenteriae type1. Another form of TMA, atypical HUS, is a rare recurrent form of the disease with a progressive course and an unfavorable prognosis, which is based on the chronic uncontrolled activation of the alternative complement pathway of a hereditary (mutations in genes encoding complement proteins) or acquired nature (antibodies to factor H), leading to the development of systemic complement-mediated form of TMA. It was found that for the implementation of aHUS, the interaction of genetic anomalies in the complement system with trigger factors of the external environment, provoking additional activation of complement in predisposed individuals, is necessary. Until now, there are no specific diagnostic markers for aHUS. Therefore, it is now known that STEC infection can also be a complement-activating state in aHUS, which allows a number of patients with initially diagnosed typical HUS to reconsider the diagnosis in favor of its atypical form after a different time interval in each case. The therapeutic approach to aHUS was radically improved with the discovery and introduction into clinical practice of eculizumab, a humanized monoclonal antibody to the C5 fraction of the terminal stage of the complement cascade, which prevents its cleavage leading to the formation of proinflammatory C5a and prothrombotic C5b-9 components (MAC), thereby eliminating their pathogenic action. Despite the obvious effect of eculizumab, the prognosis of the disease remains serious, and in some cases even unfavorable, since it is largely determined by the timely diagnosis of the disease, as well as the timing of initiation of complement inhibitory therapy. The imbalance of the alternative complement pathway as a result of genetic mutations is recognized as the main cause of the development of aHUS, however, in 40-50% of cases, the genetic nature of the disease is not confirmed, which does not exclude the diagnosis of aHUS. This is largely due to the complexity and multifactorial nature of the complement system, which is the main part of the innate immune system. According to recent studies, it has become known that in addition to the cell surface expressed and plasma regulators of complement, there is an intracellular complement system localized in complexosomes - specialized structures present in various cells, including endothelial cells. Indirect data indicate the participation of the intracellular complement system in the formation of C5b-9 (MAC) on the surface of the endothelium during relapse of aHUS. Thus, the study of the molecular mechanisms of the pathogenesis of aHUS is extremely relevant, which will allow not only to diagnose the disease in a timely manner, but also to determine the severity of the condition and the onset of relapse, which affect the prognosis of “renal” and overall survival. The objective of this project is to identify the mechanisms of activation of the intracellular complement system in endothelial cells (EC), as well as to determine its participation in the formation of C5b-9 upon contact of the endothelium with the plasma of patients with aHUS and STEC-HUS in ex vivo experiments. In accordance with this, it is planned to study the influence of neuroendocrine factors, growth factors, cytokines on the activity of the intracellular complement system in EC. The role of plasma membrane receptors and signaling systems in the exposure of endogenous factors C3 and C5, the formation of the membrane attack complex - C5b-9 will be determined. It is also expected to evaluate the effect of plasma of patients with aHUS on the viability of EC by markers of apoptosis and necrosis at two points of the study - during the period of active manifestations of aHUS (onset / relapse) and remission of the disease. The second task of the project will be to search for prognostic criteria that determine the risk of developing aHUS in patients with STEC infection, but also the likelihood of TMA recurrence after discontinuation of eculizumab therapy in patients with aHUS. The following potential prognostic signs will be considered: an indicator of the formation of a membrane-attacking complex on the surface of EC, the genetic profile of patients (or the presence of mutations in genes encoding complement proteins) using a new generation sequencing method, as well as the determination of antibodies to different epitopes of factors H and I (complement inhibitory factor). There is currently no optimal strategy for monitoring complement activity in the development of aHUS, including in patients with STEC infection in the prodrome, as well as in predicting the risk of aHUS recurrence after discontinuation of eculizumab. In addition, the mechanisms of activation of the intracellular complement system in EC under conditions of interaction with plasma of patients with aHUS have not yet been studied. Thus, the planned study will expand the understanding of the pathogenesis of HUS, increase not only the effectiveness of therapy and, but also expand the horizons of treatment for aHUS with the use (or development) of new drugs that modulate the activity of membrane receptors and signaling systems coupled with the intracellular complement system.
Expected results
Atypical HUS is a classic example of systemic TMA, in which damage develops not only to the kidneys, but also to other vital organs - the brain, heart, lungs, digestive tract, organs of vision, etc. The therapeutic approach to aHUS has been radically improved over the years. Before the introduction into practice in the 70s of the last century, the "gold standard" of therapy - fresh frozen plasma - the mortality rate was 90%. Among survivors, the incidence of end-stage renal disease at the end of an acute episode reached 20–40% in children and 48% in adults. The creation of the drug eculizumab - Soliris (Alexion company) based on nanoantibodies against complement factor C5, and now its biosimilar, Elizaria (Generium company), significantly improved the prognosis of AGUS. However, despite progress in this area, there are still many questions regarding the molecular mechanisms of the pathogenesis of this disease, predicting its course, preventing relapse, as well as choosing and developing optimal treatment protocols. In the course of the project, we intend to identify prognostic signs by which it will be possible to assess the likelihood of the transition of the disease from the latent to the acute phase, to identify the pathogenetic factors of this transition, to develop criteria for the effectiveness of complement-blocking therapy. In addition to scientific significance, the solution of these issues will contribute to the preservation of the health and life of children suffering from this serious disease. The development of optimal treatment protocols, including changing the frequency / dose of the drug, is important from an economic point of view, since complement-blocking therapy with eculizumab is expensive.
The project is planned to conduct scientific research, during which we propose to identify neuroendocrine factors, growth factors and cytokines that regulate the work of the intracellular complement system in endothelial cells. The proposed results will be important for understanding the mechanisms of endothelial damage in TMA.
According to the results of the project, it is planned to publish 10 articles in journals included in the WoS and Scopus databases. Some of these 10 articles (at least 2) will be published in journals belonging to the 1st quartile. Expected publication in the journals Kidney International (Q1), Biomedicines (Q1), Pediatric Nephrology (Q1), Therapeutic Archives, Biological Membranes.
REPORTS
Annotation of the results obtained in 2022
Atypical hemolytic uremic syndrome (aHUS) is a rare systemic disease related to thrombotic microangiopathy, which is based on uncontrolled activation of the alternative complement pathway. The mechanisms of development of aHUS remain not fully understood, but there are two main causes of the development of this disease. These are genetically determined disorders in the functioning of the factors of the complement system and autoantibodies to factor H, which block its regulatory function in the complement system. In both cases, uncontrolled activation of the alternative pathway of the complement system occurs, leading to extensive damage to the endothelium of blood vessels, primarily the kidneys, and the development of TMA. 70 DNA samples of patients diagnosed with aHUS were obtained. A search was made for mutations that may be associated with the risk of developing this disease in a panel of genes, including CD46, CFB, CFH, CFHR1, CFHR2, CFHR3, CFHR4, CFHR5, CFI, DGKE, PIGA, THBD, C2, C3, C5, CAPG , MMACHC, INF2, VTN, ADAMTS13. In the examined DNA samples, microdeletions associated with a high risk of developing aHUS, covering the CFHR1, CFHR3, and CFHR4 genes, were most widely represented. Mutations in the CFH gene were less common. The frequency of mutations in the genes CD46, CFB, CFHR2, CFHR4, CFHR5, CFI, DGKE, THBD, C3, C5, MMACHC, ADAMTS13 among the examined patients was significantly lower. Testing of identified mutations against the criteria of the American College of Medical Genetics and Genomics for clinical geneticists showed that, with the exception of identified deletions associated with the risk of developing aHUS, only 8 mutations out of 63 identified can be regarded as pathogenic. The clinical significance of the rest is assessed as undetermined.
We also analyzed the level of antibodies to factor H in 409 plasma samples of patients diagnosed with aHUS aged from a few days to 78 years. In the age group from 0 to 23 years, patients were identified whose level of antibodies to factor H exceeded 6000 AU/ml and in some cases overcame the threshold of 100,000 AU/ml with the mode Mo=616. The proportion of patients with antibody levels above 6000 AU/ml in the age group from 0 to 23 years was 19.24%. In patients older than 23 years, the level of antibodies to factor H in a single case remained in the range of up to 6000 AU/ml and averaged 1340±998 AU/ml. An analysis of the case histories and the clinical picture observed at the time of sampling did not reveal a direct relationship between the level of antibodies to factor H and the severity of the disease.
Publications
1. Emirova Kh.M., Lupan I.N., Pishchalnikov A.Y., Volyansky A.M., Vasilkova D.S., Glukhova L.V., Kudlay D.A., Muzurov A.L., Orlova O.M., Avdonin P.V., Avdonin P.P., Shatalov P.A., Kozina A.A. Комплексный подход к диагностике и тактика лечения атипичного гемолитико-уремического синдрома, ассоциированного с мутацией в гене CFH: клиническое наблюдение Педиатрия, Педиатрия, 2022, Том 6, стр.168-177. (year - 2022) https://doi.org/10.24110/0031-403X-2022-101-6-168-177
Annotation of the results obtained in 2023
Shiga toxin-producing Escherichia coli-associated hemolytic uremic syndrome (STEC-HUS) is the most common form of thrombotic microangiopathy (TMA) in children. The morbidity of STEC-HUS is 85-90%, mortality in the acute period is 2.5-12% due to damage to the central nervous system and the development of multiple organ failure syndrome. An important role in the development of STEC-HUS as one of the forms of TMA is played by changes in the activity of the metalloprotease ADAMTS13 (a disintegrin and metalloprotease with thrombospondin-1-like domains, member 13), which regulates the functional activity of von Willebrand factor (vWF), thereby limiting the growth of blood clots in the microvasculature of the circulatory system. We conducted a study of the state of the «ADAMTS13–vWF–platelet» system in STEC-HUS in children. We found that deficiency of ADAMTS13 activity in STEC-HUS is associated with a severe course of the acute period of the disease and increases the risk of developing multiple organ failure syndrome by 3.2 times (OR 3.24; 95% CI 1.38-7.6; p= 0.02), cerebral insufficiency by 3.6 times (OR 3.57; 95% CI 1.42-8.97; p=0.003), pulmonary edema by 6.5 times (OR 6.49; 95% CI 1.46-28.8; p=0.003), AKI 6 times (OR 6.1; 95% CI 2.42-15.31; p=0.0000) with the need for RRT 5.7 times ( OR 5.65; 95% CI 2.08-15.34; p=0.0008). A decrease in ADAMTS13 activity is a predictor of disease severity.
Publications
1. Emirova Kh.M., Orlova O.M., Chichuga E.M., Muzurov A.L., Avdonin P.P., Avdonin P.V. A Moderate Decrease in ADAMTS13 Activity Correlates with the Severity of STEC-HUS Biomolecules, 2023 Nov 20;13(11):1671. (year - 2023) https://doi.org/10.3390/biom13111671
2. Blinova M. S., Generalova G. A., Emirova Kh. M., Popov E. G., Tsvetaeva N. V., Vasiliev S. A., Avdonin P. P. CИСТЕМА КОМПЛЕМЕНТА КАК ОБЩЕЕ ЗВЕНО В ПАТОГЕНЕЗЕ ГЕМОЛИТИКО-УРЕМИЧЕСКОГО СИНДРОМА Биологические мембраны: Журнал мембранной и клеточной биологии, T. 40, № 4, стр. 235-258 (year - 2023) https://doi.org/10.31857/S0233475523040047