Differential contributions of the C5b-9 and C5a/C5aR pathways to microvascular and macrovascular thrombosis in complement-mediated thrombotic microangiopathy patients.

To clarify the role of the C5a/C5aR (C5a receptor) and C5b-9 pathways in macrovascular thrombosis (MAT) and renal microthrombosis (MIT), 73 renal biopsy-proven complement-mediated thrombotic microangiopathy (C-TMA) patients were enrolled; 9 patients with pure MAT and 13 patients with pure MIT were selected for further study. Twenty-five external C-TMA patients were selected as the validation cohort. Plasma C5a and sC5b-9 (soluble C5b-9) levels were significantly higher in patients with MAT than in those with MIT (P = 0.008, P = 0.041, respectively). The mean optical density of C5aR1 in the kidney was significantly higher in MAT patients than in those with MIT (P < 0.001). Both urinary sC5b-9 levels (MIT: P < 0.001, MAT: P = 0.004) and renal deposition of C5b-9 (MIT: P < 0.001, MAT: P = 0.001) were significantly higher in C-TMA patients compared to normal control, but were similar between MAT and MIT groups. In the correlation analysis within 22C-TMA patients, urinary sC5b-9 levels and renal deposition of C5b-9 were positively correlated to renal MIT formation (P = 0.009 and P = 0.031, respectively). Furthermore, the renal citrullinated histone H3 (CitH3)- and neutrophil elastase (NE)-positive area ratios were both significantly higher in the MAT group than in the MIT group (P = 0.006 and P = 0.020, respectively). Therefore, the local C5b-9 and C5a/C5aR1 pathways might have differential contributions to MIT and MAT formation in the disease.

Therapy-induced senescent cancer cells exhibit complement activation and increased complement regulatory protein expression.

Therapy-induced senescence (TIS) is a primary response to chemotherapy, contributing to untoward treatment outcomes such as evasion of immunosurveillance. Despite the established role of the complement system in the immune response to cancer, the role of complement in mediating the immune response against senescent tumor cells remains poorly understood. To explore this relationship, we exposed lung adenocarcinoma (A549), breast adenocarcinoma (MCF7) and pancreatic carcinoma (Panc-1) cell lines to sublethal doses of either etoposide or doxorubicin to trigger TIS. Identification of TIS was based on morphological changes, upregulation of the senescence-associated ß-galactosidase, p21Cip1 induction and lamin B1 downregulation. Using immunofluorescence microscopy, quantitative PCR, ELISA of conditioned media and in silico analysis, we investigated complement activation, complement protein expression, C3 levels in the conditioned media of senescent cells and secreted complement proteins as part of the senescence-associated secretory phenotype (SASP), respectively. In cell lines undergoing TIS, complement-related changes included (i) activation of the terminal pathway, evidenced by the deposition of C5b-9 on senescent cells; (ii) an increase in the expression of CD59 and complement factor H and (iii) in A549 cells, an elevation in the expression of C3 with its secretion into the medium. In addition, increased C3 expression was observed in breast cancer samples expressing TIS hallmarks following exposure to neoadjuvant chemotherapy. In conclusion, TIS led to the activation of complement, upregulation of complement regulatory proteins and increased C3 expression. Complement appears to play a role in shaping the cancer microenvironment upon senescence induction.

Endothelial dysfunction and complement activation are independently associated with disease duration in patients with systemic vasculitis.

OBJECTIVES: Systemic vasculitis is a heterogenous group of autoimmune diseases characterized by enhanced cardiovascular mortality. Endothelial dysfunction is associated with accelerated vascular damage, representing a core pathophysiologic mechanism contributing to excess CV risk. Recent studies have also shown that complement activation holds significant role in the pathogenesis of Anti-Neutrophilic Cytoplasmic Autoantibody (ANCA) -associated vasculitis (AAV). Given the potential crosstalk between the endothelium and complement, we aimed to assess, for the first time simultaneously, easily accessible biomarkers of endothelial dysfunction and complement activation in SV. METHODS: We measured circulating endothelial microvesicles (EMVs) and soluble complement components representative of alternative, classical and terminal activation (C5b-9, C1q, Bb fragments, respectively) in a meticulously selected group of patients with systemic vasculitis, but without cardiovascular disease. Individuals free from systemic diseases, who were matched with patients for cardiovascular risk factors(hypertension, diabetes, smoking, dyslipidemia), comprised the control group. RESULTS: We studied 60 individuals (30 in each group). Patients with systemic vasculitis had elevated EMVs, higher levels of C5b-9 [536.4(463.4) vs 1200.94457.3), p = 0.003] and C1q [136.2(146.5 vs 204.2(232.9), p = 0.0129], compared to controls [232.0 (243.5) vs 139.3(52.1), p < 0.001]. In multivariate analysis both EMVs and C5b-9 were independently associated with disease duration (p = 0.005 and p = 0.004 respectively), yet not with disease activity. CONCLUSION: Patients with systemic vasculitis exhibit impaired endothelial function and complement activation, both assessed by easily accessible biomarkers, even in the absence of cardiovascular disease manifestations. EMVs and soluble complement components such as C5b-9 and C1q could be used as early biomarkers of endothelial dysfunction and complement activation, respectively, in clinical practice during the course of SV, yet their predictive value in terms of future cardiovascular disease warrants further verification in appropriately designed studies.

Antiphospholipid antibody carriers and patients with quiescent antiphospholipid syndrome show persistent subclinical complement activation.

OBJECTIVES: Complement activation has been advocated as one mechanism by which antiphospholipid antibodies (aPLs) can induce thrombosis. In patients with catastrophic aPL syndrome or re-thrombosis, enhanced complement activation was shown, even in quiescent phase of the disease. We aimed to assess complement activation and to investigate its association to clinical variables in aPL positive patients with a favorable disease course. METHODS: Subjects with at least two consecutive positive aPL antibody results obtained ≥12 weeks apart were enrolled. They were subjects without history of thrombosis or pregnancy morbidity (aPL carriers), patients with pregnancy morbidity alone (OAPS), and/or with arterial, venous, or small-vessel thrombosis (TAPS); all patients should have been free of symptoms for ≥2 years. Patients affected with systemic autoimmune diseases were excluded. Healthy age and sex-matched subjects were included as controls. Plasma C5a and C5b-9 levels were assessed by commercially available ELISA assays. Non-parametric Mann-Whitney test and Spearman's correlation were applied. RESULTS: Thirty-seven OAPS, 38 TAPS, 42 aPL carriers, and 30 healthy subjects were enrolled. Median C5a and C5b-9 levels were significantly higher in quiescent aPL positive patients (OAPS, TAPS, aPL carriers) compared with controls: C5a ng/ml 10.61 (IQR 6.87-15.46) vs 4.06 (2.66-7.35), p< 0.001; C5b-9 ng/ml 283.95 (175.8-439.40) vs 165.90 (124.23-236.8), p< 0.001. Similar C5a and C5b-9 levels were observed in OAPS and TAPS patients and aPL carriers. A positive correlation between C5b-9 median levels and the number of aPL positive tests was found (p= 0.002). CONCLUSIONS: The persistence of aPL antibodies is associated to a persistent subclinical activation of the complement cascade.

Measurement of Complement Activation via Plasma-Soluble C5b-9 Comparison with Terminal Complement Complex Staining in a Series of Kidney Biopsies.

INTRODUCTION: With the emergence of therapeutic complement inhibitors, there is a need to identify patients with complement-driven inflammation. C5b-9 is the terminal product of the three complement pathways and therefore a marker of total complement activation. We present a pilot study which aims to assess whether plasma soluble C5b-9 (sC5b-9) correlates with terminal complement complex (TCC) staining in kidney tissue. The secondary aim was to assess the utility of plasma sC5b-9 as part of routine workup in kidney patients undergoing kidney biopsy. METHODS: Thirty-seven patients undergoing kidney biopsy had plasma sC5b-9 and TCC staining on kidney tissue performed. Additional blood markers including creatinine, haemoglobin, CRP, factor H, factor I, and midkine levels were also taken. These parameters were correlated with the histological diagnoses. Patients were divided into a diseased group (n = 31) and a control group (n = 6) consisting of transplanted kidneys with minor or no changes. Of the biopsies in the control group, 50% were performed as per protocol, and the other 50% were performed due to clinical need. RESULTS: There was no correlation found between plasma sC5b-9 and TCC kidney staining. Elevated sC5b-9 levels were found in a heterogeneous group of patients but were associated with higher CRP and lower haemoglobin levels. Overall, there was more TCC kidney staining in the diseased group compared with the control group, and a trend was observed of diabetic, primary membranous nephropathy, and amyloidosis patients having more intense glomerular and peritubular/interstitial staining. CONCLUSION: Plasma sC5b-9 as a marker of total complement activation does not correlate with TCC kidney staining. This discordance suggests that plasma sC5b-9 and TCC staining are distinct markers of disease. TCC staining reflects chronicity and tissue deposition of complement over time. Conversely, plasma sC5b-9 concentrations change rapidly and reflect systemic complement activation. Complement activation was present in a heterogeneous group of kidney disease, indicating the underlying role of complement in many disorders.

Lichen sclerosus: A C5B-9 mediated chronic microvascular injury syndrome potentially reflective of common adult comorbidities.

Lichen sclerosus (LS) is a cutaneous disease of unknown etiology that often involves the vulva or foreskin but also can affect extragenital sites. Regardless of the anatomic site, the histomorphology and presumably pathogenesis are similar. Perhaps a clue to the pathophysiology of LS lies in its frequent association with morphea, specifically, when occurring in an extragenital context. In our experience a striking feature evident in established lichen sclerosis (LS) is one of superficial vascular drop out whereby residual vessels exhibited endothelial cell necrosis and microvascular basement membrane zone thickening, the latter reflective of antecedent episodes of microvascular injury. We sought to understand the pathophysiology that underlies the distinct vascular changes and in doing so, shed light on the pathogenesis of LS. We examined 44 cases of LS over a period of 2019 to 2021. We were able to obtain past medical histories in 34 of the 44 cases. Regarding pathological assessment, the predominant focus was on microvascular changes. We assessed the role of C5b-9 mediated vascular injury in the pathogenesis of the vasculopathy and enhanced type I interferon signaling in vessels given the morphologic semblance to the select interferonopathy syndromes, namely fibrosing dermatomyositis and Kohlmeier Degos disease. We examined the expression of CMV DNA and protein based on prior observations in an earlier study that isolated early protein expression in the microvasculature in the setting of LS and scleroderma. From a clinical perspective, the most striking association was an older age at the time of diagnosis (mean age of 62 years and median age of 61.5 years) and the presence of vascular comorbidities of diabetes, hypertension, and hyperlipidemia in almost 80% of cases. All cases showed significant microvascular changes in the superficial corium with the most frequent findings being those of significant basement membrane zone reduplication and vascular drop out. A number of cases showed prominent microvascular deposits of C5b-9 in the zone of hyalinizing fibrosis or subjacent to the discernible table of fibroplasia in the absence of enhanced type I interferon signaling. In no case were there viral cytopathic changes associated with CMV affecting the endothelium. The studies that encode CMV DNA or protein did not show a significant role for CMV reactivation in endothelium in the majority of the studied cases. It is concluded that the pathophysiology of LS includes a microvascular injury syndrome within the papillary dermis. The mechanism of endothelial cell injury is complement mediated at least in part and could reflect an adaptive immune response targeting endothelium indicative of classic complement pathway activation when coexisting with morphea or occurring in younger individuals. A non-immune based endothelial dysfunction and complement mediated injury unrelated to antibody driven classic complement pathway activation are more likely pathogenetically in the setting of certain diseases like diabetes mellitus and hypertension. Vascular drop out can be explained by the diminished endothelial progenitor pool needed to repopulate the damaged microvessels in certain settings like hypertension and diabetes.

Overexpression of Decay Accelerating Factor Mitigates Fibrotic Responses to Lung Injury.

CD55 or decay accelerating factor (DAF), a ubiquitously expressed glycosylphosphatidylinositol (GPI)-anchored protein, confers a protective threshold against complement dysregulation which is linked to the pathogenesis of idiopathic pulmonary fibrosis (IPF). Since lung fibrosis is associated with downregulation of DAF, we hypothesize that overexpression of DAF in fibrosed lungs will limit fibrotic injury by restraining complement dysregulation. Normal primary human alveolar type II epithelial cells (AECs) exposed to exogenous complement 3a or 5a, and primary AECs purified from IPF lungs demonstrated decreased membrane-bound DAF expression with concurrent increase in the endoplasmic reticulum (ER) stress protein, ATF6. Increased loss of extracellular cleaved DAF fragments was detected in normal human AECs exposed to complement 3a or 5a, and in lungs of IPF patients. C3a-induced ATF6 expression and DAF loss was inhibited using pertussis toxin (an enzymatic inactivator of G-protein coupled receptors), in murine AECs. Treatment with soluble DAF abrogated tunicamycin-induced C3a secretion and ER stress (ATF6 and BiP expression) and restored epithelial cadherin. Bleomycin-injured fibrotic mice subjected to lentiviral overexpression of DAF demonstrated diminished levels of local collagen deposition and complement activation. Further analyses showed diminished release of DAF fragments, as well as reduction in apoptosis (TUNEL and caspase 3/7 activity), and ER stress-related transcripts. Loss-of-function studies using Daf1 siRNA demonstrated worsened lung fibrosis detected by higher mRNA levels of Col1a1 and epithelial injury-related Muc1 and Snai1, with exacerbated local deposition of C5b-9. Our studies provide a rationale for rescuing fibrotic lungs via DAF induction that will restrain complement dysregulation and lung injury.

Sublytic C5b-9 triggers glomerular mesangial cell proliferation via enhancing FGF1 and PDGFα gene transcription mediated by GCN5-dependent SOX9 acetylation in rat Thy-1 nephritis.

Rat Thy-1 nephritis (Thy-1N) is an animal model of human mesangioproliferative glomerulonephritis (MsPGN), accompanied by glomerular mesangial cell (GMC) proliferation and extracellular matrix (ECM) deposition. Although sublytic C5b-9 formed on GMC membrane could induce cell proliferation, the mechanism is still unclear. In this study, we first demonstrated that the level of SRY related HMG-BOX gene 9 (SOX9), general control nonderepressible 5 (GCN5), fibroblast growth factor 1 (FGF1) and platelet-derived growth factor α (PDGFα) was all elevated both in the renal tissues of Thy-1N rats (in vivo) and in the GMCs (in vitro) with sublytic C5b-9 stimulation. Then, we not only discovered that sublytic C5b-9 caused GMC proliferation through increasing SOX9, GCN5, FGF1 and PDGFα expression, but also proved that SOX9 and GCN5 formed a complex and combined with FGF1 and PDGFα promoters, leading to FGF1 and PDGFα gene transcription. More importantly, GCN5 could mediate SOX9 acetylation at lysine 62 (K62) to enhance SOX9 binding to FGF1 or PDGFα promoter and promote FGF1 or PDGFα synthesis and GMC proliferation. Besides, the experiments in vivo also showed that FGF1 and PDGFα expression, GMC proliferation and urinary protein secretion in Thy-1N rats were greatly reduced by silencing renal SOX9, GCN5, FGF1 or PDGFα gene. Furthermore, the renal tissues of MsPGN patients also exhibited positive expression of these genes mentioned above. Collectively, our findings indicate that GCN5, SOX9 and FGF1/PDGFα can form an axis and play an essential role in sublytic C5b-9-triggered GMC proliferation, which might provide a novel insight into the pathogenesis of Thy-1N and MsPGN.

Differences and similarities in endothelial and angiogenic profiles of preeclampsia and COVID-19 in pregnancy.

BACKGROUND: COVID-19 presents a spectrum of signs and symptoms in pregnant women that might resemble preeclampsia. Differentiation between severe COVID-19 and preeclampsia is difficult in some cases. OBJECTIVE: To study biomarkers of endothelial damage, coagulation, innate immune response, and angiogenesis in preeclampsia and COVID-19 in pregnancy in addition to in vitro alterations in endothelial cells exposed to sera from pregnant women with preeclampsia and COVID-19. STUDY DESIGN: Plasma and sera samples were obtained from pregnant women with COVID-19 infection classified into mild (n=10) or severe (n=9) and from women with normotensive pregnancies as controls (n=10) and patients with preeclampsia (n=13). A panel of plasmatic biomarkers was assessed, including vascular cell adhesion molecule-1, soluble tumor necrosis factor-receptor I, heparan sulfate, von Willebrand factor antigen (activity and multimeric pattern), α2-antiplasmin, C5b9, neutrophil extracellular traps, placental growth factor, soluble fms-like tyrosine kinase-1, and angiopoietin 2. In addition, microvascular endothelial cells were exposed to patients' sera, and changes in the cell expression of intercellular adhesion molecule 1 on cell membranes and von Willebrand factor release to the extracellular matrix were evaluated through immunofluorescence. Changes in inflammation cell signaling pathways were also assessed by of p38 mitogen-activated protein kinase phosphorylation. Statistical analysis included univariate and multivariate methods. RESULTS: Biomarker profiles of patients with mild COVID-19 were similar to those of controls. Both preeclampsia and severe COVID-19 showed significant alterations in most circulating biomarkers with distinctive profiles. Whereas severe COVID-19 exhibited higher concentrations of vascular cell adhesion molecule-1, soluble tumor necrosis factor-α receptor I, heparan sulfate, von Willebrand factor antigen, and neutrophil extracellular traps, with a significant reduction of placental growth factor compared with controls, preeclampsia presented a marked increase in vascular cell adhesion molecule-1 and soluble tumor necrosis factor-α receptor I (significantly increased compared with controls and patients with severe COVID-19), with a striking reduction in von Willebrand factor antigen, von Willebrand factor activity, and α2-antiplasmin. As expected, reduced placental growth factor, increased soluble fms-like tyrosine kinase-1 and angiopoietin 2, and a very high soluble fms-like tyrosine kinase-1 to placental growth factor ratio were also observed in preeclampsia. In addition, a significant increase in C5b9 and neutrophil extracellular traps was also detected in preeclampsia compared with controls. Principal component analysis demonstrated a clear separation between patients with preeclampsia and the other groups (first and second components explained 42.2% and 13.5% of the variance), mainly differentiated by variables related to von Willebrand factor, soluble tumor necrosis factor-receptor I, heparan sulfate, and soluble fms-like tyrosine kinase-1. Von Willebrand factor multimeric analysis revealed the absence of von Willebrand factor high-molecular-weight multimers in preeclampsia (similar profile to von Willebrand disease type 2A), whereas in healthy pregnancies and COVID-19 patients, von Willebrand factor multimeric pattern was normal. Sera from both preeclampsia and severe COVID-19 patients induced an overexpression of intercellular adhesion molecule 1 and von Willebrand factor in endothelial cells in culture compared with controls. However, the effect of preeclampsia was less pronounced than the that of severe COVID-19. Immunoblots of lysates from endothelial cells exposed to mild and severe COVID-19 and preeclampsia sera showed an increase in p38 mitogen-activated protein kinase phosphorylation. Patients with severe COVID-19 and preeclampsia were statistically different from controls, suggesting that both severe COVID-19 and preeclampsia sera can activate inflammatory signaling pathways. CONCLUSION: Although similar in in vitro endothelial dysfunction, preeclampsia and severe COVID-19 exhibit distinctive profiles of circulating biomarkers related to endothelial damage, coagulopathy, and angiogenic imbalance that could aid in the differential diagnosis of these entities.

Immune-mediated membranous nephropathy: Long term fluconazole usage caused podocyte autophagy.

The primary consequences of membranous nephropathy (MN) are the development of nephrotic syndrome including hypogammaglobulinemia, the increased infectious risk, the loss of protein-bound vitamin D, and, above all, an elevated thromboembolic incidence of up to 50% in severe proteinuria patients. Membrane nephropathy may be either idiopathic or primary, not recognized (70%-80%) or secondary (20%-30%) to pathological sicknesses such as hepatitis B, systemic lupus erythematosus, malignancies, and side-effects of medicines. The immunological responses in MN involve multiple components: immunoglobulin G (IgG), long-escaped antigens, and the membrane attachment complex, formed by the supplement to form C5b-9. In general, IgG4 is the most significant IgG subclass deposited in idiopathic membranous nephropathic disease but fluctuating IgG1 levels also are linked with immunological deposits. In contrast, IgG1, IgG2, and IgG3 deposition are greater than IgG4 deposition in secondary nephropathy. Fluconazole is a synthetic antifungal triazole that is often used. It is well tolerated in general and has never been identified as a cause of nephropathies. We report on the development of MN caused by fluconazole therapy that could potentiate podocyte autophagy.

C5b-9 mediates ferroptosis of tubular epithelial cells in trichloroethylene-sensitization mice.

Occupational medicamentose-like dermatitis due to trichloroethylene (OMDT) is a key but unresolved question. OMDT patients often present multiple organ damage, including kidney damage. However, the underlying mechanism remains unknown. The purpose of our study was to explore the effect of tubule-specific C5b-9 deposition induced by TCE sensitization on renal tubular ferroptosis and its mechanism. By analyzing pathological changes of TCE-sensitization-mice kidney, we observed a significant renal tubular ferroptosis, which was alleviated by CD59, a C5b-9 inhibitory protein. Moreover, this phenomenon was also replicated in a C5b-9-attacked HK-2 cell model. Further experiments identified that C5b-9 induced cytosolic Ca2+ overload in renal tubular epithelia cells from TCE-sensitization-mice and HK-2 cells. Furthermore, in vitro experiments showed that BAPTA-AM, an intracellular Ca2+ chelator, could rescued ferroptosis induced by C5b-9 in HK-2 cells. Taken together, TCE sensitization induced renal tubular ferroptosis is mediated by C5b-9 and cytosolic Ca2+ overload may play a key role.

Complement as the enabler of carfilzomib-induced thrombotic microangiopathy.

Carfilzomib has been associated with the development of thrombotic microangiopathy (TMA) in relapsed/refractory multiple myeloma patients, a severe disease with no currently available aetiological treatment. We evaluated the potential role of terminal complement pathway in four patients with carfilzomib-induced TMA. Membrane attack complex (C5b-9) deposition on endothelial cells in culture exposed to plasma from patients during the acute phase of the disease suggests complement overactivation as a mechanism of potential endothelial damage in three out of four patients. If confirmed in larger cohorts, C5b-9 evaluation will allow early identification of patients who could benefit from complement blockade and treatment monitoring.

Treatable, motor-sensory, axonal neuropathies with C5b-9 complement on endoneurial microvessels.

INTRODUCTION: Identification and treatment of immune-mediated polyneuropathies may lead to improved strength and function. We studied the clinical and laboratory features, and treatment response, in patients with motor-sensory axonal polyneuropathies who were found to have C5b-9 complement staining on endoneurial microvessels. METHODS: Retrospective review of 16 consecutive adults with motor-sensory axonal polyneuropathies who were then found to have C5b-9 staining of endoneurial microvessels on nerve biopsy, and subsequently treated with intravenous corticosteroids (1 g methylprednisolone for 5 consecutive days, and then weekly). Strength measurements were done using quantitative handheld dynamometry. Nerve biopsy analysis included frozen and fixed tissue. RESULTS: Patients (mean onset age, 59 ± 4 years; range, 34-83 years; 12 of 16 were males; 9 of 16 had diabetes) had progressive (median duration, 2 years), asymmetric, distal weakness, in the lower extremities (16 of 16) and/or upper extremities (7 of 16), and panmodal sensory loss. Electrodiagnostic studies showed axon loss. Nerve pathology showed abnormal C5b-9 staining on endoneurial microvessels. Axon loss was present in all nerves, often varied among fascicles. Inflammation was uncommon. Distal strength usually improved (mean improvement of 34 ± 6% of normal strength; P = .0003) with corticosteroid treatment. DISCUSSION: Motor-sensory axonal polyneuropathies having noninflammatory, humoral immune pathology with C5b-9 staining of endoneurial microvessels (HIEM) frequently manifest progressive asymmetric, distal, lower extremity with or without upper extremity weakness that improves rapidly during corticosteroid treatment. HIEM may represent a new class of noninflammatory-vasculopathic, treatable axonal motor-sensory neuropathies.

C5b-9 membrane attack complex activated NLRP3 inflammasome mediates renal tubular immune injury in trichloroethylene sensitized mice.

Trichloroethylene (TCE) induced occupational medicamentosa-like dermatitis (OMLDT) in patients is accompanied, typically, by renal damage. But the role of C5b-9 and IL-1ß in TCE-sensitized mouse renal tubular damage is unclear. This study aimed to investigate whether TCE-sensitized mouse renal tubular epithelial cell damage was induced by NLRP3 inflammasome and whether NLRP3 inflammasome was activated by sublytic C5b-9. In total, 52 specific pathogen-free BALB/c female mice, 6- to 8-week-old, were used for establishing the TCE-sensitized mouse model. Renal tubular epithelial cells were isolated and used for determining the sublytic level of C5b-9. Kidney histological examination, serum neutrophil gelatinase associated lipocalin (NGAL) level were used for kidney damage evaluation. Renal protein levels of C5b-9, NLRP3, ASC, Caspase-1, IL-1ß, and IL-18 were measured. The renal lesions, serum NGAL level, renal NLRP3, ASC, Caspase-1 and IL-1ß protein levels all increased significantly in TCE sensitized positive group. However, pretreatment with recombinant protein sCD59-Cys inhibited the expression of C5b-9, NLRP3 inflammasome, IL-1ß, IL-18, and attenuated renal tubular epithelial cell damage. The sublytic C5b-9 activated NLRP3 inflammasome and aggravated renal tubular epithelial cell damage. Pretreatment with recombinant protein sCD59-Cys blocked the expression of the NLRP3 inflammasome by inhibiting the expression of C5b-9, and alleviating renal tubular epithelial cell damage.

Changes of Serum Ficolin-3 and C5b-9 in Patients with Heart Failure.

OBJECTIVES: To investigate the correlation of serum ficolin-3 and C5b-9 with cardiac function and NT-proBNP in patients with heart failure. METHODS: Sixty patients with heart failure admitted to the Baoding First Central Hospital from May 2019 to May 2020 were selected and divided into three groups according to the classification of New York Heart Association (NYHA). Patients with NYHA grade II, III, and IV were included into group A, B, and C, respectively. Among the population undergoing physical examination at the same time, 20 cases with no significant difference in age and gender from the experimental group were selected as the control group (Group-N), and their clinical data were recorded. The serum levels of ficolin-3, C5b-9 and NT-proBNP in each group were detected and compared. RESULTS: The serum concentrations of ficolin-3 and C5b-9 in Group N were significantly different from those in Group A, B and C (p<0.05), the difference between Group C and Group A and B was statistically significant (p<0.05), there was no significant difference between group A and B (p>0.05). The correlation analysis between serum ficolin-3 and NT-proBNP showed that serum ficolin-3 was negatively correlated with NT-proBNP (r=-0.606, p<0.0001), while the correlation analysis between serum C5b-9 and NT-proBNP showed that serum C5b-9 was positively correlated with NT-proBNP (r=0.499, p<0.0001). According to the etiology of heart failure, patients with heart failure were divided into coronary heart disease (25 cases), dilated cardiomyopathy (15 cases) and others (20 cases). The differences of ficolin-3 and C5b-9 among patients were compared, and there was no statistical difference (p<0.05). CONCLUSION: Ficolin-3 was inversely associated with the severity of heart failure, while C5b-9 was positively associated with the severity of cardiac impairment. Both of them have nothing to do with the etiology of heart failure.

Sublytic C5b-9 induces proliferation of glomerular mesangial cells via ERK5/MZF1/RGC-32 axis activated by FBXO28-TRAF6 complex.

Mesangioproliferative glomerulonephritis (MsPGN) is characterized by the proliferation of glomerular mesangial cells (GMCs) and accumulation of extracellular matrix (ECM), followed by glomerulosclerosis and renal failure of patients. Although our previous studies have demonstrated that sublytic C5b-9 complex formed on the GMC membrane could trigger GMC proliferation and ECM expansion of rat Thy-1 nephritis (Thy-1N) as an animal model of MsPGN, their mechanisms are still not fully elucidated. In the present studies, we found that the levels of response gene to complement 32 (RGC-32), myeloid zinc finger 1 (MZF1), phosphorylated extracellular signal-regulated kinase 5 (phosphorylated ERK5, p-ERK5), F-box only protein 28 (FBXO28) and TNF receptor-associated factor 6 (TRAF6) were all markedly up-regulated both in the renal tissues of rats with Thy-1N (in vivo) and in the GMCs upon sublytic C5b-9 stimulation (in vitro). Further in vitro experiments revealed that up-regulated FBXO28 and TRAF6 could form protein complex binding to ERK5 and enhance ERK5 K63-ubiquitination and subsequent phosphorylation. Subsequently, ERK5 activation contributed to MZF1 expression and MZF1-dependent RGC-32 up-regulation, finally resulting in GMC proliferative response. Furthermore, the MZF1-binding element within RGC-32 promoter and the functions of FBXO28 domains were identified. Additionally, knockdown of renal FBXO28, TRAF6, ERK5, MZF1 and RGC-32 genes respectively markedly reduced GMC proliferation and ECM production in Thy-1N rats. Together, these findings indicate that sublytic C5b-9 induces GMC proliferative changes in rat Thy-1N through ERK5/MZF1/RGC-32 axis activated by the FBXO28-TRAF6 complex, which might provide a new insight into MsPGN pathogenesis.

Diagnostic roles of urinary kidney injury molecule 1 and soluble C5b-9 in acute tubulointerstitial nephritis.

Acute tubulointerstitial nephritis (ATIN) is a common cause of acute kidney injury characterized by inflammatory cells infiltrating in the interstitium. The present study aimed to explore noninvasive biomarkers that might indicate activity of pathological injuries and help direct treatment. Fifty-four patients with clinical-pathologically diagnosed ATIN from January 1, 2014, to June 30, 2016, at Peking University First Hospital were enrolled. Urine samples were collected on the morning of renal biopsy and assessed for urinary kidney injury molecule-1 (KIM-1) and urinary soluble C5b-9 (sC5b-9). Immunofluorescence staining for KIM-1 and C5b-9 was performed in biopsied kidney sections from ATIN cases. The clinical and pathological relevance of the two urinary biomarkers was analyzed. Both urinary KIM-1 and sC5b-9 values were significantly elevated in patients with ATIN compared with healthy controls. The urinary KIM-1 level positively correlated with urinary N-acetyl-ß-d-glucosaminidase (r = 0. 542, P = 0.001) and the pathological tubular injury score (r = 0.469, P < 0.001), whereas the urinary sC5b-9 level was related to pathological activity scores for tubular injury (r = 0.413, P = 0.002), interstitial inflammation (r = 0.388, P = 0.004), and treatment response (r = 0.564, P < 0.001). Urinary KIM-1 tended to have better diagnostic value for tubular injury than urinary sC5b-9, whereas only urinary sC5b-9 was able to demonstrate severe interstitial inflammation. A combination of urinary KIM-1 and sC5b-9 had an area under the receiver-operating characteristic curve of 0.864 (95% confidence interval: 0.766-0.963, P < 0.001, sensitivity: 75%, specificity: 88%) for acute tissue injury in ATIN. KIM-1 expression was markedly increased in renal tubular cells in both ATIN and acute tubular necrosis conditions, whereas a significant upregulation of C5b-9 was only detected in the tubular cells and interstitial cells in ATIN cases. Urinary KIM-1 is a specific biomarker for renal tubular injury in ATIN, whereas urinary sC5b-9 is valuable in demonstrating severe interstitial inflammation. The combination of these two biomarkers helps identify patients at an acute injury stage and, therefore, might facilitate clinical evaluation and guide immunosuppressive therapy.

An Ex Vivo Test of Complement Activation on Endothelium for Individualized Eculizumab Therapy in Hemolytic Uremic Syndrome.

RATIONALE & OBJECTIVE: Although primary atypical hemolytic uremic syndrome (aHUS) is associated with abnormalities in complement genes and antibodies to complement factor H, the role of complement in secondary aHUS remains debatable. We evaluated the usefulness of an ex vivo test to: (1) detect complement activation within the endothelium in primary and secondary aHUS, (2) differentiate active disease from remission, (3) monitor the effectiveness of eculizumab therapy, and (4) identify relapses during eculizumab dosage tapering and after discontinuation of treatment. STUDY DESIGN: Case series. SETTING & PARTICIPANTS: 121 patients with primary aHUS and 28 with secondary aHUS. Serum samples were collected during acute episodes, following remission, and during eculizumab treatment and were assessed using a serum-induced ex vivo C5b-9 endothelial deposition test. RESULTS: Serum-induced C5b-9 deposition on cultured microvascular endothelium was quantified by calculating the endothelial area covered by C5b-9 staining; values were expressed as percentage of C5b-9 deposits induced by a serum pool from healthy controls. Testing with adenosine diphosphate-activated endothelium demonstrated elevated C5b-9 deposits for all untreated patients with aHUS independent of disease activity, while testing with unstimulated endothelium demonstrated deposits only in active disease. Similar findings were observed in secondary aHUS. Serum-induced C5b-9 deposits on activated and unstimulated endothelium normalized during eculizumab treatment. 96% (22/23) of patients receiving eculizumab at extended 3- or 4-week dosing intervals demonstrated normal C5b-9 deposits on activated endothelium, despite most patients having CH50Eq (serum complement activity) > 20 UEq/mL, indicating that adequate complement control was achieved even with incomplete blockade of circulating C5. During eculizumab dosage tapering or after treatment discontinuation, all patients experiencing relapses versus only 6% (1/17) of those in stable remission had elevated C5b-9 deposits on unstimulated endothelium. LIMITATIONS: The C5b-9 endothelial deposition test can be performed in only specialized laboratories. Findings on eculizumab dosage tapering need to be confirmed with longitudinal monitoring of C5b-9 deposition. CONCLUSIONS: The C5b-9 endothelial deposition assay may represent an advance in our ability to monitor aHUS activity and individualize therapy.

Intracerebral matrix metalloproteinase 9 in fatal diabetic ketoacidosis.

There is increasing awareness that in addition to the metabolic crisis of diabetic ketoacidosis (DKA) caused by severe insulin deficiency, the immune inflammatory response is likely an active multicomponent participant in both the acute and chronic insults of this medical crisis, with strong evidence of activation for both the cytokine and complement system. Recent studies report that the matrix metalloproteinase enzymes and their inhibitors are systemically activated in young Type 1 diabetes mellitus (T1D) patients during DKA and speculate on their involvement in blood-brain barrier (BBB) disruption. Based on our previous studies, we address the question if matrix metalloproteinase 9 (MMP9) is expressed in the brain in the fatal brain edema (BE) of DKA. Our data show significant expression of MMP9 on the cells present in brain intravascular areas. The presence of MMP9 in intravascular cells and that of MMP+ cells seen passing the BBB indicates a possible role in tight junction protein disruption of the BBB, possibly leading to neurological complications including BE. We have also shown that MMP9 is expressed on neurons in the hippocampal areas of both BE/DKA cases investigated, while expression of tissue inhibitor of metalloproteinases 1 (TIMP1) was reduced in the same areas. We can speculate that intraneuronal MMP9 can be a sign of neurodegeneration. Further studies are necessary to determine the role of MMP9 in the pathogenesis of the neurologic catastrophe of the brain edema of DKA. Inhibition of MMP9 expression might be helpful in preserving neuronal function and BBB integrity during DKA.

Sepsis and septic shock: endothelial molecular pathogenesis associated with vascular microthrombotic disease.

In addition to protective "immune response", sepsis is characterized by destructive "endothelial response" of the host, leading to endotheliopathy and its molecular dysfunction. Complement activation generates membrane attack complex (MAC). MAC causes channel formation to the cell membrane of pathogen, leading to death of microorganisms. In the host, MAC also may induce channel formation to innocent bystander endothelial cells (ECs) and ECs cannot be protected. This provokes endotheliopathy, which activates two independent molecular pathways: inflammatory and microthrombotic. Activated inflammatory pathway promotes the release of inflammatory cytokines and triggers inflammation. Activated microthrombotic pathway mediates platelet activation and exocytosis of unusually large von Willebrand factor multimers (ULVWF) from ECs and initiates microthrombogenesis. Excessively released ULVWF become anchored to ECs as long elongated strings and recruit activated platelets to assemble platelet-ULVWF complexes and form "microthrombi". These microthrombi strings trigger disseminated intravascular microthrombosis (DIT), which is the underlying pathology of endotheliopathy-associated vascular microthrombotic disease (EA-VMTD). Sepsis-induced endotheliopathy promotes inflammation and DIT. Inflammation produces inflammatory response and DIT orchestrates consumptive thrombocytopenia, microangiopathic hemolytic anemia, and multiorgan dysfunction syndrome (MODS). Systemic inflammatory response syndrome (SIRS) is a combined phenotype of inflammation and endotheliopathy-associated (EA)-VMTD. Successful therapeutic design for sepsis can be achieved by counteracting the pathologic microthrombogenesis.