Proof of concept of a new plasma complement Factor H from waste plasma fraction.

Introduction: Complement factor H (FH) is a major regulator of the complement alternative pathway, its mutations predispose to an uncontrolled activation in the kidney and on blood cells and to secondary C3 deficiency. Plasma exchange has been used to correct for FH deficiency and although the therapeutic potential of purified FH has been suggested by in vivo experiments in animal models, a clinical approved FH concentrate is not yet available. We aimed to develop a purification process of FH from a waste fraction rather than whole plasma allowing a more efficient and ethical use of blood and plasma donations. Methods: Waste fractions from industrial plasma fractionation (pooled human plasma) were analyzed for FH content by ELISA. FH was purified from unused fraction III and its decay acceleration, cofactor, and C3 binding capacity were characterized in vitro. Biodistribution was assessed by high-resolution dynamic PET imaging. Finally, the efficacy of the purified FH preparation was tested in the mouse model of C3 glomerulopathy (Cfh-/- mice). Results: Our purification method resulted in a high yield of highly purified (92,07%), pathogen-safe FH. FH concentrate is intact and fully functional as demonstrated by in vitro functional assays. The biodistribution revealed lower renal and liver clearance of human FH in Cfh-/- mice than in wt mice. Treatment of Cfh-/- mice documented its efficacy in limiting C3 activation and promoting the clearance of C3 glomerular deposits. Conclusion: We developed an efficient and economical system for purifying intact and functional FH, starting from waste material of industrial plasma fractionation. The FH concentrate could therefore constitute possible treatments options of patients with C3 glomerulopathy, particularly for those with FH deficiency, but also for patients with other diseases associated with alternative pathway activation.

Venous Thromboembolism Management throughout the COVID-19 Era: Addressing Acute and Long-Term Challenges.

Background: COVID-19 increases the risk of venous thromboembolism (VTE) through a complex interplay of mechanisms collectively referred to as immunothrombosis. Limited data exist on VTE challenges in the acute setting throughout a dynamic long-term follow-up of COVID-19 patients compared to non-COVID-19 patients. The aim of the study was to investigate acute and long-term management and complications in VTE patients with and without COVID-19. Methods: A prospective, observational, single-center cohort study on VTE patients followed from the acute care stage until 24 months post-diagnosis. Results: 157 patients, 30 with COVID-19-associated VTE and 127 unrelated to COVID-19, were enrolled. The mean follow-up was 10.8 (±8.9) months. COVID-19 patients had fewer comorbidities (1.3 ± 1.29 vs. 2.26 ± 1.68, p < 0.001), a higher proportion of pulmonary embolism at baseline (96.7% vs. 76.4%, p = 0.01), and had a lower probability of remaining on anticoagulant therapy after three months (p < 0.003). The most used initial therapy was low-molecular-weight heparin in 130/157 cases, followed by long-term treatment with direct oral anticoagulants in 123/157. Two (6.7%) COVID-19 vs. three (2.4%) non-COVID-19 patients (p = 0.243) had major hemorrhagic events, all of them within the first three months. Four (3.1%) non-COVID-19 patients had VTE recurrence after six months. Three (2.4%) non-COVID-19 patients developed chronic thromboembolic pulmonary hypertension. There were no fatalities among patients with COVID-19, compared to a mortality of 12/127 (9.4%) in the non-COVID-19 subgroup (p = 0.027). Discussion: Our study offers a comprehensive overview of the evolving nature of VTE management, emphasizing the importance of personalized risk-based approaches, including a limited course of anticoagulation for most COVID-19-associated VTE cases and reduced-dose extended therapy for high-risk subsets.

A New Ultrasensitive Bioluminescence-Based Method for Assaying Monoacylglycerol Lipase.

A novel bioluminescent Monoacylglycerol lipase (MAGL) substrate 6-O-arachidonoylluciferin, a D-luciferin derivative, was synthesized, physico-chemically characterized, and used as highly sensitive substrate for MAGL in an assay developed for this purpose. We present here a new method based on the enzymatic cleavage of arachidonic acid with luciferin release using human Monoacylglycerol lipase (hMAGL) followed by its reaction with a chimeric luciferase, PLG2, to produce bioluminescence. Enzymatic cleavage of the new substrate by MAGL was demonstrated, and kinetic constants Km and Vmax were determined. 6-O-arachidonoylluciferin has proved to be a highly sensitive substrate for MAGL. The bioluminescence assay (LOD 90 pM, LOQ 300 pM) is much more sensitive and should suffer fewer biological interferences in cells lysate applications than typical fluorometric methods. The assay was validated for the identification and characterization of MAGL modulators using the well-known MAGL inhibitor JZL184. The use of PLG2 displaying distinct bioluminescence color and kinetics may offer a highly desirable opportunity to extend the range of applications to cell-based assays.

Anti-C1-Inhibitor Autoantibody Detection by ELISA.

Enzyme-linked immunosorbent assay (ELISA) is a quantitative analytical method used to measure the concentration of molecules in biological fluids through antigen-antibody reactions. Here we describe the measurement of anti-C1-inhibitor autoantibodies by an indirect ELISA. In this method patients' sera are incubated in a microplate coated with plasma derived C1-inhibitor.

IgM Autoantibodies to Complement Factor H in Atypical Hemolytic Uremic Syndrome.

BACKGROUND: Atypical hemolytic uremic syndrome (aHUS), a severe thrombotic microangiopathy, is often related to complement dysregulation, but the pathomechanisms remain unknown in at least 30% of patients. Researchers have described autoantibodies to complement factor H of the IgG class in 10% of patients with aHUS but have not reported anti-factor H autoantibodies of the IgM class. METHODS: In 186 patients with thrombotic microangiopathy clinically presented as aHUS, we searched for anti-factor H autoantibodies of the IgM class and those of the IgG and IgA classes. We used immunochromatography to purify anti-factor H IgM autoantibodies and immunoenzymatic methods and a competition assay with mapping mAbs to characterize interaction with the target protein. RESULTS: We detected anti-factor H autoantibodies of the IgM class in seven of 186 (3.8%) patients with thrombotic microangiopathy presented as aHUS. No association was observed between anti-factor H IgM and homozygous deletions involving CFHR3-CFHR1. A significantly higher proportion of patients with bone marrow transplant-related thrombotic microangiopathy had anti-factor H IgM autoantibodies versus other patients with aHUS: three of 20 (15%) versus four of 166 (2.4%), respectively. The identified IgM autoantibodies recognize the SCR domain 19 of factor H molecule in all patients and interact with the factor H molecule, inhibiting its binding to C3b. CONCLUSIONS: Detectable autoantibodies to factor H of the IgM class may be present in patients with aHUS, and their frequency is six-fold higher in thrombotic microangiopathy forms associated with bone marrow transplant. The autoantibody interaction with factor H's active site may support an autoimmune mechanism in some cases previously considered to be of unknown origin.

The central role of endothelium in hereditary angioedema due to C1 inhibitor deficiency.

An impairment of the endothelial barrier function underlies a wide spectrum of pathological conditions. Hereditary angioedema due to C1-inhibitor deficiency (C1-INH-HAE) can be considered the "pathophysiological and clinical paradigm" of Paroxysmal Permeability Diseases (PPDs), conditions characterized by recurrent transient primitively functional alteration of the endothelial sieving properties, not due to inflammatory-ischemic-degenerative injury and completely reversible after the acute flare. It is a rare yet probably still underdiagnosed disease which presents with localized, non-pitting swelling of the skin and submucosal tissues of the upper respiratory and gastrointestinal tracts, without significant wheals or pruritus. The present review addresses the pathophysiology of C1-INH-HAE with a focus on the crucial role of the endothelium during contact and kallikrein/kinin system (CAS and KKS) activation, currently available and emerging biomarkers, methods applied to get new insights into the mechanisms underlying the disease (2D, 3D and in vivo systems), new promising investigation techniques (autonomic nervous system analysis, capillaroscopy, flow-mediated dilation method, non-invasive finger plethysmography). Hints are given to the binding of C1-INH to endothelial cells. Finally, crucial issues as the local vs systemic nature of CAS/KKS activation, the episodic nature of attacks vs constant C1-INH deficiency, pros and cons as well as future perspectives of available methodologies are briefly discussed.

Fundamentos y métodos de las revisiones sistemáticas / Fundamentals and method of systematic reviews

Las revisiones sistemáticas (RS) son síntesis cualitativas y, a veces, cuantitativas sobre la evidencia disponible respecto de un tema puntual. Este trabajo contextualiza su origen en el movimiento de la medicina basada en la evidencia y las define como un tipo de investigación orientada a identificar, valorar críticamente y sintetizar conocimiento científico previo. El método de RS se presenta aquí en una serie de fases o pasos: planteo del objetivo, búsqueda y selección de estudios, evaluación de calidad, extracción y análisis de datos, metaanálisis e interpretación de resultados. Los criterios de evaluación de la evidencia científica, un pilar fundamental de este tipo de investigación, son la validez interna, la precisión y la validez externa de los resultados. La calidad de la evidencia científica, junto con la cantidad y la consistencia, son la base sobre las cuales se pueden establecer recomendaciones. El método de RS es útil en la práctica profesional, donde se requiere el manejo de evidencia científica actualizada sobre los asuntos en los que a diario se deciden diagnósticos o intervenciones, así como un tipo de investigación que puede implementarse en grado o posgrado, ya que podría llegar a resultados originales mediante la síntesis de sus antecedentes

Systematic reviews (SR) are qualitative and, sometimes, quantitative syntheses on the available evidence regarding a specific topic. This work contextualizes its origin in the evidence-based medicine movement and defines it as a type of health research aimed to identify, critically appraise and synthesize previous scientific knowledge. The SR method is presented here in a series of steps: objective, search and selection of studies, quality assessment, data extraction and analysis, meta-analysis and interpretation of results. The internal validity, precision and external validity of the results are explained as criteria to assess the quality of the evidence. The quality of the evidence, together with the quantity and consistency, are the basis on which recommendations can be made. The SR method is useful in professional practice, where the management of up-to-date scientific evidence is required to make decisions on diagnoses or interventions daily, as well as a type of research that can be implemented in undergraduate or graduate studies, since it could arrive at original results by synthesizing the previous knowledge.

Intermittent C1-Inhibitor Deficiency Associated with Recessive Inheritance: Functional and Structural Insight.

C1-inhibitor is a serine protease inhibitor (serpin) controlling complement and contact system activation. Gene mutations result in reduced C1-inhibitor functional plasma level causing hereditary angioedema, a life-threatening disorder. Despite a stable defect, the clinical expression of hereditary angioedema is unpredictable, and the molecular mechanism underlying this variability remains undisclosed. Here we report functional and structural studies on the Arg378Cys C1-inhibitor mutant found in a patient presenting reduced C1-inhibitor levels, episodically undergoing normalization. Expression studies resulted in a drop in mutant C1-innhibitor secretion compared to wild-type. Notwithstanding, the purified proteins had similar features. Thermal denaturation experiments showed a comparable denaturation profile, but the mutant thermal stability decays when tested in conditions reproducing intracellular crowding.Our findings suggest that once correctly folded, the Arg378Cys C1-inhibitor is secreted as an active, although quite unstable, monomer. However, it could bear a folding defect, occasionally promoting protein oligomerization and interfering with the secretion process, thus accounting for its plasma level variability. This defect is exacerbated by the nature of the mutation since the acquired cysteine leads to the formation of non-functional homodimers through inter-molecular disulphide bonding. All the proposed phenomena could be modulated by specific environmental conditions, rendering this mutant exceptionally vulnerable to mild stress.

Acquired Complement Regulatory Gene Mutations and Hematopoietic Stem Cell Transplant-Related Thrombotic Microangiopathy.

Hematopoietic stem cell transplant-related thrombotic microangiopathy (HSCT-TMA) is a severe complication whose pathophysiology is unknown. We describe 6 patients in which the disease was associated with complement regulatory gene abnormalities received from their respective donors. It is suggested that mutated and transplanted monocyte-derived cells are responsible for production of abnormal proteins, complement dysregulation, and, ultimately, for the disease. This observation might have important drawbacks as far as HSCT-TMA pathophysiology and treatment are concerned.

Rapid isolation of pure Complement Factor H from serum for functional studies by the use of a monoclonal antibody that discriminates FH from all the other isoforms.

Several mutations have been identified in the gene coding for Complement Factor H (FH) from patients with atypical Hemolytic Uraemic Syndrome (aHUS), Age-related Macular Degeneration (AMD) and Membranoproliferative Glomerulonephritis (MPGN). These data allow for a precise description of the structural changes affecting FH, but a simple test for specifically assessing FH function routinely is not yet of common use. We have produced and characterised a monoclonal antibody (5H5) which discriminates between FH and the smaller FH-like 1 and FH-related proteins and show here that it specifically binds to FH without detecting the smaller isoforms. We therefore used this mAb for a quick, one-step micro-purification of FH directly from control sera and showed that this affinity chromatography procedure is not disruptive of its cofactor function. We also developed a modified sheep erythrocytes haemolysis test using our antibody and affinity-purified FH. These tests can be used in conjunction for assessing the function of FH purified from patients affected by FH-related diseases. Moreover we used this mAb to develop a FH-specific ELISA test.

BDNF repairs podocyte damage by microRNA-mediated increase of actin polymerization.

Idiopathic focal segmental glomerulosclerosis (FSGS) is a progressive and proteinuric kidney disease that starts with podocyte injury. Podocytes cover the external side of the glomerular capillary by a complex web of primary and secondary ramifications. Similar to dendritic spines of neuronal cells, podocyte processes rely on a dynamic actin-based cytoskeletal architecture to maintain shape and function. Brain-derived neurotrophic factor (BDNF) is a pleiotropic neurotrophin that binds to the tropomyosin-related kinase B receptor (TrkB) and has crucial roles in neuron maturation, survival, and activity. In neuronal cultures, exogenously added BDNF increases the number and size of dendritic spines. In animal models, BDNF administration is beneficial in both central and peripheral nervous system disorders. Here we show that BDNF has a TrkB-dependent trophic activity on podocyte cell processes; by affecting microRNA-134 and microRNA-132 signalling, BDNF up-regulates Limk1 translation and phosphorylation, and increases cofilin phosphorylation, which results in actin polymerization. Importantly, BDNF effectively repairs podocyte damage in vitro, and contrasts proteinuria and glomerular lesions in in vivo models of FSGS, opening a potential new perspective to the treatment of podocyte disorders.

Podocyte glutamatergic signaling contributes to the function of the glomerular filtration barrier.

Podocytes possess the complete machinery for glutamatergic signaling, raising the possibility that neuron-like signaling contributes to glomerular function. To test this, we studied mice and cells lacking Rab3A, a small GTPase that regulates glutamate exocytosis. In addition, we blocked the glutamate ionotropic N-methyl-d-aspartate receptor (NMDAR) with specific antagonists. In mice, the absence of Rab3A and blockade of NMDAR both associated with an increased urinary albumin/creatinine ratio. In humans, NMDAR blockade, obtained by addition of ketamine to general anesthesia, also had an albuminuric effect. In vitro, Rab3A-null podocytes displayed a dysregulated release of glutamate with higher rates of spontaneous exocytosis, explained by a reduction in Rab3A effectors resulting in freedom of vesicles from the actin cytoskeleton. In addition, NMDAR antagonism led to profound cytoskeletal remodeling and redistribution of nephrin in cultured podocytes; the addition of the agonist NMDA reversed these changes. In summary, these results suggest that glutamatergic signaling driven by podocytes contributes to the integrity of the glomerular filtration barrier and that derangements in this signaling may lead to proteinuric renal diseases.

Interstitial vascular rarefaction and reduced VEGF-A expression in human diabetic nephropathy.

Diabetic nephropathy (DN) is a frequent complication in patients with diabetes. Although the majority of DN models and human studies have focused on glomeruli, tubulointerstitial damage is a major feature of DN and an important predictor of renal dysfunction. This study sought to investigate molecular markers of pathogenic pathways in the renal interstitium of patients with DN. Microdissected tubulointerstitial compartments from biopsies with established DN and control kidneys were subjected to expression profiling. Analysis of candidate genes, potentially involved in DN on the basis of common hypotheses, identified 49 genes with significantly altered expression levels in established DN in comparison with controls. In contrast to some rodent models, the growth factors vascular endothelial growth factor A (VEGF-A) and epidermal growth factor (EGF) showed a decrease in mRNA expression in DN. This was validated on an independent cohort of patients with DN by real-time reverse transcriptase-PCR. Immunohistochemical staining for VEGF-A and EGF also showed a reduced expression in DN. The decrease of renal VEGF-A expression was associated with a reduction in peritubular capillary densities shown by platelet-endothelial cell adhesion molecule-1/CD31 staining. Furthermore, a significant inverse correlation between VEGF-A and proteinuria, as well as EGF and proteinuria, and a positive correlation between VEGF-A and hypoxia-inducible factor-1alpha mRNA was found. Thus, in human DN, a decrease of VEGF-A, rather than the reported increase as described in some rodent models, may contribute to the progressive disease. These findings and the questions about rodent models in DN raise a note of caution regarding the proposal to inhibit VEGF-A to prevent progression of DN.

Glomerular podocytes contain neuron-like functional synaptic vesicles.

Although patients with chronic renal failure are increasing worldwide, many aspects of kidney biology remain to be elucidated. Recent research has uncovered several molecular properties of the glomerular filtration barrier, in which podocytes, highly differentiated, ramified cells that enwrap the glomerular basement membrane, have been reported to be mainly responsible for filter's selectivity. We previously described that podocytes express Rab3A, a GTPase restricted to cell types that are capable of highly regulated exocytosis, such as neuronal cells. Here, we first demonstrate by a proteomic study that Rab3A in podocytes coimmmunoprecipitates with molecules once thought to be synapse specific. We then show that podocytes possess structures resembling synaptic vesicles, which contain glutamate, coexpress Rab3A and synaptotagmin 1, and undergo spontaneous and stimulated exocytosis and recycling, with glutamate release. Finally, from the results of a cDNA microarray study, we describe the presence of a series of neuron- and synapse-specific molecules in normal human glomeruli and confirm the glomerular protein expression of both metabotropic and ionotropic glutamate receptors. These data point toward a synaptic-like mechanism of communication among glomerular cells, which perfectly fits with the molecular composition of the glomerular filter and puts in perspective several previous observations, proposing a different working hypothesis for understanding glomerular signaling dynamics.

Glomerular podocytes possess the synaptic vesicle molecule Rab3A and its specific effector rabphilin-3a.

Several recent studies have focused on similarities between glomerular podocytes and neurons because the two cells share a specialized cytoskeletal organization and several expression-restricted proteins, such as nephrin and synaptopodin. In neurons, the small guanosine triphosphatase Rab3A and its effector rabphilin-3A form a complex required for the correct docking of synaptic vesicles to their target membrane. Because rabphilin-3A binds in neurons to cytoskeletal proteins also important for podocyte homeostasis, and the complex rabphilin-3A-Rab3A has been demonstrated in neurons and neuroendocrine cells, the aim of our work was to investigate their possible expression and regulation in podocytes. Normal kidneys from mouse, rat, and human were studied by immunohistochemistry, Western blotting, and reverse transcriptase-polymerase chain reaction to evaluate the expression of Rab3A and rabphilin-3A. Double-staining immunohistochemistry and immunogold electron microscopy were then used to precisely localize the two proteins at the cellular and subcellular levels. Rab-3A and rabphilin-3A regulations in disease were then analyzed in growth hormone-transgenic mice, a well established model of focal and segmental glomerulosclerosis, and in human biopsies from proteinuric patients. Our results demonstrated that rabphilin-3A and Rab3A are present in normal mouse, rat, and human kidneys, with an exclusively glomerular expression and a comma-like pattern of positivity along the glomerular capillary wall, suggestive for podocyte staining. Co-localization of both molecules with synaptopodin confirmed their presence in podocytes. By immunogold electron microscopy both proteins were found around vesicles contained in podocyte foot processes. Their expression was increased in growth hormone-transgenic mice compared to their wild-type counterpart, and in a subset of biopsies from proteinuric patients. Our data, demonstrating the presence of two synaptic proteins in podocytes, further supports similarities between cytoskeletal and vesicular organization of podocytes and neurons. The altered expression observed in mouse and human proteinuric diseases suggests a possible role for these molecules in glomerulopathies.