RESUMEN
Complement-mediated diseases can be treated using systemic inhibitors. However, complement components are abundant in circulation, affecting systemic inhibitors' exposure and efficacy. Furthermore, because of complement's essential role in immunity, systemic treatments raise infection risk in patients. To address these challenges, we developed antibody fusion proteins combining the alternative-pathway complement inhibitor factor H (fH1-5) with an anti-C3d monoclonal antibody (C3d-mAb-2fH). Because C3d is deposited at sites of complement activity, this molecule localizes to tissue complement while minimizing circulating complement engagement. These fusion proteins bind to deposited complement in diseased human skin sections and localize to activated complement in a primate skin injury model. We further explored the pharmacology of C3d-mAb-2fH proteins in rodent models with robust tissue complement activation. Doses of C3d-mAb-2fH >1 mg/kg achieved >75% tissue complement inhibition in mouse and rat injury models while avoiding circulating complement blockade. Glomerular-specific complement inhibition reduced proteinuria and preserved podocyte foot-process architecture in rat membranous nephropathy, indicating disease-modifying efficacy. These data indicate that targeting local tissue complement results in durable and efficacious complement blockade in skin and kidney while avoiding systemic inhibition, suggesting broad applicability of this approach in treating a range of complement-mediated diseases.
Asunto(s)
Factor H de Complemento , Enfermedades Renales , Humanos , Ratones , Ratas , Animales , Factor H de Complemento/genética , Complemento C3d/metabolismo , Enfermedades Renales/etiología , Anticuerpos , Activación de ComplementoRESUMEN
Alternative pathway complement dysregulation with abnormal glomerular C3 deposits and glomerular damage is a key mechanism of pathology in C3 glomerulopathy (C3G). No disease-specific treatments are currently available for C3G. Therapeutics inhibiting complement are emerging as a potential strategy for the treatment of C3G. In this study, we investigated the effects of N-acetylgalactosamine (GalNAc)-conjugated small interfering RNA (siRNA) targeting the C3 component of complement that inhibits liver C3 expression in the C3G model of mice with heterozygous deficiency of factor H (Cfh +/- mice). We showed a duration of action for GalNAc-conjugated C3 siRNA in reducing the liver C3 gene expression in Cfh +/- mice that were dosed s.c. once a month for up to 7 mo. C3 siRNA limited fluid-phase alternative pathway activation, reducing circulating C3 fragmentation and activation of factor B. Treatment with GalNAc-conjugated C3 siRNA reduced glomerular C3d deposits in Cfh +/- mice to levels similar to those of wild-type mice. Ultrastructural analysis further revealed the efficacy of the C3 siRNA in slowing the formation of mesangial and subendothelial electron-dense deposits. The present data indicate that RNA interference-mediated C3 silencing in the liver may be a relevant therapeutic strategy for treating patients with C3G associated with the haploinsufficiency of complement factor H.
Asunto(s)
Glomerulonefritis Membranoproliferativa , Enfermedades Renales , Animales , Complemento C3/genética , Complemento C3/metabolismo , Factor B del Complemento/metabolismo , Factor H de Complemento/genética , Vía Alternativa del Complemento/genética , Glomerulonefritis Membranoproliferativa/patología , Humanos , Ratones , ARN Interferente Pequeño/genéticaRESUMEN
In addition to having blood glucose-lowering effects, inhibitors of sodium glucose cotransporter 2 (SGLT2) afford renoprotection in diabetes. We sought to investigate which components of the glomerular filtration barrier could be involved in the antiproteinuric and renoprotective effects of SGLT2 inhibition in diabetes. BTBR (black and tan, brachyuric) ob/ob mice that develop a type 2 diabetic nephropathy received a standard diet with or without empagliflozin for 10 weeks, starting at 8 weeks of age, when animals had developed albuminuria. Empagliflozin caused marked decreases in blood glucose levels and albuminuria but did not correct glomerular hyperfiltration. The protective effect of empagliflozin against albuminuria was not due to a reduction in podocyte damage as empagliflozin did not affect the larger podocyte filtration slit pore size nor the defective expression of nephrin and nestin. Empagliflozin did not reduce the thickening of the glomerular basement membrane. In BTBR ob/ob mice, the most profound abnormality seen using electron microscopy was in the endothelial aspect of the glomerular capillary, with significant loss of endothelial fenestrations. Remarkably, empagliflozin ameliorated the subverted microvascular endothelial ultrastructure. Caveolae and bridging diaphragms between adjacent endothelial fenestrae were seen in diabetic mice and associated with increased expression of caveolin-1 and the appearance of PV-1. These endothelial abnormalities were limited by the SGLT2 inhibitor. Although no expression of SGLT2 was found in glomerular endothelial cells, SGLT2 was expressed in the podocytes of diabetic mice. VEGF-A, which is a known stimulus for endothelial caveolin-1 and PV-1, was increased in podocytes of BTBR ob/ob mice and normalized by SGLT2 inhibitor treatment. Thus, empagliflozin's protective effect on the glomerular endothelium of diabetic mice could be due to a limitation of the paracrine signaling of podocyte-derived VEGF-A that resulted in a reduction of the abnormal endothelial caveolin-1 and PV-1, with the consequent preservation of glomerular endothelial function and permeability. © 2022 The Pathological Society of Great Britain and Ireland.
Asunto(s)
Diabetes Mellitus Experimental , Nefropatías Diabéticas , Inhibidores del Cotransportador de Sodio-Glucosa 2 , Albuminuria/tratamiento farmacológico , Albuminuria/patología , Albuminuria/prevención & control , Animales , Compuestos de Bencidrilo , Glucemia/metabolismo , Caveolina 1/metabolismo , Diabetes Mellitus Experimental/tratamiento farmacológico , Nefropatías Diabéticas/tratamiento farmacológico , Nefropatías Diabéticas/metabolismo , Nefropatías Diabéticas/prevención & control , Células Endoteliales/metabolismo , Femenino , Membrana Basal Glomerular/metabolismo , Glucósidos , Humanos , Masculino , Ratones , Transducción de Señal , Transportador 2 de Sodio-Glucosa/metabolismo , Inhibidores del Cotransportador de Sodio-Glucosa 2/farmacología , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Peritubular capillary rarefaction is a recurrent aspect of progressive nephropathies. We previously found that peritubular capillary density was reduced in BTBR ob/ob mice with type 2 diabetic nephropathy. In this model, we searched for abnormalities in the ultrastructure of peritubular capillaries, with a specific focus on the endothelial glycocalyx, and evaluated the impact of treatment with an angiotensin-converting enzyme inhibitor (ACEi). Mice were intracardially perfused with lanthanum to visualise the glycocalyx. Transmission electron microscopy analysis revealed endothelial cell abnormalities and basement membrane thickening in the peritubular capillaries of BTBR ob/ob mice compared to wild-type mice. Remodelling and focal loss of glycocalyx was observed in lanthanum-stained diabetic kidneys, associated with a reduction in glycocalyx components, including sialic acids, as detected through specific lectins. ACEi treatment preserved the endothelial glycocalyx and attenuated the ultrastructural abnormalities of peritubular capillaries. In diabetic mice, peritubular capillary damage was associated with an enhanced tubular expression of heparanase, which degrades heparan sulfate residues of the glycocalyx. Heparanase was also detected in renal interstitial macrophages that expressed tumor necrosis factor-α. All these abnormalities were mitigated by ACEi. Our findings suggest that, in experimental diabetic nephropathy, preserving the endothelial glycocalyx is important in order to protect peritubular capillaries from damage and loss.
Asunto(s)
Diabetes Mellitus Experimental , Nefropatías Diabéticas , Ratones , Animales , Nefropatías Diabéticas/metabolismo , Inhibidores de la Enzima Convertidora de Angiotensina/farmacología , Inhibidores de la Enzima Convertidora de Angiotensina/metabolismo , Diabetes Mellitus Experimental/metabolismo , Capilares/patología , Glicocálix/metabolismo , Lantano , Riñón/patología , Ratones EndogámicosRESUMEN
Sirtuin 3 (SIRT3) is the primary mitochondrial deacetylase that controls the antioxidant pathway and energy metabolism. We previously found that renal Sirt3 expression and activity were reduced in mice with type 2 diabetic nephropathy associated with oxidative stress and mitochondrial abnormalities and that a specific SIRT3 activator improved renal damage. SIRT3 is modulated by diet, and to assess whether Sirt3 deficiency aggravates mitochondrial damage and accelerates kidney disease in response to nutrient overloads, wild-type (WT) and Sirt3-/- mice were fed a high-fat-diet (HFD) or standard diet for 8 months. Sirt3-/- mice on HFD exhibited earlier and more severe albuminuria compared to WT mice, accompanied by podocyte dysfunction and glomerular capillary rarefaction. Mesangial matrix expansion, tubular vacuolization and inflammation, associated with enhanced lipid accumulation, were more evident in Sirt3-/- mice. After HFD, kidneys from Sirt3-/- mice showed more oxidative stress than WT mice, mitochondria ultrastructural damage in tubular cells, and a reduction in mitochondrial mass and energy production. Our data demonstrate that Sirt3 deficiency renders mice more prone to developing oxidative stress and mitochondrial abnormalities in response to HFD, resulting in more severe kidney diseases, and this suggests that mitochondria protection may be a method to prevent HFD-induced renal injury.
Asunto(s)
Nefropatías Diabéticas , Sirtuina 3/metabolismo , Animales , Antioxidantes/metabolismo , Dieta Alta en Grasa , Ratones , Ratones Noqueados , Estrés Oxidativo , Sirtuina 3/genéticaRESUMEN
The Renin-Angiotensin System (RAS) possesses a counter-regulatory axis composed of angiotensin converting enzyme (ACE)2, angiotensin-(1-7) [Ang-(1-7)] and the Mas receptor, which opposes many AT1-receptor-mediated effects of ligand angiotensin II. Ang-(1-7), as a ligand of the Mas receptor, has inhibitory effects on renal inflammation and fibrosis in experimental diabetes. However, Ang-(1-7) has a short half-life in plasma, which may render it unsuitable for use in clinics. Here, we investigated the effects of the lanthionine-stabilized Ang-(1-7), cyclic (c)Ang-(1-7), a lanthipeptide that is more peptidase-resistant than the linear peptide, in BTBR ob/ob mice with type 2 diabetic nephropathy. BTBR ob/ob mice received vehicle, cAng-(1-7), or the ACE inhibitor lisinopril. The treatment started at ten weeks of age, when the animals had already developed albuminuria, and ended at 19-20 weeks of age. cAng-(1-7) limited albuminuria progression, and limited podocyte dysfunction similarly to lisinopril. cAng-(1-7), unlike lisinopril, reduced glomerular fibrosis and inflammation, and counteracted glomerular capillary rarefaction. Furthermore, when cAng-(1-7) was combined with lisinopril, a superior antiproteinuric effect than with lisinopril alone was found, in association with better preservation of podocyte proteins and amelioration of capillary density. Thus, adding cAng-(1-7) to ACE-inhibitor therapy could benefit those diabetic patients who do not respond completely to ACE-inhibitor therapy.
Asunto(s)
Angiotensina I/administración & dosificación , Inhibidores de la Enzima Convertidora de Angiotensina/administración & dosificación , Diabetes Mellitus Tipo 2/complicaciones , Nefropatías Diabéticas/tratamiento farmacológico , Fragmentos de Péptidos/administración & dosificación , Proteinuria/tratamiento farmacológico , Alanina/administración & dosificación , Alanina/análogos & derivados , Alanina/química , Alanina/farmacocinética , Angiotensina I/química , Angiotensina I/farmacocinética , Animales , Diabetes Mellitus Tipo 2/genética , Nefropatías Diabéticas/diagnóstico , Nefropatías Diabéticas/etiología , Nefropatías Diabéticas/patología , Modelos Animales de Enfermedad , Quimioterapia Combinada/métodos , Semivida , Humanos , Glomérulos Renales/irrigación sanguínea , Glomérulos Renales/efectos de los fármacos , Glomérulos Renales/patología , Glomérulos Renales/ultraestructura , Lisinopril/administración & dosificación , Masculino , Ratones , Ratones Transgénicos , Microscopía Electrónica de Transmisión , Fragmentos de Péptidos/química , Fragmentos de Péptidos/farmacocinética , Péptidos Cíclicos/administración & dosificación , Péptidos Cíclicos/química , Péptidos Cíclicos/farmacocinética , Proteinuria/diagnóstico , Proteinuria/etiología , Proteinuria/patología , Sulfuros/administración & dosificación , Sulfuros/química , Sulfuros/farmacocinéticaRESUMEN
AIMS/HYPOTHESIS: Renal fibrosis is a common complication of diabetic nephropathy and is a major cause of end-stage renal disease. Despite the suggested link between renal fibrosis and microRNA (miRNA) dysregulation in diabetic nephropathy, the identification of the specific miRNAs involved is still incomplete. The aim of this study was to investigate miRNA profiles in the diabetic kidney and to identify potential downstream targets implicated in renal fibrosis. METHODS: miRNA expression profiling was investigated in the kidneys of 8-month-old Zucker diabetic fatty (ZDF) rats during overt nephropathy. Localisation of the most upregulated miRNA was established by in situ hybridisation. The candidate miRNA target was identified by in silico analysis and its expression documented in the diabetic kidney associated with fibrotic markers. Cultured tubule cells served to assess which of the profibrogenic stimuli acted as a trigger for the overexpressed miRNA, and to investigate underlying epigenetic mechanisms. RESULTS: In ZDF rats, miR-184 showed the strongest differential upregulation compared with lean rats (18-fold). Tubular localisation of miR-184 was associated with reduced expression of lipid phosphate phosphatase 3 (LPP3) and collagen accumulation. Transfection of NRK-52E cells with miR-184 mimic reduced LPP3, promoting a profibrotic phenotype. Albumin was a major trigger of miR-184 expression. Anti-miR-184 counteracted albumin-induced LPP3 downregulation and overexpression of plasminogen activator inhibitor-1. In ZDF rats, ACE-inhibitor treatment limited albuminuria and reduced miR-184, with tubular LPP3 preservation and tubulointerstitial fibrosis amelioration. Albumin-induced miR-184 expression in tubule cells was epigenetically regulated through DNA demethylation and histone lysine acetylation and was accompanied by binding of NF-κB p65 subunit to miR-184 promoter. CONCLUSIONS/INTERPRETATION: These results suggest that miR-184 may act as a downstream effector of albuminuria through LPP3 to promote tubulointerstitial fibrosis, and offer the rationale to investigate whether targeting miR-184 in association with albuminuria-lowering drugs may be a new strategy to achieve fully anti-fibrotic effects in diabetic nephropathy.
Asunto(s)
Albuminuria/metabolismo , Nefropatías Diabéticas/metabolismo , Fibrosis/metabolismo , Enfermedades Renales/metabolismo , MicroARNs/metabolismo , Albuminuria/genética , Animales , Inmunoprecipitación de Cromatina , Biología Computacional , Nefropatías Diabéticas/genética , Fibrosis/genética , Inmunohistoquímica , Hibridación in Situ , Enfermedades Renales/genética , Masculino , MicroARNs/genética , FN-kappa B/metabolismo , Fosfatidato Fosfatasa/genética , Fosfatidato Fosfatasa/metabolismo , Regiones Promotoras Genéticas/genética , Ratas , Ratas Zucker , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Shiga toxin (Stx)-producing Escherichia coli is the offending agent of postdiarrhea-associated hemolytic uremic syndrome (HUS), a disorder of glomerular ischemic damage and widespread microvascular thrombosis. We previously documented that Stx induces glomerular complement activation, generating C3a responsible for microvascular thrombosis in experimental HUS. Here, we show that the presence of C3 deposits on podocytes is associated with podocyte damage and loss in HUS mice generated by the coinjection of Stx2 and LPS. Because podocyte adhesion to the glomerular basement membrane is mediated by integrins, the relevance of integrin-linked kinase (ILK) signals in podocyte dysfunction was evaluated. Podocyte expression of ILK increased after the injection of Stx2/LPS and preceded the upregulation of Snail and downregulation of nephrin and α-actinin-4. Factor B deficiency or pretreatment with an inhibitory antibody to factor B protected mice against Stx2/LPS-induced podocyte dysregulation. Similarly, pretreatment with a C3a receptor antagonist limited podocyte loss and changes in ILK, Snail, and α-actinin-4 expression. In cultured podocytes, treatment with C3a reduced α-actinin-4 expression and promoted ILK-dependent nuclear expression of Snail and cell motility. These results suggest that Stx-induced activation of the alternative pathway of complement and generation of C3a promotes ILK signaling, leading to podocyte dysfunction and loss in Stx-HUS.
Asunto(s)
Complemento C3a/metabolismo , Vía Alternativa del Complemento/efectos de los fármacos , Síndrome Hemolítico-Urémico/patología , Glomérulos Renales/efectos de los fármacos , Podocitos/efectos de los fármacos , Toxina Shiga II/farmacología , Animales , Técnicas de Cultivo de Célula , Modelos Animales de Enfermedad , Síndrome Hemolítico-Urémico/etiología , Síndrome Hemolítico-Urémico/metabolismo , Humanos , Glomérulos Renales/metabolismo , Glomérulos Renales/patología , Lipopolisacáridos/farmacología , Masculino , Ratones , Ratones Endogámicos C57BL , Podocitos/metabolismo , Podocitos/patología , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de SeñalRESUMEN
Shiga toxin (Stx)-producing Escherichia coli is a primary cause of diarrhea-associated hemolytic uremic syndrome (HUS), a disorder of thrombocytopenia, microangiopathic hemolytic anemia, and acute renal failure. The pathophysiology of renal microvascular thrombosis in Stx-HUS is still ill-defined. Based on evidence that abnormalities in thrombomodulin (TM), an anticoagulant endothelial glycoprotein that modulates complement and inflammation, predispose to atypical HUS, we assessed whether impaired TM function may adversely affect evolution of Stx-HUS. Disease was induced by coinjection of Stx2/LPS in wild-type mice (TM(wt/wt)) and mice that lack the lectin-like domain of TM (TM(LeD/LeD)), which is critical for its anti-inflammatory and cytoprotective properties. After Stx2/LPS, TM(LeD/LeD) mice exhibited more severe thrombocytopenia and renal dysfunction than TM(wt/wt) mice. Lack of lectin-like domain of TM resulted in a stronger inflammatory reaction after Stx2/LPS with more neutrophils and monocytes/macrophages infiltrating the kidney, associated with PECAM-1 and chemokine upregulation. After Stx2/LPS, intraglomerular fibrin(ogen) deposits were detected earlier in TM(LeD/LeD) than in TM(wt/wt) mice. More abundant fibrin(ogen) deposits were also found in brain and lungs. Under basal conditions, TM(LeD/LeD) mice exhibited excess glomerular C3 deposits, indicating impaired complement regulation in the kidney that could lead to local accumulation of proinflammatory products. TM(LeD/LeD) mice with HUS had a higher mortality rate than TM(wt/wt) mice. If applicable to humans, these findings raise the possibility that genetic or acquired TM defects might have an impact on the severity of microangiopathic lesions after exposure to Stx-producing E. coli infections and raise the potential for using soluble TM in the treatment of Stx-HUS.
Asunto(s)
Síndrome Hemolítico-Urémico/genética , Síndrome Hemolítico-Urémico/inmunología , Lectinas/deficiencia , Toxina Shiga/administración & dosificación , Trombomodulina/deficiencia , Trombomodulina/genética , Animales , Síndrome Hemolítico-Urémico/inducido químicamente , Masculino , Ratones , Ratones de la Cepa 129 , Ratones Transgénicos , Estructura Terciaria de Proteína/genética , Índice de Severidad de la Enfermedad , Trombomodulina/fisiologíaRESUMEN
Complement alternative pathway (AP) dysregulation drives C3 glomerulopathy (C3G), a rare renal disorder characterized by glomerular C3 deposition and glomerular damage, for which no effective treatments are available. Blockade of complement C3 is emerging as a viable therapeutic option. In an earlier study we showed that SLN500, a small interfering RNA targeting liver C3 synthesis, was able to limit AP dysregulation and glomerular C3d deposits in mice with partial factor H (FH) deficiency (Cfh+/- mice). Here, we assessed the pharmacological effects of SLN501 - an optimized SLN500 version - in mice with complete FH deficiency (Cfh-/- mice) that exhibit a more severe C3G phenotype. SLN501 effectively prevented liver C3 synthesis, thus limiting AP dysregulation, glomerular C3d deposits and the development of ultrastructural alterations. These data provide firm evidence of the use of siRNA-mediated liver C3 gene silencing as a potential therapy for treating C3G patients with either partial or complete FH loss of function.
Asunto(s)
Factor H de Complemento/deficiencia , Glomerulonefritis Membranoproliferativa , Enfermedades por Deficiencia de Complemento Hereditario , Enfermedades Renales , Humanos , Animales , Ratones , Complemento C3/genética , Complemento C3/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/uso terapéutico , Factor H de Complemento/genética , Factor H de Complemento/uso terapéutico , Glomerulonefritis Membranoproliferativa/genética , Glomerulonefritis Membranoproliferativa/tratamiento farmacológico , Glomerulonefritis Membranoproliferativa/metabolismo , Vía Alternativa del ComplementoRESUMEN
Bardoxolone methyl is an antioxidant inflammation modulator acting through induction of Keap1-Nrf2 pathway. Results from a recent phase IIb clinical trial reported that bardoxolone methyl was associated with improvement in the estimated glomerular filtration rate in patients with advanced chronic kidney disease and Type 2 diabetes. However, increases in albuminuria, serum transaminase, and frequency of adverse events were noted. We studied the effect of 3-mo treatment with RTA 405, a synthetic triterpenoid analog of bardoxolone methyl in Zucker diabetic fatty rats with overt Type 2 diabetes. Rats were treated from 3 mo of age with vehicle, RTA 405, ramipril, or RTA 405 plus ramipril. RTA 405 caused severe changes in food intake and diuresis with decline in body weight, worsening of dyslipidemia, and increase in blood pressure. Early elevation in serum transaminase was followed by liver injury. RTA 405 worsened proteinuria, glomerulosclerosis, and tubular damage. Ramipril was renoprotective, but when given with RTA 405 it was not able to limit its worsening effects. These data could be due to degradation products in the drug substance used, as disclosed by the company once the study was concluded. To overcome such a drawback, the company offered to test dh404, a variant of RTA 405, in Zucker diabetic fatty rats. The dh404 did not display beneficial effects on proteinuria, glomerulosclerosis, and interstitial inflammation. Rather, kidneys from three rats receiving dh404 showed the presence of a granulomatous and inflammatory process reminiscent of a pseudotumor. Altogether these data raise serious concerns on the use of bardoxolone analogs in Type 2 diabetic nephropathy.
Asunto(s)
Nefropatías Diabéticas/tratamiento farmacológico , Riñón/efectos de los fármacos , Ácido Oleanólico/análogos & derivados , Triterpenos/efectos adversos , Inhibidores de la Enzima Convertidora de Angiotensina/uso terapéutico , Animales , Presión Sanguínea/efectos de los fármacos , Peso Corporal/efectos de los fármacos , Cromatografía Liquida , Nefropatías Diabéticas/patología , Modelos Animales de Enfermedad , Diuresis/efectos de los fármacos , Ingestión de Líquidos/efectos de los fármacos , Hemodinámica/efectos de los fármacos , Riñón/patología , Hígado/efectos de los fármacos , Hígado/patología , Masculino , Espectrometría de Masas , Ácido Oleanólico/efectos adversos , Ramipril/uso terapéutico , Ratas , Ratas Zucker , Circulación Renal/efectos de los fármacosRESUMEN
Shiga toxin (Stx)-producing E.coli O157:H7 has become a global threat to public health; it is a primary cause of diarrhea-associated hemolytic uremic syndrome (HUS), a disorder of thrombocytopenia, microangiopathic hemolytic anemia, and acute renal failure with thrombi occluding renal microcirculation. In this study, we explored whether Stx triggers complement-dependent microvascular thrombosis in in vitro and in vivo experimental settings of HUS. Stx induced on human microvascular endothelial cell surface the expression of P-selectin, which bound and activated C3 via the alternative pathway, leading to thrombus formation under flow. In the search for mechanisms linking complement activation and thrombosis, we found that exuberant complement activation in response to Stx generated an increased amount of C3a that caused further endothelial P-selectin expression, thrombomodulin (TM) loss, and thrombus formation. In a murine model of HUS obtained by coinjection of Stx2 and LPS and characterized by thrombocytopenia and renal dysfunction, upregulation of glomerular endothelial P-selectin was associated with C3 and fibrin(ogen) deposits, platelet clumps, and reduced TM expression. Treatment with anti-P-selectin Ab limited glomerular C3 accumulation. Factor B-deficient mice after Stx2/LPS exhibited less thrombocytopenia and were protected against glomerular abnormalities and renal function impairment, indicating the involvement of complement activation via the alternative pathway in the glomerular thrombotic process in HUS mice. The functional role of C3a was documented by data showing that glomerular fibrin(ogen), platelet clumps, and TM loss were markedly decreased in HUS mice receiving C3aR antagonist. These results identify Stx-induced complement activation, via P-selectin, as a key mechanism of C3a-dependent microvascular thrombosis in diarrhea-associated HUS.
Asunto(s)
Complemento C3a/toxicidad , Vía Alternativa del Complemento/inmunología , Endotelio Vascular/inmunología , Endotelio Vascular/patología , Síndrome Hemolítico-Urémico/inmunología , Síndrome Hemolítico-Urémico/patología , Toxina Shiga I/toxicidad , Toxina Shiga II/toxicidad , Animales , Línea Celular , Complemento C3a/biosíntesis , Complemento C3a/metabolismo , Factor B del Complemento/deficiencia , Factor B del Complemento/genética , Modelos Animales de Enfermedad , Endotelio Vascular/metabolismo , Infecciones por Escherichia coli/inmunología , Infecciones por Escherichia coli/metabolismo , Infecciones por Escherichia coli/patología , Escherichia coli O157/inmunología , Escherichia coli O157/patogenicidad , Síndrome Hemolítico-Urémico/metabolismo , Humanos , Glomérulos Renales/irrigación sanguínea , Glomérulos Renales/inmunología , Glomérulos Renales/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microcirculación/inmunología , Selectina-P/fisiología , Unión Proteica/inmunologíaRESUMEN
The suitability of chickpea, red lentil and green pea for the production of extruded products (i.e., snacks and pasta) was studied. Besides starch and technological properties, the impact of processing on some bioactive compounds was evaluated. The best results were obtained for lentil: the snacks showed the lowest porosity (21 %), the highest average pore radius (18.8 µm), and high expansion (section area: 310 mm2; inner area: 114 mm2), while pasta exhibited low cooking loss (5.7 g 100 g-1) and high firmness (924 N). Extrusion-cooking reduced the soluble phenolic acid content (-45 %) and flavonoids (-41 %), but increased the cell-wall bound phenolic acids and antioxidant activity. The different pulses did not lead to a marked difference in the antioxidant activity of the extruded products, although the lentil maintained the highest flavonoid content after both processes.
Asunto(s)
Harina , Lens (Planta) , Harina/análisis , Antioxidantes/análisis , Fenoles/análisis , Culinaria/métodos , Lens (Planta)/metabolismo , Triticum/metabolismo , FlavonoidesRESUMEN
This study investigated the impact of seed coats from peas (PC) and chickpeas (CC) (at 15 % and 30 % levels) on rice-based co-extruded snacks. Using PC and CC reduced the content of soluble (29 %) and cell-wall bound phenolic acids (21 %), but it enhanced the amount and the profile of flavonoids of rice-based snacks (up to 16 times with PC), resulting in significantly higher antioxidant activity (134 %). Snacks with 15 % CC showed a higher section area (about 335 versus 191 mm2) and a lower average pore radius (20.1 versus 23.9 mm) than PC-snacks; however, such features did not affect either texture or porosity. At 30 % level, PC resulted in a more porous structure (porosity: 73.1 versus 66.7 %) with smaller pores (17.2 versus 27.3 mm) and high firmness (55.9 versus 40.1 N). Consumers' acceptability evaluation revealed that samples containing pulse seed coat were comparable and preferred to the control (i.e., 100 % polished rice).
Asunto(s)
Cicer , Oryza , Cicer/química , Oryza/química , Pisum sativum/química , Fenoles/metabolismo , Semillas , BocadillosRESUMEN
Uncontrolled activation of the alternative pathway (AP) of complement, due to genetic and/or acquired defects, plays a primary pathogenetic role in C3 glomerulopathy (C3G), a rare and heterogeneous disease characterised by predominant C3 fragment deposition within the glomerulus, as well as glomerular damage. There are currently no approved disease-specific treatments for C3G, but new drugs that directly counteract AP dysregulation, targeting components of the pathway, have opened promising new perspectives for managing the disease. Complement factor B (FB), which is primarily synthesised by hepatocytes, is a key component of the AP, as it drives the central amplification loop of the complement system. In this study we used a GalNAc (N-Acetylgalactosamine)-conjugated siRNA to selectively target and suppress liver FB expression in two mouse models characterised by the complete (Cfh-/- mice) or partial (Cfh+/-) loss of function of complement factor H (FH). Homozygous deletion of FH induced a severe C3G phenotype, with strong dysregulation of the AP of complement, glomerular C3 deposition and almost complete C3 consumption. Mice with a heterozygous deletion of FH had intermediate C3 levels and exhibited slower disease progression, resembling human C3G more closely. Here we showed that FB siRNA treatment did not improve serum C3 levels, nor limit glomerular C3 deposition in Cfh-/- mice, while it did normalise circulating C3 levels, reduce glomerular C3 deposits, and limit mesangial electron-dense deposits in Cfh+/- mice. The present data provide important insights into the potential benefits and limitations of FB-targeted inhibition strategies and suggest RNA interference-mediated FB silencing in the liver as a possible therapeutic approach for treating C3G patients with FH haploinsufficiency.
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Glomerulonefritis Membranoproliferativa , Enfermedades Renales , Humanos , Animales , Ratones , Factor B del Complemento/genética , Factor B del Complemento/metabolismo , Complemento C3 , Homocigoto , Eliminación de Secuencia , Factor H de Complemento/genética , Hígado/metabolismo , Vía Alternativa del Complemento/genética , Glomerulonefritis Membranoproliferativa/genética , Glomerulonefritis Membranoproliferativa/terapia , Glomerulonefritis Membranoproliferativa/metabolismoRESUMEN
Diabetes mellitus and alterations in thyroid hormone (TH) signaling are closely linked. Though the role of TH signaling in cell differentiation and growth is well known, it remains unclear whether its alterations contribute to the pathobiology of diabetic cells. Here, we aim to investigate whether the administration of exogenous T3 can counteract the cellular remodeling that occurs in diabetic cardiomyocytes, podocytes, and pancreatic beta cells. Treating diabetic rats with T3 prevents dedifferentiation, pathological growth, and ultrastructural alterations in podocytes and cardiomyocytes. In vitro, T3 reverses glucose-induced growth in human podocytes and cardiomyocytes, restores cardiomyocyte cytoarchitecture, and reverses pathological alterations in kidney and cardiac organoids. Finally, T3 treatment counteracts glucose-induced transdifferentiation, cell growth, and loss in pancreatic beta cells through TH receptor alpha1 activation. Our studies indicate that TH signaling activation substantially counteracts diabetes-induced pathological remodeling, and provide a potential therapeutic approach for the treatment of diabetes and its complications.
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The spike protein of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) can interact with endothelial cells. However, no studies demonstrated the direct effect of the spike protein subunit 1 (S1) in inducing lung vascular damage and the potential mechanisms contributing to lung injury. Here, we found that S1 injection in mice transgenic for human angiotensin converting enzyme 2 (ACE2) induced early loss of lung endothelial thromboresistance at 3 days, as revealed by thrombomodulin loss and von Willebrand factor (vWF) increase. In parallel, vascular and epithelial C3 deposits and enhanced C3a receptor (C3aR) expression were observed. These changes preceded diffuse alveolar damage and lung vascular fibrin(ogen)/platelets aggregates at 7 days, as well as inflammatory cell recruitment and fibrosis. Treatment with C3aR antagonist (C3aRa) inhibited lung C3 accumulation and C3a/C3aR activation, limiting vascular thrombo-inflammation and fibrosis. Our study demonstrates that S1 triggers vascular dysfunction and activates complement system, instrumental to lung thrombo-inflammatory injury. By extension, our data indicate C3aRa as a valuable therapeutic strategy to limit S1-dependent lung pathology.
Asunto(s)
Complemento C3a , Células Endoteliales , Receptores de Complemento , Glicoproteína de la Espiga del Coronavirus , SARS-CoV-2 , Células Endoteliales/citología , Células Endoteliales/virología , Pulmón/patología , Pulmón/virología , Complemento C3a/metabolismo , Receptores de Complemento/metabolismo , Fibrosis , Ratones Transgénicos , Humanos , Animales , Ratones , COVID-19 , InflamaciónRESUMEN
Cannabigerol (CBG), a cannabinoid from Cannabis sativa L., recently attracted noteworthy attention for its dermatological applications, mainly due to its anti-inflammatory, antioxidant, and antimicrobial effectiveness similar to those of cannabidiol (CBD). In this work, based on results from studies of in vitro permeation through biomimetic membranes performed with CBG and CBD in the presence and in the absence of a randomly substituted methyl-ß-cyclodextrin (MßCD), a new CBG extemporaneous emulgel (oil-in-gel emulsion) formulation was developed by spray-drying. The powder (SDE) can be easily reconstituted with purified water, leading to a product with chemical-physical and technological characteristics that are comparable to those of the starting emulgels (E). Thermogravimetric analysis (TGA), attenuated total reflection-Fourier transformed infrared spectroscopy (ATR-FTIR), x-ray powder diffraction (XRPD), and high-performance liquid chromatography (HPLC) analyses demonstrated that the spray-drying treatment did not alter the chemical properties of CBG. This product can represent a metered-dosage form for the localized treatment of cutaneous afflictions such as acne and psoriasis.
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Strawberries and raspberries are susceptible to physiological and biological damage. Due to the consumer concern about using pesticides to control fruit rot, recent attention has been drawn to essential oils. Microbiological activity evaluations of different concentrations of tested EOs (cinnamon, clove, bergamot, rosemary and lemon; 10% DMSO-PBS solution was used as a diluent) against fruit rot fungal strains and a fruit-born human pathogen (Escherichia coli) indicated that the highest inhibition halos was found for pure cinnamon and clove oils; according to GC-MS analysis, these activities were due to the high level of the bioactive compounds cinnamaldehyde (54.5%) in cinnamon oil and eugenol (83%) in clove oil. Moreover, thermogravimetric evaluation showed they were thermally stable, with temperature peak of 232.0 °C for cinnamon and 200.6/234.9 °C for clove oils. Antibacterial activity evaluations of all tested EOs at concentrations from 5-50% (v/v) revealed a concentration of 10% (v/v) to be the minimum inhibitory concentration and minimum bactericidal concentration. The physicochemical analysis of fruits in an in vivo assay indicated that used filter papers doped with 10% (v/v) of cinnamon oil (stuck into the lids of plastic containers) were able to increase the total polyphenols and antioxidant activity in strawberries after four days, with it being easier to preserve strawberries than raspberries.
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BACKGROUND: Home parenteral nutrition (HPN) is the standard treatment for patients with chronic intestinal failure (CIF). Mortality and weaning rates of these patients differ widely among cohorts; however, these outcomes were often considered independent-rather than competing-events, leading to an upward bias of the retrieved estimates. OBJECTIVES: The aim of this retrospective cohort study was to evaluate, evaluating through a competing risk analysis, the rates and predictors of mortality and weaning in CIF patients from an Italian referral center. METHODS: All adult patients with CIF receiving > 3 mo HPN from 1985 until 2016 were enrolled. Clinical information was collected from the database of the Intestinal Failure Unit of Torino, Italy. Patients were stratified according to the presence or not of short bowel syndrome (SBS). RESULTS: The cumulative incidences of death and weaning were 27.3% and 32.3% and 39.0% and 33.7% at 5 and 10 y from HPN initiation, respectively. At multivariable competing risk analyses, mortality was predicted by age (sub-distribution hazard ratio [SHR] = 1.65 per 10-y increase; 95% CI, 1.35-2.01), type 3 SBS (SHR = 0.38; 0.15-0.94), small bowel length ≥ 100 cm (SHR = 0.42; 0.22-0.83), and reconstructive surgery (SHR = 0.11; 0.02-0.64) in SBS patients, and by age (SHR = 1.38 per 10-y increase; 1.16-1.64) and presence of stoma (SHR = 0.30; 0.12-0.78) in non-SBS patients. In the same model, weaning was predicted by type 3 SBS (SHR = 6.86; 3.10-15.16), small bowel length ≥ 100 cm (SHR = 3.54; 1.99-6.30), and reconstructive surgery (SHR = 2.86; 1.44-5.71) in SBS patients, and by age (SHR = 0.79 per 10-y increase; 0.66-0.94) and presence of stoma (SHR = 2.64; 1.38-5.07) in non-SBS patients. CONCLUSIONS: Surgical procedures strongly affected mortality and weaning risk in CIF patients.