RESUMO
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.
Assuntos
Glomerulonefrite Membranoproliferativa , Nefropatias , Animais , Complemento C3/genética , Complemento C3/metabolismo , Fator B do Complemento/metabolismo , Fator H do Complemento/genética , Via Alternativa do Complemento/genética , Glomerulonefrite Membranoproliferativa/patologia , Humanos , Camundongos , RNA Interferente Pequeno/genéticaRESUMO
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.
Assuntos
Diabetes Mellitus Experimental , Nefropatias Diabéticas , Inibidores do Transportador 2 de Sódio-Glicose , Albuminúria/tratamento farmacológico , Albuminúria/patologia , Albuminúria/prevenção & controle , Animais , Compostos Benzidrílicos , Glicemia/metabolismo , Caveolina 1/metabolismo , Diabetes Mellitus Experimental/tratamento farmacológico , Nefropatias Diabéticas/tratamento farmacológico , Nefropatias Diabéticas/metabolismo , Nefropatias Diabéticas/prevenção & controle , Células Endoteliais/metabolismo , Feminino , Membrana Basal Glomerular/metabolismo , Glucosídeos , Humanos , Masculino , Camundongos , Transdução de Sinais , Transportador 2 de Glucose-Sódio/metabolismo , Inibidores do Transportador 2 de Sódio-Glicose/farmacologia , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
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.
Assuntos
Diabetes Mellitus Experimental , Nefropatias Diabéticas , Camundongos , Animais , Nefropatias Diabéticas/metabolismo , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Inibidores da Enzima Conversora de Angiotensina/metabolismo , Diabetes Mellitus Experimental/metabolismo , Capilares/patologia , Glicocálix/metabolismo , Lantânio , Rim/patologia , Camundongos EndogâmicosRESUMO
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.
Assuntos
Nefropatias Diabéticas , Sirtuína 3/metabolismo , Animais , Antioxidantes/metabolismo , Dieta Hiperlipídica , Camundongos , Camundongos Knockout , Estresse Oxidativo , Sirtuína 3/genéticaRESUMO
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.
Assuntos
Angiotensina I/administração & dosagem , Inibidores da Enzima Conversora de Angiotensina/administração & dosagem , Diabetes Mellitus Tipo 2/complicações , Nefropatias Diabéticas/tratamento farmacológico , Fragmentos de Peptídeos/administração & dosagem , Proteinúria/tratamento farmacológico , Alanina/administração & dosagem , Alanina/análogos & derivados , Alanina/química , Alanina/farmacocinética , Angiotensina I/química , Angiotensina I/farmacocinética , Animais , Diabetes Mellitus Tipo 2/genética , Nefropatias Diabéticas/diagnóstico , Nefropatias Diabéticas/etiologia , Nefropatias Diabéticas/patologia , Modelos Animais de Doenças , Quimioterapia Combinada/métodos , Meia-Vida , Humanos , Glomérulos Renais/irrigação sanguínea , Glomérulos Renais/efeitos dos fármacos , Glomérulos Renais/patologia , Glomérulos Renais/ultraestrutura , Lisinopril/administração & dosagem , Masculino , Camundongos , Camundongos Transgênicos , Microscopia Eletrônica de Transmissão , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/farmacocinética , Peptídeos Cíclicos/administração & dosagem , Peptídeos Cíclicos/química , Peptídeos Cíclicos/farmacocinética , Proteinúria/diagnóstico , Proteinúria/etiologia , Proteinúria/patologia , Sulfetos/administração & dosagem , Sulfetos/química , Sulfetos/farmacocinéticaRESUMO
Crescentic glomerulonephritis (GN) is a devastating disease with rapidly progressive deterioration in kidney function, which, histologically, manifests as crescent formation in most glomeruli. We previously found that crescents derive from the aberrant proliferation and migration of parietal epithelial cells (PECs)/progenitor cells, and that the angiotensin (ang) II/ang II type-1 (AT1) receptor pathway may participate, together with the stromal cell-derived factor-1 (SDF-1)/C-X-C chemokine receptor 4 axis, in the development of those lesions. Herein, we elucidated sequential events and cellular and molecular interactions occurring during crescentic lesion onset and evolution. By analyzing kidney biopsy specimens of patients with extracapillary GN, divided according to the grade of glomerular lesions, we found that the accumulation of macrophages expressing matrix metalloproteinase-12 started manifesting in glomeruli affected by early-stage lesions, whereas AT1 receptor expression could not be detected. In glomeruli with advanced lesions, AT1 receptor expression increased markedly, and the up-regulation of SDF-1, and its receptor C-X-C chemokine receptor 7, was documented on podocytes and PECs, respectively. In vitro studies were instrumental to demonstrating the role of ang II in inducing podocyte SDF-1 production, which ultimately activates PECs. The present findings support the possibility that angiotensin-converting enzyme inhibitor treatment might limit PEC activation and reduce the frequency and extension of crescents in extracapillary GN.
Assuntos
Angiotensina II/metabolismo , Proliferação de Células , Glomérulos Renais/metabolismo , Podócitos/metabolismo , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/fisiologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Glomerulonefrite/tratamento farmacológico , Glomerulonefrite/patologia , Humanos , Nefropatias/patologia , Glomérulos Renais/efeitos dos fármacos , Glomérulos Renais/patologia , Macrófagos/metabolismoRESUMO
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.
Assuntos
Albuminúria/metabolismo , Nefropatias Diabéticas/metabolismo , Fibrose/metabolismo , Nefropatias/metabolismo , MicroRNAs/metabolismo , Albuminúria/genética , Animais , Imunoprecipitação da Cromatina , Biologia Computacional , Nefropatias Diabéticas/genética , Fibrose/genética , Imuno-Histoquímica , Hibridização In Situ , Nefropatias/genética , Masculino , MicroRNAs/genética , NF-kappa B/metabolismo , Fosfatidato Fosfatase/genética , Fosfatidato Fosfatase/metabolismo , Regiões Promotoras Genéticas/genética , Ratos , Ratos Zucker , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
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.
Assuntos
Complemento C3a/metabolismo , Via Alternativa do Complemento/efeitos dos fármacos , Síndrome Hemolítico-Urêmica/patologia , Glomérulos Renais/efeitos dos fármacos , Podócitos/efeitos dos fármacos , Toxina Shiga II/farmacologia , Animais , Técnicas de Cultura de Células , Modelos Animais de Doenças , Síndrome Hemolítico-Urêmica/etiologia , Síndrome Hemolítico-Urêmica/metabolismo , Humanos , Glomérulos Renais/metabolismo , Glomérulos Renais/patologia , Lipopolissacarídeos/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Podócitos/metabolismo , Podócitos/patologia , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de SinaisRESUMO
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.
Assuntos
Fator H do Complemento/deficiência , Glomerulonefrite Membranoproliferativa , Doenças da Deficiência Hereditária de Complemento , Nefropatias , Humanos , Animais , Camundongos , Complemento C3/genética , Complemento C3/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/uso terapêutico , Fator H do Complemento/genética , Fator H do Complemento/uso terapêutico , Glomerulonefrite Membranoproliferativa/genética , Glomerulonefrite Membranoproliferativa/tratamento farmacológico , Glomerulonefrite Membranoproliferativa/metabolismo , Via Alternativa do ComplementoRESUMO
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.
Assuntos
Complemento C3a/toxicidade , Via Alternativa do Complemento/imunologia , Endotélio Vascular/imunologia , Endotélio Vascular/patologia , Síndrome Hemolítico-Urêmica/imunologia , Síndrome Hemolítico-Urêmica/patologia , Toxina Shiga I/toxicidade , Toxina Shiga II/toxicidade , Animais , Linhagem Celular , Complemento C3a/biossíntese , Complemento C3a/metabolismo , Fator B do Complemento/deficiência , Fator B do Complemento/genética , Modelos Animais de Doenças , Endotélio Vascular/metabolismo , Infecções por Escherichia coli/imunologia , Infecções por Escherichia coli/metabolismo , Infecções por Escherichia coli/patologia , Escherichia coli O157/imunologia , Escherichia coli O157/patogenicidade , Síndrome Hemolítico-Urêmica/metabolismo , Humanos , Glomérulos Renais/irrigação sanguínea , Glomérulos Renais/imunologia , Glomérulos Renais/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microcirculação/imunologia , Selectina-P/fisiologia , Ligação Proteica/imunologiaRESUMO
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.
Assuntos
Glomerulonefrite Membranoproliferativa , Nefropatias , Humanos , Animais , Camundongos , Fator B do Complemento/genética , Fator B do Complemento/metabolismo , Complemento C3 , Homozigoto , Deleção de Sequência , Fator H do Complemento/genética , Fígado/metabolismo , Via Alternativa do Complemento/genética , Glomerulonefrite Membranoproliferativa/genética , Glomerulonefrite Membranoproliferativa/terapia , Glomerulonefrite Membranoproliferativa/metabolismoRESUMO
Rapidly progressive crescentic glomerulonephritis associated with anti-neutrophil cytoplasmic antibodies (ANCA-GN) is a major cause of renal failure. Current immunosuppressive therapies are associated with severe side effects, intensifying the need for new therapeutic strategies. The activation of Mas receptor/Angiotensin-(1-7) axis exerted renoprotection in chronic kidney disease. Here, we investigated the effect of adding the lanthionine-stabilized cyclic form of angiotensin-1-7 [cAng-(1-7)] to cyclophosphamide in a rat model of ANCA-GN. At the onset of proteinuria, Wistar Kyoto rats with ANCA-GN received vehicle or a single bolus of cyclophosphamide, with or without daily cAng-(1-7). Treatment with cAng-(1-7) plus cyclophosphamide reduced proteinuria by 85% vs. vehicle, and by 60% vs. cyclophosphamide, and dramatically limited glomerular crescents to less than 10%. The addition of cAng-(1-7) to cyclophosphamide protected against glomerular inflammation and endothelial rarefaction and restored the normal distribution of parietal epithelial cells. Ultrastructural analysis revealed a preserved GBM, glomerular endothelium and podocyte structure, demonstrating that combination therapy provided an additional layer of renoprotection. This study demonstrates that adding cAng-(1-7) to a partially effective dose of cyclophosphamide arrests the progression of renal disease in rats with ANCA-GN, suggesting that cAng-(1-7) could be a novel clinical approach for sparing immunosuppressants.
Assuntos
Anticorpos Anticitoplasma de Neutrófilos , Glomerulonefrite , Angiotensina I , Animais , Ciclofosfamida/farmacologia , Ciclofosfamida/uso terapêutico , Glomerulonefrite/tratamento farmacológico , Fragmentos de Peptídeos , Proteinúria/complicações , Ratos , Ratos Endogâmicos WKYRESUMO
Diabetic nephropathy is associated with cardiovascular morbidity. Angiotensin-converting enzyme (ACE) inhibitors provide imperfect renoprotection in advanced type 2 diabetes, and cardiovascular risk remains elevated. Endothelin (ET)-1 has a role in renal and cardiac dysfunction in diabetes. Here, we assessed whether combination therapy with an ACE inhibitor and ET(A) receptor antagonist provided reno- and cardioprotection in rats with overt type 2 diabetes. Four groups of Zucker diabetic fatty (ZDF) rats were treated orally from 4 (when proteinuric) to 8 mo with vehicle, ramipril (1 mg/kg), sitaxsentan (60 mg/kg), and ramipril plus sitaxsentan. Lean rats served as controls. Combined therapy ameliorated proteinuria and glomerulosclerosis mostly as a result of the action of ramipril. Simultaneous blockade of ANG II and ET-1 pathways normalized renal monocyte chemoattractant protein-1 and interstitial inflammation. Cardiomyocyte loss, volume enlargement, and capillary rarefaction were prominent abnormalities of ZDF myocardium. Myocyte volume was reduced by ramipril and sitaxsentan, which also ameliorated heart capillary density. Drug combination restored myocardial structure and reestablished an adequate capillary network in the presence of increased cardiac expression of VEGF/VEGFR-1, and significant reduction of oxidative stress. In conclusion, in type 2 diabetes concomitant blockade of ANG II synthesis and ET-1 biological activity through an ET(A) receptor antagonist led to substantial albeit not complete renoprotection, almost due to the ACE inhibitor. The drug combination also showed cardioprotective properties, which however, were mainly dependent on the contribution of the ET(A) receptor antagonist through the action of VEGF.
Assuntos
Inibidores da Enzima Conversora de Angiotensina/farmacologia , Diabetes Mellitus Tipo 2/patologia , Antagonistas do Receptor de Endotelina A , Coração/efeitos dos fármacos , Rim/patologia , Miocárdio/patologia , Animais , Glicemia/metabolismo , Peso Corporal/fisiologia , Contagem de Células , Colágeno Tipo III/metabolismo , Diabetes Mellitus Tipo 2/genética , Endotelina-1/metabolismo , Hemodinâmica/efeitos dos fármacos , Imuno-Histoquímica , Rim/efeitos dos fármacos , Rim/metabolismo , Testes de Função Renal , Masculino , Células Musculares/efeitos dos fármacos , Miocárdio/metabolismo , Ratos , Ratos Zucker , Reação em Cadeia da Polimerase em Tempo Real , Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismo , Sobrevida , Tirosina/análogos & derivados , Tirosina/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
BACKGROUND/AIM: Necrotizing crescentic glomerulonephritis (GN) associated with anti-neutrophil cytoplasmic antibodies (ANCA) against myeloperoxidase (MPO) is a devastating disease that quickly progresses to kidney failure. Current therapies are broadly immunosuppressive and associated with adverse effects. We wanted to set up a model that could be suitable for testing narrowly targeted therapies. METHODS: The model was constructed in male Wistar Kyoto rats through injections of human MPO (hMPO) and pertussis toxin, followed by a sub-nephritogenic dose of sheep anti-rat glomerular basement membrane (GBM) serum to boost the disease. Rats were monitored for 35 days. Rats given hMPO alone, saline, or human serum albumin with or without anti-GBM serum were also studied. RESULTS: Rats receiving hMPO developed circulating anti-hMPO and anti-rat MPO antibodies. Challenging hMPO-immunized rats with the anti-GBM serum led to more glomerular neutrophil infiltration and MPO release, and severe haematuria, heavy proteinuria, and higher blood urea nitrogen than hMPO alone. Pauci-immune GN developed with crescents, affecting 25% of glomeruli. The majority of crescents were fibrocellular. Necrotizing lesions and Bowman capsule ruptures were detected. Cells double positive for claudin-1 (a marker of parietal epithelial cells [PECs]) and neural cell adhesion molecule (NCAM; progenitor PECs) were present in crescents. Double staining for NCAM and Ki-67 established proliferative status of progenitor PECs. Podocyte damage was associated with endothelial and GBM changes by electron microscopy. Monocyte/macrophages and CD4+ and CD8+ T cells accumulated in glomeruli and the surrounding area and in the tubulointerstitium. Lung haemorrhage also manifested. CONCLUSION: This model reflects histological lesions of human ANCA-associated rapidly progressive GN and may be useful for investigating new therapies.
Assuntos
Anticorpos Anticitoplasma de Neutrófilos/imunologia , Glomerulonefrite/imunologia , Peroxidase/imunologia , Animais , Nitrogênio da Ureia Sanguínea , Cápsula Glomerular/patologia , Células Epiteliais/patologia , Membrana Basal Glomerular/imunologia , Hematúria/etiologia , Humanos , Glomérulos Renais/patologia , Masculino , Infiltração de Neutrófilos , Toxina Pertussis/farmacologia , Proteinúria/etiologia , Ratos , Ratos Endogâmicos WKYRESUMO
Complement factor H (FH) is the main plasma regulator of the alternative pathway of complement. Genetic and acquired abnormalities in FH cause uncontrolled complement activation amplifying, with the consequent accumulation of complement components on the renal glomeruli. This leads to conditions such as C3 glomerulopathy (C3G) and atypical hemolytic uremic syndrome (aHUS). There is no effective therapy for these diseases. Half of the patients progress to end-stage renal disease and the condition recurs frequently in transplanted kidneys. Combined liver/kidney transplantation is a valid option for these patients, but the risks of the procedure and donor organ shortages hamper its clinical application. Therefore, there is an urgent need for alternative strategies for providing a normal FH supply. Human amnion epithelial cells (hAEC) have stem cell characteristics, including the capability to differentiate into hepatocyte-like cells in vivo.Here, we administered hAEC into the livers of newborn Cfh-/- mice, which spontaneously developed glomerular complement deposition and renal lesions resembling human C3G. hAEC engrafted at low levels in the livers of Cfh-/- mice and produced sufficient human FH to prevent complement activation and glomerular C3 and C9 deposition. However, long-term engraftment was not achieved, and eventually hAEC elicited a humoral immune response in immunocompetent Cfh-/- mice.hAEC cell therapy could be a valuable therapeutic option for patients undergoing kidney transplantation in whom post-transplant immunosuppression may protect allogeneic hAEC from rejection, while allogeneic cells provide normal FH to prevent disease recurrence.
Assuntos
Âmnio , Fator H do Complemento , Animais , Complemento C3/genética , Fator H do Complemento/genética , Células Epiteliais , Humanos , Rim , CamundongosRESUMO
OBJECTIVE: CER-001 is an HDL mimetic that has been tested in different pathological conditions, but never with LCAT deficiency. This study was designed to investigate whether the absence of LCAT affects the catabolic fate of CER-001, and to evaluate the effects of CER-001 on kidney disease associated with LCAT deficiency. METHODS: Lcat-/- and wild-type mice received CER-001 (2.5, 5, 10â¯mg/kg) intravenously for 2â¯weeks. The plasma lipid/ lipoprotein profile and HDL subclasses were analyzed. In a second set of experiments, Lcat-/- mice were injected with LpX to induce renal disease and treated with CER-001 and then the plasma lipid profile, lipid accumulation in the kidney, albuminuria and glomerular podocyte markers were evaluated. RESULTS: In Lcat-/- mice a decrease in total cholesterol and triglycerides, and an increase in HDL-c was observed after CER-001 treatment. While in wild-type mice CER-001 entered the classical HDL remodeling pathway, in the absence of LCAT it disappeared from the plasma shortly after injection and ended up in the kidney. In a mouse model of renal disease in LCAT deficiency, treatment with CER-001 at 10â¯mg/kg for one month had beneficial effects not only on the lipid profile, but also on renal disease, by limiting albuminuria and podocyte dysfunction. CONCLUSIONS: Treatment with CER-001 ameliorates the dyslipidemia typically associated with LCAT deficiency and more importantly limits renal damage in a mouse model of renal disease in LCAT deficiency. The present results provide a rationale for using CER-001 in FLD patients.
Assuntos
Apolipoproteína A-I/uso terapêutico , Nefropatias/tratamento farmacológico , Deficiência da Lecitina Colesterol Aciltransferase/tratamento farmacológico , Metabolismo dos Lipídeos/efeitos dos fármacos , Lipídeos/sangue , Fosfolipídeos/uso terapêutico , Proteínas Recombinantes/uso terapêutico , Animais , Apolipoproteína A-I/farmacologia , Células Cultivadas , Modelos Animais de Doenças , Nefropatias/genética , Nefropatias/patologia , Deficiência da Lecitina Colesterol Aciltransferase/genética , Deficiência da Lecitina Colesterol Aciltransferase/metabolismo , Deficiência da Lecitina Colesterol Aciltransferase/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosfatidilcolina-Esterol O-Aciltransferase/genética , Fosfolipídeos/farmacologia , Podócitos/efeitos dos fármacos , Podócitos/patologia , Podócitos/fisiologia , Proteínas Recombinantes/farmacologiaRESUMO
Shiga toxins (Stx) are the virulence factors of enterohemorrhagic Escherichia coli O157:H7, a worldwide emerging diarrheal pathogen, which precipitates postdiarrheal hemolytic uremic syndrome, the leading cause of acute renal failure in children. In this study, we show that Stx2 triggered expression of fractalkine (FKN), a CX3C transmembrane chemokine, acting as both adhesion counterreceptor on endothelial cells and soluble chemoattractant. Stx2 caused in HUVEC expression of FKN mRNA and protein, which promoted leukocyte capture, ablated by Abs to either endothelial FKN or leukocyte CX3CR1 receptor. Exposure of human glomerular endothelial cells to Stx2 recapitulated its FKN-inducing activity and FKN-mediated leukocyte adhesion. Both processes required phosphorylation of Src-family protein tyrosine kinase and p38 MAPK in endothelial cells. Furthermore, they depended on nuclear import of NF-kappaB and other stress-responsive transcription factors. Inhibition of their nuclear import with the cell-penetrating SN50 peptide reduced FKN mRNA levels and FKN-mediated leukocyte capture by endothelial cells. Adenoviral overexpression of IkappaBalpha inhibited FKN mRNA up-regulation. The FKN-mediated responses to Stx2 were also dependent on AP-1. In mice, both virulence factors of Stx-producing E. coli, Stx and LPS, are required to elicit hemolytic uremic syndrome. In this study, FKN was detected within glomeruli of C57BL/6 mice injected with Stx2, and further increased after Stx2 plus LPS coadministration. This was associated with recruitment of CX3CR1-positive cells. Thus, in response to Stx2, FKN is induced playing an essential role in the promotion of leukocyte-endothelial cell interaction thereby potentially contributing to the renal microvascular dysfunction and thrombotic microangiopathy that underlie hemolytic uremic syndrome due to enterohemorrhagic E. coli O157:H7 infection.
Assuntos
Quimiocina CX3CL1/metabolismo , Células Endoteliais/imunologia , Síndrome Hemolítico-Urêmica/imunologia , Glomérulos Renais/imunologia , Leucócitos/imunologia , Receptores de Quimiocinas/metabolismo , Toxina Shiga II/imunologia , Animais , Receptor 1 de Quimiocina CX3C , Adesão Celular , Células Cultivadas , Quimiocina CX3CL1/imunologia , Modelos Animais de Doenças , Células Endoteliais/metabolismo , Escherichia coli O157/imunologia , Síndrome Hemolítico-Urêmica/metabolismo , Síndrome Hemolítico-Urêmica/microbiologia , Humanos , Glomérulos Renais/irrigação sanguínea , Glomérulos Renais/citologia , Glomérulos Renais/metabolismo , Lipopolissacarídeos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/imunologia , NF-kappa B/metabolismo , Receptores de Quimiocinas/imunologia , Toxina Shiga II/metabolismo , Transdução de Sinais , Fator de Transcrição AP-1/imunologia , Fator de Transcrição AP-1/metabolismo , Regulação para Cima , Proteínas Quinases p38 Ativadas por Mitógeno/imunologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
The increasing prevalence of diabetes worldwide has led to a concomitant rise in diabetic kidney disease (DKD) as a major cause of end-stage renal disease. Glomerular lesions constitute the most striking and consistent features identified in biopsies from patients with DKD, although tubulointerstitial injury has an important and often under-recognized role in the progression to overt nephropathy. In advanced stages of the disease, podocyte detachment is a pivotal event in the loss of glomerular filtration barrier integrity and may explain, at least in part, the inability of current therapies to halt renal function decline. This chapter details the systematic method that can be used to study renal tissue samples from diabetic patients, and the specific role of different imaging techniques, such as light microscopy, immunofluorescence microscopy, and transmission and scanning electron microscopy in detecting histologic lesions specific to DKD.
Assuntos
Nefropatias Diabéticas/diagnóstico , Técnicas de Preparação Histocitológica/métodos , Glomérulos Renais/patologia , Biópsia , Nefropatias Diabéticas/patologia , Progressão da Doença , Humanos , Glomérulos Renais/diagnóstico por imagem , Glomérulos Renais/ultraestrutura , Microscopia Eletrônica de Varredura/métodos , Microscopia Eletrônica de Transmissão/métodos , Microscopia de Fluorescência/métodos , Coloração e Rotulagem/métodosRESUMO
In the present study, we evaluated the effect of simultaneously blocking angiotensin II synthesis and endothelin (ET)-1 activity as a multimodal intervention to implement renoprotection in overt diabetic nephropathy. Mechanisms underlying combined therapy effectiveness were addressed by investigating podocyte structure and function and glomerular barrier size-selective properties. Uninephrectomized rats made diabetic by streptozotocin received orally placebo, lisinopril (12.5 mg/l), the ET(A) receptor antagonist avosentan (30 mg/kg), or their combination from 4 (when animals had proteinuria) to 8 mo. Proteinuria, renal damage, podocyte number, nephrin expression, and glomerular size selectivity by graded-size Ficoll molecule fractional clearance were assessed. Combined therapy normalized proteinuria, provided complete protection from tubulointerstitial damage, and induced regression of glomerular lesions, while only a partial renoprotection was achieved by each drug alone. Lisinopril plus avosentan restored to normal values the number of podocytes. Single therapies only limited podocyte depletion. Defective nephrin expression of diabetes was prevented by each drug. Altered glomerular size selectivity to large macromolecules of diabetic rats was remarkably improved by lisinopril and the combined treatment. Avosentan ameliorated peritubular capillary architecture and reduced interstitial inflammation and fibrosis. The ACE inhibitor and ET(A) receptor antagonist induced regression of glomerular lesions in overt diabetic nephropathy. Regression of renal disease was conceivably the result of the synergistic effect of the ACE inhibitor of preserving glomerular permselective properties and the ET(A) antagonist in improving tubulointerstitial changes. These findings provide mechanistic insights to explain the antiproteinuric effect of this combined therapy in diabetes.
Assuntos
Inibidores da Enzima Conversora de Angiotensina/uso terapêutico , Nefropatias Diabéticas/prevenção & controle , Lisinopril/uso terapêutico , Piridinas/uso terapêutico , Pirimidinas/uso terapêutico , Animais , Pressão Sanguínea/efeitos dos fármacos , Capilares/patologia , Contagem de Células , Diabetes Mellitus Experimental/patologia , Nefropatias Diabéticas/mortalidade , Nefropatias Diabéticas/patologia , Quimioterapia Combinada , Antagonistas do Receptor de Endotelina A , Imuno-Histoquímica , Rim/patologia , Glomérulos Renais/efeitos dos fármacos , Glomérulos Renais/patologia , Lipídeos/sangue , Metaloproteinase 9 da Matriz/biossíntese , Metaloproteinase 9 da Matriz/genética , Podócitos/efeitos dos fármacos , Podócitos/patologia , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Blockade of the renin-angiotensin system (RAS), the standard treatment for chronic proteinuric nephropathy, slows but may not halt progression of the disease, particularly when therapy is started late. Because vasopressin may also play a role in the progression of renal disease, we measured the effect of a dual V(1a) and V(2) vasopressin receptor antagonist (RWJ-676070) alone or combined with angiotensin-converting enzyme inhibition or angiotensin II type 1 receptor blockade on proteinuria and renal disease progression during overt nephropathy. Twenty-one days after renal mass reduction, a time of established injury, rats were given vehicle, RWJ-676070, enalapril, losartan, RWJ-676070 plus enalapril, or losartan in drinking water for an additional 39 days. RWJ-676070 returned the blood pressure to pre-treatment levels, which were significantly lower than those in vehicle-treated rats. Enalapril, losartan, and the combined therapies reduced blood pressure to a greater extent. RWJ-676070 afforded a partial antiproteinuric effect, which was enhanced by the addition of enalapril or losartan. Renal functional impairment, and glomerular and tubular changes were partially ameliorated by RWJ-676070; parameters significantly improved with either enalapril or losartan alone and improved to a greater extent with the combined therapies. Our findings suggest that vasopressin receptor antagonists could be of additional therapeutic value in the treatment of chronic proteinuric nephropathy.