RESUMO
The prognosis of patients undergoing hematopoietic stem cell transplantation (HSCT) depends on the rapid recovery and sustained life-long hematopoiesis. The activation of the fibrinolytic pathway promotes hematopoietic regeneration; however, the role of plasminogen activator inhibitor-1 (PAI-1), a negative regulator of the fibrinolytic pathway, has not yet been elucidated. We herein demonstrate that bone marrow (BM) stromal cells, especially osteoblasts, produce PAI-1 in response to myeloablation, which negatively regulates the hematopoietic regeneration in the BM microenvironment. Total body irradiation in mice dramatically increased the local expression levels of fibrinolytic factors, including tissue-type plasminogen activator (tPA), plasmin, and PAI-1. Genetic disruption of the PAI-1 gene, or pharmacological inhibition of PAI-1 activity, significantly improved the myeloablation-related mortality and promoted rapid hematopoietic recovery after HSCT through the induction of hematopoiesis-promoting factors. The ability of a PAI-1 inhibitor to enhance hematopoietic regeneration was abolished when tPA-deficient mice were used as recipients, thus indicating that PAI-1 represses tPA-dependent hematopoietic regeneration. The PAI-1 inhibitor not only accelerated the expansion of the donor HSCs during the early-stage of regeneration, but also supported long-term hematopoiesis. Our results indicate that the inhibition of PAI-1 activity could be a therapeutic approach to facilitate the rapid recovery and sustained hematopoiesis after HSCT.
Assuntos
Medula Óssea/metabolismo , Sobrevivência de Enxerto , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/metabolismo , Regeneração , Serpina E2/antagonistas & inibidores , Nicho de Células-Tronco , Aloenxertos , Animais , Humanos , Camundongos , Serpina E2/metabolismo , Células Estromais/metabolismoRESUMO
OBJECTIVE: Macrophage (MÏ) migration rests on the adhesion/detachment between MÏ surface components and extracellular matrixes, and the contribution of numerous inflammatory disorders. Plasminogen activator inhibitor (PAI)-1, a serine protease inhibitor, influences MÏ motility through an action distinct from its classical modulation of the plasmin-based fibrinolytic process. We rely here on a small molecule PAI-1 inhibitor (TM5275) to investigate the role of PAI-1 in MÏ migration in the pathogenesis of renal injury. APPROACH AND RESULTS: MÏ migration was inhibited both in vitro and in vivo by TM5275. It was also reduced in T-cell-deficient nude mice, but not in PAI-1-deficient mice. MÏ migration hinged on the interaction of PAI-1 with low-density lipoprotein receptor-related protein, an interaction prevented by TM5275, but not with vitronectin, urokinase-type plasminogen activator, or tissue-type plasminogen activator. Fed to rats with anti-Thy-1-induced nephritis, TM5275 significantly decreased MÏ accumulation and ameliorated the progression of renal injury. CONCLUSIONS: These findings suggest that a small molecule PAI-1 inhibitor represents a novel class of anti-inflammatory agents targeting MÏ migration by the inhibition of the interaction of PAI-1 with low-density lipoprotein receptor-related protein.
Assuntos
Macrófagos/efeitos dos fármacos , Piperazinas/farmacologia , Inibidor 1 de Ativador de Plasminogênio/fisiologia , para-Aminobenzoatos/farmacologia , Animais , Anti-Inflamatórios/farmacologia , Movimento Celular/efeitos dos fármacos , Glomerulonefrite/patologia , Isoanticorpos/farmacologia , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/fisiologia , Macrófagos/fisiologia , Camundongos , RatosRESUMO
The multipronged drug approach targeting blood pressure and serum levels of glucose, insulin, and lipids fails to fully prevent diabetic nephropathy (DN). Recently, a broad range of anomalies associated with oxygen biology, such as hypoxia, oxidative stress (OS), and dyserythropoiesis, have been implicated in DN. This review delineates the cellular mechanisms of these anomalies to pinpoint novel therapeutic approaches. The PHD-HIF system mitigates hypoxia: HIF activates a broad range of reactions against hypoxia whereas PHD is an intracellular oxygen sensor negatively regulating HIF. The Keap1-Nrf2 system mitigates OS: Nrf2 activates cellular reactions against OS whereas Keap1 negatively regulates Nrf2. Clinical trials of PHD inhibitors to correct anemia in patients with CKD as well as of a Nrf2 activator, bardoxolone methyl, for DN are under way, even if the latter has been recently interrupted. A specific PHD1 inhibitor, a Keap1 inhibitor, and an allosteric effector of hemoglobin may offer alternative, novel therapies. Erythropoietin (EPO) is critical for the development of erythroid progenitors and thus for tissue oxygen supply. Renal EPO-producing (REP) cells, originating from neural crests, but not fibroblasts from injured tubular epithelial cells, transdifferentiate into myofibroblasts and contribute to renal fibrosis. Agents restoring the initial function of REP cells might retard renal fibrosis. These newer approaches targeting oxygen biology may offer new treatments not only for DN but also for several diseases in which hypoxia and/or OS is a final, common pathway.
Assuntos
Nefropatias Diabéticas/metabolismo , Nefropatias Diabéticas/terapia , Hipóxia/metabolismo , Estresse Oxidativo/fisiologia , Oxigênio/metabolismo , Nefropatias Diabéticas/fisiopatologia , Eritropoetina/metabolismo , Humanos , Fator 1 Induzível por Hipóxia/metabolismo , Prolina Dioxigenases do Fator Induzível por Hipóxia/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteína 1 Associada a ECH Semelhante a Kelch , Fator 2 Relacionado a NF-E2/metabolismo , Transdução de Sinais/fisiologiaRESUMO
A variety of human disorders, e.g., ischemic heart disease, stroke, kidney disease, eventually share the deleterious consequences of a common, hypoxic and oxidative stress pathway. In this review, we utilize recent information on the cellular defense mechanisms against hypoxia and oxidative stress with the hope to propose new therapeutic tools. The hypoxia-inducible factor (HIF) is a key player as it activates a broad range of genes protecting cells against hypoxia. Its level is determined by its degradation rate by intracellular oxygen sensors prolyl hydroxylases (PHDs). There are three different PHD isoforms (PHD1-3). Small molecule PHD inhibitors improve hypoxic injury in experimental animals but, unfortunately, may induce adverse effects associated with PHD2 inhibition, e.g., angiogenesis. As yet, no inhibitor specific for a distinct PHD isoform is currently available. Still, the specific disruption of the PHD1 gene is known to induce hypoxic tolerance, without angiogenesis and erythrocytosis, by reprogramming basal oxygen metabolism with an attendant decreased oxidative stress in hypoxic mitochondria. A specific PHD1 inhibitor might therefore offer a novel therapy against hypoxia. The nuclear factor-erythroid 2 p45-related factor 2 (Nrf2) regulates the basal and inducible expression of numerous antioxidant stress genes. Disruption of its gene exacerbates oxidative tissue injury. Nrf2 activity is modulated by Kelch-like ECH-associated protein 1 (Keap1), an intracellular sensor for oxidative stress. Inhibitors of Keap 1 may prove therapeutic against oxidative tissue injury.
Assuntos
Hipóxia/tratamento farmacológico , Estresse Oxidativo/fisiologia , Oxigênio/metabolismo , Pró-Colágeno-Prolina Dioxigenase/antagonistas & inibidores , Indutores da Angiogênese/uso terapêutico , Animais , Humanos , Hipóxia/fisiopatologia , Fator 1 Induzível por Hipóxia/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteína 1 Associada a ECH Semelhante a Kelch , Camundongos , Mitocôndrias/metabolismo , Mitocôndrias/fisiologia , Fator 2 Relacionado a NF-E2/agonistas , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Inibidores de Proteases/uso terapêutico , RatosRESUMO
OBJECTIVE: Serine protease inhibitors (serpin) play a central role in various pathological processes including coagulation, fibrinolysis, malignancy, and inflammation. Inhibition of serpins may prove therapeutic. As yet, however, only very few small molecule serpin inhibitors have been reported. For the first time, we apply a new approach of virtual screening to discover novel, orally active, small molecule serpin inhibitors and report their effectiveness. METHODS AND RESULTS: We focused on a clinically important serpin, plasminogen activator inhibitor-1 (PAI-1), whose crystal structure has been described. We identify novel, orally active molecules able to enter into the strand 4 position (s4A) of the A beta-sheet of PAI-I as a mock compound. In vitro they specifically inhibit the PAI-1 activity and enhance fibrinolysis activity. In vivo the most effective molecule (TM5007) inhibits coagulation in 2 models: a rat arteriovenous (AV) shunt model and a mouse model of ferric chloride-induced testicular artery thrombosis. It also prevents the fibrotic process initiated by bleomycin in mouse lung. CONCLUSIONS: The present study demonstrates beneficial in vitro and in vivo effects of novel PAI-1 inhibitors. Our methodology proves to be a useful tool to obtain effective inhibitors of serpin activity.
Assuntos
Anticoagulantes/farmacologia , Inibidor 1 de Ativador de Plasminogênio/fisiologia , Serpinas/efeitos dos fármacos , Animais , Anticoagulantes/química , Derivação Arteriovenosa Cirúrgica , Sítios de Ligação , Bleomicina/toxicidade , Coagulação Sanguínea/efeitos dos fármacos , Cloretos , Simulação por Computador , Avaliação Pré-Clínica de Medicamentos/métodos , Compostos Férricos/toxicidade , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Inibidor 1 de Ativador de Plasminogênio/química , Conformação Proteica , Fibrose Pulmonar/induzido quimicamente , Fibrose Pulmonar/patologia , Fibrose Pulmonar/prevenção & controle , Ratos , Serpina E2 , Serpinas/química , Serpinas/fisiologia , Testículo/irrigação sanguínea , Trombose/tratamento farmacológico , Interface Usuário-ComputadorRESUMO
BACKGROUND: Antihypertensive angiotensin II receptor blockers (ARBs) protect the kidney, at least in part, independently of blood pressure lowering. Still, the extent to which blood pressure lowering is related to renoprotection remains unclear. METHODS AND RESULTS: 139 newly synthesized ARB-derivatives were assayed for inhibition of advanced glycation (AGEs). The 9 most powerful compounds were then tested for transition metal chelation, angiotensin II type 1 receptor (AT1R) affinity, and pharmacokinetic parameters. R-147176 was eventually selected as it strongly inhibits advanced glycation but is 6700 times less effective than olmesartan in AT1R binding. It is orally bioavailable and toxicologically safe. Despite a minimal blood pressure lowering effect, it provides significant renoprotection in 3 experimental rat models with renal injury, ie, obese, hypertensive, type 2 diabetic rats (SHR/NDmcr-cp), normotensive type 2 diabetic rats (Zucker diabetic fatty), and remnant kidney rats. CONCLUSIONS: R-147176 retains renal protective properties despite a minimal blood pressure-lowering effect. Clearly, the renal benefits of ARBs do not necessarily depend on blood pressure lowering and AT1R affinity, but rather on the inhibition of AGEs and oxidative stress inherent to their chemical structure. R-147176 opens new avenues in the treatment of cardiovascular and kidney diseases.
Assuntos
Bloqueadores do Receptor Tipo 1 de Angiotensina II/farmacologia , Diabetes Mellitus Tipo 2/tratamento farmacológico , Nefropatias Diabéticas/prevenção & controle , Imidazóis/farmacologia , Rim/efeitos dos fármacos , Tiazolidinedionas/farmacologia , Bloqueadores do Receptor Tipo 1 de Angiotensina II/farmacocinética , Bloqueadores do Receptor Tipo 1 de Angiotensina II/toxicidade , Animais , Anti-Hipertensivos/farmacologia , Antioxidantes/farmacologia , Pressão Sanguínea/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Quelantes/farmacologia , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/fisiopatologia , Nefropatias Diabéticas/etiologia , Nefropatias Diabéticas/metabolismo , Nefropatias Diabéticas/fisiopatologia , Modelos Animais de Doenças , Produtos Finais de Glicação Avançada/metabolismo , Células HeLa , Humanos , Imidazóis/farmacocinética , Rim/metabolismo , Rim/patologia , Masculino , Nefrectomia , Estresse Oxidativo/efeitos dos fármacos , Ligação Proteica , Ratos , Ratos Endogâmicos SHR , Ratos Wistar , Ratos Zucker , Receptor Tipo 1 de Angiotensina/metabolismo , Tiazolidinedionas/farmacocinética , Fatores de TempoRESUMO
AIM: Angiotensin II type 1 receptor blockers (ARB) retard the progression of hypertensive diabetic kidney disease. Clinical evidence suggests that the dose of ARB required to correct hypertension is suboptimal for renoprotection evaluated by proteinuria. No systematic, prospective study has yet evaluated separately the effect of increasing doses of ARB on blood pressure and proteinuria. METHODS: Over a period of 8 weeks, the effect of seven constant doses of an ARB, valsartan (4-160 mg/kg per day), on blood pressure and proteinuria taken as a surrogate marker of nephropathy in a hypertensive, type 2 diabetic rat model, the spontaneously hypertensive/NIH-corpulent rat (SHR/NDmcr-cp), was assessed. In this spontaneously hypertensive rat strain, a genetic mutation in the leptin receptor gene is associated with hyperphagia leading to obesity with metabolic syndrome and eventually to nephropathy. RESULTS: No additional blood pressure lowering was observed above 120 mg/kg per day of valsartan, suggesting that a dose of 80-120 mg/kg per day had a maximal effect. Nevertheless, higher doses of valsartan further reduced proteinuria in a dose-dependent fashion suggesting the absence of a maximal dose. Obesity, hyperglycaemia and hypercholesterolaemia were unaffected but hypertriglyceridaemia was partially corrected at various ARB doses. CONCLUSION: ARB improve renoprotection at doses above those required for a maximal effect on blood pressure. The mechanism of the renoprotection obtained at high doses of ARB is yet to be elucidated.
Assuntos
Bloqueadores do Receptor Tipo 1 de Angiotensina II/farmacologia , Pressão Sanguínea , Diabetes Mellitus Tipo 2/complicações , Nefropatias Diabéticas/tratamento farmacológico , Tetrazóis/farmacologia , Valina/análogos & derivados , Bloqueadores do Receptor Tipo 1 de Angiotensina II/uso terapêutico , Animais , Nefropatias Diabéticas/fisiopatologia , Modelos Animais de Doenças , Hipertensão/complicações , Modelos Lineares , Masculino , Proteinúria/tratamento farmacológico , Ratos , Ratos Endogâmicos SHR , Ratos Endogâmicos WKY , Tetrazóis/uso terapêutico , Valina/farmacologia , Valina/uso terapêutico , ValsartanaRESUMO
In diabetic nephropathy decreased activities of matrix metalloproteinase (MMP)-2, MMP-9 and plasmin contribute to mesangial matrix accumulation. Megsin, a novel member of the serine protease inhibitor superfamily, is predominantly expressed in mesangial cells and is up-regulated in diabetic nephropathy and its overexpression spontaneously induces progressive mesangial expansion in mice. High-glucose stimulated megsin mRNA expression in an in vivo model of type II diabetic nephropathy as well as in vitro in cultured mesangial cells. Megsin potentially inhibits total enzymatic activities of MMP-2 and -9 and plasmin, indicating decreased degradation of mesangial matrix. A specific monoclonal anti-megsin neutralizing antibody restored MMP activity in a transforming growth factor-beta independent manner. Our study suggests that the mesangial matrix accumulation caused by hyperglycemia in diabetes might be due at least in part to up-regulation of megsin which can inhibit plasmin and MMP activities.
Assuntos
Nefropatias Diabéticas/patologia , Matriz Extracelular/metabolismo , Células Mesangiais/patologia , Inibidores de Serina Proteinase/fisiologia , Serpinas/fisiologia , Animais , Nefropatias Diabéticas/enzimologia , Nefropatias Diabéticas/metabolismo , Matriz Extracelular/enzimologia , Fibrinolisina/metabolismo , Humanos , Rim/metabolismo , Masculino , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Células Mesangiais/enzimologia , Células Mesangiais/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Ratos , Ratos Endogâmicos SHR , Ratos Endogâmicos WKY , Ratos Sprague-Dawley , Serpinas/genética , Inibidor Tecidual de Metaloproteinase-1 , Inibidor Tecidual de Metaloproteinase-2 , Transfecção , Fator de Crescimento Transformador beta/fisiologia , Regulação para Cima/genéticaRESUMO
BACKGROUND: The reno- and cardiovascular-protective effects of angiotensin II receptor blockers (ARBs), have been ascribed, at least in part, to their ability to inhibit the formation of advanced glycation end products (AGEs), independently of their effect on blood pressure. They act through decreased oxidative stress, unlike previously reported AGE inhibitors which entrap reactive carbonyl (RCOs) precursors of AGEs. The hypotensive effects of ARBs', however, may limit their use. In the present study, we report the synthesis of a new AGE inhibitor, TM2002, and its effects in vitro and in vivo. METHODS: We screened a large chemical library (approximately 1300 compounds) including edaravone, a drug used to treat cerebral infarction, for in vitro AGE inhibitory activity. Based upon the structure-function analysis of edaravone derivatives, we synthesized a novel AGE inhibitor, 1-(5-hydroxy-3-methyl-1-phenyl-1H-pyrazol-4-yl)-6-methyl-1,3-dihydro-furo[3,4-c]pyridine-7-ol (TM2002). We delineate in vitro the biological characteristics of TM22002, evaluate in vivo its toxico-pharmacokinetics and document in animal models of rat, their renal and cardiovascular protective effectiveness. RESULTS: Screening of a large chemical library disclosed that edaravone inhibits in vitro AGE formation efficiently. Unfortunately, like most AGE inhibitors, it also traps pyridoxal, limiting its clinical usefulness. We therefore synthesized a novel AGE inhibitor, TM2002, that does not trap pyridoxal. In vitro, TM2002 shows powerful AGE inhibitory activity. Markers of oxidation, i.e. o-tyrosine formation and transition metal chelation, are efficiently inhibited by TM2002-like ARBs. TM2002 does not bind to the angiotensin II type 1 receptor. It is readily bioavailable and non-toxic. In vivo, TM2002, given acutely or for 8 weeks, has no adverse effects. In four different rat models of renal injury (anti-Thy1 and ischaemia-reperfusion) and cardiovascular injury (carotid artery balloon injury and angiotensin II-induced cardiac fibrosis), TM2002 improves renal and cardiovascular lesions without modification of blood pressure. CONCLUSIONS: TM2002 is a novel, non-toxic AGE inhibitor acting through ARB-like mechanisms, able to prevent renal and cardiovascular diseases independently of blood pressure lowering.
Assuntos
Doenças Cardiovasculares/prevenção & controle , Furanos/uso terapêutico , Produtos Finais de Glicação Avançada/antagonistas & inibidores , Nefropatias/prevenção & controle , Piridinas/uso terapêutico , Animais , Doenças Cardiovasculares/metabolismo , Modelos Animais de Doenças , Nefropatias/metabolismo , Masculino , Estresse Oxidativo , Ratos , Ratos WistarRESUMO
OBJECTIVE: Hypoxia inducible factor (HIF) plays a pivotal role in the adaptation to ischemic conditions. Its activity is modulated by an oxygen-dependent hydroxylation of proline residues by prolyl hydroxylases (PHD). METHODS AND RESULTS: We discovered 2 unique compounds (TM6008 and TM6089), which inhibited PHD and stabilized HIF activity in vitro. Our docking simulation studies based on the 3-dimensional structure of human PHD2 disclosed that they preferentially bind to the active site of PHD. Whereas PHD inhibitors previously reported inhibit PHD activity via iron chelation, TM6089 does not share an iron chelating motif and is devoid of iron chelating activity. In vitro Matrigel assays and in vivo sponge assays demonstrated enhancement of angiogenesis by local administration of TM6008 and TM6089. Their oral administration stimulated HIF activity in various organs of transgenic rats expressing a hypoxia-responsive reporter vector. No acute toxicity was observed up to 2 weeks after a single oral dose of 2000 mg/kg for TM6008. Oral administration of TM6008 protected neurons in a model of cerebrovascular disease. The protection was associated with amelioration of apoptosis but independent of enhanced angiogenesis. CONCLUSIONS: The present study uncovered beneficial effects of novel PHD inhibitors preferentially binding to the active site of PHD.
Assuntos
Indutores da Angiogênese/farmacologia , Isquemia Encefálica/prevenção & controle , Inibidores Enzimáticos/farmacologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Neovascularização Fisiológica/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Pró-Colágeno-Prolina Dioxigenase/antagonistas & inibidores , Administração Oral , Indutores da Angiogênese/administração & dosagem , Indutores da Angiogênese/química , Indutores da Angiogênese/farmacocinética , Indutores da Angiogênese/toxicidade , Animais , Animais Geneticamente Modificados , Apoptose/efeitos dos fármacos , Sítios de Ligação , Isquemia Encefálica/patologia , Células Cultivadas , Quelantes/farmacologia , Simulação por Computador , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Desenho de Fármacos , Inibidores Enzimáticos/administração & dosagem , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacocinética , Inibidores Enzimáticos/toxicidade , Genes Reporter , Gerbillinae , Coração/efeitos dos fármacos , Humanos , Hidroxilação , Prolina Dioxigenases do Fator Induzível por Hipóxia , Rim/efeitos dos fármacos , Rim/metabolismo , Dose Letal Mediana , Fígado/efeitos dos fármacos , Fígado/metabolismo , Modelos Moleculares , Estrutura Molecular , Miocárdio/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/patologia , Fármacos Neuroprotetores/administração & dosagem , Fármacos Neuroprotetores/química , Fármacos Neuroprotetores/farmacocinética , Fármacos Neuroprotetores/toxicidade , Pró-Colágeno-Prolina Dioxigenase/química , Pró-Colágeno-Prolina Dioxigenase/metabolismo , Ligação Proteica , Conformação Proteica , RatosRESUMO
AIM: Antihypertensive agents inhibiting the renin-angiotensin system (RAS), such as angiotensin II type 1 receptor blockers (ARB), are now part of the standard treatment of patients with diabetic nephropathy, regardless of the presence of systemic hypertension. Whether ARB achieve better renoprotection than other RAS-independent antihypertensive drugs has been an issue of controversy. Several lines of large clinical studies provided better renoprotection of ARB. However, a recent meta-analysis argued against additional benefits of ARB beyond blood pressure. We generated a novel mouse model of diabetic nephropathy; that is, megsin/receptor for advanced glycation end products/inducible nitric oxide synthase triple transgenic mice. This model is normotensive but progressively develops severe diabetic nephropathy that resembles those observed in humans. METHODS: In the present study, we tested whether olmesartan (ARB) achieves better renoprotection than amlodipine (calcium channel blocker). Drug treatment was initiated at the age of 6 weeks and lasted for 12 weeks. RESULTS: This model develops significant glomerular lesions and albuminuria even at the age of 5 weeks. Despite equal blood pressure lowering, only olmesartan suppressed the progression of albuminuria. Neither olmesartan nor amlodipine modified histological lesions. CONCLUSION: Proteinuria and its reduction are known to predict the progression of diabetic nephropathy. Our results support the additional benefit of ARB beyond blood pressure lowering.
Assuntos
Albuminúria/tratamento farmacológico , Bloqueadores do Receptor Tipo 1 de Angiotensina II/uso terapêutico , Pressão Sanguínea/efeitos dos fármacos , Nefropatias Diabéticas/tratamento farmacológico , Imidazóis/uso terapêutico , Óxido Nítrico Sintase Tipo II/fisiologia , Receptores Imunológicos/fisiologia , Serpinas/fisiologia , Tetrazóis/uso terapêutico , Animais , Modelos Animais de Doenças , Rim/patologia , Masculino , Camundongos , Camundongos Endogâmicos ICR , Camundongos Transgênicos , Óxido Nítrico Sintase Tipo II/genética , Receptor para Produtos Finais de Glicação Avançada , Receptores Imunológicos/genética , Serpinas/genéticaRESUMO
Many factors are involved in the pathogenesis of diabetic nephropathy. A single gene abnormality may be prerequisite but insufficient to the disease to manifest. It is therefore only when a second or sometimes a third damage is associated that the consequences of pathogenic phenotypes become evident. We generated the triple transgenic mice overexpressing megsin (a novel glomerular-specific serpin), a receptor for advanced glycation end products (RAGE), and inducible nitric oxide synthase (iNOS). Compared with the single- or two-gene transgenic mice, the triple transgenic mice developed, at an early age (16 weeks), severe albuminuria and renal damage with all of the characteristics of human diabetic nephropathy (i.e., glomerular hypertrophy, diffuse mesangial expansion, inflammatory cell infiltration, and interstitial fibrosis). Interestingly, 30-40% of glomeruli exhibit nodule-like lesions. Oxidative and carbonyl stress makers (pentosidine, N(epsilon)-carboxymethyllysine, and 8-hydroxy-deoxyguanosine) were significantly higher in the triple transgenic mice. The iNOS transgenic mice have a diabetes phenotype, the renal consequences of which are moot, and the superimposition of RAGE leads to more conspicuous manifestations. By additional overexpression of megsin, a gene known to be involved in mesangial proliferation and expansion, these local consequences become dramatically manifest and approximate those observed in human pathology. This multiple hit approach is of interest in consideration of the sequential events during development of diabetic nephropathy.
Assuntos
Nefropatias Diabéticas/metabolismo , Óxido Nítrico Sintase Tipo II/genética , Receptores Imunológicos/metabolismo , Serpinas/genética , Serpinas/metabolismo , Animais , Nefropatias Diabéticas/genética , Modelos Animais de Doenças , Regulação da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Produtos Finais de Glicação Avançada , Rim/citologia , Rim/patologia , Camundongos , Camundongos Transgênicos , Óxido Nítrico Sintase Tipo II/metabolismo , Receptor para Produtos Finais de Glicação Avançada , Receptores Imunológicos/genéticaRESUMO
We have developed a novel, non-toxic inhibitor of advanced glycation and oxidative stress, TM2002, devoid of effect on blood pressure. In transient focal ischemia, TM2002 significantly decreased infarct volume compared with vehicle (79.5+/-18.7 vs. 183.3+/-22.9 mm3, p<0.01). In permanent focal ischemia, TM2002 (2.79, 5.58, and 11.16 mg/kg twice a day) dose-dependently reduced infarct volume (242.1+/-32.3, 201.3+/-15.1, and 171.3+/-15.2 mm3, respectively), and improved neurological deficits. Reduction of infarct volume is demonstrable, provided that TM2002 was administered within 1.5 h after the occlusion. To unravel TM2002's mechanism of action, we examined its in vitro effect on endoplasmic reticulum (ER) stress, using aortic smooth muscle cells isolated from ORP 150(+/-) mice and F9 Herp null mutated cells. Cell death induced by ER stress (tunicamycin or hypoxia) was dose-dependently prevented by TM2002. In vivo immunohistochemical study demonstrated a significant reduction of ORP- and TUNEL-positive apoptotic cells, especially in the penumbra. Inhibition of advanced glycation and oxidative stress was confirmed by a significantly reduced number of cells positive for advanced glycation end products and heme oxygenase-1. TM2002 reduced the levels of protein carbonyl formation in ischemic caudate. The efficacy of TM2002 is equivalent to that of a known neuroprotective agent, NXY-059. In conclusion, TM2002 significantly ameliorates ischemic cerebral damage through reduction of ER stress, advanced glycation, and oxidative stress, independently of blood pressure lowering.
Assuntos
Antioxidantes/farmacologia , Isquemia Encefálica/complicações , Retículo Endoplasmático/fisiologia , Furanos/farmacologia , Produtos Finais de Glicação Avançada/antagonistas & inibidores , Infarto da Artéria Cerebral Média/prevenção & controle , Fármacos Neuroprotetores , Estresse Oxidativo/fisiologia , Piridinas/farmacologia , Animais , Antipirina/análogos & derivados , Antipirina/farmacologia , Benzenossulfonatos/farmacologia , Pressão Sanguínea/efeitos dos fármacos , Isquemia Encefálica/patologia , Morte Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Edaravone , Retículo Endoplasmático/efeitos dos fármacos , Imuno-Histoquímica , Indicadores e Reagentes , Infarto da Artéria Cerebral Média/etiologia , Infarto da Artéria Cerebral Média/patologia , Trombose Intracraniana/patologia , Trombose Intracraniana/prevenção & controle , Masculino , Camundongos , Camundongos Knockout , Oxirredução , Estresse Oxidativo/efeitos dos fármacos , Desnaturação Proteica , Ratos , Ratos Sprague-DawleyRESUMO
OBJECTIVE: Heat sterilization of glucose peritoneal dialysis (PD) fluid generates reactive carbonyl compounds (RCOs), which have been implicated in the formation of advanced glycation end products (AGEs) on peritoneal proteins, with an attendant deterioration of peritoneal permeability in PD patients. To reduce their levels in PD fluid, we had previously devised beads coupled with RCO-trapping agents. The hazards linked to the diffusion of RCO-trapping compounds in the systemic circulation are avoided. Hydrazine-epoxy beads proved the most effective. Still, the amount needed to trap all RCOs remained relatively large. METHODS: We developed a novel agent linking a powerful RCO-trapping AGE inhibitor, pyrazolinone-polyethyleneimine, with cellulose beads (PPCBs). We tested its effectiveness to lower RCOs and AGE formation. RESULTS: Mixed with glucose PD fluid, PPCBs markedly lowered RCOs (alpha-dicarbonyls and aldehydes) and inhibited the generation of pentosidine, an AGE, to levels similar to those of filter-sterilized PD fluid. Their effectiveness is more than one order of magnitude above those of previously developed beads. The PPCBs markedly improved PD fluid biocompatibility. Incubation of 1 L commercial glucose PD fluid at 25 degrees C for 24 hours with 10 or 30 g of wet PPCBs reduced RCO content by 75% - 90% and 100% respectively, without altering the pH or glucose and electrolyte contents of the PD fluid. CONCLUSIONS: We developed a high-affinity adsorption bead to reduce the toxic RCO content and AGE formation potential (carbonyl stress) of PD fluid.
Assuntos
Soluções para Diálise , Produtos Finais de Glicação Avançada/antagonistas & inibidores , Cetonas/isolamento & purificação , Adsorção , Linhagem Celular , Produtos Finais de Glicação Avançada/isolamento & purificação , HumanosRESUMO
The paper describes potassium toxicity as it emerged in the 19th century following the studies of J.N. Comhaire (1778-1837), to these of V. Feltz (1835-1893) and F. Ritter (1837-1884).
Assuntos
Nefrectomia/história , Potássio/toxicidade , Ureia/toxicidade , Animais , Cães , História do Século XIX , Humanos , Nefrectomia/efeitos adversos , Ureia/metabolismoRESUMO
Oxidative tissue damage in vivo is a complex phenomenon involving many factors and pathways. Proteins are particularly attractive targets for oxidative products analysis in order to understand better the physiopathology of human diseases. Protein modifications serve as footprints of biochemical processes. They also help ascertain the mechanism of anti-oxidative action of medical drugs and further search for novel agents that inhibit efficiently oxidative protein damage. Several drugs already used clinically interfere with oxidative protein damage through different mechanisms characteristic of their chemical structure. This review delineates the oxidative protein modifications existing in diabetic nephropathy and their regression in association with renoprotective anti-hypertensive agents. Our hypothetical approach will require further testing. Nevertheless, the insights gained on the biochemistry of protein modifications open new avenues towards the development of new classes of renoprotective agents for diabetic nephropathy.
Assuntos
Pegada de DNA , Nefropatias Diabéticas/genética , Nefropatias Diabéticas/terapia , Animais , Antioxidantes/farmacologia , Nefropatias Diabéticas/metabolismo , Produtos Finais de Glicação Avançada/antagonistas & inibidores , Humanos , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/fisiologia , RatosAssuntos
Febre Hemorrágica com Síndrome Renal/epidemiologia , Animais , Arvicolinae/virologia , Clima , Surtos de Doenças , Reservatórios de Doenças/virologia , Ecossistema , Europa (Continente)/epidemiologia , Fagus , Feminino , Alimentos , Orthohantavírus/classificação , Orthohantavírus/isolamento & purificação , Febre Hemorrágica com Síndrome Renal/transmissão , Febre Hemorrágica com Síndrome Renal/virologia , Humanos , Masculino , Camundongos , Filogenia , Estações do AnoRESUMO
Multiple sclerosis (MS) is characterized by inflammatory demyelination and deposition of fibrinogen in the central nervous system (CNS). Elevated levels of a critical inhibitor of the mammalian fibrinolitic system, plasminogen activator inhibitor 1 (PAI-1) have been demonstrated in human and animal models of MS. In experimental studies that resemble neuroinflammatory disease, PAI-1 deficient mice display preserved neurological structure and function compared to wild type mice, suggesting a link between the fibrinolytic pathway and MS. We previously identified a series of PAI-1 inhibitors on the basis of the 3-dimensional structure of PAI-1 and on virtual screening. These compounds have been reported to provide a number of in vitro and in vivo benefits but none was tested in CNS disease models because of their limited capacity to penetrate the blood-brain barrier (BBB). The existing candidates were therefore optimized to obtain CNS-penetrant compounds. We performed an in vitro screening using a model of BBB and were able to identify a novel, low molecular PAI-1 inhibitor, TM5484, with the highest penetration ratio among all other candidates. Next, we tested the effects on inflammation and demyelination in an experimental allergic encephalomyelitis mice model. Results were compared to either fingolimod or 6α-methylprednisolone. Oral administration of TM5484 from the onset of signs, ameliorates paralysis, attenuated demyelination, and axonal degeneration in the spinal cord of mice. Furthermore, it modulated the expression of brain-derived neurotrophic factor, which plays a protective role in neurons against various pathological insults, and choline acetyltransferase, a marker of neuronal density. Taken together, these results demonstrate the potential benefits of a novel PAI-1 inhibitor, TM5484, in the treatment of MS.
Assuntos
Doenças Desmielinizantes/patologia , Esclerose Múltipla/patologia , Neurônios/efeitos dos fármacos , Neurônios/patologia , Inibidor 1 de Ativador de Plasminogênio/farmacologia , Animais , Axônios , Disponibilidade Biológica , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Colina O-Acetiltransferase/metabolismo , Colágeno Tipo IV/metabolismo , Doenças Desmielinizantes/tratamento farmacológico , Modelos Animais de Doenças , Encefalomielite Autoimune Experimental/tratamento farmacológico , Encefalomielite Autoimune Experimental/patologia , Camundongos , Esclerose Múltipla/tratamento farmacológico , Paralisia/tratamento farmacológico , Paralisia/etiologia , Permeabilidade , Desempenho Psicomotor/efeitos dos fármacosRESUMO
BACKGROUND: Advanced glycation end products (AGEs) and their reactive carbonyl precursors accumulate in renal failure ("carbonyl stress"). Carbonyl stress derives from a broad derangement in the nonenzymatic biochemistry of both carbohydrates and lipids. We tested the influence of dialysate quality on plasma level of pentosidine, an AGE moiety taken as a surrogate marker of carbonyl stress, in hemodialysis patients. METHODS: Plasma pentosidine is measured by means of high-performance liquid chromatography in patients hemodialyzed successively with a conventional and an ultrapure endotoxin-free dialysate. RESULTS: Dialysate endotoxin level decreased from 0.040 +/- 0.017 EU/mL before the switch to the new water supply system to less than the detection limit (<0.001 EU/mL) after the switch. Plasma pentosidine levels decreased from 1.55 +/- 0.61 nmol/mL before the switch to 1.38 +/- 0.52 nmol/mL (P < 0.0001) and 1.31 +/- 0.50 nmol/mL (P < 0.0001) 3 and 6 months after the switch to an ultrapure dialysate, respectively. Decreases in plasma pentosidine levels were similar regardless of dialyzer membrane type; ie, high-flux polysulfone, high-flux polymethylmethacrylate, and low-flux cellulose acetate membranes. Unexpectedly, plasma triglyceride levels decreased from 150 +/- 116 mg/dL (1.69 +/- 1.31 mmol/L) before the switch to 124 +/- 79 mg/dL (1.40 +/- 0.89 mmol/L; P < 0.01) and 119 +/- 75 mg/dL (1.34 +/- 0.85 mmol/L; P < 0.01) 3 and 6 months after the switch despite unchanged total cholesterol levels, respectively. Changes in pentosidine levels were unrelated to those in triglycerides, and both were unrelated to C-reactive protein levels, which remained stable throughout the study. CONCLUSION: Ultrapure dialysate reduces plasma pentosidine levels and improves plasma triglyceride levels in hemodialysis patients through still undefined mechanisms.