RESUMO
Ischemic acute kidney injury is common, deadly, and accelerates the progression of chronic kidney disease, yet has no specific therapy. After ischemia, reperfusion is patchy with early and persistent impairment in regional renal blood flow and cellular injury. We tested the hypothesis that intrarenal coagulation results in sustained renal ischemia following reperfusion, using a well-characterized model. Markedly decreased, but heterogeneous, microvascular plasma flow with microthrombi was found postischemia by intravital microscopy. Widespread tissue factor expression and fibrin deposition were also apparent. Clotting was accompanied by complement activation and inflammation. Treatment with exosomes derived from renal tubular cells or with the fibrinolytic urokinase, given 24 h postischemia when renal failure was established, significantly improved microvascular flow, coagulation, serum creatinine, and histological evidence of injury. These data support the hypothesis that intrarenal clotting occurs early and the resultant sustained ischemia is a critical determinant of renal failure following ischemia; they demonstrate that the coagulation abnormalities are amenable to therapy and that therapy results in improvement in both function and postischemic inflammation.NEW & NOTEWORTHY Ischemic renal injury carries very high morbidity and mortality, yet has no specific therapy. We found markedly decreased, heterogeneous microvascular plasma flow, tissue factor induction, fibrin deposition, and microthrombi after renal ischemia-reperfusion using a well-characterized model. Renal exosomes or the fibrinolytic urokinase, administered after renal failure was established, improved microvascular flow, coagulation, renal function, and histology. Data demonstrate that intrarenal clotting results in sustained ischemia amenable to therapy that improves both function and postischemic inflammation.
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Injúria Renal Aguda , Traumatismo por Reperfusão , Animais , Creatinina , Traumatismo por Reperfusão/patologia , Tromboplastina/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Rim/metabolismo , Isquemia/metabolismo , Injúria Renal Aguda/metabolismo , Reperfusão , Modelos Animais de Doenças , Inflamação/metabolismo , Fibrina/metabolismoRESUMO
INTRODUCTION: Increased oxidative stress is associated with vascular calcification in patients with chronic kidney disease (CKD). We have previously demonstrated that cellular-derived matrix vesicles (MV), but not media-derived MV, are endocytosed in the presence of phosphorus by recipient normal rat vascular smooth muscle cells (VSMC) and induce calcification through ERK1/2 and [Ca2+]i signaling. We hypothesized that these changes were mediated by increased reactive oxygen species (ROS) production. METHODS: MV were co-cultured with recipient VSMC in the presence of high phosphorus and ROS production and cell signaling assessed. RESULTS: The results demonstrated MV endocytosis led to increased ROS production in recipient VSMC with no increase in mitochondrial oxygen consumption or oxidative phosphorylation (OXPHOS), indicating the ROS was not from the mitochondria. The use of inhibitors demonstrated that endocytosis of these MV by VSMC led to a signaling cascade in the cytoplasm beginning with ERK1/2 signaling, then increased [Ca2+]i and stimulation of ROS production, mediated by nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX)1/4. Media-derived MV did not induce this cascade, indicating endocytosis itself was not a factor. Furthermore, inhibition of either ERK1/2 activation or [Ca2+]i reduced vascular calcification. CONCLUSION: We conclude that endocytosis of pro-mineralizing MV can induce a series of signaling events in normal VSMC that culminate in generation of ROS via activation of NOX1/4. Understanding these pathways will allow the development of future targeted therapeutics.
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Músculo Liso Vascular , Calcificação Vascular , Animais , Células Cultivadas , Humanos , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Ratos , Espécies Reativas de Oxigênio/metabolismo , Calcificação Vascular/metabolismoRESUMO
The brain and muscle aryl hydrocarbon receptor nuclear translocator-like protein (BMAL)-1 constitutes a major transcriptional regulator of the circadian clock. Here, we explored the impact of conditional deletion of Bmal1 in endothelium and hematopoietic cells in murine models of microvascular and macrovascular injury. We used two models of Bmal1fx/fx;Tek-Cre mice, a retinal ischemia/reperfusion model and a neointimal hyperplasia model of the femoral artery. Eyes were enumerated for acellular capillaries and were stained for oxidative damage markers using nitrotyrosine immunohistochemistry. LSK (lineage-negative, stem cell antigen-1-positive, c-Kit-positive) cells were quantified and proliferation assessed. Hematopoiesis is influenced by innervation to the bone marrow, which we assessed using IHC analysis. The number of acellular capillaries increased threefold, and nitrotyrosine staining increased 1.5-fold, in the retinas of Bmal1fx/fx;Tek-Cre mice. The number of LSK cells from the Bmal1fx/fx;Tek-Cre mice decreased by 1.5-fold and was accompanied by a profound decrease in proliferative potential. Bmal1fx/fx;Tek-Cre mice also exhibited evidence of bone marrow denervation, demonstrating a loss of neurofilament-200 staining. Injured femoral arteries showed a 20% increase in neointimal hyperplasia compared with similarly injured wild-type controls. Our study highlights the importance of the circadian clock in maintaining vascular homeostasis and demonstrates that specific deletion of BMAL1 in endothelial and hematopoietic cells results in phenotypic features similar to those of diabetes.
Assuntos
Fatores de Transcrição ARNTL/fisiologia , Neointima/patologia , Traumatismo por Reperfusão/metabolismo , Vasos Retinianos/metabolismo , Fatores de Transcrição ARNTL/deficiência , Fatores de Transcrição ARNTL/genética , Animais , Medula Óssea/metabolismo , Medula Óssea/patologia , Capilares/patologia , Proliferação de Células , Ritmo Circadiano/fisiologia , Modelos Animais de Doenças , Células Endoteliais/metabolismo , Artéria Femoral/lesões , Artéria Femoral/patologia , Deleção de Genes , Células-Tronco Hematopoéticas/patologia , Hiperplasia , Antígenos Comuns de Leucócito/análise , Contagem de Leucócitos , Camundongos Transgênicos , Óxido Nítrico Sintase Tipo III/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Traumatismo por Reperfusão/patologia , Retina/metabolismo , Vasos Retinianos/patologiaRESUMO
Ischemic renal injury is a complex syndrome; multiple cellular abnormalities cause accelerating cycles of inflammation, cellular damage, and sustained local ischemia. There is no single therapy that effectively resolves the renal damage after ischemia. However, infusions of normal adult rat renal cells have been a successful therapy in several rat renal failure models. The sustained broad renal benefit achieved by relatively few donor cells led to the hypothesis that extracellular vesicles (EV, largely exosomes) derived from these cells are the therapeutic effector in situ We now show that EV from adult rat renal tubular cells significantly improved renal function when administered intravenously 24 and 48 hours after renal ischemia in rats. Additionally, EV treatment significantly improved renal tubular damage, 4-hydroxynanoneal adduct formation, neutrophil infiltration, fibrosis, and microvascular pruning. EV therapy also markedly reduced the large renal transcriptome drift observed after ischemia. These data show the potential utility of EV to limit severe renal ischemic injury after the occurrence.
Assuntos
Vesículas Extracelulares , Túbulos Renais/metabolismo , Rim/metabolismo , Traumatismo por Reperfusão/patologia , Injúria Renal Aguda/patologia , Aldeídos/química , Animais , Comunicação Celular , Modelos Animais de Doenças , Exossomos/metabolismo , Feminino , Perfilação da Expressão Gênica , Genótipo , Hipóxia/patologia , Rim/patologia , Microcirculação , Neutrófilos/metabolismo , Fenótipo , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Insuficiência Renal , Fatores de TempoRESUMO
Angiotensin-converting enzyme (ACE)-2 is the primary enzyme of the vasoprotective axis of the renin angiotensin system that regulates the classic renin angiotensin system axis. We aimed to determine whether local retinal overexpression of adenoassociated virus (AAV)-ACE2 prevents or reverses diabetic retinopathy. Green fluorescent protein (GFP)-chimeric mice were generated to distinguish resident (retinal) from infiltrating bone marrow-derived inflammatory cells and were made diabetic using streptozotocin injections. Retinal digestion using trypsin was performed and acellular capillaries enumerated. Capillary occlusion by GFP(+) cells was used to measure leukostasis. Overexpression of ACE2 prevented (prevention cohort: untreated diabetic, 11.3 ± 1.4; ACE2 diabetic, 6.4 ± 0.9 per mm(2)) and partially reversed (reversal cohort: untreated diabetic, 15.7 ± 1.9; ACE2 diabetic, 6.5 ± 1.2 per mm(2)) the diabetes-associated increase of acellular capillaries and the increase of infiltrating inflammatory cells into the retina (F4/80(+)) (prevention cohort: untreated diabetic, 24.2 ± 6.7; ACE2 diabetic, 2.5 ± 1.6 per mm(2); reversal cohort: untreated diabetic, 56.8 ± 5.2; ACE2 diabetic, 5.6 ± 2.3 per mm(2)). In both study cohorts, intracapillary bone marrow-derived cells, indicative of leukostasis, were only observed in diabetic animals receiving control AAV injections. These results indicate that diabetic retinopathy, and possibly other diabetic microvascular complications, can be prevented and reversed by locally restoring the balance between the classic and vasoprotective renin angiotensin system.
Assuntos
Retinopatia Diabética/enzimologia , Peptidil Dipeptidase A/metabolismo , Enzima de Conversão de Angiotensina 2 , Animais , Dependovirus , Diabetes Mellitus Experimental/enzimologia , Diabetes Mellitus Experimental/patologia , Diabetes Mellitus Tipo 1 , Retinopatia Diabética/patologia , Terapia Genética/métodos , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Human bone marrow mesenchymal progenitor cells (MPCs) are multipotent cells that play an essential role in endogenous repair and the maintenance of the stem cell niche. We have recently shown that high levels of glucose, conditions mimicking diabetes, cause impairment of MPCs, resulting in enhanced adipogenesis and suppression of osteogenesis. This implies that diabetes may lead to reduced endogenous repair mechanisms through altering the differentiation potential of MPCs and, consequently, disrupting the stem cell niche. Phenotypic alterations in the bone marrow of long-term diabetic patients closely resemble this observation. Here, we show that high levels of glucose selectively enhance autogenous Wnt11 expression in MPCs to stimulate adipogenesis through the Wnt/protein kinase C noncanonical pathway. This novel mechanism may account for increased bone marrow adipogenesis, severe bone loss, and reduced vascular stem cells leading to chronic secondary complications of diabetes.
Assuntos
Adipogenia/efeitos dos fármacos , Glucose/farmacologia , Via de Sinalização Wnt/efeitos dos fármacos , Antígeno AC133 , Idoso , Angiopoietina-2/metabolismo , Animais , Antígenos CD/metabolismo , Medula Óssea/patologia , Diferenciação Celular/efeitos dos fármacos , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patologia , Glicoproteínas/metabolismo , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Pessoa de Meia-Idade , Modelos Biológicos , Peptídeos/metabolismo , Proteína Quinase C/metabolismo , Ratos Sprague-Dawley , Bibliotecas de Moléculas Pequenas/farmacologia , Proteínas Wnt/metabolismo , beta Catenina/metabolismoRESUMO
By using pseudorabies virus expressing green fluorescence protein, we found that efferent bone marrow-neural connections trace to sympathetic centers of the central nervous system in normal mice. However, this was markedly reduced in type 1 diabetes, suggesting a significant loss of bone marrow innervation. This loss of innervation was associated with a change in hematopoiesis toward generation of more monocytes and an altered diurnal release of monocytes in rodents and patients with type 1 diabetes. In the hypothalamus and granular insular cortex of mice with type 1 diabetes, bone marrow-derived microglia/macrophages were activated and found at a greater density than in controls. Infiltration of CD45(+)/CCR2(+)/GR-1(+)/Iba-1(+) bone marrow-derived monocytes into the hypothalamus could be mitigated by treatment with minocycline, an anti-inflammatory agent capable of crossing the blood-brain barrier. Our studies suggest that targeting central inflammation may facilitate management of microvascular complications.
Assuntos
Medula Óssea/inervação , Medula Óssea/patologia , Sistema Nervoso Central/patologia , Diabetes Mellitus Tipo 1/complicações , Diabetes Mellitus Tipo 1/patologia , Inflamação/patologia , Animais , Medula Óssea/efeitos dos fármacos , Sistema Nervoso Central/efeitos dos fármacos , Sistema Nervoso Central/metabolismo , Citocinas/metabolismo , Ensaio de Imunoadsorção Enzimática , Proteínas de Fluorescência Verde/metabolismo , Hematopoese/efeitos dos fármacos , Herpesvirus Suídeo 1/efeitos dos fármacos , Herpesvirus Suídeo 1/fisiologia , Humanos , Inflamação/complicações , Inflamação/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microglia/efeitos dos fármacos , Microglia/metabolismo , Microglia/patologia , Minociclina/farmacologia , Modelos Biológicos , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Monócitos/patologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/patologia , Neurotransmissores/metabolismo , Proteínas Proto-Oncogênicas c-fos/metabolismo , Ratos , Ratos Wistar , Sistema Nervoso Simpático/efeitos dos fármacos , Sistema Nervoso Simpático/metabolismo , Sistema Nervoso Simpático/patologiaRESUMO
Following exposure to microgravity, there is a reduced ability of astronauts to augment peripheral vascular resistance, often resulting in orthostatic hypotension. The purpose of this study was to test the hypothesis that mesenteric arteries and veins will exhibit diminished vasoconstrictor responses after spaceflight. Mesenteric arteries and veins from female mice flown on the Space Transportation System (STS)-131 (n=11), STS-133 (n=6), and STS-135 (n=3) shuttle missions and respective ground-based control mice (n=30) were isolated for in vitro experimentation. Vasoconstrictor responses were evoked in arteries via norepinephrine (NE), potassium chloride (KCl), and caffeine, and in veins through NE across a range of intraluminal pressures (2-12 cmH(2)O). Vasoconstriction to NE was also determined in mesenteric arteries at 1, 5, and 7 d postlanding. In arteries, maximal constriction to NE, KCl, and caffeine were reduced immediately following spaceflight and 1 d postflight. Spaceflight also reduced arterial ryanodine receptor-3 mRNA levels. In mesenteric veins, there was diminished constriction to NE after flight. The results indicate that the impaired vasoconstriction following spaceflight occurs through the ryanodine receptor-mediated intracellular Ca(2+) release mechanism. Such vascular changes in astronauts could compromise the maintenance of arterial pressure during orthostatic stress.
Assuntos
Adaptação Fisiológica , Artérias Mesentéricas/fisiologia , Veias Mesentéricas/fisiologia , Voo Espacial , Vasoconstrição , Animais , Feminino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Nonunion describes bone fractures that fail to heal, resulting in the fracture callus failing to fully ossify or, in atrophic cases, not forming altogether. Fracture healing is regulated, in part, by the balance of proinflammatory and anti-inflammatory processes occurring within the bone marrow and surface cell populations. We sought to further understand the role of osteoimmunology (i.e., study of the close relationship between the immune system and bone) by examining immune cell gene expression via single-cell RNA sequencing of intramedullary canal tissue obtained from human patients with femoral nonunions. Intramedullary canal tissue samples obtained by reaming were collected at the time of surgical repair for femur fracture nonunion (n = 5) or from native bone controls when harvesting autologous bone graft (n = 4). Cells within the samples were isolated and analyzed using the Chromium Single-Cell System (10x Genomics Inc.) and Illumina sequencers. Twenty-three distinct cell clusters were identified, with higher cell proportions in the nonunion samples for monocytes and CD14 + dendritic cells (DCs), and lower proportions of T cells, myelocytes, and promyelocytes in nonunion samples. Gene expression differences were identified in each of the cell clusters from cell types associated with osteoimmunology, including CD14 + DC, monocytes, T cells, promyelocytes, and myelocytes. These results provide human-derived gene profiles that can further our understanding of pathways that may be a cause or a consequence of nonunion, providing the clinical rationale to focus on specific components of osteoimmunology. Clinical significance: The novel single-cell approach may lead to clinically relevant diagnostic biomarkers during earlier stages of nonunion development and/or investigation into therapeutic options.
Assuntos
Fraturas do Fêmur , Fraturas não Consolidadas , Humanos , Análise da Expressão Gênica de Célula Única , Calo Ósseo , Consolidação da Fratura , Osteogênese , Fraturas não Consolidadas/terapia , Resultado do Tratamento , Estudos RetrospectivosRESUMO
Dietary phosphorus restriction and phosphorus binders are commonly prescribed for patients with chronic kidney disease (CKD). However, occurrences of non-adherence to these interventions are common. As low-phosphorus (LP) diets have been consistently experimentally shown in vitro to increase intestinal phosphorus absorption efficiency, a bout of non-adherence to diet or binders may cause an unintended consequence of enhanced intestinal phosphorus absorption. Thus, we aimed to determine the effect of a single bout of high-phosphorus (HP) intake after acclimation to a LP diet. Male Sprague Dawley rats with 5/6 nephrectomy (n = 36) or sham operation (n = 36) were block-randomized to 1 of 3 diets: LP (0.1% P w/w), HP (1.2%), or LP followed by acute HP (LPHP 0.1% then 1.2%). Phosphorus absorption tests were conducted using 33P radioisotope administrated by oral gavage or intravenously (iv). Although the overall two-way ANCOVA model for intestinal fractional phosphorus absorption was non-significant, exploratory comparisons showed intestinal fractional phosphorus absorption efficiency tended to be higher in rats in the LP compared with HP or LPHP groups. Rats in the HP or LPHP groups had higher plasma phosphorus compared with rats in the LP group, but the LPHP group was not different from the HP group. Gene expression of the major intestinal phosphate transporter, NaPi-2b, was lower in the jejunum of rats in the LPHP group compared with rats in the HP group but not different in the duodenum. These results demonstrate that an acute HP load after acclimation to a LP diet does not lead to enhanced intestinal fractional phosphorus absorption efficiency in 5/6 nephrectomized male rats. These data provide evidence against the notion that dietary phosphorus restriction or binder use adversely increases absorption efficiency after a single instance of dietary or binder non-adherence. However, other adverse consequences of fluctuating dietary phosphorus intake cannot be ruled out. © 2022 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.
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Testicular function and associated testosterone concentration decline with advancing age, and an impaired O2 supply may contribute, in part, to this reduction. We hypothesized that there would be a reduced microvascular Po2 (Po2(m)) in the testes from aged rats, and this reduced Po2(m) would be associated with impaired vasomotor control in isolated resistance arterioles. In addition, given the positive effect of exercise on microvascular Po2 and arteriolar function, we further hypothesized that there would be an enhanced Po2(m) in the testes from aged animals after aerobic exercise training. Testicular Po2(m) was measured in vivo via phosphorescence quenching in young and aged sedentary (SED) and exercise-trained (ET; 15 m/min treadmill walking, 15-degree incline, 5 days/wk for 10 wk) male Fischer-344 rats. Vasoconstriction to α-adrenergic [norepinephrine (NE) and phenylephrine (PE)] and myogenic stimuli in testicular arterioles was assessed in vitro. In the SED animals, testicular Po2(m) was reduced by â¼50% with old age (aged SED 11.8 ± 1.9 vs. young SED 22.1 ± 1.1 mmHg; P = 0.0001). Contrary to our hypothesis, exercise training did not alter Po2(m) in the aged group and reduced testicular Po2(m) in the young animals, abolishing age-related differences (young ET, 10.0 ± 0.8 vs. aged ET, 10.7 ± 0.9 mmHg; P = 0.37). Vasoconstrictor responsiveness to NE and PE was diminished in aged compared with young (NE: young SED, 58 ± 2 vs. aged SED, 47 ± 2%; P = 0.001) (PE: young SED, 51 ± 3 vs. aged SED, 36 ± 5%; P = 0.008). Exercise training did not alter maximal vasoconstriction to NE in young or aged groups. In summary, advancing age is associated with a reduced testis Po2(m) and impaired adrenergic vasoconstriction. The diminished testicular microvascular driving pressure of O2 and associated vascular dysfunction provides mechanistic insight into the old age-related decrease in testicular function, and a reduced Po2(m) may contribute, in part, to reduced fertility markers after exercise training.
Assuntos
Envelhecimento , Microcirculação , Oxigênio/sangue , Resistência Física , Testículo/irrigação sanguínea , Vasoconstrição , Agonistas Adrenérgicos/farmacologia , Fatores Etários , Análise de Variância , Animais , Arteríolas/metabolismo , Relação Dose-Resposta a Droga , Fertilidade , Masculino , Microcirculação/efeitos dos fármacos , Ratos , Ratos Endogâmicos F344 , Vasoconstrição/efeitos dos fármacos , Vasoconstritores/farmacologiaRESUMO
The ability to study the bone microenvironment of failed fracture healing may lead to biomarkers for fracture nonunion. Herein the authors describe a technique for isolating individual cells suitable for single-cell RNA sequencing analyses from intramedullary canal tissue collected by reaming during surgery. The purpose was to detail challenges and solutions inherent to the collection and processing of intramedullary canal tissue samples. The authors then examined single-cell RNA sequencing data from fresh and reanimated samples to demonstrate the feasibility of this approach for prospective studies.
Assuntos
Fixação Intramedular de Fraturas , Análise de Sequência de RNA , Biologia , Pinos Ortopédicos , Fraturas Ósseas , Estudos ProspectivosRESUMO
Aging diminishes myogenic tone in arterioles from skeletal muscle. Recent evidence indicates that both large-conductance Ca2+-activated (BKCa) and voltage-dependent (KV) K+ channels mediate negative feedback control of the myogenic response. Thus we tested the hypothesis that aging increases the contributions of KV and BKCa channels to myogenic regulation of vascular tone. Because myogenic responsiveness differs between oxidative and glycolytic muscles, we predicted that KV and BKCa channel contributions to myogenic responsiveness vary with fiber type. Myogenic responses of first-order arterioles from the gastrocnemius and soleus muscles of 4- and 24-mo-old Fischer 344 rats were evaluated in the presence and absence of 4-aminopyridine (5 mM) or iberiotoxin (30 nM), inhibitors of KV and BKCa, respectively. 4-Aminopyridine enhanced myogenic tone with aging and normalized age-related differences in both muscle types. By contrast, iberiotoxin eliminated age-related differences in soleus arterioles and had no effect in gastrocnemius vessels. KV1.5 is an integral component of KV channels in vascular smooth muscle; therefore, we determined the relative protein expression of KV1.5, as well as BKCa, in soleus and gastrocnemius arterioles. Immunoblot analysis revealed no differences in KV1.5 protein with aging or between variant fiber types, whereas BKCa protein levels declined with age in arterioles from both muscle groups. Collectively, these results suggest that the contribution of BKCa to myogenic regulation of vascular tone changes with age in soleus muscle arterioles, whereas increased KV channel expression and negative feedback regulation of myogenic tone increases with advancing age in arterioles from both oxidative and glycolytic muscles.
Assuntos
Envelhecimento/metabolismo , Canais de Potássio Ativados por Cálcio de Condutância Alta/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/irrigação sanguínea , Músculo Liso Vascular/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Potássio/metabolismo , Vasoconstrição , Fatores Etários , Animais , Arteríolas/metabolismo , Relação Dose-Resposta a Droga , Retroalimentação Fisiológica , Glicólise , Técnicas In Vitro , Canal de Potássio Kv1.5/antagonistas & inibidores , Canal de Potássio Kv1.5/metabolismo , Canais de Potássio Ativados por Cálcio de Condutância Alta/antagonistas & inibidores , Masculino , Músculo Esquelético/metabolismo , Músculo Liso Vascular/efeitos dos fármacos , Oxirredução , Bloqueadores dos Canais de Potássio/farmacologia , Canais de Potássio de Abertura Dependente da Tensão da Membrana/antagonistas & inibidores , Ratos , Ratos Endogâmicos F344 , Vasoconstrição/efeitos dos fármacosRESUMO
Hypoxic acute kidney injury, a major unresolved problem, initiates, or aggravates, renal functional and structural decline. There is no treatment for hypoxic acute renal injury and its sequelae. We tested the hypothesis that human kidney tubular cells, or their extracellular vesicles (exosomes), prevent renal injury when infused intravenously 24 hours after 50 minutes of bilateral renal ischemia in Nude rats. Cells and their exosomes were from harvested human kidneys declined for transplantation. Injections of either cells or exosomes, given after 24 and 48 hours of reperfusion, preserved renal function and structure in both treatment groups. However, exosomes were superior to cells; and maintained renal vascular and epithelial networks, prevented renal oxidant stress, and apoptosis; and restrained activation of pro-inflammatory and pro-fibrogenic pathways. Exosomes worked in 24 hours, consistent with functional rather than regenerative activity. Comprehensive proteomic analysis identified 6152 renal proteins from all cellular compartments; and 628 were altered by ischemia at all cell levels, while 377 were significantly improved by exosome infusions. We conclude that renal damage from severe ischemia was broad, and human renal exosomes prevented most protein alterations. Thus, exosomes seem to acutely correct a critical and consequential abnormality during reperfusion. In their absence, renal structure and cells transition to a chronic state of fibrosis and extensive renal cell loss.
Assuntos
Injúria Renal Aguda/terapia , Micropartículas Derivadas de Células/transplante , Túbulos Renais/transplante , Traumatismo por Reperfusão/terapia , Injúria Renal Aguda/patologia , Animais , Apoptose , Exossomos/transplante , Vesículas Extracelulares/transplante , Humanos , Masculino , Ratos , Traumatismo por Reperfusão/patologiaRESUMO
UNLABELLED: We determined whether aging diminishes bone blood flow and impairs endothelium-dependent vasodilation. Femoral perfusion was lower in old animals, as well as endothelium-dependent vasodilation and NO bioavailability. These effects could contribute to old age-related bone loss and the increased risk of fracture. INTRODUCTION: Aging has been shown to diminish bone blood flow in rats and humans. The purpose of this study was to determine whether blood flow to regions of the femur perfused primarily through the principal nutrient artery (PNA) are diminished with aging and whether this putative reduction in flow is associated with impaired endothelium-dependent vasodilation. MATERIALS AND METHODS: Blood flow was measured in conscious young adult (4-6 mo old) and aged (24-26 mo old) male Fischer-344 rats using radiolabeled microspheres. Endothelium-dependent vasodilation of the PNA was assessed in vitro using acetylcholine (ACh), whereas the contribution of the NO synthase (NOS) and cyclooxygenase (COX) signaling pathways to endothelium-dependent vasodilation was determined using the NOS and COX inhibitors L-NAME and indomethacin, respectively. RESULTS: Femoral blood flow in the aged rats was 21% and 28% lower in the proximal and distal metaphyses, respectively, and 45% lower in the diaphyseal marrow. Endothelium-dependent vasodilation was reduced with old age (young: 83 +/- 6% maximal relaxation; aged: 62 +/- 5% maximal relaxation), whereas endothelium-independent vasodilation (sodium nitroprusside) was unaffected by age. The reduction in endothelium-dependent vasodilation was mediated through impairment of the NOS signaling pathway, which resulted in lower NO bioavailability (young: 168 +/- 56 nM; aged: 50 +/- 7 nM). CONCLUSIONS: These data show that reductions in metaphyseal bone and diaphyseal marrow perfusion with old age are associated with diminished endothelium-dependent vasodilation through an impairment of the NOS mechanism. Such age-related changes in bone perfusion and vascular NO signaling could impact clinical bone loss, increase risk of fracture, and impair fracture healing in the elderly.
Assuntos
Envelhecimento/fisiologia , Fêmur/irrigação sanguínea , Fêmur/metabolismo , Óxido Nítrico/metabolismo , Vasodilatação , Acetilcolina/farmacologia , Animais , Ciclo-Oxigenase 1/genética , Ciclo-Oxigenase 1/metabolismo , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/metabolismo , Inibidores de Ciclo-Oxigenase/farmacologia , Endotélio/irrigação sanguínea , Endotélio/metabolismo , Regulação Enzimológica da Expressão Gênica , Masculino , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Nitroprussiato/farmacologia , RNA Mensageiro/genética , Ratos , Ratos Endogâmicos F344 , Vasodilatação/efeitos dos fármacosRESUMO
Purpose: Diabetic patients often are affected by vision problems. We previously identified diabetic retinopathy (DR) as a disease of clock gene dysregulation. TNF-α, a proinflammatory cytokine, is known to be elevated in DR. Müller cells maintain retinal water homeostasis and K+ concentration via Kir4.1 channels. Notably, Kir4.1 expression is reduced in diabetes; however, the interplay of TNF-α, Kir4.1, and clock genes in Müller cells remains unknown. We hypothesize that the Kir4.1 in Müller cells is under clock regulation, and increase in TNF-α is detrimental to Kir4.1. Methods: Long-Evans rats were made diabetic using streptozotocin (STZ). Retinal Kir4.1 expression was determined at different time intervals. Rat Müller (rMC-1) cells were transfected with siRNA for Per2 or Bmal1 and in parallel treated with TNF-α (5-5000 pM) to determine Kir4.1 expression. Results: Kir4.1 expression exhibited a diurnal rhythm in the retina; however, with STZ-induced diabetes, Kir4.1 was reduced overall. Kir4.1 rhythm was maintained in vitro in clock synchronized rMC-1 cells. Clock gene siRNA-treated rMC-1 exhibited a decrease in Kir4.1 expression. TNF-α treatment of rMCs lead to a profound decrease in Kir4.1 due to reduced colocalization of Kir4.1 channels with synapse-associated protein (SAP97) and disorganization of the actin cytoskeleton. Conclusions: Our findings demonstrate that Kir4.1 channels possess a diurnal rhythm, and this rhythm is dampened with diabetes, thereby suggesting that the increase in TNF-α is detrimental to normal Kir4.1 rhythm and expression.
Assuntos
Diabetes Mellitus Experimental , Retinopatia Diabética/genética , Células Ependimogliais/metabolismo , Regulação da Expressão Gênica , Canais de Potássio Corretores do Fluxo de Internalização/genética , RNA Mensageiro/genética , Fator de Necrose Tumoral alfa/uso terapêutico , Animais , Western Blotting , Células Cultivadas , Ritmo Circadiano , Retinopatia Diabética/tratamento farmacológico , Retinopatia Diabética/metabolismo , Células Ependimogliais/efeitos dos fármacos , Células Ependimogliais/patologia , Imuno-Histoquímica , Canais de Potássio Corretores do Fluxo de Internalização/biossíntese , Ratos , Ratos Long-Evans , Reação em Cadeia da Polimerase em Tempo Real , Retina/efeitos dos fármacos , Retina/metabolismo , Retina/patologiaRESUMO
Purpose: We investigated whether subthreshold retinal phototherapy (SRPT) was associated with recruitment of bone marrow (BM)-derived cells to the neurosensory retina (NSR) and RPE layer. Methods: GFP chimeric mice and wild-type (WT) mice were subjected to SRPT using a slit-lamp infrared laser. Duty cycles of 5%, 10%, 15%, and 20% (0.1 seconds, 250 mW, spot size 50 µm) with 30 applications were placed 50 to 100 µm from the optic disc. In adoptive transfer studies, GFP+ cells were given intravenously immediately after WT mice received SRPT. Immunohistochemistry was done for ionized calcium-binding adapter molecule-1 (IBA-1+), CD45, Griffonia simplicifolia lectin isolectin B4, GFP or cytokeratin). Expression of Ccl2, Il1b, Il6, Hspa1a, Hsp90aa1, Cryab, Hif1a, Cxcl12, and Cxcr4 mRNA and flow cytometry of the NSR and RPE-choroid were performed. Results: Within 12 to 24 hours of SRPT, monocytes were detected in the NSR and RPE-choroid. Detection of reparative progenitors in the RPE occurred at 2 weeks using flow cytometry. Recruitment of GFP+ cells to the RPE layer occurred in a duty cycle-dependent manner in chimeric mice and in mice undergoing adoptive transfer. Hspa1a, Hsp90aa1, and Cryab mRNAs increased in the NSR at 2 hours post laser; Hif1a, Cxcl12, Hspa1a increased at 4 hours in the RPE-choroid; and Ccl2, Il1b, Ifng, and Il6 increased at 12 to 24 hours in the RPE-choroid. Conclusions: SRPT induces monocyte recruitment to the RPE followed by hematopoietic progenitor cell homing at 2 weeks. Recruitment occurs in a duty cycle-dependent manner and potentially could contribute to the therapeutic efficacy of SRPT.
Assuntos
Células da Medula Óssea/fisiologia , Movimento Celular/fisiologia , Fototerapia , Retina/citologia , Epitélio Pigmentado da Retina/citologia , Transferência Adotiva , Animais , Biomarcadores/metabolismo , Células Cultivadas , Quimiocina CXCL12/metabolismo , Corioide/citologia , Corioide/metabolismo , Feminino , Citometria de Fluxo , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Choque Térmico/metabolismo , Transplante de Células-Tronco Hematopoéticas , Imuno-Histoquímica , Terapia a Laser , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Monócitos/fisiologia , Receptores CXCR4/metabolismo , Retina/metabolismo , Retina/cirurgia , Epitélio Pigmentado da Retina/metabolismoRESUMO
In this study, the role of CX3CR1 in the progression of diabetic retinopathy (DR) was investigated. The retinas of wild-type (WT), CX3CR1 null (CX3CR1gfp/gfp, KO), and heterozygous (CX3CR1+/gfp, Het) mice were compared in the presence and absence of streptozotocin (STZ)-induced diabetes. CX3CR1 deficiency in STZ-KO increased vascular pathology at 4 months of diabetes, as a significant increase in acellular capillaries was observed only in the STZ-KO group. CX3CR1 deficiency and diabetes had similar effects on retinal neurodegeneration measured by an increase in DNA fragmentation. Retinal vascular pathology in STZ-KO mice was associated with increased numbers of monocyte-derived macrophages in the retina. Furthermore, compared to STZ-WT, STZ-KO mice exhibited increased numbers of inflammatory monocytes in the bone marrow and impaired homing of monocytes to the spleen. The induction of retinal IL-10 expression by diabetes was significantly less in KO mice, and when bone marrow-derived macrophages from KO mice were maintained in high glucose, they expressed significantly less IL-10 and more TNF-α in response to LPS stimulation. These findings support that CX3CR1 deficiency accelerates the development of vascular pathology in DR through increased recruitment of proinflammatory myeloid cells that demonstrate reduced expression of anti-inflammatory IL-10. KEY MESSAGES: ⢠CX3CR1 deletion in STZ-diabetic mice accelerated the onset of diabetic retinopathy (DR). ⢠The early onset of DR was associated with increased retinal cell apoptosis. ⢠The early onset of DR was associated with increased recruitment of bone marrow-derived macrophages to the retina. ⢠Bone marrow-derived macrophages from CX3CR1 KO diabetic mice expressed more TNF-α and less IL-10. ⢠The role of IL-10 in protection from progression of DR is highlighted.
Assuntos
Receptor 1 de Quimiocina CX3C/deficiência , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 1/patologia , Retinopatia Diabética/metabolismo , Retinopatia Diabética/patologia , Animais , Apoptose , Peso Corporal , Células da Medula Óssea/metabolismo , Receptor 1 de Quimiocina CX3C/metabolismo , Modelos Animais de Doenças , Deleção de Genes , Hemoglobinas Glicadas/metabolismo , Homeostase , Hipotálamo/metabolismo , Inflamação/patologia , Mediadores da Inflamação/metabolismo , Interleucina-10/metabolismo , Macrófagos/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Monócitos/metabolismo , Células Mieloides/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Retina/metabolismo , Retina/patologia , EstreptozocinaRESUMO
We previously showed that peripheral neuropathy of the bone marrow was associated with loss of circadian rhythmicity of stem/progenitor cell release into the circulation. Bone marrow neuropathy results in dramatic changes in hematopoiesis that lead to microvascular complications, inflammation, and reduced endothelial repair. This series of events represents early pathogenesis before development of diabetic retinopathy. In this study we characterized early alterations within the bone marrow of streptozotocin (STZ)-induced diabetic rats following treatments that prevent experimental peripheral neuropathy. We asked whether bone marrow neuropathy and the associated bone marrow pathology were reversed with treatments that prevent peripheral neuropathy. Three strategies were tested: inhibition of neutral endopeptidase, inhibition of aldose reductase plus lipoic acid supplementation, and insulin therapy with antioxidants. All strategies prevented loss of nerve conduction velocity resulting from STZ-induced diabetes and corrected the STZ-induced diabetes-associated increase of immunoreactivity of neuropeptide Y, tyrosine hydroxylase, and somatostatin. The treatments also reduced concentrations of interleukin-1ß, granulocyte colony-stimulating factor, and matrix metalloproteinase 2 in STZ-induced diabetic bone marrow supernatant and decreased the expression of NADPH oxidase 2, nitric oxide synthase 2, and nuclear factor-κB1 mRNA in bone marrow progenitor cells. These therapies represent novel approaches to attenuate the diabetic phenotype within the bone marrow and may constitute an important therapeutic strategy for diabetic microvascular complications.
Assuntos
Medula Óssea/patologia , Diabetes Mellitus Experimental/terapia , Neuropatias Diabéticas/prevenção & controle , Compostos Heterocíclicos com 3 Anéis/uso terapêutico , Hipoglicemiantes/uso terapêutico , Inflamação/prevenção & controle , Tecido Adiposo/fisiologia , Animais , Citocinas/genética , Citocinas/metabolismo , Óleos de Peixe/administração & dosagem , Óleos de Peixe/uso terapêutico , Regulação da Expressão Gênica , Compostos Heterocíclicos com 3 Anéis/administração & dosagem , Hipoglicemiantes/administração & dosagem , Imidazolidinas/uso terapêutico , Insulina/administração & dosagem , Insulina/uso terapêutico , Masculino , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , NADPH Oxidase 2 , NADPH Oxidases/genética , NADPH Oxidases/metabolismo , Neuropeptídeo Y , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Medição da Dor , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Somatostatina , Células-Tronco , Estreptozocina , Ácido Tióctico/uso terapêutico , Tirosina 3-Mono-OxigenaseRESUMO
To investigate whether exercise training can reverse age-related impairment of myogenic vasoconstriction in skeletal muscle arterioles, young (4 mo) and old (22 mo) male Fischer 344 rats were randomly assigned to either sedentary or exercise-trained groups. The roles of the endothelium and Kv1 channels in age- and exercise training-induced adaptations of myogenic responses were assessed through evaluation of pressure-induced constriction in endothelium-intact and denuded soleus muscle arterioles in the presence and absence of the Kv1 channel blocker, correolide. Exercise training enhanced myogenic constriction in arterioles from both old and young rats. In arterioles from old rats, exercise training restored myogenic constriction to a level similar to that of arterioles from young sedentary rats. Removal of the endothelium did not alter myogenic constriction of arterioles from young sedentary rats, but reduced myogenic constriction in arterioles from young exercise-trained rats. In contrast, endothelial removal had no effect on myogenic constriction of arterioles from old exercise-trained rats, but increased myogenic vasoconstriction in old sedentary rats. The effect of Kv1 channel blockade was also dependent on age and training status. In arterioles from young sedentary rats, Kv1 blockade had little effect on myogenic constriction, whereas in old sedentary rats Kv1 blockade increased myogenic constriction. After exercise training, Kv1 channel blockade increased myogenic constriction in arterioles from both young and old rats. Thus exercise training restores myogenic constriction of arterioles from old rats and enhances myogenic constriction from young rats through adaptations of the endothelium and smooth muscle Kv1 channels.