RESUMO
OBJECTIVES: Glucagon-like peptide-1 receptor (GLP-1R) agonists reduce the rates of major cardiovascular events, including myocardial infarction in people with type 2 diabetes, and decrease infarct size while preserving ventricular function in preclinical studies. Nevertheless, the precise cellular sites of GLP-1R expression that mediate the cardioprotective actions of GLP-1 in the setting of ischemic cardiac injury are uncertain. METHODS: Publicly available single cell RNA sequencing (scRNA-seq) datasets on mouse and human heart cells were analyzed for Glp1r/GLP1R expression. Fluorescent activated cell sorting was used to localize Glp1r expression in cell populations from the mouse heart. The importance of endothelial and hematopoietic cells for the cardioprotective response to liraglutide in the setting of acute myocardial infarction (MI) was determined by inactivating the Glp1r in Tie2+ cell populations. Cardiac gene expression profiles regulated by liraglutide were examined using RNA-seq to interrogate mouse atria and both infarcted and non-infarcted ventricular tissue after acute coronary artery ligation. RESULTS: In mice, cardiac Glp1r mRNA transcripts were exclusively detected in endocardial cells by scRNA-seq. In contrast, analysis of human heart by scRNA-seq localized GLP1R mRNA transcripts to populations of atrial and ventricular cardiomyocytes. Moreover, very low levels of GIPR, GCGR and GLP2R mRNA transcripts were detected in the human heart. Cell sorting and RNA analyses detected cardiac Glp1r expression in endothelial cells (ECs) within the atria and ventricle in the ischemic and non-ischemic mouse heart. Transcriptional responses to liraglutide administration were not evident in wild type mouse ventricles following acute MI, however liraglutide differentially regulated genes important for inflammation, cardiac repair, cell proliferation, and angiogenesis in the left atrium, while reducing circulating levels of IL-6 and KC/GRO within hours of acute MI. Inactivation of the Glp1r within the Tie2+ cell expression domain encompassing ECs revealed normal cardiac structure and function, glucose homeostasis and body weight in Glp1rTie2-/- mice. Nevertheless, the cardioprotective actions of liraglutide to reduce infarct size, augment ejection fraction, and improve survival after experimental myocardial infarction (MI), were attenuated in Glp1rTie2-/- mice. CONCLUSIONS: These findings identify the importance of the murine Tie2+ endothelial cell GLP-1R as a target for the cardioprotective actions of GLP-1R agonists and support the importance of the atrial and ventricular endocardial GLP-1R as key sites of GLP-1 action in the ischemic mouse heart. Hitherto unexplored species-specific differences in cardiac GLP-1R expression challenge the exclusive use of mouse models for understanding the mechanisms of GLP-1 action in the normal and ischemic human heart.
Assuntos
Fibrilação Atrial , Receptor do Peptídeo Semelhante ao Glucagon 1 , Liraglutida , Infarto do Miocárdio , Animais , Humanos , Camundongos , Diabetes Mellitus Tipo 2/tratamento farmacológico , Células Endoteliais/metabolismo , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Receptor do Peptídeo Semelhante ao Glucagon 1/efeitos dos fármacos , Receptor do Peptídeo Semelhante ao Glucagon 1/metabolismo , Liraglutida/farmacologia , Infarto do Miocárdio/tratamento farmacológico , RNA Mensageiro , Modelos Animais de Doenças , Receptor TIE-2/metabolismoRESUMO
The causes of the increased risk of severe coronavirus disease 2019 (COVID-19) in people with diabetes are unclear. It has been speculated that renin-angiotensin system (RAS) blockers may promote COVID-19 by increasing ACE2, which severe acute respiratory syndrome coronavirus 2 uses to enter host cells, along with the host protease TMPRSS2. Taking a reverse translational approach and by combining in situ hybridization, primary cell isolation, immunoblotting, quantitative RT-PCR, and liquid chromatography-tandem mass spectrometry, we studied lung and kidney ACE2 and TMPRSS2 in diabetic mice mimicking host factors linked to severe COVID-19. In healthy young mice, neither the ACE inhibitor ramipril nor the AT1 receptor blocker telmisartan affected lung or kidney ACE2 or TMPRSS2, except for a small increase in kidney ACE2 protein with ramipril. In contrast, mice with comorbid diabetes (aging, high-fat diet, and streptozotocin-induced diabetes) had heightened lung ACE2 and TMPRSS2 protein levels and increased lung ACE2 activity. None of these parameters were affected by RAS blockade. ACE2 was similarly upregulated in the kidneys of mice with comorbid diabetes compared with aged controls, whereas TMPRSS2 (primarily distal nephron) was highest in telmisartan-treated animals. Upregulation of lung ACE2 activity in comorbid diabetes may contribute to an increased risk of severe COVID-19. This upregulation is driven by comorbidity and not by RAS blockade.
Assuntos
Enzima de Conversão de Angiotensina 2/genética , Diabetes Mellitus Experimental/metabolismo , Dieta Hiperlipídica , Rim/metabolismo , Pulmão/metabolismo , Serina Endopeptidases/genética , Fatores Etários , Bloqueadores do Receptor Tipo 1 de Angiotensina II/farmacologia , Enzima de Conversão de Angiotensina 2/efeitos dos fármacos , Enzima de Conversão de Angiotensina 2/metabolismo , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Animais , COVID-19 , Immunoblotting , Hibridização In Situ , Rim/efeitos dos fármacos , Pulmão/efeitos dos fármacos , Masculino , Camundongos , Ramipril/farmacologia , Receptores de Coronavírus/efeitos dos fármacos , Receptores de Coronavírus/genética , Receptores de Coronavírus/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , SARS-CoV-2 , Serina Endopeptidases/efeitos dos fármacos , Serina Endopeptidases/metabolismo , Telmisartan/farmacologiaRESUMO
Pulmonary arterial hypertension (PAH) is a rare, but progressive and devastating vascular disease with few treatment options to prevent the advancement to right ventricular dysfunction hypertrophy and failure. Empagliflozin, a sodium-glucose cotransporter 2 (SGLT2) inhibitor, enhances urinary glucose excretion as well as reduces cardiovascular events and mortality in individuals with type 2 diabetes. While empagliflozin has been reported to lower systemic hypertension due to increased diuresis, the effect of empagliflozin on PAH is unknown. We used monocrotaline (MCT)-treated Sprague-Dawley rats to determine if empagliflozin alters PAH-associated outcomes. Compared to vehicle control, daily empagliflozin administration significantly improved survival in rats with severe MCT-induced PAH. Hemodynamic assessments showed that empagliflozin treatment significantly reduced mean pulmonary artery pressure, right ventricular systolic pressure, and increased pulmonary acceleration time. Empagliflozin treatment resulted in reduced right ventricular hypertrophy and fibrosis. Histological and molecular assessments of lung vasculature revealed significantly reduced medial wall thickening and decreased muscularization of pulmonary arterioles after empagliflozin treatment compared to vehicle-treated rats. In summary, SGLT2 inhibition with empagliflozin lowered mortality, reduced right ventricle systolic pressure, and attenuated maladaptive pulmonary remodeling in MCT-induced PAH. Clinical studies evaluating the efficacy of SGLT-2 inhibition should be considered for patients with PAH.
Assuntos
Compostos Benzidrílicos/farmacologia , Glucosídeos/farmacologia , Hipertrofia Ventricular Direita/prevenção & controle , Hipertensão Arterial Pulmonar/tratamento farmacológico , Inibidores do Transportador 2 de Sódio-Glicose/farmacologia , Animais , Compostos Benzidrílicos/metabolismo , Pressão Sanguínea/efeitos dos fármacos , Diabetes Mellitus Tipo 2/patologia , Fibrose/tratamento farmacológico , Glucosídeos/metabolismo , Ventrículos do Coração/efeitos dos fármacos , Hemodinâmica/efeitos dos fármacos , Humanos , Pulmão/patologia , Masculino , Modelos Animais , Monocrotalina/efeitos adversos , Mortalidade , Artéria Pulmonar/patologia , Ratos Sprague-Dawley , Medição de Risco , Inibidores do Transportador 2 de Sódio-Glicose/metabolismo , Remodelação Vascular/efeitos dos fármacosRESUMO
Blood glucose-lowering therapies can positively or negatively affect heart function in type 2 diabetes, or they can have neutral effects. Dipeptidyl peptidase 4 (DPP-4) inhibitors lower blood glucose by preventing the proteolytic inactivation of glucagon-like peptide 1 (GLP-1). However, GLP-1 is not the only peptide substrate of DPP-4. Here, we investigated the GLP-1-independent cardiac effects of DPP-4 substrates. Pointing to GLP-1 receptor (GLP-1R)-independent actions, DPP-4 inhibition prevented systolic dysfunction equally in pressure-overloaded wild-type and GLP-1R knockout mice. Likewise, DPP-4 inhibition or the DPP-4 substrates substance P or C-X-C motif chemokine ligand 12 (CXCL12) improved contractile recovery after no-flow ischemia in the hearts of otherwise healthy young adult mice. Either DPP-4 inhibition or CXCL12 increased phosphorylation of the Ca2+ regulatory protein phospholamban (PLN), and CXCL12 directly enhanced cardiomyocyte Ca2+ flux. In contrast, hearts of aged obese diabetic mice (which may better mimic the comorbid patient population) had diminished levels of PLN phosphorylation. In this setting, CXCL12 paradoxically impaired cardiac contractility in a phosphoinositide 3-kinase γ-dependent manner. These findings indicate that the cardiac effects of DPP-4 inhibition primarily occur through GLP-1R-independent processes and that ostensibly beneficial DPP-4 substrates can paradoxically worsen heart function in the presence of comorbid diabetes.
Assuntos
Cálcio/metabolismo , Quimiocina CXCL12/metabolismo , Diabetes Mellitus/metabolismo , Coração/fisiopatologia , Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Animais , Quimiocina CXCL12/genética , Diabetes Mellitus/fisiopatologia , Dieta Hiperlipídica , Receptor do Peptídeo Semelhante ao Glucagon 1/genética , Receptor do Peptídeo Semelhante ao Glucagon 1/metabolismo , Camundongos , Camundongos Knockout , FosforilaçãoRESUMO
Background: Heart failure (HF) is associated with reduced expression of plasma membrane Ca2+-ATPase 4 (PMCA4). Cardiac-specific overexpression of human PMCA4b in mice inhibited nNOS activity and reduced cardiac hypertrophy by inhibiting calcineurin. Here we examine temporally regulated cardiac-specific overexpression of hPMCA4b in mouse models of myocardial ischemia reperfusion injury (IRI) ex vivo, and HF following experimental myocardial infarction (MI) in vivoMethods and results: Doxycycline-regulated cardiomyocyte-specific overexpression and activity of hPMCA4b produced adaptive changes in expression levels of Ca2+-regulatory genes, and induced hypertrophy without significant differences in Ca2+ transients or diastolic Ca2+ concentrations. Total cardiac NOS and nNOS-specific activities were reduced in mice with cardiac overexpression of hPMCA4b while nNOS, eNOS and iNOS protein levels did not differ. hMPCA4b-overexpressing mice also exhibited elevated systolic blood pressure vs. controls, with increased contractility and lusitropy in vivo In isolated hearts undergoing IRI, hPMCA4b overexpression was cardioprotective. NO donor-treated hearts overexpressing hPMCA4b showed reduced LVDP and larger infarct size versus vehicle-treated hearts undergoing IRI, demonstrating that the cardioprotective benefits of hPMCA4b-repressed nNOS are lost by restoring NO availability. Finally, both pre-existing and post-MI induction of hPMCA4b overexpression reduced infarct expansion and improved survival from HF.Conclusions: Cardiac PMCA4b regulates nNOS activity, cardiac mass and contractility, such that PMCA4b overexpression preserves cardiac function following IRI, heightens cardiac performance and limits infarct progression, cardiac hypertrophy and HF, even when induced late post-MI. These data identify PMCA4b as a novel therapeutic target for IRI and HF.
Assuntos
Infarto do Miocárdio/prevenção & controle , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Miócitos Cardíacos/enzimologia , ATPases Transportadoras de Cálcio da Membrana Plasmática/metabolismo , Animais , Sinalização do Cálcio , Modelos Animais de Doenças , Insuficiência Cardíaca/enzimologia , Insuficiência Cardíaca/fisiopatologia , Insuficiência Cardíaca/prevenção & controle , Humanos , Hipertrofia Ventricular Esquerda/enzimologia , Hipertrofia Ventricular Esquerda/fisiopatologia , Hipertrofia Ventricular Esquerda/prevenção & controle , Preparação de Coração Isolado , Camundongos Transgênicos , Contração Miocárdica , Infarto do Miocárdio/enzimologia , Infarto do Miocárdio/genética , Infarto do Miocárdio/fisiopatologia , Traumatismo por Reperfusão Miocárdica/enzimologia , Traumatismo por Reperfusão Miocárdica/genética , Traumatismo por Reperfusão Miocárdica/fisiopatologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/patologia , Óxido Nítrico/metabolismo , Doadores de Óxido Nítrico/farmacologia , Óxido Nítrico Sintase Tipo I/metabolismo , ATPases Transportadoras de Cálcio da Membrana Plasmática/genética , Regulação para Cima , Função Ventricular Esquerda , Pressão VentricularRESUMO
To contend with the deleterious effects of accumulating misfolded protein aggregates or damaged organelles cells rely on a system of quality control processes, among them the autophagy-lysosome pathway. This pathway is itself controlled by a master regulator transcription factor termed transcription factor EB (TFEB). When TFEB localizes to the cell nucleus it promotes the expression of a number of genes involved in protein clearance. Here, we set out to determine (1) whether TFEB expression is altered in chronic kidney disease (CKD); (2) whether inhibition of the cytosolic deacetylase histone deacetylase 6 (HDAC6) affects TFEB acetylation and nuclear localization; and (3) whether HDAC6 inhibition, in turn, alters the natural history of experimental CKD. TFEB mRNA and protein levels were observed to be diminished in the kidneys of humans with diabetic kidney disease, accompanied by accumulation of the protein aggregate adaptor protein p62 in tubule epithelial cells. In cultured NRK-52E cells, HDAC6 inhibition with the small molecule inhibitor Tubastatin A acetylated TFEB, increasing TFEB localization to the nucleus and attenuating cell death. In a rat model of CKD, Tubastatin A prevented the accumulation of misfolded protein aggregates in tubule epithelial cells, attenuated proteinuria progression, limited tubule cell death and diminished tubulointerstitial collagenous matrix deposition. These findings point to the common occurrence of dysregulated quality control processes in CKD and they suggest that TFEB downregulation may contribute to tubule injury in CKD. They also identify a regulatory relationship between HDAC6 and TFEB. HDAC6 inhibitors and TFEB activators both warrant further investigation as treatments for CKD.
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Focal segmental glomerulosclerosis (FSGS) is an important cause of nondiabetic chronic kidney disease (CKD). Sodium-glucose cotransporter 2 inhibition (SGLT2i) therapy attenuates the progression of diabetic nephropathy, but it remains unclear whether SGLT2i provides renoprotection in nondiabetic CKD such as FSGS. The primary aim of this pilot study was to determine the effect of 8 wk of dapagliflozin on glomerular filtration rate (GFR) in humans and in experimental FSGS. Secondary end points were related to changes in renal hemodynamic function, proteinuria, and blood pressure (BP). GFR (inulin) and renal plasma flow (para-aminohippurate), proteinuria, and BP were measured in patients with FSGS ( n = 10), and similar parameters were measured in subtotally nephrectomized (SNx) rats. In response to dapagliflozin, changes in GFR, renal plasma flow, and 24-h urine protein excretion were not statistically significant in humans or rats. Systolic BP (SBP) decreased in SNx rats (196 ± 26 vs. 165 ± 33 mmHg; P < 0.001), whereas changes were not statistically significant in humans (SBP 112.7 ± 8.5 to 112.8 ± 11.2 mmHg, diastolic BP 71.8 ± 6.5 to 69.6 ± 8.4 mmHg; P = not significant), although hematocrit increased (0.40 ± 0.05 to 0.42 ± 0.05%; P = 0.03). In archival kidney tissue from a separate patient cohort, renal parenchymal SGLT2 mRNA expression was decreased in individuals with FSGS compared with controls. Short-term treatment with the SGLT2i dapagliflozin did not modify renal hemodynamic function or attenuate proteinuria in humans or in experimental FSGS. This may be related to downregulation of renal SGLT2 expression. Studies examining the impact of SGLT2i on markers of kidney disease in patients with other causes of nondiabetic CKD are needed.
Assuntos
Arteríolas/efeitos dos fármacos , Compostos Benzidrílicos/uso terapêutico , Taxa de Filtração Glomerular/efeitos dos fármacos , Glomerulosclerose Segmentar e Focal/tratamento farmacológico , Glucosídeos/uso terapêutico , Rim/irrigação sanguínea , Inibidores do Transportador 2 de Sódio-Glicose/uso terapêutico , Vasoconstrição/efeitos dos fármacos , Adulto , Animais , Arteríolas/metabolismo , Arteríolas/fisiopatologia , Modelos Animais de Doenças , Feminino , Glomerulosclerose Segmentar e Focal/diagnóstico , Glomerulosclerose Segmentar e Focal/metabolismo , Glomerulosclerose Segmentar e Focal/fisiopatologia , Humanos , Masculino , Pessoa de Meia-Idade , Projetos Piloto , Estudo de Prova de Conceito , Proteinúria/tratamento farmacológico , Proteinúria/metabolismo , Proteinúria/fisiopatologia , Ratos Sprague-Dawley , Transportador 2 de Glucose-Sódio/genética , Transportador 2 de Glucose-Sódio/metabolismo , Fatores de Tempo , Resultado do TratamentoRESUMO
Histone protein modifications control fate determination during normal development and dedifferentiation during disease. Here, we set out to determine the extent to which dynamic changes to histones affect the differentiated phenotype of ordinarily quiescent adult glomerular podocytes. To do this, we examined the consequences of shifting the balance of the repressive histone H3 lysine 27 trimethylation (H3K27me3) mark in podocytes. Adriamycin nephrotoxicity and subtotal nephrectomy (SNx) studies indicated that deletion of the histone methylating enzyme EZH2 from podocytes decreased H3K27me3 levels and sensitized mice to glomerular disease. H3K27me3 was enriched at the promoter region of the Notch ligand Jag1 in podocytes, and derepression of Jag1 by EZH2 inhibition or knockdown facilitated podocyte dedifferentiation. Conversely, inhibition of the Jumonji C domain-containing demethylases Jmjd3 and UTX increased the H3K27me3 content of podocytes and attenuated glomerular disease in adriamycin nephrotoxicity, SNx, and diabetes. Podocytes in glomeruli from humans with focal segmental glomerulosclerosis or diabetic nephropathy exhibited diminished H3K27me3 and heightened UTX content. Analogous to human disease, inhibition of Jmjd3 and UTX abated nephropathy progression in mice with established glomerular injury and reduced H3K27me3 levels. Together, these findings indicate that ostensibly stable chromatin modifications can be dynamically regulated in quiescent cells and that epigenetic reprogramming can improve outcomes in glomerular disease by repressing the reactivation of developmental pathways.
Assuntos
Nefropatias Diabéticas/metabolismo , Histonas/metabolismo , Podócitos/metabolismo , Animais , Nefropatias Diabéticas/patologia , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Feminino , Histona Desmetilases/metabolismo , Humanos , Proteína Jagged-1/metabolismo , Histona Desmetilases com o Domínio Jumonji/metabolismo , Masculino , Metilação , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Proteínas Nucleares/metabolismo , Podócitos/patologiaRESUMO
Incretin hormones exert pleiotropic metabolic actions beyond the pancreas. Although the heart expresses both incretin receptors, the cardiac biology of GIP receptor (GIPR) action remains incompletely understood. Here we show that GIPR agonism did not impair the response to cardiac ischemia. In contrast, genetic elimination of the Gipr reduced myocardial infarction (MI)-induced ventricular injury and enhanced survival associated with reduced hormone sensitive lipase (HSL) phosphorylation; it also increased myocardial triacylglycerol (TAG) stores. Conversely, direct GIPR agonism in the isolated heart reduced myocardial TAG stores and increased fatty acid oxidation. The cardioprotective phenotype in Gipr-/- mice was partially reversed by pharmacological activation or genetic overexpression of HSL. Selective Gipr inactivation in cardiomyocytes phenocopied Gipr-/- mice, resulting in improved survival and reduced adverse remodeling following experimental MI. Hence, the cardiomyocyte GIPR regulates fatty acid metabolism and the adaptive response to ischemic cardiac injury. These findings have translational relevance for developing GIPR-based therapeutics.
Assuntos
Infarto do Miocárdio/patologia , Receptores dos Hormônios Gastrointestinais/metabolismo , Adenilil Ciclases/metabolismo , Animais , Ativação Enzimática , Polipeptídeo Inibidor Gástrico/metabolismo , Células HEK293 , Insuficiência Cardíaca/patologia , Humanos , Sistema de Sinalização das MAP Quinases , Camundongos , Camundongos Endogâmicos C57BL , Infarto do Miocárdio/diagnóstico por imagem , Infarto do Miocárdio/fisiopatologia , Infarto do Miocárdio/prevenção & controle , Miocárdio/metabolismo , Miocárdio/patologia , Miócitos Cardíacos/metabolismo , Fosforilação , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores dos Hormônios Gastrointestinais/deficiência , Receptores dos Hormônios Gastrointestinais/genética , Transdução de Sinais , Esterol Esterase/metabolismo , Triglicerídeos/metabolismo , Remodelação VentricularRESUMO
OBJECTIVES: Glucagon-like peptide-1 (GLP-1) is secreted from enteroendocrine cells and exerts a broad number of metabolic actions through activation of a single GLP-1 receptor (GLP-1R). The cardiovascular actions of GLP-1 have garnered increasing attention as GLP-1R agonists are used to treat human subjects with diabetes and obesity that may be at increased risk for development of heart disease. Here we studied mechanisms linking GLP-1R activation to control of heart rate (HR) in mice. METHODS: The actions of GLP-1R agonists were examined on the control of HR in wild type mice (WT) and in mice with cardiomyocyte-selective disruption of the GLP-1R (Glp1rCM-/-). Complimentary studies examined the effects of GLP-1R agonists in mice co-administered propranolol or atropine. The direct effects of GLP-1R agonism on HR and ventricular developed pressure were examined in isolated perfused mouse hearts ex vivo, and atrial depolarization was quantified in mouse hearts following direct application of liraglutide to perfused atrial preparations ex vivo. RESULTS: Doses of liraglutide and lixisenatide that were equipotent for acute glucose control rapidly increased HR in WT and Glp1rCM-/- mice in vivo. The actions of liraglutide to increase HR were more sustained relative to lixisenatide, and diminished in Glp1rCM-/- mice. The acute chronotropic actions of GLP-1R agonists were attenuated by propranolol but not atropine. Neither native GLP-1 nor lixisenatide increased HR or developed pressure in perfused hearts ex vivo. Moreover, liraglutide had no direct effect on sinoatrial node firing rate in mouse atrial preparations ex vivo. Despite co-localization of HCN4 and GLP-1R in primate hearts, HCN4-directed Cre expression did not attenuate levels of Glp1r mRNA transcripts, but did reduce atrial Gcgr expression in the mouse heart. CONCLUSIONS: GLP-1R agonists increase HR through multiple mechanisms, including regulation of autonomic nervous system function, and activation of the atrial GLP-1R. Surprisingly, the isolated atrial GLP-1R does not transduce a direct chronotropic effect following exposure to GLP-1R agonists in the intact heart, or isolated atrium, ex vivo. Hence, cardiac GLP-1R circuits controlling HR require neural inputs and do not function in a heart-autonomous manner.
Assuntos
Receptor do Peptídeo Semelhante ao Glucagon 1/fisiologia , Frequência Cardíaca/fisiologia , Animais , Sistema Nervoso Autônomo/fisiologia , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Receptor do Peptídeo Semelhante ao Glucagon 1/agonistas , Receptor do Peptídeo Semelhante ao Glucagon 1/metabolismo , Frequência Cardíaca/efeitos dos fármacos , Liraglutida/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Peptídeos/farmacologiaRESUMO
The therapeutic targeting of prostanoid subtype receptors may slow the development of chronic kidney disease (CKD) through mechanisms that are distinct from those of upstream COX inhibition. Here, employing multiple experimental models of CKD, we studied the effects of inhibition of the EP4 receptor, one of four receptor subtypes for the prostanoid prostaglandin E2. In streptozotocin-diabetic endothelial nitric oxide synthase knockout mice, EP4 inhibition attenuated the development of albuminuria, whereas the COX inhibitor indomethacin did not. In Type 2 diabetic db/db mice, EP4 inhibition lowered albuminuria to a level comparable with that of the ACE inhibitor captopril. However, unlike captopril, EP4 inhibition had no effect on blood pressure or hyperfiltration although it did attenuate mesangial matrix accumulation. Indicating a glucose-independent mechanism of action, EP4 inhibition also attenuated proteinuria development and glomerular scarring in non-diabetic rats subjected to surgical renal mass ablation. Finally, in vitro, EP4 inhibition prevented transforming growth factor-ß1 induced dedifferentiation of glomerular podocytes. In rodent models of diabetic and non-diabetic CKD, EP4 inhibition attenuated renal injury through mechanisms that were distinct from either broadspectrum COX inhibition or "standard of care" renin angiotensin system blockade. EP4 inhibition may represent a viable repurposing opportunity for the treatment of CKD.
Assuntos
Nefropatias Diabéticas/tratamento farmacológico , Naftalenos/farmacologia , Fenilbutiratos/farmacologia , Receptores de Prostaglandina E Subtipo EP4/antagonistas & inibidores , Insuficiência Renal Crônica/tratamento farmacológico , Animais , Células Cultivadas , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Naftalenos/uso terapêutico , Óxido Nítrico Sintase Tipo III/genética , Óxido Nítrico Sintase Tipo III/metabolismo , Fenilbutiratos/uso terapêutico , Podócitos/efeitos dos fármacos , Podócitos/metabolismo , Ratos , Ratos Sprague-Dawley , Fator de Crescimento Transformador beta/metabolismoRESUMO
OBJECTIVE: Glucagon is a hormone with metabolic actions that maintains normoglycemia during the fasting state. Strategies enabling either inhibition or activation of glucagon receptor (Gcgr) signaling are being explored for the treatment of diabetes or obesity. However, the cardiovascular consequences of manipulating glucagon action are poorly understood. METHODS: We assessed infarct size and the following outcomes following left anterior descending (LAD) coronary artery ligation; cardiac gene and protein expression, acylcarnitine profiles, and cardiomyocyte survival in normoglycemic non-obese wildtype mice, and in newly generated mice with selective inactivation of the cardiomyocyte Gcgr. Complementary experiments analyzed Gcgr signaling and cell survival in cardiomyocyte cultures and cell lines, in the presence or absence of exogenous glucagon. RESULTS: Exogenous glucagon administration directly impaired recovery of ventricular pressure in ischemic mouse hearts ex vivo, and increased mortality from myocardial infarction after LAD coronary artery ligation in mice in a p38 MAPK-dependent manner. In contrast, cardiomyocyte-specific reduction of glucagon action in adult Gcgr (CM-/-) mice significantly improved survival, and reduced hypertrophy and infarct size following myocardial infarction. Metabolic profiling of hearts from Gcgr (CM-/-) mice revealed a marked reduction in long chain acylcarnitines in both aerobic and ischemic hearts, and following high fat feeding, consistent with an essential role for Gcgr signaling in the control of cardiac fatty acid utilization. CONCLUSIONS: Activation or reduction of cardiac Gcgr signaling in the ischemic heart produces substantial cardiac phenotypes, findings with implications for therapeutic strategies designed to augment or inhibit Gcgr signaling for the treatment of metabolic disorders.
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GLP-1R agonists improve outcomes in ischemic heart disease. Here we studied GLP-1R-dependent adaptive and cardioprotective responses to ventricular injury. Glp1r (-/-) hearts exhibited chamber-specific differences in gene expression, but normal mortality and left ventricular (LV) remodeling after myocardial infarction (MI) or experimental doxorubicin-induced cardiomyopathy. Selective disruption of the cardiomyocyte GLP-1R in Glp1r (CM-/-) mice produced no differences in survival or LV remodeling following LAD coronary artery occlusion. Unexpectedly, the GLP-1R agonist liraglutide still produced robust cardioprotection and increased survival in Glp1r (CM-/-) mice following LAD coronary artery occlusion. Although liraglutide increased heart rate (HR) in Glp1r (CM-/-) mice, basal HR was significantly lower in Glp1r (CM-/-) mice. Hence, endogenous cardiomyocyte GLP-1R activity is not required for adaptive responses to ischemic or cardiomyopathic injury, and is dispensable for GLP-1R agonist-induced cardioprotection or enhanced chronotropic activity. However the cardiomyocyte GLP-1R is essential for the control of HR in mice.
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PURPOSE: Heart failure with preserved ejection fraction (HFpEF) is a common comorbidity in people with chronic kidney disease (CKD) for which no evidence-based treatment currently exists. Recently, a group of anti-hyperglycemic agents used in the treatment of Type 2 diabetes, termed incretin-based therapies, have come under scrutiny for their putative glucose-independent effects on cardiac function. In the present study, the actions of the dipeptidyl peptidase-4 (DPP-4) inhibitor class of incretin-based therapy in preventing HFpEF induced by chronic renal impairment were investigated. METHODS: Sham-operated and subtotally-nephrectomized rats were randomized to receive the DPP-4 inhibitors, linagliptin or sitagliptin for seven weeks before assessment of cardiac and renal structure and function. RESULTS: Analysis of pressure-volume loops revealed that both linagliptin and sitagliptin prevented the development of cardiac diastolic dysfunction, with cardiac collagen I synthesis also being reduced by DPP-4 inhibition. These attenuating cardiac effects occurred without change in renal function or structure where, in the doses administered, neither linagliptin nor sitagliptin affected GFR decline, proteinuria, renal fibrosis or the increased urinary excretion of biomarkers of renal toxicity. CONCLUSION: The beneficial cardiac effects of DPP-4 inhibition, in the absence of a concurrent improvement in renal dysfunction, raise the possibility that these agents may confer cardiovascular advantages in the CKD population.
Assuntos
Inibidores da Dipeptidil Peptidase IV/uso terapêutico , Insuficiência Renal Crônica/tratamento farmacológico , Função Ventricular Esquerda/efeitos dos fármacos , Animais , Feminino , Masculino , Ratos Wistar , Insuficiência Renal Crônica/cirurgiaRESUMO
The progressive decline of renal function in chronic kidney disease (CKD) is characterized by both disruption of the microvascular architecture and the accumulation of fibrotic matrix. One angiogenic pathway recently identified as playing an essential role in renal vascular development is the stromal cell-derived factor-1α (SDF-1)/CXCR4 pathway. Because similar developmental processes may be recapitulated in the disease setting, we hypothesized that the SDF-1/CXCR4 system would regulate microvascular health in CKD. Expression of CXCR4 was observed to be increased in the kidneys of subtotally nephrectomized (SNx) rats and in biopsies from patients with secondary focal segmental glomerulosclerosis (FSGS), a rodent model and human correlate both characterized by aberration of the renal microvessels. A reno-protective role for local SDF-1/CXCR4 signaling was indicated by i) CXCR4-dependent glomerular eNOS activation following acute SDF-1 administration; and ii) acceleration of renal function decline, capillary loss and fibrosis in SNx rats treated with chronic CXCR4 blockade. In contrast to the upregulation of CXCR4, SDF-1 transcript levels were decreased in SNx rat kidneys as well as in renal fibroblasts exposed to the pro-fibrotic cytokine transforming growth factor ß (TGF-ß), the latter effect being attenuated by histone deacetylase inhibition. Increased renal SDF-1 expression was, however, observed following the treatment of SNx rats with the ACE inhibitor, perindopril. Collectively, these observations indicate that local SDF-1/CXCR4 signaling functions to preserve microvascular integrity and prevent renal fibrosis. Augmentation of this pathway, either purposefully or serendipitously with either novel or existing therapies, may attenuate renal decline in CKD.
Assuntos
Capilares/patologia , Quimiocina CXCL12/metabolismo , Rim/irrigação sanguínea , Receptores CXCR4/metabolismo , Insuficiência Renal Crônica/metabolismo , Insuficiência Renal Crônica/fisiopatologia , Transdução de Sinais , Adulto , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Inibidores da Enzima Conversora de Angiotensina/uso terapêutico , Animais , Benzilaminas , Biópsia , Capilares/efeitos dos fármacos , Capilares/metabolismo , Linhagem Celular , Quimiocina CXCL12/genética , Ciclamos , Fibrose , Glomerulosclerose Segmentar e Focal/tratamento farmacológico , Glomerulosclerose Segmentar e Focal/genética , Glomerulosclerose Segmentar e Focal/patologia , Compostos Heterocíclicos/farmacologia , Compostos Heterocíclicos/uso terapêutico , Humanos , Imuno-Histoquímica , Rim/enzimologia , Rim/patologia , Rim/cirurgia , Testes de Função Renal , Nefrectomia , Óxido Nítrico Sintase Tipo III/metabolismo , Perindopril/farmacologia , Perindopril/uso terapêutico , Fosforilação/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Endogâmicos F344 , Reação em Cadeia da Polimerase em Tempo Real , Receptores CXCR4/antagonistas & inibidores , Receptores CXCR4/genética , Insuficiência Renal Crônica/tratamento farmacológico , Insuficiência Renal Crônica/patologia , Serina/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
AIMS: The aim of this study was to investigate anti-apoptotic gene therapy using ultrasound-mediated plasmid delivery of survivin, an inhibitor of apoptosis protein, to prevent apoptosis and to attenuate left ventricular (LV) systolic dysfunction in a model of heart failure induced by doxorubicin. METHODS AND RESULTS: Effect of survivin transduction was investigated in vitro in rat cardiomyoblasts. After survivin transduction, survivin protein was detected in cell culture supernate confirming secretion of extracellular survivin. Under doxorubicin stimulation, survivin-transduced cells had significantly reduced apoptosis; however, incubation with survivin-conditioned media also showed reduced apoptosis that was absent with null-conditioned media. Doxorubicin-induced cardiomyopathy was established in Fischer rats. Subsets of animals underwent ultrasound-mediated survivin gene delivery or empty vector gene delivery at Week 3. Control rats received doxorubicin alone. Animals were studied using PCR, immunohistochemistry, echocardiography, and invasive haemodynamic studies out to Week 6. By Week 6, LV % fractional shortening by echocardiography and systolic function by pressure-volume loops were greater in survivin treated when compared with control- and empty-treated animals. There was reduced apoptosis by TUNEL and caspase activity in survivin-treated animals compared with control and empty treated at Week 4, with reduced interstitial fibrosis at Week 6. CONCLUSION: Survivin gene therapy can attenuate the progression of LV systolic dysfunction in doxorubicin cardiomyopathy. This effect can be attributed to decreased myocyte apoptosis and prevention of maladaptive LV remodelling, by both direct myocyte transfection and potentially by paracrine mechanisms.
Assuntos
Apoptose/efeitos dos fármacos , Doxorrubicina/toxicidade , Fibrose/metabolismo , Proteínas Inibidoras de Apoptose/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Repressoras/metabolismo , Disfunção Ventricular Esquerda/terapia , Animais , Apoptose/fisiologia , Cardiomiopatias/tratamento farmacológico , Cardiomiopatias/genética , Linhagem Celular , Terapia Genética/métodos , Proteínas Inibidoras de Apoptose/genética , Camundongos , Proteínas Associadas aos Microtúbulos/genética , Miocárdio/metabolismo , Ratos , Proteínas Repressoras/genética , Survivina , Disfunção Ventricular Esquerda/induzido quimicamente , Disfunção Ventricular Esquerda/genética , Disfunção Ventricular Esquerda/metabolismoRESUMO
Endothelial nitric oxide synthase (eNOS) deficiency may contribute to the pathogenesis of diabetic nephropathy in both experimental models and humans, but the underlying mechanism is not fully understood. Here, we studied two common sequelae of endothelial dysfunction in diabetes: glomerular capillary growth and effects on neighboring podocytes. Streptozotocin-induced diabetes increased glomerular capillary volume in both C57BL/6 and eNOS(-/-) mice. Inhibiting the vascular endothelial growth factor receptor attenuated albuminuria in diabetic C57BL/6 mice but not in diabetic eNOS(-/-) mice, even though it inhibited glomerular capillary enlargement in both. In eNOS(-/-) mice, an acute podocytopathy and heavy albuminuria occurred as early as 2 weeks after inducing diabetes, but treatment with either captopril or losartan prevented these effects. In vitro, serum derived from diabetic eNOS(-/-) mice augmented actin filament rearrangement in cultured podocytes. Furthermore, conditioned medium derived from eNOS(-/-) glomerular endothelial cells exposed to both high glucose and angiotensin II activated podocyte RhoA. Taken together, these results suggest that the combined effects of eNOS deficiency and hyperglycemia contribute to podocyte injury, highlighting the importance of communication between endothelial cells and podocytes in diabetes. Identifying mediators of this communication may lead to the future development of therapies targeting endothelial dysfunction in albuminuric individuals with diabetes.
Assuntos
Nefropatias Diabéticas/metabolismo , Nefropatias Diabéticas/patologia , Óxido Nítrico Sintase Tipo III/deficiência , Podócitos/metabolismo , Podócitos/patologia , Albuminúria/etiologia , Albuminúria/prevenção & controle , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Animais , Capilares/patologia , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patologia , Nefropatias Diabéticas/etiologia , Modelos Animais de Doenças , Glucose/metabolismo , Humanos , Glomérulos Renais/irrigação sanguínea , Glomérulos Renais/metabolismo , Glomérulos Renais/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Óxido Nítrico Sintase Tipo III/genética , Podócitos/efeitos dos fármacos , Sistema Renina-Angiotensina/efeitos dos fármacos , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Proteínas rho de Ligação ao GTP/metabolismo , Proteína rhoA de Ligação ao GTPRESUMO
UNLABELLED: BACKGROUND/AIMSBACKGROUND/AIMS: While experimental models that emulate diabetic nephropathy are valuable tools for elucidating pathogenetic mechanisms and developing novel therapies, existing models imperfectly recapitulate human disease. In diabetes, hyperglycemia and hemodynamic forces act in concert to induce renal injury. Accordingly, in the present study, we combined streptozotocin-induced diabetes with surgical ablation of 5/6 of the kidney mass with the aim of evaluating their additive effects on renal function and glomerular morphology. METHODS: Female F344 rats were randomized to undergo subtotal nephrectomy (SNx) either at baseline or following 4 weeks of diabetes. RESULTS: In comparison to sham rats, rats with diabetes or rats after SNx surgery, diabetic subtotally nephrectomized (DM-SNx) rats demonstrated an increase in systolic blood pressure, glomerular volume and mesangial matrix. Albuminuria was synergistically increased by hyperglycemia and renal mass ablation associated with decreased nephrin expression. In contrast, glomerular capillary rarefaction and glomerular filtration rate were similarly reduced in SNx and DM-SNx rats. CONCLUSION: The DM-SNx rat recapitulates some of the features of human disease, most notably augmented albuminuria. Since this model avoids the deletion or overexpression of gene(s) linked to the pathogenesis of nephropathy, the DM-SNx rat model represents a complementary tool for the trial of novel therapies.
RESUMO
Cell therapy has been extensively investigated in heart disease but less so in the kidney. We considered whether cell therapy also might be useful in diabetic kidney disease. Cognizant of the likely need for autologous cell therapy in humans, we sought to assess the efficacy of donor cells derived from both healthy and diabetic animals. Eight-week-old db/db mice were randomized to receive a single intravenous injection of PBS or 0.5 × 10(6) early-outgrowth cells (EOCs) from db/m or db/db mice. Effects were assessed 4 weeks after cell infusion. Untreated db/db mice developed mesangial matrix expansion and tubular epithelial cell apoptosis in association with increased reactive oxygen species (ROS) and overexpression of thioredoxin interacting protein (TxnIP). Without affecting blood glucose or blood pressure, EOCs not only attenuated mesangial and peritubular matrix expansion, as well as tubular apoptosis, but also diminished ROS and TxnIP overexpression in the kidney of db/db mice. EOCs derived from both diabetic db/db and nondiabetic db/m mice were equally effective in ameliorating kidney injury and oxidative stress. The similarly beneficial effects of cells from healthy and diabetic donors highlight the potential of autologous cell therapy in the related clinical setting.
Assuntos
Antioxidantes/uso terapêutico , Transplante de Medula Óssea , Diabetes Mellitus Tipo 2/terapia , Nefropatias Diabéticas/prevenção & controle , Rim/fisiopatologia , Albuminúria/prevenção & controle , Animais , Antioxidantes/farmacologia , Apoptose/efeitos dos fármacos , Células da Medula Óssea/fisiologia , Proteínas de Transporte/biossíntese , Células Cultivadas , Meios de Cultivo Condicionados/farmacologia , Diabetes Mellitus Tipo 2/patologia , Diabetes Mellitus Tipo 2/fisiopatologia , Nefropatias Diabéticas/patologia , Túbulos Renais Proximais/efeitos dos fármacos , Masculino , Camundongos , Espécies Reativas de Oxigênio/metabolismo , Rodaminas , Tiorredoxinas/biossíntese , Regulação para Cima/efeitos dos fármacosRESUMO
Rarefaction of the renal microvasculature correlates with declining kidney function. However, current technologies commonly used for its evaluation are limited by their reliance on endothelial cell antigen expression and assessment in two dimensions. We set out to establish a widely applicable and unbiased optical sectioning method to enable three dimensional imaging and reconstruction of the renal microvessels based on their luminal filling. The kidneys of subtotally nephrectomized (SNx) rats and their sham-operated counterparts were subjected to either routine two-dimensional immunohistochemistry or the novel technique of fluorescent microangiography (FMA). The latter was achieved by perfusion of the kidney with an agarose suspension of fluorescent polystyrene microspheres followed by optical sectioning of 200 µm thick cross-sections using a confocal microscope. The fluorescent microangiography method enabled the three-dimensional reconstruction of virtual microvascular casts and confirmed a reduction in both glomerular and peritubular capillary density in the kidneys of SNx rats, despite an overall increase in glomerular volume. FMA is an uncomplicated technique for evaluating the renal microvasculature that circumvents many of the limitations imposed by conventional analysis of two-dimensional tissue sections.