RESUMO
The Epstein-Barr virus (EBV) has a very high prevalence (>90% in adults), establishes a lifelong latency after primary infection, and exerts an oncogenic potential. This dsDNA virus encodes for various molecules, including microRNAs (miRs), which can be detected in the latent and lytic phases with different expression levels and affect, among others, immune evasion and malignant transformation. In this study, the different EBV miRs are quantified in EBV-positive lymphomas, and the impact on the host cell transcriptome of the most abundant EBV miRs will be analyzed using comparative RNA sequencing analyses. The EBV miRs ebv-miR-BART1, -BART4, -BART17, and -BHRF1-1 were most highly expressed, and their selective overexpression in EBV-negative human cells resulted in a large number of statistically significantly down- and up-regulated host cell genes. Functional analyses showed that these dysregulated target genes are involved in important cellular processes, including growth factor pathways such as WNT, EGF, FGF, and PDGF, as well as cellular processes such as apoptosis regulation and inflammation. Individual differences were observed between these four analyzed EBV miRs. In particular, ebv-miR-BHRF1-1 appears to be more important for malignant transformation and immune evasion than the other EBV miRs.
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Infecções por Vírus Epstein-Barr , Herpesvirus Humano 4 , MicroRNAs , Transcriptoma , Humanos , MicroRNAs/genética , Herpesvirus Humano 4/genética , Infecções por Vírus Epstein-Barr/virologia , Infecções por Vírus Epstein-Barr/genética , RNA Viral/genética , Regulação Viral da Expressão Gênica , Interações Hospedeiro-Patógeno/genética , Perfilação da Expressão GênicaRESUMO
Under healthy conditions, foot processes of neighbouring podocytes are interdigitating and connected by an electron-dense slit diaphragm. Besides slit diaphragm proteins, typical adherens junction proteins are also found to be expressed at this cell-cell junction. It is therefore considered as a highly specialized type of adherens junction. During podocyte injury, podocyte foot processes lose their characteristic 3D structure and the filtration slits typical meandering structure gets linearized. It is still under debate how this change of structure leads to the phenomenon of proteinuria. Using super-resolution 3D-structured illumination microscopy, we observed a spatially restricted up-regulation of the tight junction protein claudin-5 (CLDN5) in areas where podocyte processes of patients suffering from minimal change disease (MCD), focal and segmental glomerulosclerosis (FSGS) as well as in murine nephrotoxic serum (NTS) nephritis and uninephrectomy DOCA-salt hypertension models, were locally injured. CLDN5/nephrin ratios in human glomerulopathies and NTS-treated mice were significantly higher compared to controls. In patients, the CLDN5/nephrin ratio is significantly correlated with the filtration slit density as a foot process effacement marker, confirming a direct association of local CLDN5 up-regulation in injured foot processes. Moreover, CLDN5 up-regulation was observed in some areas of high filtration slit density, suggesting that CLND5 up-regulation preceded the changes of foot processes. Therefore, CLDN5 could serve as a biomarker predicting early foot process effacement.
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Claudina-5/metabolismo , Glomerulosclerose Segmentar e Focal/patologia , Nefropatias/patologia , Glomérulos Renais/metabolismo , Proteínas de Membrana/metabolismo , Podócitos/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Modelos Animais de Doenças , Feminino , Glomerulosclerose Segmentar e Focal/metabolismo , Humanos , Nefropatias/metabolismo , Glomérulos Renais/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Podócitos/metabolismoRESUMO
Cerebral blood supply is finely tuned by regulatory mechanisms depending on vessel caliber the disruption of which contributes to the development of diseases such as vascular dementia, Alzheimer's and Parkinson 's diseases. This study scopes whether cAMP-mimetic-ligands relax young and aged murine cerebral arteries, whether this relates to the activation of PKA or Epac signaling pathways and is changed with advanced age. The hormone Urocortin-1 relaxed submaximally contracted young and old basilar arteries with a similar pD2 and DMAX (~ -8.5 and ~ 90% in both groups). In permeabilized arteries, PKA activation by 6-Bnz-cAMP or Epac activation by 8-pCPT-2'- O-Me-cAMP also induced relaxation with pD2 of -6.3 vs. -5.8 in old for PKA-ligands, and -4.4 and -4.0 in old for Epac-ligands. Furthermore, aging significantly increased submaximal Ca2+-induced force. The effect of 8-pCPT-2'-O-Me-cAMP on intact arteries was attenuated by aging or nitric oxide synthase inhibition. No relaxing effect in both age-groups was observed after treatment with PKAactivator, Sp-6-Phe-cAMPS. In conclusion, our results suggest that in intact basilar arteries relaxation induced by cAMP-mimetics refers only to the activation of Epac and is impaired by smooth muscle and endothelial aging. The study presents an interesting option allowing therapeutic discrimination between both pathways, possibly for the exclusive activation of Epac in brain circulatory system.
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Envelhecimento , Artéria Basilar/fisiologia , AMP Cíclico/fisiologia , Endotélio/fisiologia , Fatores de Troca do Nucleotídeo Guanina/fisiologia , Vasodilatação , Animais , Permeabilidade da Membrana Celular , AMP Cíclico/análogos & derivados , Proteínas Quinases Dependentes de AMP Cíclico , Camundongos , Músculo Liso/fisiologiaAssuntos
Potenciais de Ação , Canal de Sódio Disparado por Voltagem NAV1.7 , Sistema Nervoso Simpático , Canal de Sódio Disparado por Voltagem NAV1.7/fisiologia , Canal de Sódio Disparado por Voltagem NAV1.7/genética , Canal de Sódio Disparado por Voltagem NAV1.7/metabolismo , Animais , Potenciais de Ação/fisiologia , Sistema Nervoso Simpático/fisiologia , HumanosRESUMO
Endothelin-1 (ET-1) stimulates contractions in isolated rat renal pelves. The signal transduction mechanisms that mediate ET-1-induced renal pelvic contractions and the role of ET-1 for the in vivo regulation of renal pelvic function are not well characterized. We tested if ET-1 stimulates contractions in murine and human renal pelves, if ET-1 activates the renal pelvic RhoA/ROCK pathway, and if low renal ET-1 formation or ET receptor blockade reduce renal pelvic contractile activity. ET-1 increased contraction frequency and force in murine renal pelves. The majority of human renal pelvic tissue samples showed tonic contractions in response to ET-1. Seven out of 20 human tissue samples showed phasic contractions. In four samples, they were elicited by ET-1 at 10-33 nmol/l. ET-1 increased renal pelvic RhoA-GTP content and myosin phosphatase target subunit 1 phosphorylation in isolated rat renal pelves. Renal pelvic contraction frequency (29 ± 2 vs. 29 ± 3 min(-1)) and renal pelvic pressure (7.1 ± 0.9 vs. 5.9 ± 1.7 mmHg) were similar in collecting duct-specific ET-1 knockout mice and in ET-1 floxed controls in vivo. ET-1 sensitivity of isolated renal pelves was similar in both groups. ET receptor blockade did not significantly affect pelvic contraction frequency and pressure in rats. We conclude that ET-1 stimulates phasic contractions in murine, rat, and, to a lesser extent, in human renal pelves. ET-1 activates the RhoA/ROCK pathway in the renal pelvic wall. Endogenous, kidney-derived ET-1 does not play a major role for the regulation of renal pelvic contractions in vivo.
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Endotelina-1/metabolismo , Pelve Renal/metabolismo , Idoso , Animais , Feminino , Humanos , Masculino , Camundongos , Camundongos Knockout , Contração Muscular/fisiologia , Músculo Liso/metabolismo , Ratos , Transdução de Sinais/fisiologia , Quinases Associadas a rho/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
Osteopontin (OPN) expression has been reported to be elevated in experimental models of renal injury such as arterial hypertension or diabetic nephropathy finally leading to focal segmental glomerulosclerosis (FSGS). FSGS is characterized by glomerular matrix deposition and loss or damage of podocytes that represent the main constituents of the glomerular filtration barrier. To evaluate the role of OPN in the kidney we investigated WT and OPN knockout mice (OPN-/-) without treatment, after uninephrectomy (UNX), as well as after UNX and desoxycorticosterone acetate (DOCA)-salt treatment with respect to urine parameters, glomerular morphology, and expression of podocyte markers. OPN-/- mice showed normal urine parameters while a thickening of the glomerular basement membrane was evident. Intriguingly, following UNX, OPN-/- mice exhibited prominent FSGS, proteinuria, and glomerular matrix deposition. Electron microscopy revealed bulgings of the glomerular basement membrane and occasionally an effacement of podocytes. After UNX and DOCA-salt treatment, severe glomerular lesions as well as proteinuria and albuminuria were seen in WT and OPN-/- mice. Moreover, we found a reduction of specific markers such as Wilm's tumor-1, podocin, and synaptopodin in both experimental groups indicating a loss of podocytes. Podocyte damage was accompanied by increased number of Ki-67-positive cells in the parietal epithelium of Bowman's capsule. We conclude that OPN plays a crucial role in adaptation of podocytes following renal ablation and is renoprotective when glomerular mechanical load is increased.
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Glomerulosclerose Segmentar e Focal/etiologia , Rim/fisiologia , Osteopontina/deficiência , Podócitos/fisiologia , Actinas/biossíntese , Animais , Autofagia , Desoxicorticosterona/farmacologia , Membrana Basal Glomerular/patologia , Glomerulosclerose Segmentar e Focal/patologia , Glomerulosclerose Segmentar e Focal/fisiopatologia , Rim/patologia , Glomérulos Renais/metabolismo , Glomérulos Renais/patologia , Ativação de Macrófagos , Masculino , Camundongos , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/biossíntese , Nefrectomia , Podócitos/patologiaRESUMO
The study was undertaken to compare antitumor efficacy of electrochemotherapy (ECT) with cold plasma therapy (CP) in a melanoma mouse model. After melanoma implantation into the flank of C57BL/6N mice, CP by two different plasma sources (APPJ and DBD) was applied directly to the tumor surface. ECT was performed with bleomycin intravenously at a field strength of 1000 V/cm without or combined with CP. Primary endpoints were tumor growth acceleration (TGA), daily volume progression (DVP) and survival after treatment. Both plasma sources as single treatment showed a significant TGA delay, which proved less effective than ECT. CP (APPJ) combined with ECT (ECJ) significantly improved per cent mouse survival, with significant superiority compared with ECT. Plasma therapy alone albeit less effective seems a potential alternative to ECT in patients with melanoma and can be applied manifold in a session without general anaesthesia. Accordingly, CP alone and combined with ECT may serve as new option in palliative skin melanoma therapy.
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Eletroquimioterapia , Melanoma Experimental/tratamento farmacológico , Melanoma Experimental/terapia , Gases em Plasma/uso terapêutico , Neoplasias Cutâneas/tratamento farmacológico , Neoplasias Cutâneas/terapia , Animais , Terapia Combinada , Feminino , Melanoma Experimental/patologia , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias Cutâneas/patologia , Resultado do TratamentoRESUMO
Arterial hypertension is a major health problem, accounting for 12 % of the global death rate. A large proportion of patients treated for high blood pressure do not reach target blood pressure values. The question arises if new antihypertensive drugs could improve present hypertension treatment. Rho-kinases (ROCKs) are ubiquitously expressed serine/threonine kinases and involved in a variety of cell functions. They contribute to the pathogenesis of human and experimental hypertension. Pharmacological ROCK inhibition has been shown to effectively lower blood pressure in patients and experimental animals. Progress has been made towards the understanding on how non-selective ROCK inhibitors lower arterial pressure and efforts are currently undertaken to develop ROCK inhibitors to improve their specificity and isoenzyme selectivity. If introduction of ROCK inhibitors for the treatment of high blood pressure can significantly advance currently available options of antihypertensive pharmacotherapy awaits further experimental and clinical research.
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Anti-Hipertensivos/uso terapêutico , Hipertensão/tratamento farmacológico , Inibidores de Proteínas Quinases/uso terapêutico , Quinases Associadas a rho/antagonistas & inibidores , Animais , Humanos , Transdução de Sinais/efeitos dos fármacosRESUMO
OBJECTIVE: Obesity is associated with hyperleptinemia but it is not clear whether leptin protects vascular function or promotes dysfunction. We therefore studied the consequences of hyperleptinemia in lean mice. METHODS AND RESULTS: Wild-type and endothelial NO synthase (eNOS)(-/-) mice were infused with leptin (0.4 mg/kg per day, 7 days), and endothelium-dependent relaxation was studied in aortic segments. Leptin had no effect on acetylcholine-induced endothelium-dependent relaxation in normal wild-type mice but restored endothelium-dependent relaxation in wild-type mice treated with angiotensin II (0.7 mg/kg per day, 7 days) to induce endothelial dysfunction. Leptin also sensitized aortae from eNOS(-/-) mice to acetylcholine, an effect blocked by neuronal NOS (nNOS) inhibition and not observed in eNOS-nNOS double(-/-) mice. Consistent with these findings, leptin induced nNOS expression in murine and human vessels and human endothelial but not smooth muscle cells. Aortic nNOS expression was also induced in mice by a high-fat diet. Mechanistically, leptin increased endothelial Janus kinase 2 and signal transducer and activator of transcription 3 phosphorylation, and inhibition of Janus kinase 2 prevented nNOS induction in cultured cells and leptin-induced relaxations in eNOS(-/-) mice. CONCLUSIONS: Leptin induces endothelial nNOS expression, which compensates, in part, for a lack of NO production by eNOS to maintain endothelium-dependent relaxation.
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Endotélio Vascular/fisiologia , Leptina/farmacologia , Óxido Nítrico Sintase Tipo III/fisiologia , Óxido Nítrico Sintase Tipo I/fisiologia , Vasodilatação/efeitos dos fármacos , Acetilcolina/farmacologia , Animais , Aorta/enzimologia , Humanos , Janus Quinase 2/fisiologia , Camundongos , Camundongos Knockout , Fator de Transcrição STAT3/fisiologia , Transdução de SinaisRESUMO
Vascular smooth muscle voltage-gated potassium (Kv) channels have been proposed to contribute to myogenic autoregulation. Surprisingly, in initial experiments, we observed that the Kv2 channel inhibitor stromatoxin induced vasomotion without affecting myogenic tone. Thus, we tested the hypothesis that Kv2 channels contribute to myogenic autoregulation by fine-tuning the myogenic response. Expression of Kv2 channel mRNA was determined using real-time PCR and 'multiplex' single-cell RT-PCR. Potassium currents were measured using the patch-clamp technique. Contractile responses of intact arteries were studied using isobaric myography. Expression of Kv2.1 but not Kv2.2 channels was detected in intact rat superior cerebellar arteries and in single smooth muscle cells. Stromatoxin, a high-affinity inhibitor of Kv2 channels, reduced smooth muscle Kv currents by 61% at saturating concentrations (EC50 36 nmol/L). Further, stromatoxin (10-100 nmol/L) induced pronounced vasomotion in 48% of the vessels studied. In vessels not exhibiting vasomotion, stromatoxin did not affect myogenic reactivity. Notably, in vessels exhibiting stromatoxin-induced vasomotion, pressure increases evoked two effects: First, they facilitated the occurrence of random vasodilations and/or vasoconstrictions, disturbing the myogenic response (24% of the vessels). Second, they modified the vasomotion by decreasing its amplitude and increasing its frequency, thereby destabilizing myogenic tone (76% of the vessels). Our study demonstrates that (i) Kv2.1 channels are the predominantly expressed Kv channels in smooth muscle cells of rat superior cerebellar arteries, and (ii) Kv2.1 channels provide a novel type of negative feedback mechanism in myogenic autoregulation by preventing vasomotion and thereby safeguarding the myogenic response.
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Artérias , Canais de Potássio Shab , Animais , Ratos , Artérias/metabolismo , Potássio/metabolismo , Ratos Sprague-Dawley , Canais de Potássio Shab/metabolismo , VasoconstriçãoRESUMO
OBJECTIVE: Small arteries from different organs vary with regard to the mechanisms that regulate vasoconstriction. This study investigated the impact of advanced age on the regulation of vasoconstriction in isolated human small arteries from kidney cortex and periintestinal mesenteric tissue. METHODS: Renal and mesenteric tissues were obtained from patients (mean age 71â±â9âyears) undergoing elective surgery. Furthermore, intrarenal and mesenteric arteries from young and aged mice were studied. Arteries were investigated by small vessel myography and western blot. RESULTS: Human intrarenal arteries (h-RA) showed higher stretch-induced tone and higher reactivity to α 1 adrenergic receptor stimulation than human mesenteric arteries (h-MA). Rho-kinase (ROK) inhibition resulted in a greater decrease in Ca 2+ and depolarization-induced tone in h-RA than in h-MA. Basal and α 1 adrenergic receptor stimulation-induced phosphorylation of the regulatory light chain of myosin (MLC 20 ) was higher in h-RA than in h-MA. This was associated with higher ROK-dependent phosphorylation of the regulatory subunit of myosin light-chain-phosphatase (MLCP), MYPT1-T853. In h-RA phosphorylation of ribosomal S6-kinase II (RSK2-S227) was significantly higher than in h-MA. Stretch-induced tone and RSK2 phosphorylation was also higher in interlobar arteries (m-IAs) from aged mice than in respective vessels from young mice and in murine mesenteric arteries (m-MA) from both age groups. CONCLUSION: Vasoconstriction in human intrarenal arteries shows a greater ROK-dependence than in mesenteric arteries. Activation of RSK2 may contribute to intrarenal artery tone dysregulation associated with aging. Compared with h-RA, h-MA undergo age-related remodeling leading to a reduction of the contractile response to α 1 adrenergic stimulation.
Assuntos
Receptores Adrenérgicos alfa 1 , Quinases Associadas a rho , Humanos , Camundongos , Animais , Pessoa de Meia-Idade , Idoso , Idoso de 80 Anos ou mais , Quinases Associadas a rho/metabolismo , Receptores Adrenérgicos alfa 1/metabolismo , Artérias Mesentéricas/metabolismo , Transdução de Sinais , Vasoconstrição , Miosinas/metabolismo , Fosforilação , Fosfatase de Miosina-de-Cadeia-Leve/metabolismoRESUMO
This review is focused on the mechanisms that regulate health, disease and aging redox status, the signal pathways that counteract oxidative and reductive stress, the role of food components and additives with antioxidant properties (curcumin, polyphenols, vitamins, carotenoids, flavonoids, etc.), and the role of the hormones irisin and melatonin in the redox homeostasis of animal and human cells. The correlations between the deviation from optimal redox conditions and inflammation, allergic, aging and autoimmune responses are discussed. Special attention is given to the vascular system, kidney, liver and brain oxidative stress processes. The role of hydrogen peroxide as an intracellular and paracrine signal molecule is also reviewed. The cyanotoxins ß-N-methylamino-l-alanine (BMAA), cylindrospermopsin, microcystins and nodularins are introduced as potentially dangerous food and environment pro-oxidants.
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Background: We investigated the association between leukocyte telomere length, mitochondrial DNA copy number, and endothelial function in patients with aging-related cardiovascular disease (CVD). Methods: In total 430 patients with CVD and healthy persons were enrolled in the current study. Peripheral blood was drawn by routine venipuncture procedure. Plasma and peripheral blood mononuclear cells (PBMCs) were collected. Cell-free genomic DNA (cfDNA) and leukocytic genomic DNA (leuDNA) were extracted from plasma and PBMCs, respectively. Relative telomere length (TL) and mitochondrial DNA copy number (mtDNA-CN) were analyzed using quantitative polymerase chain reaction. Endothelial function was evaluated by measuring flow-mediated dilation (FMD). The correlation between TL of cfDNA (cf-TL), mtDNA-CN of cfDNA (cf-mtDNA), TL of leuDNA (leu-TL), mtDNA-CN of leuDNA (leu-mtDNA), age, and FMD were analyzed based on Spearman's rank correlation. The association between cf-TL, cf-mtDNA, leu-TL, leu-mtDNA, age, gender, and FMD were explored using multiple linear regression analysis. Results: cf-TL positively correlated with cf-mtDNA (r = 0.1834, P = 0.0273), and leu-TL positively correlated with leu-mtDNA (r = 0.1244, P = 0.0109). In addition, both leu-TL (r = 0.1489, P = 0.0022) and leu-mtDNA (r = 0.1929, P < 0.0001) positively correlated with FMD. In a multiple linear regression analysis model, both leu-TL (ß = 0.229, P = 0.002) and leu-mtDNA (ß = 0.198, P = 0.008) were positively associated with FMD. In contrast, age was inversely associated with FMD (ß = -0.426, P < 0.0001). Conclusion: TL positively correlates mtDNA-CN in both cfDNA and leuDNA. leu-TL and leu-mtDNA can be regarded as novel biomarkers of endothelial dysfunction.
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Stretch-induced vascular tone is an important element of autoregulatory adaptation of cerebral vasculature to maintain cerebral flow constant despite changes in perfusion pressure. Little is known as to the regulation of tone in senescent basilar arteries. We tested the hypothesis, that thin filament mechanisms in addition to smooth muscle myosin-II regulatory-light-chain-(MLC20)-phosphorylation and non-muscle-myosin-II, contribute to regulation of stretch-induced tone. In young BAs (y-BAs) mechanical stretch does not lead to spontaneous tone generation. Stretch-induced tone in y-BAs appeared only after inhibition of NO-release by L-NAME and was fully prevented by treatment with 3 µmol/L RhoA-kinase (ROK) inhibitor Y27632. L-NAME-induced tone was reduced in y-BAs from heterozygous mice carrying a point mutation of the targeting-subunit of the myosin phosphatase, MYPT1 at threonine696 (MYPT1-T696A/+). In y-BAs, MYPT1-T696A-mutation also blunted the ability of L-NAME to increase MLC20-phosphorylation. In contrast, senescent BAs (s-BAs; >24 months) developed stable spontaneous stretch-induced tone and pharmacological inhibition of NO-release by L-NAME led to an additive effect. In s-BAs the MYPT1-T696A mutation also blunted MLC20-phosphorylation, but did not prevent development of stretch-induced tone. In s-BAs from both lines, Y27632 completely abolished stretch- and L-NAME-induced tone. In s-BAs phosphorylation of non-muscle-myosin-S1943 and PAK1-T423, shown to be down-stream effectors of ROK was also reduced by Y27632 treatment. Stretch- and L-NAME tone were inhibited by inhibition of non-muscle myosin (NM-myosin) by blebbistatin. We also tested whether the substrate of PAK1 the thin-filament associated protein, caldesmon is involved in the regulation of stretch-induced tone in advanced age. BAs obtained from heterozygotes Cald1+/- mice generated stretch-induced tone already at an age of 20-21 months old BAs (o-BA). The magnitude of stretch-induced tone in Cald1+/- o-BAs was similar to that in s-BA. In addition, truncation of caldesmon myosin binding Exon2 (CaD-âµEx2-/-) did not accelerate stretch-induced tone. Our study indicates that in senescent cerebral vessels, mechanisms distinct from MLC20 phosphorylation contribute to regulation of tone in the absence of a contractile agonist. While in y-and o-BA the canonical pathways, i.e., inhibition of MLCP by ROK and increase in pMLC20, predominate, tone regulation in senescence involves ROK regulated mechanisms, involving non-muscle-myosin and thin filament linked mechanisms involving caldesmon.
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OBJECTIVE: Multidrug resistance-related protein 2 (Mrp2) is expressed in apical membranes of renal proximal tubular cells and contributes to the renal secretion of cyclosporine A (CsA). Mrp2â»/â» deficiency may lead to local renal CsA accumulation. We investigated whether kidney-specific Mrp2 deficiency enhances acute CsA nephrotoxicity in rats. METHODS: Kidney-specific Mrp2 deletion was achieved by bilateral nephrectomy and transplantation of a congenic Mrp2-deficient kidney into wild-type recipients. Controls received a wild-type kidney. Animals were treated with CsA (10 or 30 mg/kg/day) for 7 days. Renal hemodynamics and renal cortical mRNA expression profile, oxidative stress, and the abundance of multidrug resistance protein 1 (Mdr1) and Mrp2 were assessed. RESULTS: CsA accumulation and CsA-induced reduction in glomerular filtration rate were similar in wild-type and Mrp2â»/â» kidneys. Renal vascular resistance and agonist-induced renal vascular responses were similar in both groups. A PCR array on 84 genes involved in the biotransformation and antioxidant defense revealed increased CsA-induced mRNA expression of genes involved in oxidative and metabolic stress, inflammation, and apoptosis. This gene expression pattern was similar in wild-type and Mrp2â»/â» kidneys. CsA increased the renal cortical oxidized glutathione, did not affect xanthine oxidase-dependent superoxide formation, and decreased renal cortical NADPH oxidase-dependent superoxide formation. Furthermore, CsA increased Mdr1 protein abundance to a greater extent in Mrp2â»/â» than in wild-type kidneys. CONCLUSION: Mrp2 is not critical for renal CsA disposition and its deficiency does not enhance acute CsA nephrotoxicity. The high Mdr1 abundance may at least in part prevent exaggerated CsA accumulation in Mrp2â»/â» kidneys.
Assuntos
Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Ciclosporina/efeitos adversos , Deleção de Genes , Nefropatias/induzido quimicamente , Nefropatias/genética , Rim/metabolismo , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Animais , Pressão Sanguínea/efeitos dos fármacos , Ciclosporina/sangue , Regulação da Expressão Gênica/efeitos dos fármacos , Taxa de Filtração Glomerular/efeitos dos fármacos , Dissulfeto de Glutationa/metabolismo , Hemodinâmica/efeitos dos fármacos , Rim/efeitos dos fármacos , Rim/patologia , Rim/fisiopatologia , Córtex Renal/metabolismo , Córtex Renal/patologia , Córtex Renal/fisiopatologia , Nefropatias/patologia , Nefropatias/fisiopatologia , Masculino , Especificidade de Órgãos/efeitos dos fármacos , Especificidade de Órgãos/genética , Reação em Cadeia da Polimerase , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Superóxidos/metabolismoRESUMO
This work explored the mechanism of augmented stress-induced vascular reactivity of senescent murine femoral arteries (FAs). Mechanical and pharmacological reactivity of young (12-25 weeks, y-FA) and senescent (>104 weeks, s-FAs) femoral arteries was measured by wire myography. Expression and protein phosphorylation of selected regulatory proteins were studied by western blotting. Expression ratio of the Exon24 in/out splice isoforms of the regulatory subunit of myosin phosphatase, MYPT1 (MYPT1-Exon24 in/out), was determined by polymerase chain reaction (PCR). While the resting length-tension relationship showed no alteration, the stretch-induced-tone increased to 8.3 ± 0.9 mN in s-FA versus only 4.6 ± 0.3 mN in y-FAs. Under basal conditions, phosphorylation of the regulatory light chain of myosin at S19 was 19.2 ± 5.8% in y-FA versus 49.2 ± 12.6% in s-FA. Inhibition of endogenous NO release raised tone additionally to 10.4 ± 1.2 mN in s-FA, whereas this treatment had a negligible effect in y-FAs (4.8 ± 0.3 mN). In s-FAs, reactivity to NO donor was augmented (pD2 = -4.5 ± 0.3 in y-FA vs. -5.2 ± 0.1 in senescent). Accordingly, in s-FAs, MYPT1-Exon24-out-mRNA, which is responsible for expression of the more sensitive to protein-kinase G, leucine-zipper-positive MYPT1 isoform, was increased. The present work provides evidence that senescent murine s-FA undergoes vascular remodelling associated with increases in stretch-activated contractility and sensitivity to NO/cGMP/PKG system.
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Artéria Femoral/metabolismo , Óxido Nítrico/metabolismo , Estresse Fisiológico/fisiologia , Remodelação Vascular/fisiologia , Fatores Etários , Animais , GMP Cíclico/metabolismo , Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Fosfatase de Miosina-de-Cadeia-Leve/metabolismo , Doadores de Óxido Nítrico/farmacologia , Fosforilação , Reação em Cadeia da Polimerase , RNA Mensageiro/metabolismo , Rigidez Vascular/fisiologiaRESUMO
Background: We investigated the pleiotropic effects of an angiotensin receptor-neprilysin inhibitor (ARNi) on collateral-dependent myocardial perfusion in a rat model of coronary arteriogenesis, and performed comprehensive analyses to uncover the underlying molecular mechanisms. Methods: A rat model of coronary arteriogenesis was established by implanting an inflatable occluder on the left anterior descending coronary artery followed by a 7-day repetitive occlusion procedure (ROP). Coronary collateral perfusion was measured by using a myocardial particle infusion technique. The putative ARNi-induced pro-arteriogenic effects were further investigated and compared with an angiotensin-converting enzyme inhibitor (ACEi). Expression of the membrane receptors and key enzymes in the natriuretic peptide system (NPS), renin-angiotensin-aldosterone system (RAAS) and kallikrein-kinin system (KKS) were analyzed by quantitative polymerase chain reaction (qPCR) and immunoblot assay, respectively. Protein levels of pro-arteriogenic cytokines were measured by enzyme-linked immunosorbent assay, and mitochondrial DNA copy number was assessed by qPCR due to their roles in arteriogenesis. Furthermore, murine heart endothelial cells (MHEC5-T) were treated with a neprilysin inhibitor (NEPi) alone, or in combination with bradykinin receptor antagonists. MHEC5-T proliferation was analyzed by colorimetric assay. Results: The in vivo study showed that ARNis markedly improved coronary collateral perfusion, regulated the gene expression of KKS, and increased the concentrations of relevant pro-arteriogenic cytokines. The in vitro study demonstrated that NEPis significantly promoted MHEC5-T proliferation, which was diminished by bradykinin receptor antagonists. Conclusion: ARNis improve coronary collateral perfusion and exert pro-arteriogenic effects via the bradykinin receptor signaling pathway.
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Upper urinary tract peristalsis is generated in the proximal renal pelvis that connects to the renal parenchyma at the pelvis-kidney junction. It may be exposed to the high renal endothelin-1 (ET-1) concentrations. Dietary NaCl restriction increases renal pelvic ET(A) receptor expression. We investigated the contribution of ET(A) and ET(B) receptors to ET-1-stimulated rat renal pelvic contractions and whether the sensitivity of renal pelvic contractile activity to ET-1 stimulation increases with dietary NaCl restriction. We tested whether ET-1-induced contractile activity depends on cyclooxygenase (COX)-1 or -2 and to what extent spontaneous as well as agonist-induced peristalsis depends on Rho kinases (ROCK). Contractions of isolated renal pelvises were investigated by myography. ET-1 concentration-dependently increased pelvic contractile activity up to 400% of basal activity. ET(A) but not ET(B) receptor blockade inhibited ET-1-induced pelvic contractions. Basal and ET-1-stimulated contractions were similar in renal pelvises from rats on a high-NaCl diet or on a NaCl-deficient diet. COX-1 inhibition reduced spontaneous and almost completely blocked the ET-1-induced pelvic contractions. ROCK inhibition reduced spontaneous and ET-1 stimulated pelvic contractile activity by 90%. RT-PCR revealed that both ROCK isoenzymes are present in the renal pelvic wall. Western blot analyses did not show increased phosphorylation of ROCK substrates myosin phosphatase target subunit 1, ezrin, radixin, and moesin in ET-1-treated isolated renal pelvises. ET-1 is a powerful ET(A) receptor-dependent activator of renal pelvic contractions. COX-1 and ROCK activity are required for the ET-1 effects on pelvic contractions, which are not significantly affected by dietary NaCl intake.
Assuntos
Ciclo-Oxigenase 1/metabolismo , Endotelina-1/farmacologia , Pelve Renal/efeitos dos fármacos , Peristaltismo/efeitos dos fármacos , Quinases Associadas a rho/metabolismo , Análise de Variância , Animais , Western Blotting , Relação Dose-Resposta a Droga , Pelve Renal/metabolismo , Masculino , Miografia , Peristaltismo/fisiologia , Fosforilação/efeitos dos fármacos , Ratos , Ratos Endogâmicos F344 , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Cloreto de Sódio na DietaRESUMO
Acute kidney injury (AKI) is frequently accompanied by activation of the sympathetic nervous system (SNS). This may result from pre-exisiting chronic diseases associated with sympathetic activation prior to AKI or it may be induced by stressors that ultimately lead to AKI such as endotoxins and arterial hypotension in circulatory shock. Conversely, sympathetic activation may also result from acute renal injury. Focusing on studies in experimental renal ischaemia and reperfusion (IR), this review summarizes the current knowledge on how the SNS is activated in IR-induced AKI and on the consequences of sympathetic activation for the development of acute renal damage. Experimental studies show beneficial effects of sympathoinhibitory interventions on renal structure and function in response to IR. However, few clinical trials obtained in scenarios that correspond to experimental IR, namely major elective surgery, showed that peri-operative treatment with centrally acting sympatholytics reduced the incidence of AKI. Apparently, discrepant findings on how sympathetic activation influences renal responses to acute IR-induced injury are discussed and future areas of research in this field are identified.
Assuntos
Injúria Renal Aguda/fisiopatologia , Traumatismo por Reperfusão/fisiopatologia , Sistema Nervoso Simpático/fisiopatologia , Simpatolíticos/farmacologia , Injúria Renal Aguda/tratamento farmacológico , Animais , HumanosRESUMO
Increasing efferent renal sympathetic nerve activity (ERSNA) increases afferent renal nerve activity (ARNA), which in turn decreases ERSNA via activation of the renorenal reflexes in the overall goal of maintaining low ERSNA. We now examined whether the ERSNA-induced increases in ARNA are modulated by dietary sodium and the role of endothelin (ET). The ARNA response to reflex increases in ERSNA was enhanced in high (HNa)- vs. low-sodium (LNa) diet rats, 7,560 +/- 1,470 vs. 900 +/- 390%.s. The norepinephrine (NE) concentration required to increase PGE(2) and substance P release from isolated renal pelvises was 10 pM in HNa and 6,250 pM in LNa diet rats. In HNa diet pelvises 10 pM NE increased PGE(2) release from 67 +/- 6 to 150 +/- 13 pg/min and substance P release from 6.7 +/- 0.8 to 12.3 +/- 1.8 pg/min. In LNa diet pelvises 6,250 pM NE increased PGE(2) release from 64 +/- 5 to 129 +/- 22 pg/min and substance P release from 4.5 +/- 0.4 to 6.6 +/- 0.7 pg/min. In the renal pelvic wall, ETB-R are present on unmyelinated Schwann cells close to the afferent nerves and ETA-R on smooth muscle cells. ETA-receptor (R) protein expression in the renal pelvic wall is increased in LNa diet. In HNa diet, renal pelvic administration of the ETB-R antagonist BQ788 reduced ERSNA-induced increases in ARNA and NE-induced release of PGE(2) and substance P. In LNa diet, the ETA-R antagonist BQ123 enhanced ERSNA-induced increases in ARNA and NE-induced release of substance P without altering PGE(2) release. In conclusion, activation of ETB-R and ETA-R contributes to the enhanced and suppressed interaction between ERSNA and ARNA in conditions of HNa and LNa diet, respectively, suggesting a role for ET in the renal control of ERSNA that is dependent on dietary sodium.