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Advanced glycation endproducts (AGEs) contribute to cellular damage of various pathologies, including kidney diseases. Acute kidney injury (AKI) represents a syndrome seldom characterized by a single, distinct pathophysiological cause. Rhabdomyolysis-induced acute kidney injury (RIAKI) constitutes roughly 15% of AKI cases, yet its underlying pathophysiology remains poorly understood. Using a murine model of RIAKI induced by muscular glycerol injection, we observed elevated levels of AGEs and the AGE receptor galectin-3 (LGALS3) in the kidney. Immunofluorescence localized LGALS3 to distal nephron segments. According to transcriptomic profiling via next-generation sequencing, RIAKI led to profound changes in kidney metabolism, oxidative stress, and inflammation. Cellular stress was evident in both proximal and distal tubules, as shown by kidney injury markers KIM-1 and NGAL. However, only proximal tubules exhibited overt damage and apoptosis, as detected by routine morphology, active Caspase-3, and TUNEL assay, respectively. In vitro, distal convoluted tubule (DCT) cells challenged with AGEs underwent apoptosis, which was markedly enhanced by Lgals3 siRNA treatment. Thus, in RIAKI, the upregulation of LGALS3 may protect the distal nephron from AGE-mediated damage, while proximal tubules lacking LGALS3 stay at risk. Thus, stimulating LGALS3 in the proximal nephron, if achievable, may attenuate RIAKI.
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Introduction: Mechanic power output (MPO) and oxygen consumption (VO2) reflect endurance capacity and are often stated relative to body mass (BM) but less often per skeletal muscle mass (SMM). Rating of perceived exertion (RPE) has previously shown conflicting results between sexes at submaximal intensities. Individual body composition, however, largely differs due to sex and training status. It was the aim of this study to evaluate RPE of untrained and trained individuals of both sexes considering body composition and to estimate whether RPE could be improved as a tool to determine endurance capacity. Methods: The study included 34 untrained adults (age 26.18 ± 6.34 years, 18 women) and 29 endurance trained (age 27.86 ± 5.19, 14 women) who were measured for body composition (InBody 770, InBody Europe B.V., Germany) and tested on a treadmill (Pulsar, H/P/Cosmos, Germany) for aerobic capacity (Metalyzer 3B, Cortex Biophysik GmbH, Germany) in an all-out exercise test applying the Bruce-protocol. VO2, MPO, heart rate (HR), and RPE were obtained at each exercise stage. VO2 and MPO were calculated per BM and SMM. RPE values were correlated with absolute VO2 and MPO, as well as relative to BM, and SMM. HR values and the parameters' standardized values served for comparison to standard procedures. Results: VO2 and MPO were higher in men compared to women and in trained compared to untrained participants. No differences between groups and sexes exist when VO2 and MPO were calculated per BM. When calculated per SMM, VO2 and MPO indicate opposite results already at low intensity stages of exercise test. RPE values had highest correlation with MPO per SMM (R2 = 0.8345) compared to absolute MPO (R2 = 0.7609), or MPO per BM (R2 = 0.8176). Agreement between RPE and MPO per SMM was greater than between RPE and HR (p = 0.008). Conclusion: Although RPE represents a subjective value at first glance, it was shown that RPE constitutes a valuable tool to estimate endurance capacity, which can be further enhanced if individual body composition is considered. Furthermore, MPO and VO2 should be considered relative to SMM. These findings might help to avoid over-exertion, especially among untrained people, by adjusting the training intensity for each subject according to the individual strain evaluated in an exercise test based on individual body composition.
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The polyamines spermidine and spermine and their common precursor molecule putrescine are involved in tissue injury and repair. Here, we test the hypothesis that impaired polyamine homeostasis contributes to various kidney pathologies in mice during experimental models of ischemia-reperfusion, transplantation, rhabdomyolysis, cyclosporine treatment, arterial hypertension, diabetes, unilateral ureteral obstruction, high oxalate feeding, and adenine-induced injuries. We found a remarkably similar pattern in most kidney pathologies with reduced expression of enzymes involved in polyamine synthesis together with increased expression of polyamine degrading enzymes. Transcript levels of amine oxidase copper-containing 1 (Aoc1), an enzyme which catalyzes the breakdown of putrescine, were barely detectable by in situ mRNA hybridization in healthy kidneys. Aoc1 was highly expressed upon various experimental kidney injuries resulting in a significant reduction of kidney putrescine content. Kidney levels of spermine were also significantly reduced, whereas spermidine was increased in response to ischemia-reperfusion injury. Increased Aoc1 expression in injured kidneys was mainly accounted for by an Aoc1 isoform that harbors 22 additional amino acids at its N-terminus and shows increased secretion. Mice with germline deletion of Aoc1 and injured kidneys showed no decrease of kidney putrescine content; although they displayed no overt phenotype, they had fewer tubular casts upon ischemia-reperfusion injury. Hyperosmotic stress stimulated AOC1 expression at the transcriptional and post-transcription levels in metanephric explants and kidney cell lines. AOC1 expression was also significantly enhanced after kidney transplantation in humans. These data demonstrate that the kidneys respond to various forms of injury with down-regulation of polyamine synthesis and activation of the polyamine breakdown pathway. Thus, an imbalance in kidney polyamines may contribute to various etiologies of kidney injury.
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Amina Oxidase (contendo Cobre) , Traumatismo por Reperfusão , Humanos , Camundongos , Animais , Poliaminas/metabolismo , Espermidina/metabolismo , Putrescina/metabolismo , Espermina/metabolismo , Espermina/farmacologia , Acetiltransferases/genética , Acetiltransferases/metabolismo , Rim/patologia , Amina Oxidase (contendo Cobre)/metabolismo , Traumatismo por Reperfusão/patologia , Expressão GênicaRESUMO
Chronic Cyclosporine-A treatment is associated with serious side effects, including kidney toxicity and anemia. Although pathophysiology of Cyclosporine-A-induced kidney injury remains incompletely understood, hypoxia is likely involved. Here, we investigated the effect of the hypoxia inducible factor activator daprodustat on Cyclosporine-A -induced kidney toxicity. As Cyclosporine-A profoundly alters protein phosphorylation by inhibiting the phosphatase calcineurin, special attention was directed towards the kidney phospho-proteome. Mice received Cyclosporine-A with or without daprodustat for up to eight weeks. In kidney homogenates, 1360 selected proteins were analyzed at expression and phosphorylation levels. Of these, Cyclosporine-A changed the expression of 79 and the phosphorylation of 86 proteins. However, when Cyclosporine-A treatment was combined with daprodustat, the expression of 95 proteins and phosphorylation of only six proteins was altered suggesting that daprodustat prevented most protein phosphorylation brought about by Cyclosporine-A. Although daprodustat showed only marginal effect on its own, angiogenesis-related pathways were among the most profoundly impacted by daprodustat when given on top of Cyclosporine-A. Additionally, Cyclosporine-A lowered the blood hemoglobin concentration and caused kidney capillary rarefaction, which daprodustat prevented. Thus, combined daprodustat/Cyclosporine-A treatment prevented deleterious Cyclosporine-A effects on microcirculation and hemoglobin, and the protective action of daprodustat involves suppression of broad protein phosphorylation changes caused by Cyclosporine-A.
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Anemia , Ciclosporina , Anemia/induzido quimicamente , Anemia/prevenção & controle , Animais , Barbitúricos , Calcineurina , Ciclosporina/toxicidade , Glicina/análogos & derivados , Hemoglobinas/metabolismo , Hipóxia/complicações , Camundongos , ProteomaRESUMO
Broxbam, also known as N-hydroxy-4-{1-methoxy-4-[4'-(3'-bromo-4',5'-dimethoxyphenyl)-oxazol-5'-yl]-2-phenoxy} butanamide, is a novel chimeric inhibitor that contains two distinct pharmacophores in its molecular structure. It has been previously demonstrated to inhibit the activity of histone deacetylases (HDAC) and tubulin polymerisation, two critical components required for cancer growth and survival. In the present study, the potential suitability of broxbam for the treatment of liver cancer was investigated. The effects of broxbam on cell proliferation and apoptosis, in addition to the underlying molecular mechanism of action, were first investigated in primary liver cancer cell lines Huh7, HepG2, TFK1 and EGI1. Real-time proliferation measurements made using the iCELLigence system and viable cell number counting following crystal violet staining) revealed that broxbam time- and dose-dependently reduced the proliferation of liver cancer cell lines with IC50 values <1 µM. In addition, a significant inhibition of the growth of hepatoblastoma microtumours on the chorioallantoic membranes (CAM) of fertilised chicken eggs by broxbam was observed according to results from the CAM assay, suggesting antineoplastic potency in vivo. Broxbam also exerted apoptotic effects through p53- and mitochondria-driven caspase-3 activation in Huh7 and HepG2 cells according to data from western blotting (p53 and phosphorylated p53), mitochondrial membrane potential measurements (JC-1 assay) and fluorometric capsase-3 measurements. Notably, no contribution of unspecific cytotoxic effects mediated by broxbam were observed from LDH-release measurements. HDAC1, -2, -4 and -6 expression was measured by western blotting and the HDAC inhibitory potency of broxbam was next evaluated using subtype-specific HDAC enzymatic assays, which revealed a largely pan-HDAC inhibitory activity with the most potent inhibition observed on HDAC6. Silencing HDAC6 expression in Huh7 cells led to a drop in the expression of the proliferation markers Ki-67 and E2F3, suggesting that HDAC6 inhibition by broxbam may serve a predominant role in their antiproliferative effects on liver cancer cells. Immunofluorescence staining of cytoskeletal proteins (α-tubulin & actin) of broxbam-treated HepG2 cells revealed a pronounced inhibition of tubulin polymerisation, which was accompanied by reduced cell migration as determined by wound healing scratch assays. Finally, data from zebrafish angiogenesis assays revealed marked antiangiogenic effects of broxbam in vivo, as shown by the suppression of subintestinal vein growth in zebrafish embryos. To conclude, the pleiotropic anticancer activities of this novel chimeric HDAC- and tubulin inhibitor broxbam suggest that this compound is a promising candidate for liver cancer treatment, which warrants further pre-clinical and clinical evaluation.
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Antineoplásicos , Neoplasias Hepáticas , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Citoesqueleto/metabolismo , Inibidores de Histona Desacetilases/farmacologia , Inibidores de Histona Desacetilases/uso terapêutico , Histona Desacetilases/metabolismo , Humanos , Neoplasias Hepáticas/tratamento farmacológico , Tubulina (Proteína)/metabolismo , Proteína Supressora de Tumor p53 , Peixe-Zebra/metabolismoRESUMO
Fatigue is a key factor that affects human motion and modulates physiology, biochemistry, and performance. Prolonged cyclic human movements (locomotion primarily) are characterized by a regular pattern, and this extended activity can induce fatigue. However, the relationship between fatigue and regularity has not yet been extensively studied. Wearable sensor methodologies can be used to monitor regularity during standardized treadmill tests (e.g., the widely used Bruce test) and to verify the effects of fatigue on locomotion regularity. Our study on 50 healthy adults [27 males and 23 females; <40 years; five dropouts; and 22 trained (T) and 23 untrained (U) subjects] showed how locomotion regularity follows a parabolic profile during the incremental test, without exception. At the beginning of the trial, increased walking speed in the absence of fatigue is associated with increased regularity (regularity index, RI, a. u., null/unity value for aperiodic/periodic patterns) up until a peak value (RI = 0.909 after 13.8 min for T and RI = 0.915 after 13.4 min for U subjects; median values, n. s.) and which is then generally followed (after 2.8 and 2.5 min, respectively, for T/U, n. s.) by the walk-to-run transition (at 12.1 min for both T and U, n. s.). Regularity then decreases with increased speed/slope/fatigue. The effect of being trained was associated with significantly higher initial regularity [0.845 (T) vs 0.810 (U), p < 0.05 corrected], longer test endurance [23.0 min (T) vs 18.6 min (U)], and prolonged decay of locomotor regularity [8.6 min (T) vs 6.5 min (U)]. In conclusion, the monitoring of locomotion regularity can be applied to the Bruce test, resulting in a consistent time profile. There is evidence of a progressive decrease in regularity following the walk-to-run transition, and these features unveil significant differences among healthy trained and untrained adult subjects.
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Eritropoetina , Oxigênio , Humanos , Neovascularização Patológica , Pericitos , Prolil HidroxilasesRESUMO
Oxygen affinity to haemoglobin is indicated by the p50 value (pO2 at 50% O2Hb) and critically determines cellular oxygen availability. Although high Hb-O2 affinity can cause tissue hypoxia under conditions of well O2 saturated blood, individual differences in p50 are commonly not considered in clinical routine. Here, we investigated the diversity in Hb-O2 affinity in the context of physiological relevance. Oxyhaemoglobin dissociation curves (ODCs) of 60 volunteers (18-40 years, both sexes, either endurance trained or untrained) were measured at rest and after maximum exercise (VO2max) test. At rest, p50 values of all participants ranged over 7 mmHg. For comparison, right shift of ODC after VO2max test, representing the maximal physiological range to release oxygen to the tissue, indicated a p50 difference of up to 10 mmHg. P50 at rest differs significantly between women and men, with women showing lower Hb-O2 affinity that is determined by higher 2,3-BPG and BPGM levels. Regular endurance exercise did not alter baseline Hb-O2 affinity. Thus, p50 diversity is already high at baseline level and needs to be considered under conditions of impaired tissue oxygenation. For fast prediction of Hb-O2 affinity by blood gas analysis, only venous but not capillary blood samples can be recommended.
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Hemoglobinas/metabolismo , Oxigênio/sangue , Oxigênio/metabolismo , Adolescente , Adulto , Gasometria/métodos , Exercício Físico/fisiologia , Feminino , Humanos , Hipóxia/sangue , Hipóxia/metabolismo , Masculino , Consumo de Oxigênio/fisiologia , Oxiemoglobinas/metabolismo , Adulto JovemAssuntos
Injúria Renal Aguda , Serina , Injúria Renal Aguda/diagnóstico , Biomarcadores , Humanos , RimRESUMO
Protein modification with ISG15 (ISGylation) represents a major type I IFN-induced antimicrobial system. Common mechanisms of action and species-specific aspects of ISGylation, however, are still ill defined and controversial. We used a multiphasic coxsackievirus B3 (CV) infection model with a first wave resulting in hepatic injury of the liver, followed by a second wave culminating in cardiac damage. This study shows that ISGylation sets nonhematopoietic cells into a resistant state, being indispensable for CV control, which is accomplished by synergistic activity of ISG15 on antiviral IFIT1/3 proteins. Concurrent with altered energy demands, ISG15 also adapts liver metabolism during infection. Shotgun proteomics, in combination with metabolic network modeling, revealed that ISG15 increases the oxidative capacity and promotes gluconeogenesis in liver cells. Cells lacking the activity of the ISG15-specific protease USP18 exhibit increased resistance to clinically relevant CV strains, therefore suggesting that stabilizing ISGylation by inhibiting USP18 could be exploited for CV-associated human pathologies.
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Infecções por Coxsackievirus/metabolismo , Citocinas/metabolismo , Enterovirus Humano B/metabolismo , Fígado/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Infecções por Coxsackievirus/genética , Citocinas/genética , Feminino , Gluconeogênese , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Fígado/patologia , Fígado/virologia , Camundongos , Camundongos Knockout , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Ubiquitina Tiolesterase/genética , Ubiquitina Tiolesterase/metabolismo , Ubiquitinas/genética , Ubiquitinas/metabolismoRESUMO
Chimeric inhibitors, which merge two drug pharmacophores in a single molecule have become a prominent approach for the design of novel anticancer compounds. Here, we examined animacroxam, which combines histone deacetylase (HDAC) inhibitory and cytoskeleton-interfering pharmacophores, in testicular germ cell tumors (TGCT). The effectiveness of animacroxam was compared to that of the commonly applied chemotherapeutic cisplatin as well as the clinically approved HDAC inhibitor vorinostat. The antineoplastic and antiangiogenic effects of animacroxam on TGCT in vivo were assessed through exploratory animal studies and a modified chorioallantoic membrane assay, revealing that animacroxam has significant antitumor activity in TGCT. A novel positron emission tomography/MR-imaging approach was applied to determine tumor volume and glucose [2-fluoro-2-deoxy-d-glucose (18F-FDG)] uptake in TGCT tumors, revealing reduced glucose uptake in animacroxam-treated TGCTs and showing a dose-dependent suppression of glycolytic enzymes, which led to a breakdown in glycolytic energy production. Furthermore, the observed antiangiogenic effects of animacroxam were related to its ability to inhibit endothelial cell-cell communication, as the expression of gap junction-forming connexin 43 was strongly suppressed, and gap-junctional intercellular mass transport was reduced. Our data suggest that the chimeric HDAC inhibitor animacroxam may become a promising candidate for the treatment of solid cancers and may serve as an interesting alternative to platinum-based therapies.
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Antineoplásicos/farmacologia , Cinamatos/farmacologia , Inibidores de Histona Desacetilases/farmacologia , Imidazóis/farmacologia , Neoplasias Embrionárias de Células Germinativas , Neoplasias Testiculares , Animais , Linhagem Celular Tumoral , Humanos , Masculino , Camundongos , Camundongos Nus , Neoplasias Embrionárias de Células Germinativas/tratamento farmacológico , Neoplasias Embrionárias de Células Germinativas/metabolismo , Neoplasias Embrionárias de Células Germinativas/patologia , Neoplasias Testiculares/tratamento farmacológico , Neoplasias Testiculares/metabolismo , Neoplasias Testiculares/patologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Antagonists of the V1a vasopressin receptor (V1aR) are emerging as a strategy for slowing progression of CKD. Physiologically, V1aR signaling has been linked with acid-base homeostasis, but more detailed information is needed about renal V1aR distribution and function. METHODS: We used a new anti-V1aR antibody and high-resolution microscopy to investigate Va1R distribution in rodent and human kidneys. To investigate whether V1aR activation promotes urinary H+ secretion, we used a V1aR agonist or antagonist to evaluate V1aR function in vasopressin-deficient Brattleboro rats, bladder-catheterized mice, isolated collecting ducts, and cultured inner medullary collecting duct (IMCD) cells. RESULTS: Localization of V1aR in rodent and human kidneys produced a basolateral signal in type A intercalated cells (A-ICs) and a perinuclear to subapical signal in type B intercalated cells of connecting tubules and collecting ducts. Treating vasopressin-deficient Brattleboro rats with a V1aR agonist decreased urinary pH and tripled net acid excretion; we observed a similar response in C57BL/6J mice. In contrast, V1aR antagonist did not affect urinary pH in normal or acid-loaded mice. In ex vivo settings, basolateral treatment of isolated perfused medullary collecting ducts with the V1aR agonist or vasopressin increased intracellular calcium levels in ICs and decreased luminal pH, suggesting V1aR-dependent calcium release and stimulation of proton-secreting proteins. Basolateral treatment of IMCD cells with the V1aR agonist increased apical abundance of vacuolar H+-ATPase in A-ICs. CONCLUSIONS: Our results show that activation of V1aR contributes to urinary acidification via H+ secretion by A-ICs, which may have clinical implications for pharmacologic targeting of V1aR.
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Equilíbrio Ácido-Base/efeitos dos fármacos , Receptores de Vasopressinas/efeitos dos fármacos , Vasopressinas/farmacologia , Equilíbrio Ácido-Base/genética , Animais , Células Cultivadas/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Imunofluorescência , Células HEK293/efeitos dos fármacos , Células HEK293/metabolismo , Humanos , Concentração de Íons de Hidrogênio/efeitos dos fármacos , Imuno-Histoquímica , Túbulos Renais Coletores/citologia , Túbulos Renais Coletores/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Ratos Brattleboro , Ratos Wistar , Reação em Cadeia da Polimerase em Tempo Real/métodos , Receptores de Vasopressinas/genética , Sensibilidade e Especificidade , Urinálise/métodosRESUMO
BACKGROUND/AIMS: Recently, we have demonstrated that episodic hypoxia occurs in kidneys of mice challenged repetitively with the immunosuppressant cyclosporine A (CsA), in analogy to humans on CsA treatment. However, the molecular consequences of episodic hypoxia remain poorly defined, as is its impact on cell survival. Here, we systematically study cell response to episodic, as compared to single course hypoxia. METHODS: In vivo, kidneys of mice challenged daily with CsA for one week were analyzed by microarray analysis, gene ontology analysis, and qPCR. In vitro, renal cells were subjected to hypoxia (1 % O2) which was either episodic (4 h for 6 consecutive days), short-term (4 h), or sustained (24 h). Western blot analysis quantified hypoxia-inducible factor-1α (HIF-1α). 2',7'-dichlorofluorescein diacetate detected intracellular ROS. After re-oxygenation, staurosporine served to induce apoptosis, quantified by active caspase-3. RESULTS: In vivo, HIF target gene expression was suppressed by daily CsA treatment. Yet, we found up-regulation of genes involved in defence against cellular stress, notably against ROS. Renal cells in vitro behaved largely different under episodic and sustained hypoxia, while their response to short-term hypoxia oscillated between the previous two. Episodic hypoxia exhibited the highest total HIF-1α protein level, lowest nucleus-to-cytoplasm ratio, and lowest HIF target gene expression. When compared with normoxia, re-oxygenation after sustained hypoxia increased ROS by 3.04 ± 1.04 fold (p<0.001), and re-oxygenation after episodic hypoxia by 1.26 ± 0.16 fold (p<0.01). Staurosporine-induced active caspase-3 was highest after sustained, and lowest after episodic hypoxia. CONCLUSION: In vitro episodic hypoxia mimics the largely HIF-independent transcriptome observed after repetitive CsA treatment in vivo. In vitro preconditioning with episodic hypoxia protects against stress-induced apoptosis. Despite of its long-term adverse effects, CsA derived episodic hypoxia induces a unique renal hypoxia response that provides adaptation to re-oxygenation mediated ROS damage.
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Adaptação Fisiológica , Apoptose , Hipóxia , Rim , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismo , Animais , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Ciclosporina/farmacologia , Hipóxia/metabolismo , Hipóxia/patologia , Hipóxia/fisiopatologia , Rim/irrigação sanguínea , Rim/metabolismo , Rim/patologia , Rim/fisiopatologia , Camundongos , Camundongos TransgênicosRESUMO
The aim was to identify new targets that regulate gene expression at the posttranscriptional level in angiotensin II (ANGII)-mediated hypertension. Heparin affinity chromatography was used to enrich nucleic acid-binding proteins from kidneys of two-kidney, one-clip (2K1C) hypertensive Wistar rats. The experiment was repeated with 14-day ANGII infusion using Alzet osmotic mini pumps, with or without ANGII receptor AT1a inhibition using losartan in the drinking water. Mean arterial pressure increased after 2K1C or ANGII infusion and was inhibited with losartan. Heparin affinity chromatography and mass spectrometry were used to identify Annexin-A2 (ANXA2) as having differential nucleic acid-binding activity. Total Annexin-A2 protein expression was unchanged, whereas nucleic acid-binding activity was increased in both kidneys of 2K1C and after ANGII infusion through AT1a stimulation. Costaining of Annexin-A2 with α-smooth muscle actin and aquaporin 2 showed prominent expression in the endothelia of larger arteries and the cells of the inner medullary collecting duct. The nuclear factor of activated T cells (NFAT) transcription factor was identified as a likely Annexin-A2 target using enrichment analysis on a 2K1C microarray data set and identifying several binding sites in the regulatory region of the mRNA. Expression analysis showed that ANGII increases NFAT5 protein but not mRNA level and, thus, indicated that NFAT5 is regulated by posttranscriptional regulation, which correlates with activation of the RNA-binding protein Annexin-A2. In conclusion, we show that ANGII increases Annexin-A2 nucleic acid-binding activity that correlates with elevated protein levels of the NFAT5 transcription factor. NFAT signaling appears to be a major contributor to renal gene regulation in high-renin states.
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Angiotensina II , Anexina A2/metabolismo , Pressão Arterial , Hipertensão/metabolismo , Rim/metabolismo , Processamento Pós-Transcricional do RNA , Fatores de Transcrição/metabolismo , Animais , Anexina A2/genética , Modelos Animais de Doenças , Hipertensão/genética , Hipertensão/patologia , Hipertensão/fisiopatologia , Rim/patologia , Rim/fisiopatologia , Masculino , Ratos Wistar , Receptor Tipo 1 de Angiotensina/metabolismo , Transdução de Sinais , Fatores de Transcrição/genética , Regulação para CimaRESUMO
MicroRNAs (miRNAs) are non-coding RNAs originally involved in RNA silencing and post-transcriptional regulation of gene expression. We have shown in previous work that the miRNA let-7b can act as a signalling molecule for Toll-like receptor 7, thereby initiating innate immune pathways and apoptosis in the central nervous system. Here, we investigated whether different members of the miRNA family let-7, abundantly expressed in the brain, are released into the human cerebrospinal fluid (CSF) and whether quantitative differences in let-7 copies exist in neurodegenerative diseases. RNA isolated from CSF of patients with Alzheimer´s disease (AD) and from control patients with frontotemporal lobe dementia (FTLD), major depressive episode (MDE) without clinical or neurobiological signs of AD, and healthy individuals, was reverse transcribed with primers against nine let-7 family members, and miRNAs were quantified and analyzed comparatively by quantitative PCR. let-7 miRNAs were present in CSF from patients with AD, FTLD, MDE, and healthy controls. However, the amount of individual let-7 miRNAs in the CSF varied substantially. CSF from AD patients contained higher amounts of let-7b and let-7e compared to healthy controls, while no differences were observed regarding the other let-7 miRNAs. No increase in let-7b and let-7e was detected in CSF from FTLD patients, while in CSF from MDE patients, let-7b and let-7e copy levels were elevated. In CSF from AD patients, let-7b and let-7e were associated with extracellular vesicles. let-7 family members present in the CSF mediated neurotoxicity in vitro, albeit to a variable extent. Taken together, neurotoxic let-7 miRNAs are differentially and specifically released in AD, but also in MDE patients. Thus, these miRNAs may mirror common neuropathological paths and by this serve to unscramble mechanisms of different neurodegenerative diseases.
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Doença de Alzheimer/líquido cefalorraquidiano , Micropartículas Derivadas de Células/metabolismo , Regulação da Expressão Gênica , MicroRNAs/líquido cefalorraquidiano , Idoso , Idoso de 80 Anos ou mais , Transtorno Depressivo Maior/líquido cefalorraquidiano , Feminino , Demência Frontotemporal/líquido cefalorraquidiano , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
Contrast-induced acute kidney injury (CIAKI) occurs in up to 30% of patients who receive iodinated contrast media and is generally considered to be the third most common cause of hospital-acquired AKI. Accurate assessment of the incidence of CIAKI is obscured, however, by the use of various definitions for diagnosis, the different populations studied and the prophylactic measures put in place. A deeper understanding of the mechanisms that underlie CIAKI is required to enable reliable risk assessment for individual patients, as their medical histories will determine the specific pathways by which contrast media administration might lead to kidney damage. Here, we highlight common triggers that prompt the development of CIAKI and the subsequent mechanisms that ultimately cause kidney damage. We also discuss effective protective measures, such as rapidly acting oral hydration schemes and loop diuretics, in the context of CIAKI pathophysiology. Understanding of how CIAKI arises in different patient groups could enable a marked reduction in incidence and improved outcomes. The ultimate goal is to shape CIAKI prevention strategies for individual patients.
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Injúria Renal Aguda/induzido quimicamente , Injúria Renal Aguda/prevenção & controle , Meios de Contraste/efeitos adversos , Injúria Renal Aguda/diagnóstico , Injúria Renal Aguda/fisiopatologia , Hidratação , HumanosRESUMO
Neuroblastoma, the major cause of infant cancer deaths, results from fast proliferation of undifferentiated neuroblasts. Treatment of high-risk neuroblastoma includes differentiation with retinoic acid (RA); however, the resistance of many of these tumors to RA-induced differentiation poses a considerable challenge. Human achaete-scute homolog 1 (hASH1) is a proneural basic helix-loop-helix transcription factor essential for neurogenesis and is often upregulated in neuroblastoma. Here, we identified a novel function for hASH1 in regulating the differentiation phenotype of neuroblastoma cells. Global analysis of 986 human neuroblastoma datasets revealed a negative correlation between hASH1 and neuron differentiation that was independent of the N-myc (MYCN) oncogene. Using RA to induce neuron differentiation in two neuroblastoma cell lines displaying high and low levels of hASH1 expression, we confirmed the link between hASH1 expression and the differentiation defective phenotype, which was reversed by silencing hASH1 or by hypoxic preconditioning. We further show that hASH1 suppresses neuronal differentiation by inhibiting transcription at the RA receptor element. Collectively, our data indicate hASH1 to be key for understanding neuroblastoma resistance to differentiation therapy and pave the way for hASH1-targeted therapies for augmenting the response of neuroblastoma to differentiation therapy.
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Protein synthesis is a primary energy-consuming process in the cell. Therefore, under hypoxic conditions, rapid inhibition of global mRNA translation represents a major protective strategy to maintain energy metabolism. How some mRNAs, especially those that encode crucial survival factors, continue to be efficiently translated in hypoxia is not completely understood. By comparing specific transcript levels in ribonucleoprotein complexes, cytoplasmic polysomes and endoplasmic reticulum (ER)-bound ribosomes, we show that the synthesis of proteins encoded by hypoxia marker genes is favoured at the ER in hypoxia. Gene expression profiling revealed that transcripts particularly increased by the HIF-1 transcription factor network show hypoxia-induced enrichment at the ER. We found that mRNAs favourably translated at the ER have higher conservation scores for both the 5'- and 3'-untranslated regions (UTRs) and contain less upstream initiation codons (uAUGs), indicating the significance of these sequence elements for sustained mRNA translation under hypoxic conditions. Furthermore, we found enrichment of specific cis-elements in mRNA 5'- as well as 3'-UTRs that mediate transcript localization to the ER in hypoxia. We conclude that transcriptome partitioning between the cytoplasm and the ER permits selective mRNA translation under conditions of energy shortage.