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With antimicrobial resistance (AMR) rapidly evolving in pathogens, quick and accurate identification of genetic determinants of phenotypic resistance is essential for improving surveillance, stewardship, and clinical mitigation. Machine learning (ML) models show promise for AMR prediction in diagnostics but require a deep understanding of internal processes to use effectively. Our study utilized AMR gene, pangenomic, and predicted plasmid features from 647 Enterococcus faecium and Enterococcus faecalis genomes across the One Health continuum, along with corresponding resistance phenotypes, to develop interpretive ML classifiers. Vancomycin resistance could be predicted with 99% accuracy with AMR gene features, 98% with pangenome features, and 96% with plasmid clusters. Top pangenome features overlapped with the resistance genes of the vanA operon, which are often laterally transmitted via plasmids. Doxycycline resistance prediction achieved approximately 92% accuracy with pangenome features, with the top feature being elements of Tn916 conjugative transposon, a tet(M) carrier. Erythromycin resistance prediction models achieved about 90% accuracy, but top features were negatively correlated with resistance due to the confounding effect of population structure. This work demonstrates the importance of reviewing ML models' features to discern biological relevance even when achieving high-performance metrics. Our workflow offers the potential to propose hypotheses for experimental testing, enhancing the understanding of AMR mechanisms, which are crucial for combating the AMR crisis.
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Antimicrobial resistance (AMR) is a global health crisis that poses a great threat to modern medicine. Effective prevention strategies are urgently required to slow the emergence and further dissemination of AMR. Given the availability of data sets encompassing hundreds or thousands of pathogen genomes, machine learning (ML) is increasingly being used to predict resistance to different antibiotics in pathogens based on gene content and genome composition. A key objective of this work is to advocate for the incorporation of ML into front-line settings but also highlight the further refinements that are necessary to safely and confidently incorporate these methods. The question of what to predict is not trivial given the existence of different quantitative and qualitative laboratory measures of AMR. ML models typically treat genes as independent predictors, with no consideration of structural and functional linkages; they also may not be accurate when new mutational variants of known AMR genes emerge. Finally, to have the technology trusted by end users in public health settings, ML models need to be transparent and explainable to ensure that the basis for prediction is clear. We strongly advocate that the next set of AMR-ML studies should focus on the refinement of these limitations to be able to bridge the gap to diagnostic implementation.
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Antibacterianos , Farmacorresistência Bacteriana , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Farmacorresistência Bacteriana/genética , Aprendizado de MáquinaRESUMO
In this retrospective, analytical study, we developed a deep learning-based diagnostic model that can be applied to canine stifle joint diseases and compared its accuracy with that achieved by veterinarians to verify its potential as a reliable diagnostic method. A total of 2382 radiographs of the canine stifle joint from cooperative animal hospitals were included in a dataset. Stifle joint regions were extracted from the original images using the faster region-based convolutional neural network (R-CNN) model, and the object detection accuracy was evaluated. Four radiographic findings: patellar deviation, drawer sign, osteophyte formation, and joint effusion, were observed in the stifle joint and used to train a residual network (ResNet) classification model. Implant and growth plate groups were analyzed to compare the classification accuracy against the total dataset. All deep learning-based classification models achieved target accuracies exceeding 80%, which is comparable to or slightly less than those achieved by veterinarians. However, in the case of drawer signs, further research is necessary to improve the low sensitivity of the model. When the implant group was excluded, the classification accuracy significantly improved, indicating that the implant acted as a distraction. These results indicate that deep learning-based diagnoses can be expected to become useful diagnostic models in veterinary medicine.
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Aprendizado Profundo , Doenças do Cão , Artropatias , Cães , Animais , Joelho de Quadrúpedes/diagnóstico por imagem , Estudos Retrospectivos , Artropatias/diagnóstico por imagem , Artropatias/veterinária , Redes Neurais de Computação , Doenças do Cão/diagnóstico por imagemRESUMO
Primary cilia on kidney tubular cells play crucial roles in maintaining structure and physiological function. Emerging evidence indicates that the absence of primary cilia, and their length, are associated with kidney diseases. The length of primary cilia in kidney tubular epithelial cells depends, at least in part, on oxidative stress and extracellular signal-regulated kinase 1/2 (ERK) activation. Hydrogen sulfide (H2S) is involved in antioxidant systems and the ERK signaling pathway. Therefore, in this study, we investigated the role of H2S in primary cilia elongation and the downstream pathway. In cultured Madin-Darby Canine Kidney cells, the length of primary cilia gradually increased up to 4 days after the cells were grown to confluent monolayers. In addition, the expression of H2S-producing enzyme increased concomitantly with primary cilia length. Treatment with NaHS, an exogenous H2S donor, accelerated the elongation of primary cilia whereas DL-propargylglycine (a cystathionine γ-lyase inhibitor) and hydroxylamine (a cystathionine-ß-synthase inhibitor) delayed their elongation. NaHS treatment increased ERK activation and Sec10 and Arl13b protein expression, both of which are involved in cilia formation and elongation. Treatment with U0126, an ERK inhibitor, delayed elongation of primary cilia and blocked the effect of NaHS-mediated primary cilia elongation and Sec10 and Arl13b upregulation. Finally, we also found that H2S accelerated primary cilia elongation after ischemic kidney injury. These results indicate that H2S lengthens primary cilia through ERK activation and a consequent increase in Sec10 and Arl13b expression, suggesting that H2S and its downstream targets could be novel molecular targets for regulating primary cilia.
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Mitochondrial NADP+-dependent isocitrate dehydrogenase 2 (IDH2) produces NADPH, which is known to inhibit mitochondrial oxidative stress. Ureteral obstruction induces kidney inflammation and fibrosis via oxidative stress. Here, we investigated the role and underlying mechanism of IDH2 in unilateral ureteral obstruction (UUO)-induced kidney inflammation using IDH2 gene deleted mice (IDH2-/-). Eight- to 10-week-old female IDH2-/- mice and wild type (IDH2+/+) littermates were subjected to UUO and kidneys were harvested 5 days after UUO. IDH2 was not detected in the kidneys of IDH2-/- mice, while UUO decreased IDH2 in IDH2+/+ mice. UUO increased the expressions of markers of oxidative stress in both IDH2+/+ and IDH2-/- mice, and these changes were greater in IDH2-/- mice compared to IDH2+/+ mice. Bone marrow-derived macrophages of IDH2-/- mice showed a more migrating phenotype with greater ruffle formation and Rac1 distribution than that of IDH2+/+ mice. Correspondently, UUO-induced infiltration of monocytes/macrophages was greater in IDH2-/- mice compared to IDH2+/+ mice. Taken together, these data demonstrate that IDH2 plays a protective role against UUO-induced inflammation through inhibition of oxidative stress and macrophage infiltration.
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Orexin A (OXA) is a neuropeptide associated with plasma insulin and leptin levels involved in body weight and appetite regulation. However, little is known about the effect of OXA on leptin secretion in adipocytes and its physiological roles. Leptin secretion and expression were analysed in 3T3-L1 adipocytes. Plasma leptin, adiponectin and insulin levels were measured by ELISA assay. Phosphorylated signal transducer and activator of transcription 3 (pSTAT3) levels in the hypothalamus were evaluated by western blotting. OXA dose-dependently suppressed leptin secretion from 3T3-L1 adipocytes by inhibiting its gene expression while facilitating adiponectin secretion. The leptin inhibition by OXA was mediated via orexin receptors (OXR1 and OXR2). In addition to the pathway via extracellular signal-regulated kinases, OXA triggered adenylyl cyclase-induced cAMP elevation, which results in protein kinase A-mediated activation of cAMP response element-binding proteins (CREB). Accordingly, CREB inhibition restored the OXA-induced downregulation of leptin gene expression and secretion. Exogenous OXA for 4 weeks decreased fasting plasma leptin levels and increased hypothalamic pSTAT3 levels in high-fat diet-fed mice, regardless of increase in body weight and food intake. These results suggest that high dose of OXA directly inhibits leptin mRNA expression and thus secretion in adipocytes, which may be a peripheral mechanism of OXA for its role in appetite drive during fasting. It may be also critical for lowering basal plasma leptin levels and thus maintaining postprandial hypothalamic leptin sensitivity.
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Adipócitos/efeitos dos fármacos , Adipócitos/metabolismo , Hipotálamo/efeitos dos fármacos , Hipotálamo/metabolismo , Leptina/sangue , Leptina/metabolismo , Orexinas/farmacologia , Células 3T3-L1 , Animais , Apetite/efeitos dos fármacos , Peso Corporal/efeitos dos fármacos , Linhagem Celular , Dieta Hiperlipídica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neuropeptídeos/metabolismo , Receptores de Orexina/metabolismoRESUMO
Primary cilium is a microtubule-based non-motile organelle that plays critical roles in kidney pathophysiology. Our previous studies revealed that the lengths of primary cilia decreased upon renal ischemia/reperfusion injury and oxidative stress, and restored with recovery. Here, we tested the hypothesis that lack of primary cilium causes epithelial to mesenchymal transition (EMT) of kidney tubule cells. We investigated the alteration of length of primary cilia in TGF-ß-induced EMT via visualization of primary cilia by fluorescence staining against acetylated α-tubulin. EMT was determined by measuring mesenchymal protein expression using quantitative PCR and indirect fluorescence staining. As a result, TGF-ß treatment decreased ciliary length along with EMT. To test whether defect of primary cilia trigger onset of EMT, cilia formation was disturbed by knock down of ciliary protein using siRNA along with/without TGF-ß treatment. Knock down of Arl13b and Ift20 reduced cilia elongation and increased expression of EMT markers such as fibronectin, α-SMA, and collagen III. TGF-ß-induced EMT was greater as well in Arl13b and Ift20-knock down cells compared to control cells. Taken together, deficiency of primary cilia trigger EMT and exacerbates it under pro-fibrotic signals.
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Cílios/efeitos dos fármacos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Fator de Crescimento Transformador beta/farmacologia , Tubulina (Proteína)/genética , Fatores de Ribosilação do ADP/antagonistas & inibidores , Fatores de Ribosilação do ADP/genética , Fatores de Ribosilação do ADP/metabolismo , Actinas/genética , Actinas/metabolismo , Animais , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Tamanho Celular , Cílios/metabolismo , Cílios/ultraestrutura , Colágeno Tipo III/genética , Colágeno Tipo III/metabolismo , Cães , Transição Epitelial-Mesenquimal/genética , Fibronectinas/genética , Fibronectinas/metabolismo , Regulação da Expressão Gênica , Células Madin Darby de Rim Canino , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Tubulina (Proteína)/metabolismoRESUMO
Migration of surviving kidney tubule cells after sub-lethal injury, for example ischemia/reperfusion (I/R), plays a critical role in recovery. Exocytosis is known to be involved in cell migration, and a key component in exocytosis is the highly-conserved eight-protein exocyst complex. We investigated the expression of a central exocyst complex member, Sec10, in kidneys following I/R injury, as well as the role of Sec10 in wound healing following scratch injury of cultured Madin-Darby canine kidney (MDCK) cells. Sec10 overexpression and knockdown (KD) in MDCK cells were used to investigate the speed of wound healing and the mechanisms underlying recovery. In mice, Sec10 decreased after I/R injury, and increased during the recovery period. In cell culture, Sec10 OE inhibited ruffle formation and wound healing, while Sec10 KD accelerated it. Sec10 OE cells had higher amounts of diacylglycerol kinase (DGK) gamma at the leading edge than did control cells. A DGK inhibitor reversed the inhibition of wound healing and ruffle formation in Sec10 OE cells. Conclusively, downregulation of Sec10 following I/R injury appears to accelerate recovery of kidney tubule cells through activated ruffle formation and enhanced cell migration.
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Diacilglicerol Quinase/antagonistas & inibidores , Túbulos Renais/metabolismo , Traumatismo por Reperfusão/prevenção & controle , Proteínas de Transporte Vesicular/genética , Animais , Bioensaio , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Diacilglicerol Quinase/genética , Diacilglicerol Quinase/metabolismo , Cães , Inibidores Enzimáticos/farmacologia , Exocitose , Regulação da Expressão Gênica , Túbulos Renais/patologia , Células Madin Darby de Rim Canino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Piperidinas/farmacologia , Quinazolinonas/farmacologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Traumatismo por Reperfusão/genética , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/patologia , Proteínas de Transporte Vesicular/agonistas , Proteínas de Transporte Vesicular/antagonistas & inibidores , Proteínas de Transporte Vesicular/metabolismo , Cicatrização/fisiologiaRESUMO
Mitochondrial NADP+-dependent isocitrate dehydrogenase (IDH2) catalyzes the oxidative decarboxylation of isocitrate to α-ketoglutarate, synthesizing NADPH, which is essential for mitochondrial redox balance. Ischemia-reperfusion (I/R) is one of most common causes of AKI. I/R disrupts the mitochondrial redox balance, resulting in oxidative damage to mitochondria and cells. Here, we investigated the role of IDH2 in I/R-induced AKI. I/R injury in mice led to the inactivation of IDH2 in kidney tubule cells. Idh2 gene deletion exacerbated the I/R-induced increase in plasma creatinine and BUN levels and the histologic evidence of tubule injury, and augmented the reduction of NADPH levels and the increase in oxidative stress observed in the kidney after I/R. Furthermore, Idh2 gene deletion exacerbated I/R-induced mitochondrial dysfunction and morphologic fragmentation, resulting in severe apoptosis in kidney tubule cells. In cultured mouse kidney proximal tubule cells, Idh2 gene downregulation enhanced the mitochondrial damage and apoptosis induced by treatment with hydrogen peroxide. This study demonstrates that Idh2 gene deletion exacerbates mitochondrial damage and tubular cell death via increased oxidative stress, suggesting that IDH2 is an important mitochondrial antioxidant enzyme that protects cells from I/R insult.
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Morte Celular , Isocitrato Desidrogenase/deficiência , Túbulos Renais/patologia , Rim/irrigação sanguínea , Rim/enzimologia , Mitocôndrias/enzimologia , Traumatismo por Reperfusão/enzimologia , Injúria Renal Aguda/enzimologia , Injúria Renal Aguda/etiologia , Animais , Apoptose , Células Cultivadas , Deleção de Genes , Isocitrato Desidrogenase/genética , Masculino , Camundongos , Camundongos KnockoutRESUMO
The most significant barrier to success in human activity recognition is extracting and selecting the right features. In traditional methods, the features are chosen by humans, which requires the user to have expert knowledge or to do a large amount of empirical study. Newly developed deep learning technology can automatically extract and select features. Among the various deep learning methods, convolutional neural networks (CNNs) have the advantages of local dependency and scale invariance and are suitable for temporal data such as accelerometer (ACC) signals. In this paper, we propose an efficient human activity recognition method, namely Iss2Image (Inertial sensor signal to Image), a novel encoding technique for transforming an inertial sensor signal into an image with minimum distortion and a CNN model for image-based activity classification. Iss2Image converts real number values from the X, Y, and Z axes into three color channels to precisely infer correlations among successive sensor signal values in three different dimensions. We experimentally evaluated our method using several well-known datasets and our own dataset collected from a smartphone and smartwatch. The proposed method shows higher accuracy than other state-of-the-art approaches on the tested datasets.
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Acelerometria/métodos , Técnicas Biossensoriais/métodos , Exercício Físico/fisiologia , Reconhecimento Automatizado de Padrão/métodos , Adulto , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Redes Neurais de Computação , SmartphoneRESUMO
Personalized emotion recognition provides an individual training model for each target user in order to mitigate the accuracy problem when using general training models collected from multiple users. Existing personalized speech emotion recognition research has a cold-start problem that requires a large amount of emotionally-balanced data samples from the target user when creating the personalized training model. Such research is difficult to apply in real environments due to the difficulty of collecting numerous target user speech data with emotionally-balanced label samples. Therefore, we propose the Robust Personalized Emotion Recognition Framework with the Adaptive Data Boosting Algorithm to solve the cold-start problem. The proposed framework incrementally provides a customized training model for the target user by reinforcing the dataset by combining the acquired target user speech with speech from other users, followed by applying SMOTE (Synthetic Minority Over-sampling Technique)-based data augmentation. The proposed method proved to be adaptive across a small number of target user datasets and emotionally-imbalanced data environments through iterative experiments using the IEMOCAP (Interactive Emotional Dyadic Motion Capture) database.
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Algoritmos , Emoções/fisiologia , Fala/fisiologia , Bases de Dados Factuais , HumanosRESUMO
Acute kidney injury (AKI) is a major complication of hepatic surgeries. The primary cilium protrudes to the lumen of kidney tubules and plays an important role in renal functions. Disruption of primary cilia homeostasis is highly associated with human diseases including AKI. Here, we investigated whether transient hepatic ischemia induces length change and deciliation of kidney primary cilia, and if so, whether reactive oxygen species (ROS)/oxidative stress regulates those. HIR induced damages to the liver and kidney with increases in ROS/oxidative stress. HIR shortened the cilia of kidney epithelial cells and caused them to shed into the urine. This shortening and shedding of cilia was prevented by Mn(III) tetrakis(1-methyl-4-pyridyl) porphyrin (MnTMPyP, an antioxidant). The urine of patient undergone liver resection contained ciliary proteins. These findings indicate that HIR induces shortening and deciliation of kidney primary cilia into the urine via ROS/oxidative stress, suggesting that primary cilia is associated with HIR-induced AKI and that the presence of ciliary proteins in the urine could be a potential indication of kidney injury.
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Injúria Renal Aguda/metabolismo , Homeostase , Fígado/metabolismo , Estresse Oxidativo , Traumatismo por Reperfusão/metabolismo , Injúria Renal Aguda/etiologia , Injúria Renal Aguda/patologia , Animais , Antioxidantes/farmacologia , Cílios/metabolismo , Cílios/patologia , Fígado/patologia , Masculino , Metaloporfirinas/farmacologia , Camundongos , Traumatismo por Reperfusão/complicações , Traumatismo por Reperfusão/patologiaRESUMO
Obesity is a critical risk factor for the hypertension. Although angiotensin II (Ang II) in obese individuals is known to be upregulated in obesity-induced hypertension, direct evidence that explains the underlying mechanism for increased vascular tone and consequent increase in blood pressure (BP) is largely unknown. The purpose of this study is to investigate the novel mechanism underlying Ang II-induced hyper-contractility and hypertension in obese rats. Eight-week old male Sprague-Dawley rats were fed with 60% fat diet or normal diet for 4 months. Body weight, plasma lipid profile, plasma Ang II level, BP, Ang II-induced vascular contraction, and expression of regulatory proteins modulating vascular contraction with/without Ang II stimulation were measured. As a result, high fat diet (HFD) accelerated age-dependent body weight gaining along with increased plasma Ang II concentration. It also increased BP and Ang II-induced aortic contraction. Basal expression of p-CPI-17 and myosin light chain (MLC) kinase was increased by HFD along with increased phosphorylation of MLC. Ang II-induced phosphorylation of CPI-17 and MLC were also higher in HFD group than control group. In conclusion HFD-induced hypertension is through at least in part by increased vascular contractility via increased expression and activation of contractile proteins and subsequent MLC phosphorylation induced by increased Ang II.
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C/EBP homologous protein (CHOP), a transcription factor for the expression of apoptosis-related genes, plays an important role in endoplasmic reticulum (ER) stress-related organ diseases, including diseases of the kidney. Here, we investigated the role of CHOP in ischemia/reperfusion (I/R)-induced acute kidney injury using CHOP-knockout (CHOP(-/-)) and wild type (CHOP(+/+)) mice. Fifteen or thirty minutes of bilateral renal ischemia (I/R) insult resulted in necrotic and apoptotic tubular epithelial cell death, together with increases in plasma creatinine (PCr) and blood urea nitrogen (BUN) concentrations. After I/R, BiP/GRP78 and CHOP expressions in the kidney gradually increased over time. CHOP expression was greater in the outer medulla than that in the cortex and localized intensely in the nucleus. I/R caused apoptosis of tubular epithelial cells in both CHOP(-/-) and CHOP(+/+) mice. The number of apoptotic cells after I/R was lower in CHOP(-/-) mice than that in CHOP(+/+) mice. Consistent with the degree of apoptosis, I/R-induced kidney morphological and functional damages were milder in CHOP(-/-) than that in CHOP(+/+) mice. The cleavage of procaspase-3 and the induction of Bax protein after I/R were lower in CHOP(-/-) than that in CHOP(+/+) mice. In contrast, the expression levels of Bcl-2, Bcl-xL, cIAP2, Mcl-1, and XIAP were higher in CHOP(-/-) than that in CHOP(+/+) mice. These results indicate that I/R induces ER stress, leading to the activation of CHOP-associated apoptosis signals, resulting in renal functional and histological damages.
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During passage through the gastrointestinal tract, enterohaemorrhagic Escherichia coli (EHEC) encounters numerous stresses, each producing unique antimicrobial conditions. Beyond surviving these stresses, EHEC may also use them as cues about the local microenvironment to modulate its virulence. Of particular interest is how exposure to changing concentrations of short-chain fatty acids (SCFAs) associated with passage through the small and large intestines affects EHEC virulence, as well as flagella expression and motility specifically. In this study, we investigate the impact of exposure to SCFA mixes simulating concentrations and compositions within the small and large intestines on EHEC flagella expression and function. Using a combination of DNA microarray, quantitative real-time PCR, immunoblot analysis, flow cytometry and motility assays, we show that there is a marked, significant upregulation of flagellar genes, the flagellar protein, FliC, and motility when EHEC is exposed to SCFA mixes representative of the small intestine. By contrast, when EHEC is exposed to SCFA mixes representative of the large intestine, there is a significant downregulation of flagellar genes, FliC and motility. Our results demonstrate that EHEC modulates flagella expression and motility in response to SCFAs, with differential responses associated with SCFA mixes typical of the small and large intestines. This research contributes to our understanding of how EHEC senses and responds to host environmental signals and the mechanisms it uses to successfully infect the human host. Significantly, it also suggests that EHEC is using this key gastrointestinal chemical signpost to cue changes in flagella expression and motility in different locations within the host intestinal tract.
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Infecções por Escherichia coli/microbiologia , Escherichia coli O157/metabolismo , Ácidos Graxos Voláteis/metabolismo , Flagelos/metabolismo , Infecções por Escherichia coli/metabolismo , Escherichia coli O157/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Flagelos/genética , Regulação Bacteriana da Expressão Gênica , HumanosRESUMO
The regulator of G protein signaling 2 (RGS2) is a potent negative regulator of Gq protein signals including the angiotensin II (AngII)/AngII receptor signal, which plays a critical role in the progression of fibrosis. However, the role of RGS2 on the progression of kidney fibrosis has not been assessed. Here, we investigated the role of RGS2 in kidney fibrosis induced by unilateral ureteral obstruction (UUO) in mice. UUO resulted in increased expression of RGS2 mRNA and protein in the kidney along with increases of AngII and its type 1 receptor (AT1R) signaling and fibrosis. Furthermore, UUO increased the levels of F4/80, Ly6G, myeloperoxidase, and CXCR4 in the kidneys. RGS2 deficiency significantly enhanced these changes in the kidney. RGS2 deletion in the bone marrow-derived cells by transplanting the bone marrow of RGS2 knock-out mice into wild type mice enhanced UUO-induced kidney fibrosis. Overexpression of RGS2 in HEK293 cells, a human embryonic kidney cell line, and RAW264.7 cells, a monocyte/macrophage line, inhibited the AngII-induced activation of ERK and increase of CXCR4 expression. These findings provide the first evidence that RGS2 negatively regulates the progression of kidney fibrosis following UUO, likely by suppressing fibrogenic and inflammatory responses through the inhibition of AngII/AT1R signaling.
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Angiotensina II/metabolismo , Fibrose/etiologia , Nefropatias/etiologia , Proteínas RGS/fisiologia , Receptores de Angiotensina/metabolismo , Obstrução Ureteral/complicações , Angiotensina II/genética , Animais , Western Blotting , Células Cultivadas , Progressão da Doença , Feminino , Fibrose/metabolismo , Fibrose/patologia , Humanos , Técnicas Imunoenzimáticas , Nefropatias/metabolismo , Nefropatias/patologia , Camundongos , Camundongos Knockout , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Receptor Tipo 1 de Angiotensina , Receptores de Angiotensina/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
BACKGROUND: Progression of acute kidney injury to chronic kidney disease (CKD) is associated with inadequate recovery of damaged kidney. Hydrogen sulfide (H2S) regulates a variety of cellular signals involved in cell death, differentiation and proliferation. This study aimed to identify the role of H2S and its producing enzymes in the recovery of kidney following ischemia/reperfusion (I/R) injury. METHODS: Mice were subjected to 30 min of bilateral renal ischemia. Some mice were administered daily NaHS, an H2S donor, and propargylglycine (PAG), an inhibitor of the H2S-producing enzyme cystathionine gamma-lyase (CSE), during the recovery phase. Cell proliferation was assessed via 5'-bromo-2'-deoxyuridine (BrdU) incorporation assay. RESULTS: Ischemia resulted in decreases in CSE and cystathionine beta-synthase (CBS) expression and activity, and H2S level in the kidney. These decreases did not return to sham level until 8 days after ischemia when kidney had fibrotic lesions. NaHS administration to I/R-injured mice accelerated the recovery of renal function and tubule morphology, whereas PAG delayed that. Furthermore, PAG increased mortality after ischemia. NaHS administration to I/R-injured mice accelerated tubular cell proliferation, whereas it inhibited interstitial cell proliferation. In addition, NaHS treatment reduced post-I/R superoxide formation, lipid peroxidation, level of GSSG/GSH and Nox4 expression, whereas it increased catalase and MnSOD expression. CONCLUSIONS: Our findings demonstrate that H2S accelerates the recovery of I/R-induced kidney damage, suggesting that the H2S-producing transsulfuration pathway plays an important role in kidney repair after acute injury.
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Poluentes Atmosféricos/farmacologia , Proliferação de Células/efeitos dos fármacos , Nefropatias/tratamento farmacológico , Túbulos Renais/efeitos dos fármacos , Traumatismo por Reperfusão/tratamento farmacológico , Sulfetos/farmacologia , Alcinos/farmacologia , Animais , Western Blotting , Cistationina beta-Sintase/antagonistas & inibidores , Cistationina beta-Sintase/metabolismo , Cistationina gama-Liase/antagonistas & inibidores , Cistationina gama-Liase/metabolismo , Glicina/análogos & derivados , Glicina/farmacologia , Técnicas Imunoenzimáticas , Nefropatias/metabolismo , Nefropatias/patologia , Túbulos Renais/metabolismo , Túbulos Renais/patologia , Peroxidação de Lipídeos/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/patologia , Transdução de Sinais/efeitos dos fármacos , Superóxidos/metabolismoRESUMO
Extracellular signal-regulated kinase (ERK) signals play important roles in cell death and survival. However, the role of ERK in the repair process after injury remains to be defined in the kidney. Here, we investigated the role of ERK in proliferation and differentiation of tubular epithelial cells, and proliferation of interstitial cells following ischemia/reperfusion (I/R) injury in the mouse kidney. Mice were subjected to 30min of renal ischemia. Some mice were administered with U0126, a specific upstream inhibitor of ERK, daily during the recovery phase, beginning at 1day after ischemia until sacrifice. I/R caused severe tubular cell damage and functional loss in the kidney. Nine days after ischemia, the kidney was restored functionally with a partial restoration of damaged tubules and expansion of fibrotic lesions. ERK was activated by I/R and the activated ERK was sustained for 9days. U0126 inhibited the proliferation, basolateral relocalization of Na,K-ATPase and lengthening of primary cilia in tubular epithelial cells, whereas it enhanced the proliferation of interstitial cells and accumulation of extracellular matrix. Furthermore, U0126 elevated the expression of cell cycle arrest-related proteins, p21 and phospholylated-chk2 in the post-ischemic kidney. U0126 mitigated the post-I/R increase of Sec10 which is a crucial component of exocyst complex and an important factor in ciliogenesis and tubulogenesis. U0126 also enhanced the expression of fibrosis-related proteins, TGF-ß1 and phosphorylated NF-κB after ischemia. Our findings demonstrate that activation of ERK is required for both the restoration of damaged tubular epithelial cells and the inhibition of fibrosis progression following injury.
Assuntos
Células Epiteliais/citologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Fibrose/prevenção & controle , Túbulos Renais/citologia , Traumatismo por Reperfusão/complicações , Animais , Western Blotting , Butadienos/farmacologia , Cílios/metabolismo , Cílios/patologia , Creatinina/sangue , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Células Epiteliais/metabolismo , Matriz Extracelular/metabolismo , Fibrose/etiologia , Fibrose/metabolismo , Imunofluorescência , Técnicas Imunoenzimáticas , Túbulos Renais/metabolismo , MAP Quinase Quinase 1/antagonistas & inibidores , MAP Quinase Quinase 1/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/metabolismo , Nitrilas/farmacologia , Fosforilação , Espécies Reativas de Oxigênio/metabolismo , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/patologia , Transdução de Sinais , Fator de Crescimento Transformador beta1/metabolismoRESUMO
Increase of interstitial cell population, resulting in the expansion of interstitium, excessive production of extracellular matrix, and reduction of functioning tubules, is critical in fibrotic progression in the kidney of patients suffering from chronic renal diseases. Here, we investigated the contribution of bone marrow-derived cells (BMDC) in kidney fibrosis caused by ureteral obstruction (UO) using eGFP bone marrow-reconstituted chimeric mice. UO caused dramatic increases in the numbers of interstitial cells and expansion of the interstitium. Most kidney interstitial cells expressed GFP. Twenty nine percent of interstitial cells were cells that had proliferated and approximately 89% among them were BMDCs. Proliferation of fibroblasts differentiated from BMDCs significantly occurred in the interstitium of UO-kidney. Removal of BMDCs by whole body irradiation after UO resulted in reduction of kidney fibrosis, while injection of RAW264.7 cells, monocytes/macrophages, into irradiated mice induced a reversal of this reduction. Treatment with apocynin, an inhibitor of NADPH oxidase, reduced infiltration of BMDCs into the UO-kidney, leading to reduction of kidney fibrosis. In addition, only a few slow-cycling cells were observed in the interstitium of normal kidney. Even after UO, no change in the number of those cells was observed. Our findings demonstrate that BMDCs are a major source for interstitial expansion during kidney fibrosis via infiltration into damaged sites, differentiation to fibroblasts, and subsequent proliferation, contributing kidney fibrosis. These data provide a clear therapeutic target for treatment of chronic kidney disease.
Assuntos
Células da Medula Óssea/metabolismo , Diferenciação Celular , Proliferação de Células , Fibroblastos/metabolismo , Insuficiência Renal Crônica/metabolismo , Acetofenonas/farmacologia , Animais , Células da Medula Óssea/patologia , Transplante de Medula Óssea , Linhagem Celular , Inibidores Enzimáticos/farmacologia , Fibroblastos/patologia , Fibrose/metabolismo , Fibrose/patologia , Fibrose/terapia , Masculino , Camundongos , NADPH Oxidases/antagonistas & inibidores , NADPH Oxidases/metabolismo , Insuficiência Renal Crônica/patologia , Insuficiência Renal Crônica/terapia , Quimeras de Transplante , Transplante Homólogo , Obstrução Ureteral/complicações , Obstrução Ureteral/metabolismo , Obstrução Ureteral/patologiaRESUMO
Hydrogen sulfide (H2S) produced by cystathionine ß-synthase (CBS) and cystathionine γ-lyase (CSE) in the transsulfuration pathway of homocysteine plays a number of pathophysiological roles. Hyperhomocysteinemia is involved in kidney fibrosis. However, the role of H2S in kidney fibrosis remains to be defined. Here, we investigated the role of H2S and its acting mechanism in unilateral ureteral obstruction (UO)-induced kidney fibrosis in mice. UO decreased expressions of CBS and CSE in the kidney with decrease of H2S concentration. Treatment with sodium hydrogen sulfide (NaHS, a H2S producer) during UO reduced UO-induced oxidative stress with preservations of catalase, copper-zinc superoxide dismutase (CuZnSOD), and manganese superoxide dismutase (MnSOD) expression, and glutathione level. In addition, NaHS mitigated decreases of CBS and CSE expressions, and H2S concentration in the kidney. NaHS treatment attenuated UO-induced increases in levels of TGF-ß1, activated Smad3, and activated NF-κB. This study provided the first evidence of involvement of the transsulfuration pathway and H2S in UO-induced kidney fibrosis, suggesting that H2S and its transsulfuration pathway may be a potential target for development of therapeutics for fibrosis-related diseases.