RESUMO
Metalloproteins play fundamental roles in organisms and are utilized as starting points for the directed evolution of artificial enzymes. Knowing the strategies of metalloproteins, by which they exquisitely tune their activities, will not only lead to an understanding of biochemical phenomena but also contribute to various applications. The blue copper protein (BCP) has been a renowned model system to understand the biology, chemistry, and physics of metalloproteins. Pseudoazurin (Paz), a blue copper protein, mediates electron transfer in the bacterial anaerobic respiratory chain. Its redox potential is finely tuned by hydrogen (H) bond networks; however, difficulty in visualizing H atom positions in the protein hinders the detailed understanding of the protein's structure-function relationship. We here used neutron and sub-ångström resolution X-ray crystallography to directly observe H atoms in Paz. The 0.86-Å-resolution X-ray structure shows that the peptide bond between Pro80 and the His81 Cu ligand deviates from the ideal planar structure. The 1.9-Å-resolution neutron structure confirms a long-overlooked H bond formed by the amide of His81 and the S atom of another Cu ligand Cys78. Quantum mechanics/molecular mechanics calculations show that this H bond increases the redox potential of the Cu site and explains the experimental results well. Our study demonstrates the potential of neutron and sub-ångström resolution X-ray crystallography to understand the chemistry of metalloproteins at atomic and quantum levels.
Assuntos
Cobre , Metaloproteínas , Cobre/metabolismo , Cristalografia por Raios X , Ligação de Hidrogênio , Ligantes , Modelos Moleculares , Metaloproteínas/metabolismo , NêutronsRESUMO
This study aimed to investigate the role of bone marrow stromal cell antigen-1 (Bst1; also known as CD157) in acute kidney injury (AKI). Bst1 is a cell surface molecule with various enzymatic activities and downstream intracellular signaling pathways that modulate the immune response. Previous research has linked Bst1 to diseases such as ovarian cancer, Parkinson's disease, and rheumatoid arthritis. We used bilateral ischemia-reperfusion injury (IRI) as an AKI model and created bone marrow chimeric mice to evaluate the role of Bst1 in bone marrow-derived cells. We also used flow cytometry to identify Bst1/CD157 expression in hematopoietic cells and evaluate immune cell dynamics in the kidney. The findings showed that Bst1-deficient (Bst1-/-) mice were protected against renal bilateral IRI. Bone marrow chimera experiments revealed that Bst1 expression on hematopoietic cells, but not parenchymal cells, induced renal IRI. Bst1 was mainly found in B cells and neutrophils by flow cytometry of the spleen and bone marrow. In vitro, migration of neutrophils from Bst1-/- mice was suppressed, and adoptive transfer of neutrophils from wild-type Bst1+/+ mice abolished the renal protective effect in Bst1 knockout mice. In conclusion, the study demonstrated that Bst1-/- mice are protected against renal IRI and that Bst1 expression in neutrophils plays a crucial role in inducing renal IRI. These findings suggest that targeting Bst1 in neutrophils could be a potential therapeutic strategy for AKI.NEW & NOTEWORTHY Acute kidney injury (AKI), a serious disease for which there is no effective Federal Drug Administration-approved treatment, is associated with high mortality rates. Bone marrow stromal cell antigen-1 (Bst1) is a cell surface molecule that can cause kidney fibrosis, but its role in AKI is largely unknown. Our study showed that Bst1-/- mice revealed a protective effect against renal bilateral ischemia-reperfusion injury (IRI). Adoptive transfer studies confirmed that Bst1 expression in hematopoietic cells, especially neutrophils, contributed to renal bilateral IRI.
Assuntos
Injúria Renal Aguda , Células-Tronco Mesenquimais , Traumatismo por Reperfusão , Camundongos , Animais , Injúria Renal Aguda/genética , Injúria Renal Aguda/prevenção & controle , Rim/metabolismo , Traumatismo por Reperfusão/genética , Traumatismo por Reperfusão/prevenção & controle , Neutrófilos/metabolismo , Camundongos Knockout , Células-Tronco Mesenquimais/metabolismo , Camundongos Endogâmicos C57BLRESUMO
Histone H3 lysine-9 di-methylation (H3K9me2) and lysine-27 tri-methylation (H3K27me3) are linked to repression of gene expression, but the functions of repressive histone methylation dynamics during inflammatory responses remain enigmatic. Here, we report that lysine demethylases 7A (KDM7A) and 6A (UTX) play crucial roles in tumor necrosis factor (TNF)-α signaling in endothelial cells (ECs), where they are regulated by a novel TNF-α-responsive microRNA, miR-3679-5p. TNF-α rapidly induces co-occupancy of KDM7A and UTX at nuclear factor kappa-B (NF-κB)-associated elements in human ECs. KDM7A and UTX demethylate H3K9me2 and H3K27me3, respectively, and are both required for activation of NF-κB-dependent inflammatory genes. Chromosome conformation capture-based methods furthermore uncover increased interactions between TNF-α-induced super enhancers at NF-κB-relevant loci, coinciding with KDM7A and UTX recruitments. Simultaneous pharmacological inhibition of KDM7A and UTX significantly reduces leukocyte adhesion in mice, establishing the biological and potential translational relevance of this mechanism. Collectively, these findings suggest that rapid erasure of repressive histone marks by KDM7A and UTX is essential for NF-κB-dependent regulation of genes that control inflammatory responses of ECs.
Assuntos
Células Endoteliais/imunologia , Histona Desmetilases/metabolismo , Histonas/metabolismo , Histona Desmetilases com o Domínio Jumonji/metabolismo , MicroRNAs/genética , Animais , Adesão Celular , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Histonas/química , Células Endoteliais da Veia Umbilical Humana , Humanos , Lisina/metabolismo , Masculino , Metilação , Camundongos , Transdução de Sinais , Fator de Necrose Tumoral alfa/metabolismoRESUMO
The immune system is involved in hypertension development with different immune cells reported to have either pro or anti-hypertensive effects. In hypertension, immune cells have been thought to infiltrate blood pressure-regulating organs, resulting in either elevation or reduction of blood pressure. There is controversy over whether macrophages play a detrimental or beneficial role in the development of hypertension, and the few existing studies have yielded conflicting results. This study aimed to determine the effects of angiotensin II (Ang II) salt-induced hypertension on renal immune cells and to determine whether renal macrophages are involved in the induction of hypertension. Hypertension was induced by administration of Ang II and saline for two weeks. The effects of hypertension on kidney immune cells were assessed using flow cytometry. Macrophage infiltration in the kidney was assessed by immunohistochemistry and kidney fibrosis was assessed using trichrome stain and kidney real time-qPCR. Liposome encapsulated clodronate was used to deplete macrophages in C57BL/6J mice and investigate the direct role of macrophages in hypertension induction. Ang II saline mice group developed hypertension, had increased renal macrophages, and had increased expression of Acta2 and Col1a1 and kidney fibrotic areas. Macrophage depletion blunted hypertension development and reduced the expression of Acta2 and Col1a1 in the kidney and kidney fibrotic areas in Ang II saline group. The results of this study demonstrate that macrophages infiltrate the kidneys and increase kidney fibrosis in Ang II salt-induced hypertension, and depletion of macrophages suppresses the development of hypertension and decreases kidney fibrosis. This indicates that macrophages play a direct role in hypertension development. Hence macrophages have a potential to be considered as therapeutic target in hypertension management.
Assuntos
Angiotensina II , Modelos Animais de Doenças , Fibrose , Hipertensão , Rim , Macrófagos , Animais , Camundongos , Angiotensina II/farmacologia , Pressão Sanguínea/efeitos dos fármacos , Hipertensão/induzido quimicamente , Hipertensão/patologia , Hipertensão/metabolismo , Rim/patologia , Rim/metabolismo , Rim/efeitos dos fármacos , Nefropatias/induzido quimicamente , Nefropatias/patologia , Nefropatias/metabolismo , Nefropatias/etiologia , Macrófagos/metabolismo , Macrófagos/patologia , Macrófagos/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Cloreto de Sódio na Dieta/efeitos adversosRESUMO
Acute kidney injury is highly prevalent and associated with high morbidity and mortality, and there are no approved drugs for its prevention and treatment. Vagus nerve stimulation (VNS) alleviates inflammatory diseases including kidney disease; however, neural circuits involved in VNS-induced tissue protection remain poorly understood. The vagus nerve, a heterogeneous group of neural fibers, innervates numerous organs. VNS broadly stimulates these fibers without specificity. We used optogenetics to selectively stimulate vagus efferent or afferent fibers. Anterograde efferent fiber stimulation or anterograde (centripetal) sensory afferent fiber stimulation both conferred kidney protection from ischemia-reperfusion injury. We identified the C1 neurons-sympathetic nervous system-splenic nerve-spleen-kidney axis as the downstream pathway of vagus afferent fiber stimulation. Our study provides a map of the neural circuits important for kidney protection induced by VNS, which is critical for the safe and effective clinical application of VNS for protection from acute kidney injury.
Assuntos
Injúria Renal Aguda/etiologia , Suscetibilidade a Doenças , Neuroimunomodulação , Baço/imunologia , Baço/inervação , Estimulação do Nervo Vago , Injúria Renal Aguda/metabolismo , Injúria Renal Aguda/patologia , Animais , Camundongos , Neurônios , Sistema Nervoso Simpático/fisiologiaRESUMO
There is still no established treatment for acute kidney injury (AKI), and the intervention of AKI remains limited to supportive treatments. Li et al. demonstrated the mechanism by which immune tolerance by dendritic cell ameliorates AKI in a mouse ischemia-reperfusion injury model. The phase I/II clinical trials of tolerogenic dendritic cell therapy have been conducted for kidney transplantation, so it is expected to have potential as a cell therapy for AKI in the future.
Assuntos
Injúria Renal Aguda , Transplante de Rim , Animais , Camundongos , Injúria Renal Aguda/terapia , Terapia Baseada em Transplante de Células e Tecidos , Modelos Animais de Doenças , Células DendríticasRESUMO
The autonomic nervous system plays an important role in the regulation of peripheral inflammation. Sympathetic nervous activation stimulates inflammatory gene expression and cytokines, whereas parasympathetic nervous activation suppresses the production of inflammatory cytokines by immune cells. However, most studies on the relationship between the autonomic nervous system and immune processes have analyzed a single branch of the autonomic nerves in isolation. Therefore, this study aimed to examine the effects of sympathetic and parasympathetic stimulation on macrophages, which are controlled by autonomic regulation. Macrophages were stimulated with lipopolysaccharide (LPS) to induce TNF-α. Then, the effects of ß2 adrenergic receptor and α7 nicotinic acetylcholine receptor activation on TNF-α production were assessed using concentration-dependent assays. RNA-seq data were also used to identify genes whose expression was enhanced by parasympathetic and sympathetic stimulation. The simultaneous activation of ß2 adrenergic receptors and α7 nicotinic acetylcholine receptors suppressed LPS-induced TNF-α production in a concentration-dependent manner. Moreover, simultaneous activation of these receptors had synergistic anti-inflammatory effects and induced Tspan13 expression, thereby contributing to anti-inflammatory mechanisms in macrophages. Our study revealed the synergistic anti-inflammatory effects of the parasympathetic and sympathetic stimulation of macrophages. Our results suggest that targeting both sympathetic and parasympathetic signaling is a promising therapeutic approach for inflammatory diseases.
Assuntos
Receptores Nicotínicos , Fator de Necrose Tumoral alfa , Lipopolissacarídeos/farmacologia , Receptor Nicotínico de Acetilcolina alfa7 , Macrófagos , Citocinas , Anti-Inflamatórios , TetraspaninasRESUMO
The immune system is known to be controlled by the autonomic nervous system including sympathetic and parasympathetic (vagus) nerves. C1 neurons in the medulla oblongata, which participate in the control of the autonomic nervous system, are responders to stressors and regulate the immune system. Short-term activation of C1 neurons suppresses inflammation, while the effect of a long-term activation of these neurons on the inflammatory reflex is unclear. We, herein, demonstrate that the coactivation of both the splenic sympathetic nerves and the adrenal gland adrenergic response are indispensable for the prognosis of acute lung injury. The chemogenetic activation of C1 neurons increased plasma catecholamine including adrenaline and noradrenaline levels. The deletion of catecholaminergic cells using local injections of viral vector in the adrenal gland abolished the protective effect against acute lung injury when the C1 neurons were stimulated by either chemogenetic or optogenetic tools. Furthermore, repeated activation of C1 neurons using chemogenetic tool inhibited the adrenal response without affecting the plasma noradrenaline levels, eliminated the protective effect against acute lung injury. This was rescued by the isoprenaline administration. We concluded that the maintenance of an adrenergic response via C1 neurons in the adrenal gland is a prerequisite for the delivery of an effective anti-inflammatory response.
Assuntos
Adrenérgicos , Neurônios , Adrenérgicos/farmacologia , Bulbo/fisiologia , Glândulas Suprarrenais , Norepinefrina/farmacologia , Anti-Inflamatórios/farmacologiaRESUMO
Copper-containing nitrite reductases (CuNIRs) transform nitrite to gaseous nitric oxide, which is a key process in the global nitrogen cycle. The catalytic mechanism has been extensively studied to ultimately achieve rational control of this important geobiochemical reaction. However, accumulated structural biology data show discrepancies with spectroscopic and computational studies; hence, the reaction mechanism is still controversial. In particular, the details of the proton transfer involved in it are largely unknown. This situation arises from the failure of determining positions of hydrogen atoms and protons, which play essential roles at the catalytic site of CuNIRs, even with atomic resolution X-ray crystallography. Here, we determined the 1.50 Å resolution neutron structure of a CuNIR from Geobacillus thermodenitrificans (trimer molecular mass of â¼106 kDa) in its resting state at low pH. Our neutron structure reveals the protonation states of catalytic residues (deprotonated aspartate and protonated histidine), thus providing insights into the catalytic mechanism. We found that a hydroxide ion can exist as a ligand to the catalytic Cu atom in the resting state even at a low pH. This OH-bound Cu site is unexpected from previously given X-ray structures but consistent with a reaction intermediate suggested by computational chemistry. Furthermore, the hydrogen-deuterium exchange ratio in our neutron structure suggests that the intramolecular electron transfer pathway has a hydrogen-bond jump, which is proposed by quantum chemistry. Our study can seamlessly link the structural biology to the computational chemistry of CuNIRs, boosting our understanding of the enzymes at the atomic and electronic levels.
Assuntos
Cobre/química , Cristalografia/métodos , Geobacillus/enzimologia , Nitrito Redutases/química , Nitrito Redutases/metabolismo , Domínio Catalítico , Cristalização , Regulação Bacteriana da Expressão Gênica/fisiologia , Regulação Enzimológica da Expressão Gênica/fisiologia , Geobacillus/genética , Geobacillus/metabolismo , Modelos Moleculares , Nitrito Redutases/genética , Conformação ProteicaRESUMO
BACKGROUND: Oral care with gel is a common method for preventing aspiration in high-risk patients. An oral care gel is used to clean and moisturize the oral cavity. However, the effects of gel care on the oral bacteria remain unclear. In this pilot study, we described a matching transformation system (MA-T) for elderly high-risk patients. MA-T is an on-demand aqueous chlorine dioxide solution that provides excellent safety and has various antimicrobial activities, even in the presence of abundant organic compounds. This study investigated the effects of MA-T gel in patients requiring nursing care. MATERIALS AND METHODS: Patients who were hospitalized for nursing care were included in this study. No drugs and foods were administered orally. Oral bacteria and intraoral humidity were examined by daily care using MA-T gel. Moreover, oral membranous substances were analyzed and material from the oral cavity was cultured on selective media for identifying opportunistic organisms. RESULTS: Membranous substances were present in the oral cavities of all patients. The number of bacteria decreased, and oral moisture improved, after treatment with MA-T gel. Moreover, oral humidity was also controlled with the continued use of MA-T gel. MA-T gels should be used not only for professional care but also on a daily basis for better oral care. Furthermore, the results of bacterial cultures showed that MA-T controls the propagation of opportunistic bacterial infections. CONCLUSION: Membranous substances may be observed in the oral cavity of individuals requiring nursing care for tube feeding. The results of this pilot study suggest that MA-T, a novel disinfectant, can be used for oral care in the elderly to reduce the risk of aspiration-pneumonia.
Assuntos
Desinfetantes , Idoso , Humanos , Projetos Piloto , Géis , Boca , PacientesRESUMO
Some patients with diabetic kidney disease (DKD) show a fast progression of kidney dysfunction and are known as a "fast decliner" (FD). Therefore, it is critical to understand pathomechanisms specific for fast decline. Here, we performed a comprehensive metabolomic analysis of patients with stage G3 DKD and identified increased urinary lysophosphatidylcholine (LPC) in fast decline. This was confirmed by quantification of urinary LPC using mass spectrometry and identified urinary LPC containing saturated fatty acids palmitic (16:0) and stearic (18:0) acids was increased in FDs. The upsurge in urinary LPC levels was correlated with a decline in estimated glomerular filtration rate after 2.5 years. To clarify a pathogenic role of LPC in FD, we studied an accelerated rat model of DKD and observed an increase in LPC (16:0) and (18:0) levels in the urine and kidney tubulointerstitium as the disease progressed. These findings suggested that local dysregulation of lipid metabolism resulted in excessive accumulation of this LPC species in the kidney. Our in vitro studies also confirmed LPC-mediated lipotoxicity in cultured proximal tubular cells. LPC induced accumulation of lipid droplets via activation of peroxisome proliferator-activated receptor-δ followed by upregulation of the lipid droplet membrane protein perilipin 2 and decreased autophagic flux, thereby inducing organelle stress and subsequent apoptosis. Thus, LPC (16:0) and (18:0) may mediate a fast progression of DKD and may serve as a target for novel therapeutic approaches.
Assuntos
Diabetes Mellitus , Nefropatias Diabéticas , Insuficiência Renal , Animais , Diabetes Mellitus/metabolismo , Nefropatias Diabéticas/patologia , Taxa de Filtração Glomerular , Humanos , Rim/patologia , Lisofosfatidilcolinas/metabolismo , RatosRESUMO
Neurogenic pulmonary oedema (NPE) is a life-threatening complication that develops rapidly and dramatically after an injury to the central nervous system (CNS). The autonomic system imbalance produced by severe brain damage may play an important role in the development of NPE. Activation of the sympathetic nervous system and inhibition of the vagus nerve system are essential prerequisites for autonomic system imbalance. The more severe the damage, the more pronounced the phenomenon. Sympathetic hyperactivity is associated with increased release of catecholamines from peripheral sympathetic nerve endings, which can cause dramatic changes in haemodynamics and cause pulmonary oedema. On the other hand, the abnormal inflammatory response caused by vagus nerve inhibition may also play an important role in the pathogenesis of NPE. The perspective of autonomic system imbalance seems to perfectly integrate the existing pathogenesis of NPE and can explain the entire development progression of NPE.
Assuntos
Edema Pulmonar , Sistema Nervoso Central , Humanos , Edema Pulmonar/etiologia , Sistema Nervoso Simpático , Nervo VagoRESUMO
Cholinergic anti-inflammatory pathway (CAP) describes a neuronal-inflammatory reflex centered on systemic cytokine regulation by α7 nicotinic acetylcholine receptor (α7nAChR) activation of spleen-residue macrophage. However, the CAP mechanism attenuating distal tissue inflammation, inducing a low level of systemic inflammation, is lesser known. In this study, we hypothesized that CAP regulates monocyte accessibility by influencing their adhesion to endothelial cells. Using RNA-seq analysis, we identified that α1,3-Fucosyltransferase 7 (FucT-VII), the enzyme required for processing selectin ligands, was significantly downregulated by α7nAChR agonist among other cell-cell adhesion genes. The α7nAChR agonist inhibited monocytic cell line U-937 binding to P-selectin and adhesion to endothelial cells. Furthermore, α7nAChR agonist selectivity was confirmed by α7nAChR knockdown assays, showing that FUT7 inhibition and adhesion attenuation by the agonist was abolished by siRNA targeting α7nAChR encoding gene. Consistently, FUT7 knockdown inhibited the adhesive properties of U-937 and prevented them to adhere to endothelial cells. Overexpression of FUT7 also abrogated the adhesion attenuation induced by GTS-21 indicating that FUT7 inhibition was sufficient for inhibiting adhesion by α7nAChR activation. Our work demonstrated that α7nAChR activation regulates monocyte adhesion to endothelial cells through FUT7 inhibition, providing a novel insight into the CAP mechanism.
Assuntos
Fucosiltransferases/antagonistas & inibidores , Células Endoteliais da Veia Umbilical Humana/citologia , Monócitos/citologia , Receptor Nicotínico de Acetilcolina alfa7/metabolismo , Compostos de Benzilideno/farmacologia , Adesão Celular/efeitos dos fármacos , Adesão Celular/genética , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Fucosiltransferases/metabolismo , Técnicas de Silenciamento de Genes , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Piridinas/farmacologia , Células U937 , Receptor Nicotínico de Acetilcolina alfa7/antagonistas & inibidoresRESUMO
Matching transformation system (MA-T), an on-demand aqueous chlorine dioxide solution, is an excellent safety disinfectant, because chlorine dioxide is not detected during storage or before use. The production of chlorine dioxide in MA-T is induced by a catalytic reaction in the presence of target microorganisms. In this study, we investigated MA-T disinfection of masks as a reuse method to eliminate mask shortages. After spraying Escherichia coli on sterilized surgical mask, samples (factitiously contaminated masks) were treated with MA-T spraying or immersion, and the bactericidal efficacy was assessed by culturing. Used surgical masks were also sprayed with MA-T or were immersed in MA-T, and then were cultured to verify the bactericidal effect. The performance of N95 masks was assessed before and after application of MA-T. After spraying with MA-T, the numbers of bacteria of factitiously contaminated masks and used masks were drastically reduced compared with control samples (not applicable and p = 0.002, respectively). After MA-T immersion, the bacterial counts of both masks (factitiously contaminated masks and used masks) were significantly reduced (both p = 0.002). Taken together, the disinfection test on factitiously contaminated with E. coli and used surgical masks showed that masks can be disinfected by MA-T spray and sterilized by immersion, respectively. The N95 mask performance test after 30 min of immersion in MA-T showed that MA-T disinfected the mask without degrading the performance of the mask. In conclusion, MA-T is useful for the reuse of masks because of its decontamination effect and safety while maintaining the function of the mask.
Assuntos
Desinfetantes , Escherichia coli , Antibacterianos , Bactérias , Desinfetantes/farmacologiaRESUMO
Although thousands of long noncoding RNAs (lncRNAs) are localized in the nucleus, only a few dozen have been functionally characterized. Here we show that nuclear enriched abundant transcript 1 (NEAT1), an essential lncRNA for the formation of nuclear body paraspeckles, is induced by influenza virus and herpes simplex virus infection as well as by Toll-like receptor3-p38 pathway-triggered poly I:C stimulation, resulting in excess formation of paraspeckles. We found that NEAT1 facilitates the expression of antiviral genes including cytokines such as interleukin-8 (IL8). We found that splicing factor proline/glutamine-rich (SFPQ), a NEAT1-binding paraspeckle protein, is a repressor of IL8 transcription, and that NEAT1 induction relocates SFPQ from the IL8 promoter to the paraspeckles, leading to transcriptional activation of IL8. Together, our data show that NEAT1 plays an important role in the innate immune response through the transcriptional regulation of antiviral genes by the stimulus-responsive cooperative action of NEAT1 and SFPQ.
Assuntos
Imunidade Inata/genética , Interleucina-8/genética , RNA Longo não Codificante/fisiologia , Proteínas de Ligação a RNA/metabolismo , Regulação da Expressão Gênica , Células HeLa , Herpesvirus Humano 1/imunologia , Humanos , Vírus do Sarampo/imunologia , Orthomyxoviridae/imunologia , Fator de Processamento Associado a PTB , Regiões Promotoras Genéticas , Transporte Proteico , RNA Longo não Codificante/genética , Transcrição GênicaRESUMO
BACKGROUND: The sympathetic nervous system regulates immune cell dynamics. However, the detailed role of sympathetic signaling in inflammatory diseases is still unclear because it varies according to the disease situation and responsible cell types. This study focused on identifying the functions of sympathetic signaling in macrophages in LPS-induced sepsis and renal ischemia-reperfusion injury (IRI). METHODS: We performed RNA sequencing of mouse macrophage cell lines to identify the critical gene that mediates the anti-inflammatory effect of ß2-adrenergic receptor (Adrb2) signaling. We also examined the effects of salbutamol (a selective Adrb2 agonist) in LPS-induced systemic inflammation and renal IRI. Macrophage-specific Adrb2 conditional knockout (cKO) mice and the adoptive transfer of salbutamol-treated macrophages were used to assess the involvement of macrophage Adrb2 signaling. RESULTS: In vitro, activation of Adrb2 signaling in macrophages induced the expression of T cell Ig and mucin domain 3 (Tim3), which contributes to anti-inflammatory phenotypic alterations. In vivo, salbutamol administration blocked LPS-induced systemic inflammation and protected against renal IRI; this protection was mitigated in macrophage-specific Adrb2 cKO mice. The adoptive transfer of salbutamol-treated macrophages also protected against renal IRI. Single-cell RNA sequencing revealed that this protection was associated with the accumulation of Tim3-expressing macrophages in the renal tissue. CONCLUSIONS: The activation of Adrb2 signaling in macrophages induces anti-inflammatory phenotypic alterations partially via the induction of Tim3 expression, which blocks LPS-induced systemic inflammation and protects against renal IRI.
RESUMO
The relevance of primary cilia shortening in kidney disease and its pathomechanism are largely unknown. Tubular damage in acute kidney injury (AKI) is strongly associated with mitochondrial dysfunction. Thus, we investigated the interaction between primary cilia and mitochondria in cisplatin-induced AKI mouse models. We observed that the expression of intraflagellar transport 88 (IFT88), a ciliary maintenance protein, was decreased in the renal cortex following tubular damage due to cisplatin-induced AKI. This result was consistent with the decreased IFT88 expression in cisplatin-treated RPTEC/TERT1 cells (human primary proximal tubular cells) parallel to the shortening of primary cilia, suggesting a causative link between tubular damage and IFT88-mediated cilia regulation. To address the effect of impaired primary cilia with decreased IFT88 expression on tubular function, RPTEC/TERT1 cells treated with cisplatin and knocked down for IFT88 using siRNA (IFT88-KD) were assessed for phenotypic changes and mitochondrial metabolic function. Both cisplatin and IFT88-KD caused primary cilia shortening, downregulated mitochondrial oxidative phosphorylation capacity, and had defective fatty acid oxidation and decreased ATP production. Furthermore, IFT88 overexpression enhanced mitochondrial respiration, which partially counteracted cisplatin-induced defective fatty acid oxidation. These results are indicative of the contribution of IFT88 to mitochondrial homeostasis. Our findings suggest that tubular mitochondrial dysfunction in cisplatin-induced AKI is mediated, at least in part, by a decrease in IFT88 expression with primary cilia shortening. That is, tubular mitochondrial damage followed by tubular injury in AKI may occur through alteration of IFT88 expression and subsequent ciliary shortening in tubular cells.NEW & NOTEWORTHY Here, we demonstrated organelle cross-talk between primary cilia and mitochondria of proximal tubular cells in cisplatin-induced acute kidney injury. The primary cilia-mitochondria interaction may open new avenues for the development of novel therapeutic approaches in the treatment of acute kidney injury.
Assuntos
Injúria Renal Aguda/metabolismo , Cílios/metabolismo , Cisplatino/farmacologia , Proteínas Supressoras de Tumor/metabolismo , Injúria Renal Aguda/induzido quimicamente , Animais , Apoptose/genética , Apoptose/fisiologia , Cílios/genética , Cisplatino/metabolismo , Células Epiteliais/metabolismo , Túbulos Renais/metabolismo , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Proteínas Supressoras de Tumor/genéticaRESUMO
The autonomic nervous system plays an important role in maintaining homeostasis in organisms. Recent studies have shown that it also controls inflammation by directly altering the function of the immune system. The cholinergic anti-inflammatory pathway (CAP) is one of the neural circuits operating through the vagus nerve. Acetylcholine released from the terminal of the vagus nerve, which is a parasympathetic nerve, acts on the α7 nicotinic acetylcholine receptor of macrophages and reduces inflammation in the body. Previous animal studies demonstrated that vagus nerve stimulation reduced renal ischemia-reperfusion injury. Furthermore, restraint stress and pulsed ultrasound had similar protective effects against kidney injury, which were mainly thought to be mediated by the CAP. Using optogenetics, which can stimulate specific nerves, it was also revealed that activation of the CAP by restraint stress was mediated by C1 neurons in the medulla oblongata. Nevertheless, there still remain many unclear points regarding the role of the nervous and immune systems in controlling renal diseases, and further research is needed.
Assuntos
Injúria Renal Aguda/fisiopatologia , Rim/inervação , Neuroimunomodulação , Neurônios/fisiologia , Acetilcolina/metabolismo , Animais , Humanos , Ondas Ultrassônicas , Estimulação do Nervo VagoRESUMO
The glycosylation of small hydrophobic compounds is catalyzed by uridine diphosphate glycosyltransferases (UGTs). Because glycosylation is an invaluable tool for improving the stability and water solubility of hydrophobic compounds, UGTs have attracted attention for their application in the food, cosmetics, and pharmaceutical industries. However, the ability of UGTs to accept and glycosylate a wide range of substrates is not clearly understood due to the existence of a large number of UGTs. PaGT2, a UGT from Phytolacca americana, can regioselectively glycosylate piceatannol but has low activity toward other stilbenoids. To elucidate the substrate specificity and catalytic mechanism, we determined the crystal structures of PaGT2 with and without substrates and performed molecular docking studies. The structures have revealed key residues involved in substrate recognition and suggest the presence of a nonconserved catalytic residue (His81) in addition to the highly conserved catalytic histidine in UGTs (His18). The role of the identified residues in substrate recognition and catalysis is elucidated with the mutational assay. Additionally, the structure-guided mutation of Cys142 to other residues, Ala, Phe, and Gln, allows PaGT2 to glycosylate resveratrol with high regioselectivity, which is negligibly glycosylated by the wild-type enzyme. These results provide a basis for tailoring an efficient glycosyltransferase.
Assuntos
Cristalografia por Raios X/métodos , Glicosiltransferases/metabolismo , Simulação de Acoplamento Molecular/métodos , Phytolacca americana/enzimologia , Proteínas de Plantas/metabolismo , Polifenóis/metabolismo , Difosfato de Uridina/metabolismo , Sequência de Aminoácidos , Glicosilação , Glicosiltransferases/genética , Mutação , Filogenia , Proteínas de Plantas/genética , Elementos Estruturais de Proteínas , Especificidade por SubstratoRESUMO
The ketogenic diet treatment is effective for drug-resistant epilepsy. Because its antiepileptic effect is associated with lactate dehydrogenase (LDH), drug development is possible by targeting LDH enzymes. Seizures in rodent models are suppressed by inhibiting LDH; however, it remains unclear whether LDH in the brain is changed by seizures. In the present study, we examined the expression of LDH subunits (LDHA and LDHB) in a chronic model of temporal lobe epilepsy, in which seizures were induced by the microinjection of kainate into the mouse hippocampus. Using Western blot analyses, we found that LDHA expression was increased in the hippocampus of the chronic seizure model, whereas LDHB expression was not. Lactate levels in the hippocampus were also increased in this seizure model, suggesting elevated LDH enzymatic activities. Furthermore, the inhibition of LDHA suppressed spontaneous paroxysmal discharges in vivo in the chronic seizure model. In conclusion, our results show that chronic seizures increase LDHA, and conversely, the inhibition of LDHA suppresses seizures, which supports LDHA as a molecular target for the development of new antiepileptic drugs.