RESUMO
The transition of fibroblasts into myofibroblasts is a crucial step in kidney fibrosis. However, the biological processes involved in this transdifferentiation are incompletely understood. In this study, we discovered that the midbody plays a role in the fibroblast-myofibroblast transition by mediating TGF-ß/Smad signaling. Combining bulk RNA-seq, histology, and the western blot of unilateral ureteral obstruction kidneys, we demonstrated that the pathway related to microtubules is implicated in kidney fibrosis, and the blocking of microtubule dynamics by colchicine improved kidney fibrosis. Subsequently, to explore microtubule-based organelles in detail, we cultured NRK-49F (rat kidney fibroblast cell line) and HKC-8 (human proximal tubule cell line) under transforming growth factor-ß1 (TGF-ß1) stimulation, which caused deciliation in both cell lines during epithelial-mesenchymal and fibroblast-myofibroblast transition. We identified another microtubule-based organelle, the midbody, whose formation is promoted by TGF-ß1 in fibroblasts as a result of proliferation in contrast to tubular cells. Notably, TGF-ß receptors were present in the midbody of both cell lines. In TGF-ß1-treated fibroblasts, colchicine or Hedgehog pathway inhibitor 4 impaired the midbody formation, and attenuated the upregulation of canonical TGF-ß/Smad signaling and α-SMA expression. These findings offer novel insight into the midbody as an active organelle involved in fibroblast-myofibroblast transition by mediating TGF-ß/Smad signaling, which could be a potential therapeutic target.
Assuntos
Nefropatias , Miofibroblastos , Animais , Colchicina/farmacologia , Transição Epitelial-Mesenquimal , Feminino , Fibroblastos/metabolismo , Fibrose , Proteínas Hedgehog/metabolismo , Humanos , Nefropatias/patologia , Masculino , Miofibroblastos/metabolismo , Ratos , Transdução de Sinais/fisiologia , Fator de Crescimento Transformador beta1/metabolismoRESUMO
Renal ischemia-reperfusion injury (IRI) is involved in the majority of clinical conditions that manifest as renal function deterioration; however, specific treatment for this type of injury is still far from clinical use. Since Toll-like receptor (TLR)-mediated signaling is a key mediator of IRI, we examined the effect of a multiple-TLR-blocking peptide named TLR-inhibitory peptide 1 (TIP1), which exerts the strongest action on TLR4, on renal IRI. We subjected C57BL/6 mice to 23 min of renal pedicle clamping preceded by intraperitoneal injection with a vehicle or TIP1. Sham control mice underwent flank incision only. Mouse kidneys were harvested after 24 h of reperfusion for histology, western blot, RT-PCR, and flow cytometry analysis. Pretreatment with TIP1 lowered the magnitude of elevated plasma creatinine levels and attenuated tubular injury. TIP1 treatment also reduced mRNA expression of inflammatory cytokines and decreased apoptotic cells and oxidative stress in post-ischemic kidneys. In kidneys pretreated with TIP1, the infiltration of macrophages and T helper 17 cells was less abundant than those in the IRI only group. These results suggest that TIP1 has a potential beneficial effect in attenuating the degree of kidney damage induced by IRI.
Assuntos
Injúria Renal Aguda/prevenção & controle , Peptídeos Penetradores de Células/administração & dosagem , Traumatismo por Reperfusão/prevenção & controle , Transdução de Sinais/efeitos dos fármacos , Injúria Renal Aguda/genética , Injúria Renal Aguda/metabolismo , Animais , Peptídeos Penetradores de Células/farmacologia , Creatinina/sangue , Citocinas/genética , Modelos Animais de Doenças , Regulação da Expressão Gênica/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Estresse Oxidativo/efeitos dos fármacos , Traumatismo por Reperfusão/genética , Traumatismo por Reperfusão/metabolismo , Receptores Toll-Like/genética , Receptores Toll-Like/metabolismoRESUMO
BACKGROUND: Oxidative stress induced by chronic hyperglycemia is recognized as a significant mechanistic contributor to the development of diabetic kidney disease (DKD). Nonphagocytic nicotinamide adenine dinucleotide phosphate oxidase 4 (Nox4) is a major source of reactive oxygen species (ROS) in many cell types and in the kidney tissue of diabetic animals. We designed this study to explore the therapeutic potential of chloroquine (CQ) and amodiaquine (AQ) for inhibiting mitochondrial Nox4 and diabetic tubular injury. METHODS: Human renal proximal tubular epithelial cells (hRPTCs) were cultured in high-glucose media (30 mM D-glucose), and diabetes was induced with streptozotocin (STZ, 50 mg/kg i.p. for 5 days) in male C57BL/6J mice. CQ and AQ were administered to the mice via intraperitoneal injection for 14 weeks. RESULTS: CQ and AQ inhibited mitochondrial Nox4 and increased mitochondrial mass in hRPTCs under high-glucose conditions. Reduced mitochondrial ROS production after treatment with the drugs resulted in decreased endoplasmic reticulum (ER) stress, suppressed inflammatory protein expression and reduced cell apoptosis in hRPTCs under high-glucose conditions. Notably, CQ and AQ treatment diminished Nox4 activation and ER stress in the kidneys of STZ-induced diabetic mice. In addition, we observed attenuated inflammatory protein expression and albuminuria in STZ-induced diabetic mice after CQ and AQ treatment. CONCLUSION: We substantiated the protective actions of CQ and AQ in diabetic tubulopathy associated with reduced mitochondrial Nox4 activation and ER stress alleviation. Further studies exploring the roles of mitochondrial Nox4 in the pathogenesis of DKD could suggest new therapeutic targets for patients with DKD.
Assuntos
Amodiaquina/farmacologia , Cloroquina/farmacologia , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Mitocôndrias/metabolismo , NADPH Oxidase 4/metabolismo , Amodiaquina/química , Amodiaquina/metabolismo , Amodiaquina/uso terapêutico , Animais , Apoptose/efeitos dos fármacos , Células Cultivadas , Cloroquina/química , Cloroquina/metabolismo , Cloroquina/uso terapêutico , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Experimental/patologia , Diabetes Mellitus Tipo 1/patologia , Glucose/farmacologia , Humanos , Túbulos Renais Proximais/citologia , Túbulos Renais Proximais/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , NADPH Oxidase 4/antagonistas & inibidores , Espécies Reativas de Oxigênio/metabolismoRESUMO
INTRODUCTION: A recent study showed that early renal tubular injury is ameliorated in Nod-like receptor pyrin domain-containing protein 3 (NLRP3) KO mice with rhabdomyolysis-induced acute kidney injury (RIAKI). However, the precise mechanism has not been determined. Therefore, we investigated the role of NLRP3 in renal tubular cells in RIAKI. METHODS: Glycerol-mediated RIAKI was induced in NLRP3 KO and wild-type (WT) mice. The mice were euthanized 24 h after glycerol injection, and both kidneys and plasma were collected. HKC-8 cells were treated with ferrous myoglobin to mimic a rhabdomyolytic environment. RESULTS: Glycerol injection led to increase serum creatinine, aspartate aminotransferase (AST), and renal kidney injury molecule-1 (KIM-1) level; renal tubular necrosis; and apoptosis. Renal injury was attenuated in NLRP3 KO mice, while muscle damage and renal neutrophil recruitment did not differ between NLRP3 KO mice and WT mice. Following glycerin injection, increases in cleaved caspase-3, poly (ADP-ribose) polymerase (PARP), and a decrease in the glutathione peroxidase 4 (GPX-4) level were observed in the kidneys of mice with RIAKI, and these changes were alleviated in the kidneys of NLRP3 KO mice. NLRP3 was upregulated, and cell viability was suppressed in HKC-8 cells treated with ferrous myoglobin. Myoglobin-induced apoptosis and lipid peroxidation were significantly decreased in siNLRP3-treated HKC-8 cells compared to ferrous myoglobin-treated HKC-8 cells. Myoglobin reduced the mitochondrial membrane potential and increased mitochondrial fission and reactive oxygen species (ROS) and lipid peroxidation levels, which were restored to normal levels in NLRP3-depleted HKC-8 cells. CONCLUSIONS: NLRP3 depletion ameliorated renal tubular injury in a murine glycerol-induced acute kidney injury (AKI) model. A lack of NLRP3 improved tubular cell viability via attenuation of myoglobin-induced mitochondrial injury and lipid peroxidation, which might be the critical factor in protecting the kidney.
Assuntos
Injúria Renal Aguda , Túbulos Renais , Peroxidação de Lipídeos , Mitocôndrias , Proteína 3 que Contém Domínio de Pirina da Família NLR/deficiência , Rabdomiólise , Injúria Renal Aguda/etiologia , Injúria Renal Aguda/genética , Injúria Renal Aguda/metabolismo , Injúria Renal Aguda/patologia , Animais , Túbulos Renais/metabolismo , Túbulos Renais/patologia , Camundongos , Camundongos Knockout , Mitocôndrias/genética , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Dinâmica Mitocondrial/genética , Mioglobina/genética , Mioglobina/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Rabdomiólise/complicações , Rabdomiólise/genética , Rabdomiólise/metabolismo , Rabdomiólise/patologiaRESUMO
During aging, the kidney undergoes functional and physiological changes that are closely affiliated with chronic kidney disease (CKD). There is increasing evidence supporting the role of lipid or lipid-derived mediators in the pathogenesis of CKD and other aging-related diseases. To understand the role of lipids in various metabolic processes during kidney aging, we conducted matrix-assisted laser desorption/ionization-imaging mass spectrometry (MALDI-IMS) analysis in kidneys harvested from young (2 months old, n = 3) and old mice (24 months old, n = 3). MALDI-IMS analysis showed an increase in ceramide level and a decrease in sphingomyelin (SM) and phosphatidylcholine (PC) levels in kidneys of old mice. The increased expression of cPLA2 and SMPD1 protein in aged kidney was confirmed by immunohistochemistry and Western blot analysis. Our MALDI-IMS data showed the altered distribution of lipids in aged kidney as indicative of aging-related functional changes of the kidney. Combined analysis of MALDI-IMS and IHC confirmed lipidomic changes and expression levels of responsible enzymes as well as morphological changes.
Assuntos
Envelhecimento , Rim/química , Lipidômica/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Animais , Ceramidas/metabolismo , Imuno-Histoquímica , Rim/diagnóstico por imagem , Camundongos , Fosfatidilcolinas/metabolismo , Fosfolipases A2/metabolismo , Esfingomielina Fosfodiesterase/metabolismo , Esfingomielinas/metabolismoRESUMO
We examined the effects of empagliflozin, a selective inhibitor of Na+-glucose cotransporter 2, on mitochondrial quality control and autophagy in renal tubular cells in a diabetic environment in vivo and in vitro. Human renal proximal tubular cells (hRPTCs) were incubated under high-glucose conditions. Diabetes was induced with streptozotocin in male C57BL/6J mice. Improvements in mitochondrial biogenesis and balanced fusion-fission protein expression were noted in hRPTCs after treatment with empagliflozin in high-glucose media. Empagliflozin also increased autophagic activities in renal tubular cells in the high-glucose environment, which was accompanied with mammalian target of rapamycin inhibition. Moreover, reduced mitochondrial reactive oxygen species production and decreased apoptotic and fibrotic protein expression were observed in hRPTCs after treatment with empagliflozin, even in the hyperglycemic circumstance. Importantly, empagliflozin restored AMP-activated protein kinase-α phosphorylation and normalized levels of AMP-to-ATP ratios in hRPTCs subjected to a high-glucose environment, which suggests the way that empagliflozin is involved in mitochondrial quality control. Empagliflozin effectively suppressed Na+-glucose cotransporter 2 expression and ameliorated renal morphological changes in the kidneys of streptozotocin-induced diabetic mice. Electron microscopy analysis showed that mitochondrial fragmentation was decreased and 8-hydroxy-2'-deoxyguanosine content was low in renal tubular cells of empagliflozin treatment groups compared with those of the diabetic control group. We suggest one mechanism related to the renoprotective actions of empagliflozin, which reverse mitochondrial dynamics and autophagy.
Assuntos
Autofagia/efeitos dos fármacos , Compostos Benzidrílicos/uso terapêutico , Nefropatias Diabéticas/tratamento farmacológico , Nefropatias Diabéticas/patologia , Glucosídeos/uso terapêutico , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/patologia , Inibidores do Transportador 2 de Sódio-Glicose/uso terapêutico , Monofosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Proteínas Reguladoras de Apoptose/antagonistas & inibidores , Linhagem Celular , Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/patologia , Expressão Gênica/efeitos dos fármacos , Humanos , Túbulos Renais Proximais/efeitos dos fármacos , Túbulos Renais Proximais/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Biogênese de Organelas , Espécies Reativas de Oxigênio/metabolismo , Transportador 2 de Glucose-Sódio/metabolismoRESUMO
The intratubular renin-angiotensin system (RAS) is thought to play an essential role in hypertensive renal disease, but information regarding sex-related differences in this system is limited. The present study investigated sex differences in the intratubular RAS in two-kidney, one-clip (2K1C) rats. A 2.5-mm clip was placed on the left renal artery of Sprague-Dawley rats, and rats were euthanized 3 or 5 wk after the operation. Systolic blood pressure increased in 2K1C rats in both sexes but was significantly higher in male rats than in female rats, and an antihypertensive effect was not observed in 2K1C ovariectomized (OVX) female rats. Compared with male 2K1C rats, intratubular angiotensin-converting enzyme (ACE) and ANG II were repressed, and intratubular ACE2, angiotensin (1-7), and Mas receptor were increased in both kidneys in female 2K1C rats 5 wk after surgery. Comparison with male and female rats and intratubular mRNA levels of ACE and ANG II type 1 receptor were augmented in OVX female rats, regardless of the clipping surgery 3 wk postoperation. ANG II type 2 receptor was upregulated in female rats with or without OVX; thus, the ANG II type 1-to-type 2 receptor ratio was higher in male rats than in female rats. In conclusion, female rats were protected from hypertensive renal and cardiac injury after renal artery clipping. An increase in the intratubular nonclassic RAS [ACE2/angiotensin (1-7)/Mas receptor] and a decrease in the ANG II type 1-to-type 2 receptor ratio could limit the adverse effects of the classic RAS during renovascular hypertension in female rats, and estrogen is suggested to play a primary role in the regulation of intratubular RAS components.
Assuntos
Pressão Sanguínea , Estrogênios/metabolismo , Hipertensão/metabolismo , Túbulos Renais/metabolismo , Artéria Renal/cirurgia , Sistema Renina-Angiotensina , Angiotensina I/metabolismo , Angiotensina II/metabolismo , Enzima de Conversão de Angiotensina 2 , Animais , Constrição , Modelos Animais de Doenças , Feminino , Hipertensão/etiologia , Hipertensão/genética , Hipertensão/fisiopatologia , Hipertrofia Ventricular Esquerda/etiologia , Hipertrofia Ventricular Esquerda/metabolismo , Hipertrofia Ventricular Esquerda/fisiopatologia , Túbulos Renais/fisiopatologia , Macrófagos/metabolismo , Masculino , Ovariectomia , Fragmentos de Peptídeos/metabolismo , Peptidil Dipeptidase A/genética , Peptidil Dipeptidase A/metabolismo , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Ratos Sprague-Dawley , Receptor Tipo 1 de Angiotensina/genética , Receptor Tipo 1 de Angiotensina/metabolismo , Receptor Tipo 2 de Angiotensina/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Artéria Renal/fisiopatologia , Fatores Sexuais , Transdução de SinaisRESUMO
BACKGROUND: Peritoneal fibrosis is a devastating complication of peritoneal dialysis. However, its precise mechanism is unclear, and specific treatments have not yet been established. Recent evidence suggests that the sonic hedgehog (SHH) signaling pathway is involved in tissue fibrogenesis. Drugs that inhibit this pathway are emerging in the field of anti-fibrosis therapy. Itraconazole, an anti-fungal agent, was also recently recognized as an inhibitor of the SHH signaling pathway. In this study, we used a mouse model to investigate whether the SHH signaling pathway is involved in the development of peritoneal fibrosis and the effects of itraconazole on peritoneal fibrosis. METHODS: Peritoneal fibrosis was induced by intraperitoneal (IP) injection of 0.1% chlorhexidine gluconate (CG) solution every other day for 4 weeks, with or without itraconazole treatment (20 mg/kg, IP injection on a daily basis). Male C57BL/6 mice were divided into 4 groups: saline group, saline plus itraconazole group, CG group, and CG plus itraconazole group. Isotonic saline was administered intraperitoneally to the control group. The peritoneal tissues were evaluated for histological changes, expression of fibrosis markers, and the main components of the SHH signaling pathway. RESULTS: Peritoneal thickening was evident in the CG group and was significantly decreased by itraconazole administration (80.4 ± 7.7 vs. 28.2 ± 3.8 µm, p < 0.001). The expression of the following SHH signaling pathway components was upregulated in the CG group and suppressed by itraconazole treatment: SHH, patched, smoothened, and glioma-associated oncogene transcription factor 1. The IP injection of CG solution increased the expression of fibrosis markers such as α-smooth muscle actin and transforming growth factor-ß1 in the peritoneal tissues. Itraconazole treatment significantly decreased the expression of these markers. CONCLUSION: Our study provides the first evidence that the SHH signaling pathway may be implicated in peritoneal fibrosis. It also demonstrates that itraconazole treatment has protective effects on peritoneal fibrosis through the regulation of the SHH signaling pathway. These findings suggest that blockage of the SHH signaling pathway is a potential therapeutic strategy for peritoneal fibrosis.
Assuntos
Proteínas Hedgehog/metabolismo , Itraconazol/farmacologia , Diálise Peritoneal/efeitos adversos , Fibrose Peritoneal/tratamento farmacológico , Transdução de Sinais/efeitos dos fármacos , Animais , Clorexidina/administração & dosagem , Clorexidina/análogos & derivados , Clorexidina/toxicidade , Modelos Animais de Doenças , Humanos , Injeções Intraperitoneais , Itraconazol/uso terapêutico , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fibrose Peritoneal/induzido quimicamente , Fibrose Peritoneal/patologia , Peritônio/efeitos dos fármacos , Peritônio/patologia , Resultado do TratamentoRESUMO
Early diagnosis of infectious diseases is important for treatment; therefore, selective and rapid detection of pathogenic bacteria is essential for human health. We report a strategy for highly selective detection and rapid separation of pathogenic microorganisms using magnetic nanoparticle clusters. Our approach to develop probes for pathogenic bacteria, including Salmonella, is based on a theoretically optimized model for the size of clustered magnetic nanoparticles. The clusters were modified to provide enhanced aqueous solubility and versatile conjugation sites for antibody immobilization. The clusters with the desired magnetic property were then prepared at critical micelle concentration (CMC) by evaporation-induced self-assembly (EISA). Two different types of target-specific antibodies for H- and O-antigens were incorporated on the cluster surface for selective binding to biological compartments of the flagella and cell body, respectively. For the two different specific binding properties, Salmonella were effectively captured with the O-antibody-coated polysorbate 80-coated magnetic nanoclusters (PCMNCs). The synergistic effect of combining selective targeting and the clustered magnetic probe leads to both selective and rapid detection of infectious pathogens.
Assuntos
Técnicas Bacteriológicas/métodos , Nanopartículas/química , Salmonella/isolamento & purificação , Anticorpos Antibacterianos/química , Técnicas Bacteriológicas/instrumentação , Espectroscopia de Ressonância Magnética , Magnetismo/instrumentação , Magnetismo/métodos , Polissorbatos/química , Salmonella/imunologia , Sorogrupo , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
Mitochondrial dysfunction plays a pivotal role in diabetic kidney disease initiation and progression. PTEN-induced serine/threonine kinase 1 (PINK1) is a core organizer of mitochondrial quality control; however, its function in diabetic kidney disease remains controversial. Here, we aimed to investigate the pathophysiological roles of PINK1 in diabetic tubulopathy, focusing on its effects on mitochondrial homeostasis and tubular cell necroptosis, which is a specialized form of regulated cell death. PINK1-knockout mice showed more severe diabetes-induced tubular injury, interstitial fibrosis, and albuminuria. The expression of profibrotic cytokines significantly increased in the kidneys of diabetic Pink1-/- mice, which eventually culminated in aggravated interstitial fibrosis. Additionally, the knockdown of PINK1 in HKC-8 cells upregulated the fibrosis-associated proteins, and these effects were rescued by PINK1 overexpression. PINK1 deficiency was also associated with exaggerated hyperglycemia-induced mitochondrial dysfunction and defective mitophagic activity, whereas PINK1 overexpression ameliorated these negative effects and restored mitochondrial homeostasis. Mitochondrial reactive oxygen species triggered tubular cell necroptosis under hyperglycemic conditions, which was aggravated by PINK1 deficiency and improved by its overexpression. In conclusion, PINK1 plays a pivotal role in suppressing mitochondrial dysfunction and tubular cell necroptosis under high glucose conditions and exerts protective effects in diabetic kidney disease.
Assuntos
Diabetes Mellitus , Nefropatias Diabéticas , Camundongos , Animais , Nefropatias Diabéticas/genética , Nefropatias Diabéticas/metabolismo , Necroptose/genética , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Mitocôndrias/metabolismo , Camundongos Knockout , Fibrose , Diabetes Mellitus/metabolismoRESUMO
The adsorption of peptides and proteins on hydrophobic solid surfaces has received considerable research attention owing to their wide applications to biocompatible nanomaterials and nanodevices, such as biosensors and cell adhesion materials with reduced nanomaterial toxicity. However, fundamental understandings about physicochemical hydrophobic interactions between peptides and hydrophobic solid surfaces are still unknown. In this study, we investigate the effect of secondary structures on adsorption energies between peptides and hydrophobic solid surfaces via experimental and theoretical analyses using surface-assisted laser desorption/ionization-time-of-flight (SALDI-TOF) and molecular dynamics (MD) simulations. The hydrophobic interactions between peptides and hydrophobic solid surfaces measured via SALDI-TOF and MD simulations indicate that the hydrophobic interaction of peptides with random coil structures increased more than that of peptides with an α-helix structure when polar amino acids are replaced with hydrophobic amino acids. Additionally, our study sheds new light on the fundamental understanding of the hydrophobic interaction between hydrophobic solid surfaces and peptides that have diverse secondary structures.
RESUMO
This Letter describes the novel radiosensitizing agents based on nucleoside base modification. In addition to the known 5-phenylselenide derivative, 5-methylselenide modified thymidine, which has a van der Waals radius smaller than the phenyl group, was newly synthesized. The similar monomer activity of 5-methylselenide derivative under oxidation condition was confirmed by NMR experiments. The cytotoxicity tests and radiosensitizing experiments of both compounds were carried out using the H460 lung cancer cell line. Both the 5-phenylselenide and the 5-methylselenide derivatives showed a relatively low toxicity to the cells. However, in combination with γ-radiolysis, both exerted good radiosensitizing effects to the lung cancer cell lines in vitro. This result confirms that 5-methylselenide modified thymidine could be a useful candidate as a potential radiosensitizing agent in vivo.
Assuntos
Desoxiuridina/análogos & derivados , Compostos Organosselênicos/química , Radiossensibilizantes/química , Linhagem Celular Tumoral , Desoxiuridina/síntese química , Desoxiuridina/química , Desoxiuridina/toxicidade , Raios gama , Humanos , Espectroscopia de Ressonância Magnética , Compostos Organosselênicos/síntese química , Compostos Organosselênicos/toxicidade , Oxirredução , Radiossensibilizantes/síntese química , Radiossensibilizantes/toxicidadeRESUMO
[This corrects the article DOI: 10.3389/fimmu.2021.656632.].
RESUMO
Urine has been regarded as a good resource based on the assumption that urine can directly reflect the state of the allograft or ongoing injury in kidney transplantation. Previous studies, suggesting the usefulness of urinary mRNA as a biomarker of acute rejection, imply that urinary mRNA mirrors the transcriptional activity of the kidneys. We selected 14 data-driven candidate genes through a meta-analysis and measured the candidate genes using quantitative PCR without pre-amplification in the cross-sectional specimens from Korean kidney transplant patients. Expression of 9/14 genes (CXCL9, CD3ϵ, IP-10, LCK, C1QB, PSMB9, Tim-3, Foxp3, and FAM26F) was significantly different between acute rejection and stable graft function with normal pathology and long-term graft survival in 103 training samples. CXCL9 was also distinctly expressed in allografts with acute rejection in in situ hybridization analysis. This result, consistent with the qPCR result, implies that urinary mRNA could reflect the magnitude of allograft injury. We developed an AR prediction model with the urinary mRNAs by a binary logistic regression and the AUC of the model was 0.89 in the training set. The model was validated in 391 independent samples, and the AUC value yielded 0.84 with a fixed manner. In addition, the decision curve analysis indicated a range of reasonable threshold probabilities for biopsy. Therefore, we suggest the urine mRNA signature could be used as a non-invasive monitoring tool of acute rejection for clinical application and could help determine whether to perform a biopsy in a recipient with increased creatinine.
Assuntos
Aloenxertos/imunologia , Biomarcadores , Rejeição de Enxerto/diagnóstico , Rejeição de Enxerto/etiologia , Transplante de Rim/efeitos adversos , Biópsia Líquida/métodos , RNA Mensageiro/genética , Doença Aguda , Adulto , Biomarcadores/urina , Ácidos Nucleicos Livres , Feminino , Humanos , Imuno-Histoquímica , Transplante de Rim/métodos , Masculino , Pessoa de Meia-Idade , Curva ROC , Reprodutibilidade dos TestesRESUMO
Approximately 5% of patients with IgA nephropathy (IgAN) exhibit mild mesangial lesions with acute onset nephrotic syndrome and diffuse foot process effacement representative of minimal change disease (MCD). It is not clear whether these unusual cases of IgAN with MCD (IgAN-MCD) are variant types of IgAN or coincidental deposition of IgA in patients with MCD. In a retrospective multicenter cohort study of 18 hospitals in Korea, we analyzed 46 patients with IgAN-MCD. Patients with endocapillary proliferation, segmental sclerosis, and crescent were excluded, and the clinical features and prognosis of IgAN-MCD were compared with those of pure MCD. In addition, we performed galactose-deficient IgA1 (KM55) staining to characterize IgAN-MCD. Among the 21,697 patients with glomerulonephritis enrolled in the database, 46 patients (0.21%) were diagnosed with IgAN-MCD, and 1610 patients (7.4%) with pure MCD. The 46 patients with IgAN-MCD accounted for 0.6% of primary IgAN patients (n = 7584). There was no difference in prognosis between patients with IgAN-MCD and those with only MCD. IgA and KM55 showed double positivity in all patients with IgAN-MCD (n = 4) or primary IgAN (n = 5) under double immunofluorescent staining. However, in four patients with lupus nephritis, mesangial IgA was deposited, but galactose-deficient-IgA1 (Gd-IgA1) was not. These findings suggest that IgAN-MCD is a dual glomerulopathy in which MCD was superimposed on possibly indolent IgAN. We confirmed by KM55 staining that IgAN-MCD is true IgAN, enabling better characterizations of the disease. Furthermore, IgAN-MCD shows a good prognosis when treated according to the usual MCD treatment modality.
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PURPOSE: The poor survival rate of hepatocellular carcinoma (HCC) is in part due to the inability to diagnose patients at an early stage. Therefore, the aim of this study was to search for candidate serum marker for HCC and to test their ability to distinguish a HCC from benign liver disease. EXPERIMENTAL DESIGN: Genome-wide analysis by a microarray in 40 HCC patients was done between HCC and paired nontumor liver tissues. Expression of cystatin B (CSTB) was examined by mRNA expression analysis and immunohistochemistry. The serum CSTB levels were measured using a sandwich ELISA method in four groups, including normal healthy subjects (group 1, n = 52) and patients with noncirrhotic chronic hepatitis (group 2, n = 53), cirrhosis (group 3, n = 43), and HCC (group 4, n = 62). RESULTS: Microarray and statistical analyses identified 248 genes that were expressed differently between HCC and nontumor liver tissues. One of them, CSTB, was expressed preferentially in the HCCs compared with the nontumor tissues, 36 of 45 specimens (80%) by Northern blot and semiquantitative reverse transcription-PCR analyses. The serum CSTB level was much higher in HCC patients than in those with nonmalignant chronic liver disease (groups 2 and 3; P < 0.0001). The receiver operating characteristic curve indicated 5.34 ng/mL to be the optimal value for CSTB, and the sensitivity and specificity for this CSTB value were 85.5% (95% confidence interval, 74.2-93.1%) and 53.1% (95% confidence interval, 42.7-63.4%), respectively, in distinguishing between patients with HCC and those with nonmalignant chronic liver disease. CONCLUSION: CSTB is specifically overexpressed in most HCCs and is also elevated in the serum of a large proportion of HCC patients. CSTB or the combination of CSTB and alpha-fetoprotein may be a useful marker for diagnosing patients with HCC with a high sensitivity.
Assuntos
Biomarcadores Tumorais/sangue , Carcinoma Hepatocelular/sangue , Cistatinas/sangue , Neoplasias Hepáticas/sangue , Adulto , Biomarcadores Tumorais/genética , Northern Blotting , Western Blotting , Carcinoma Hepatocelular/genética , Linhagem Celular Tumoral , Doença Crônica , Cistatina B , Cistatinas/biossíntese , Cistatinas/genética , Ensaio de Imunoadsorção Enzimática , Feminino , Expressão Gênica , Perfilação da Expressão Gênica , Humanos , Imuno-Histoquímica , Hepatopatias/sangue , Neoplasias Hepáticas/genética , Masculino , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/análise , Curva ROC , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sensibilidade e Especificidade , alfa-Fetoproteínas/análiseRESUMO
Binding of epidermal growth factor (EGF) to its cell surface receptor induces production of H2O2, which serves as an intracellular messenger. We have shown that exogenous H2O2 reversibly inactivates the phosphatidylinositol 4-phosphate [PtdIns(4)P] phosphatase Sac1 (suppressor of actin 1) at the Golgi complex of mammalian cells by oxidizing its catalytic cysteine residue and thereby increases both the amount of Golgi PtdIns(4)P and the rate of protein secretion. Here we investigated the effects of EGF on Sac1 oxidation and PtdIns(4)P abundance at the Golgi in A431 cells. EGF induced a transient increase in Golgi PtdIns(4)P as well as a transient oxidation of Sac1 in a manner dependent on elevation of the intracellular Ca2+ concentration and on H2O2. Oxidation of Sac1 occurred at the Golgi, as revealed with the use of the Golgi-confined Sac1-K2A mutant. Knockdown of Duox enzymes implicated these Ca2+-dependent members of the NADPH oxidase family as the major source of H2O2 for Sac1 oxidation. Expression of a Golgi-targeted H2O2 probe revealed transient EGF-induced H2O2 production at this organelle. Our findings have thus uncovered a previously unrecognized EGF signaling pathway that links intracellular Ca2+ mobilization to events at the Golgi including Duox activation, H2O2 production, Sac1 oxidation, and PtdIns(4)P accumulation.
Assuntos
Cálcio/metabolismo , Complexo de Golgi/efeitos dos fármacos , Peróxido de Hidrogênio/metabolismo , Proteínas de Membrana/genética , Fosfatos de Fosfatidilinositol/metabolismo , Linhagem Celular Tumoral , Oxidases Duais/antagonistas & inibidores , Oxidases Duais/genética , Oxidases Duais/metabolismo , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Receptores ErbB/farmacologia , Regulação da Expressão Gênica , Complexo de Golgi/metabolismo , Humanos , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/metabolismo , Oxirredução , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de SinaisRESUMO
Endocan is a water-soluble proteoglycan exclusively secreted by vascular endothelium. Endocan levels may be elevated in kidney transplant recipients experiencing antibody-mediated rejection (ABMR), which is characterized by vascular inflammation in transplanted kidney. We evaluated the clinical relevance of endocan as markers of microvascular inflammation in patients who underwent kidney transplantation. Plasma and urinary endocan levels were measured in 203 kidney transplant recipients and were compared across different etiologies of allograft dysfunction and various pathologic scores. Both plasma and urinary endocan levels were significantly higher in patients with acute ABMR than those in patients with normal pathology, acute tubular necrosis (ATN), acute pyelonephritis, BK virus associated nephropathy (BKVN), and T-cell mediated rejection (TCMR). Patients with chronic active ABMR also exhibited significantly higher plasma and urinary endocan levels than patients with long-term graft survival. Scores of glomerulitis and peritubular capillaritis, which are typical features of microvascular inflammation, were significantly elevated in patients with higher plasma and/or urinary endocan levels. Furthermore, plasma and urinary endocan levels could effectively discriminate ABMR from ATN, BKVN, and TCMR. Finally, patients exhibiting high urinary and plasma endocan levels in acute ABMR group showed significantly worse renal survival. Altogether, plasma and urinary endocan levels may serve as potential markers of microvascular inflammation in kidney transplant recipients.
Assuntos
Inflamação/imunologia , Falência Renal Crônica/cirurgia , Transplante de Rim , Microcirculação/imunologia , Proteínas de Neoplasias/sangue , Proteínas de Neoplasias/urina , Proteoglicanas/sangue , Proteoglicanas/urina , Adulto , Área Sob a Curva , Biópsia , Feminino , Rejeição de Enxerto , Humanos , Masculino , Pessoa de Meia-Idade , Necrose , Infecções por Polyomavirus/metabolismo , Pielonefrite/imunologia , Curva ROC , Estudos Retrospectivos , Linfócitos T/citologia , Transplantados , Resultado do Tratamento , Infecções Tumorais por Vírus/imunologiaRESUMO
PURPOSE: Cyclin B2, a G(2)-M cyclin, is overexpressed in colorectal adenocarcinomas compared with the normal mucosa. This study examined the level of cyclin B2 overexpression according to the histologic findings and investigated the mechanism(s) and clinical implications of cyclin B2 overexpression in colorectal adenocarcinomas. EXPERIMENTAL DESIGN: The immunoreactivity of the polyclonal antibodies to cyclin B2 was determined in colorectal cancer cells. The transcriptional regulation of cyclin B2 by NF-Y was analyzed using an in vitro transfection assay and an in vivo chromatin immunoprecipitation assay. The proliferative activity of the colorectal cancer cells in relation to cyclin B2 overexpression was further examined. RESULTS: The cytoplasmic distribution of cyclin B2 immunoreactivity was positive in 42 of 65 (64.6%) cases of colorectal adenocarcinoma, and the level was similar regardless of the histologic type. A dominant-negative form of NF-YA effectively inhibited the cyclin B2 promoter activity, and NF-Y was found to bind three conserved CCAAT boxes in the cyclin B2 promoter in colorectal adenocarcinoma cells. Tumor cells with a higher functional cyclin B2 activity grew faster than those with a lower activity. Furthermore, there was a correlation between the cells showing immunoreactivity to cyclin B2 and those containing the proliferating cell nuclear antigen, a G1-S cyclin, which is also downstream of NF-Y in colorectal adenocarcinoma cells. CONCLUSIONS: Cyclin B2 seems to be a molecular marker of a colorectal adenocarcinoma and that its up-regulation and coordinate expression of the other cell cycle-related genes by NF-Y might contribute to tumor cell proliferation by accelerating cell cycle progression.
Assuntos
Fator de Ligação a CCAAT/fisiologia , Neoplasias Colorretais/metabolismo , Ciclina B/biossíntese , Regulação Neoplásica da Expressão Gênica , Adulto , Idoso , Fator de Ligação a CCAAT/metabolismo , Linhagem Celular Tumoral , Neoplasias Colorretais/patologia , Ciclina B2 , Citoplasma/metabolismo , Feminino , Neoplasias Gastrointestinais/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Regiões Promotoras Genéticas , Distribuição Tecidual , Transcrição GênicaRESUMO
The NOD-like receptor family, pyrin domain containing-3 (NLRP3) inflammasome has been implicated in renal inflammation and fibrosis. However, the biological function of inflammasome-independent NLRP3 in non-immune cells is still unclear. We evaluated the role of inflammasome-independent NLRP3 in renal tubular cells and assessed the value of NLRP3 as a therapeutic target for acute kidney injury (AKI). Various renal tubular cell lines and primary cultured tubular cells from NLRP3 knockout (KO) mice were used for in vitro studies. We also tested the role of tubular NLRP3 in AKI with a unilateral ureter obstruction model (UUO). Hypoxia induced significant increase of NLRP3 independent of ASC, caspase-1, and IL-1ß. NLRP3 in renal tubular cells relocalized from the cytosol to the mitochondria during hypoxia and bound to mitochondrial antiviral signal protein (MAVS). The deletion of NLRP3 or MAVS in renal tubular cells attenuated mitochondrial reactive oxygen species (ROS) production and depolarization of the mitochondrial membrane potentials under hypoxia. In response to UUO, NLRP3 KO mice showed less fibrosis, apoptosis, and ROS injury than wild type (WT) mice. Compared with WT kidney, mitophagy was up-regulated in NLRP3 KO kidney relative to the baseline and it was protective against AKI. Our results indicate that inflammasome-independent NLRP3 in renal tubular cells plays important role in mitochondrial ROS production and injury by binding to MAVS after hypoxic injury. This mitochondrial regulation in the absence of NLRP3 increases autophagy and attenuates apoptosis after UUO. We suggest that inflammasome-independent NLRP3 could be a therapeutic target of AKI to prevent the progression of chronic kidney disease.