RESUMO
BACKGROUND: The protection of the diabetic kidney by Empagliflozin (EMPA) is attributed to its interaction with the sodium glucose cotransporter 2 located on proximal tubular epithelial cells (PTECs). Estrogen-related receptor α (ESRRA), known for its high expression in PTECs and association with mitochondrial biogenesis, plays a crucial role in this process. This study aimed to explore the impact of ESRRA on mitochondrial mass in diabetic tubular injury and elucidate the mechanism underlying the protective effects of EMPA. METHODS: Mitochondrial changes in PTECs of 16-week-old diabetic mice were assessed using transmission electron microscopy (TEM) and RNA-sequences. In vivo, EMPA administration was carried out in db/db mice for 8 weeks, while in vitro experiments involved modifying ESRRA expression in HK2 cells using pcDNA-ESRRA or EMPA. RESULTS: Evaluation through TEM revealed reduced mitochondrial mass and swollen mitochondria in PTECs, whereas no significant changes were observed under light microscopy. Analysis of RNA-sequences identified 110 downregulated genes, including Esrra, associated with mitochondrial function. Notably, ESRRA overexpression rescued the loss of mitochondrial mass induced by high glucose (HG) in HK2 cells. EMPA treatment ameliorated the ultrastructural alterations and mitigated the downregulation of ESRRA both in db/db mice and HG-treated HK2 cells. CONCLUSION: The diminished expression of ESRRA is implicated in the decline of mitochondrial mass in PTECs during the early stages of diabetes, highlighting it as a key target of EMPA for preventing the progression of diabetic kidney injury.
Assuntos
Compostos Benzidrílicos , Diabetes Mellitus Experimental , Nefropatias Diabéticas , Glucosídeos , Mitocôndrias , Animais , Glucosídeos/farmacologia , Glucosídeos/uso terapêutico , Mitocôndrias/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/ultraestrutura , Compostos Benzidrílicos/farmacologia , Camundongos , Nefropatias Diabéticas/tratamento farmacológico , Nefropatias Diabéticas/metabolismo , Nefropatias Diabéticas/patologia , Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Experimental/patologia , Diabetes Mellitus Experimental/metabolismo , Masculino , Receptor ERRalfa Relacionado ao Estrogênio , Humanos , Túbulos Renais Proximais/efeitos dos fármacos , Túbulos Renais Proximais/patologia , Túbulos Renais Proximais/metabolismo , Túbulos Renais Proximais/ultraestrutura , Camundongos Endogâmicos C57BL , Linhagem Celular , Receptores de Estrogênio/metabolismoRESUMO
Tris(2-chloroethyl) phosphate (TCEP) and tris(1-chloro-2-propyl) phosphate (TCPP) are common chlorinated organophosphorus flame retardants (OPFRs) used in industry. They have been frequently detected together in aquatic environments and associated with various hazardous effects. However, the ecological risks of prolonged exposure to these OPFRs at environmentally relevant concentrations in non-model aquatic organisms remain unexplored. This study investigated the effects of long-term exposure (up to 25 days) to TCEP and TCPP on metamorphosis, hepatic antioxidants, and endocrine function in Polypedates megacephalus tadpoles. Exposure concentrations were set at 3, 30, and 90 µg/L for each substance, conducted independently and in equal-concentration combinations, with a control group included for comparison. The integrated biomarker response (IBR) method developed an optimal linear model for predicting the overall ecological risks of TCEP and TCPP to tadpoles in potential distribution areas of Polypedates species. Results showed that: (1) Exposure to environmentally relevant concentrations of TCEP and TCPP elicited variable adverse effects on tadpole metamorphosis time, hepatic antioxidant enzyme activity and related gene expression, and endocrine-related gene expression, with their combined exposure exacerbating these effects. (2) The IBR value of TCEP was consistently greater than that of TCPP at each concentration, with an additive effect observed under their combined exposure. (3) The ecological risk of tadpoles exposed to the combined presence of TCEP and TCPP was highest in China's Taihu Lake and Vietnam's Hanoi than in other distribution locations. In summary, prolonged exposure to environmentally relevant concentrations of TCEP and TCPP presents potential ecological risks to amphibian tadpoles, offering insights for the development of policies and strategies to control TCEP and TCPP pollution in aquatic ecosystems. Furthermore, the methodology employed in establishing the IBR prediction model provides a methodological framework for assessing the overall ecological risks of multiple OPFRs.
Assuntos
Biomarcadores , Retardadores de Chama , Larva , Organofosfatos , Compostos Organofosforados , Poluentes Químicos da Água , Animais , Larva/efeitos dos fármacos , Larva/crescimento & desenvolvimento , Poluentes Químicos da Água/toxicidade , Retardadores de Chama/toxicidade , Biomarcadores/metabolismo , Medição de Risco , Compostos Organofosforados/toxicidade , Organofosfatos/toxicidade , Exposição Ambiental , Anuros , Metamorfose Biológica/efeitos dos fármacos , Monitoramento Ambiental/métodosRESUMO
Renal tubular epithelial cells (TECs) undergo an energy-related metabolic shift from fatty acid oxidation to glycolysis during chronic kidney disease (CKD) progression. However, the mechanisms underlying this burst of glycolysis remain unclear. Herein, a new critical glycolysis regulator, the transcription factor forkhead box protein K1 (FOXK1) that is expressed in TECs during renal fibrosis and exhibits fibrogenic and metabolism-rewiring capacities is reported. Genetic modification of the Foxk1 locus in TECs alters glycolytic metabolism and fibrotic lesions. A surge in the expression of a set of glycolysis-related genes following FOXK1 protein activation contributes to the energy-related metabolic shift. Nuclear-translocated FOXK1 forms condensate through liquid-liquid phase separation (LLPS) to drive the transcription of target genes. Core intrinsically disordered regions within FOXK1 protein are mapped and validated. A therapeutic strategy is explored by targeting the Foxk1 locus in a murine model of CKD by the renal subcapsular injection of a recombinant adeno-associated virus 9 vector encoding Foxk1-short hairpin RNA. In summary, the mechanism of a FOXK1-mediated glycolytic burst in TECs, which involves the LLPS to enhance FOXK1 transcriptional activity is elucidated.
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Tris(2-chloroethyl) phosphate (TCEP), a chlorinated organophosphate ester, is commonly found in aquatic environments. Due to its various toxic effects, it may pose a risk to the health of aquatic organisms. However, the potential impacts of TCEP exposure on the intestinal microbiota and hepatic function in amphibians have not been reported. This study investigated the impact of long-term exposure to environmentally relevant concentrations of TCEP (0, 3, and 90 µg/L) on the intestinal microbiota and hepatic transcriptome of Polypedates megacephalus tadpoles. The results showed that the body size of the tadpoles decreased significantly with an increase in TCEP concentration. Additionally, TCEP exposure affected the diversity and composition of the intestinal microbiota in tadpoles, leading to significant changes in the relative abundance of certain bacterial groups (the genera Aeromonas decreased and Citrobacter increased) and potentially promoting a more even distribution of microbial species, as indicated by a significant increase in the Simpson index. Moreover, the impact of TCEP on hepatic gene expression profiles in tadpoles was significant, with the majority of differentially expressed genes (DEGs) (709 out of 906 total DEGs in 3 µg/L of TCEP versus control, and 344 out of 387 DEGs in 90 µg/L of TCEP versus control) being significantly down-regulated, which were primarily related to immune response and immune system process. Notably, exposure to TCEP significantly reduced the relative abundance of the genera Aeromonas and Cetobacterium in the tadpole intestine. This reduction was positively correlated with the down-regulated expression of immune-related genes in the liver of corresponding tadpoles. In summary, these findings provide empirical evidence of the potential health risks to tadpoles exposed to TCEP at environmentally relevant concentrations.
Assuntos
Microbioma Gastrointestinal , Larva , Fígado , Transcriptoma , Poluentes Químicos da Água , Animais , Microbioma Gastrointestinal/efeitos dos fármacos , Transcriptoma/efeitos dos fármacos , Poluentes Químicos da Água/toxicidade , Larva/efeitos dos fármacos , Fígado/efeitos dos fármacos , Anuros , OrganofosfatosRESUMO
Tris(2-chloroethyl) phosphate (TCEP) and tris(1chloro-2-propyl) phosphate (TCPP) are widely used as chlorinated organophosphate flame retardants (OPFRs) due to their fire-resistance capabilities. However, their extensive use has led to their permeation and pollution in aquatic environments. Using amphibians, which are non-model organisms, to test the toxic effects of OPFRs is relatively uncommon. This study examined the acute and chronic toxicity differences between TCEP and TCPP on Polypedates megacephalus tadpoles and evaluated the potential ecological risks to tadpoles in different aquatic environments using the risk quotient (RQ). In acute toxicity assay, the tadpole survival rates decreased with increased exposure time and concentrations, with TCEP exhibiting higher LC50 values than TCPP, at 305.5 mg/L and 70 mg/L, respectively. In the chronic assay, prolonged exposure to 300 µg/L of both substances resulted in similar adverse effects on tadpole growth, metamorphosis, and hepatic antioxidant function. Based on RQ values, most aquatic environments did not pose an ecological risk to tadpoles. However, the analysis showed that wastewater presented higher risks than rivers and drinking water, and TCPP posed a higher potential risk than TCEP in all examined aquatic environments. These findings provide empirical evidence to comprehend the toxicological effects of OPFRs on aquatic organisms and to assess the safety of aquatic environments.
Assuntos
Anuros , Retardadores de Chama , Larva , Organofosfatos , Compostos Organofosforados , Poluentes Químicos da Água , Animais , Retardadores de Chama/toxicidade , Larva/efeitos dos fármacos , Larva/crescimento & desenvolvimento , Poluentes Químicos da Água/toxicidade , Compostos Organofosforados/toxicidade , Medição de Risco , Organofosfatos/toxicidade , Anuros/crescimento & desenvolvimento , Metamorfose Biológica/efeitos dos fármacos , Testes de Toxicidade Aguda , Dose Letal MedianaRESUMO
Activation of the renin-angiotensin system is associated with podocyte injury and has been well demonstrated as a pivotal factor in the progression of chronic kidney disease. Podocyte energy metabolism is crucial for maintaining their physiological functions. However, whether renin-angiotensin system activation promotes chronic kidney disease progression by disturbing the energy metabolism of podocytes has not been elucidated. Angiotensin II, the main active molecule of the renin-angiotensin system, plays a crucial role in chronic kidney disease initiation and progression, but its impact on podocyte metabolism remains unclear. Here, we demonstrate a rapid decrease in the expression of pyruvate kinase M2, a key glycolytic enzyme, and reduced glycolytic flux in podocytes exposed to angiotensin II in vivo and in vitro. Podocyte-specific deletion of pyruvate kinase M2 in mice aggravated angiotensin II-induced glomerular and podocyte injury with foot process effacement and proteinuria. The inhibition of glycolysis was accompanied by adenosine triphosphate deficiency, cytoskeletal remodeling and podocyte apoptosis. Mechanistically, we found that angiotensin II-induced glycolysis impairment contributed to an insufficient energy supply to the foot process, leading to podocyte injury. Additionally, pyruvate kinase M2 expression was found to be reduced in podocytes from kidney biopsies of patients with hypertensive nephropathy and diabetic kidney disease. Thus, our findings suggest that glycolysis activation is a potential therapeutic strategy for podocyte injury.
Assuntos
Nefropatias Diabéticas , Podócitos , Insuficiência Renal Crônica , Camundongos , Animais , Podócitos/patologia , Angiotensina II/metabolismo , Anaerobiose , Piruvato Quinase/genética , Piruvato Quinase/metabolismo , Nefropatias Diabéticas/patologia , Insuficiência Renal Crônica/patologia , GlicóliseRESUMO
Lymphoblastic lymphoma (LBL) is a highly malignant form of lymphoma with rapid progression and high mortality. According to the World Health Organization immunophenotype, it is classified into T-lymphoblastic lymphoma (T-LBL) and B-lymphoblastic lymphoma (B-LBL). B-LBL often involves lymph nodes and extranodal locations, such as the skin, bones, and soft tissues. However, renal damage as an initial symptom is very rare in B-LBL. The present study presented a rare case of renal involvement in a 30-year-old male patient with B-LBL presenting with acute renal failure with bilateral renal enlargement. Renal involvement is rare in B-LBL, and nephrologists should improve the understanding of this disease.
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Hepcidin is a cysteine-rich antimicrobial peptide that serves an important role in the immunity system of fishes. It exhibits antibacterial, antifungal, antiviral, and antitumor activities. However, the exact role of fish hepcidin in the regulation of the intestinal flora still remains a mystery. In our study, we sequenced and characterized hepcidin from the liver of Acrossocheilus fasciatus. Phylogenetic tree analysis showed that A. fasciatus hepcidin and Gobiocypris rarus hepcidin were the most closely related, and both belonged to the fish HAMP1 cluster. Studies conducted on in vivo tissue distribution showed that the expression of hepcidin was highest in healthy A. fasciatus liver. Aeromonas hydrophila infection was confirmed by the increased expression of pro-inflammatory cytokine genes and bacterial loads in A. fasciatus tissues. After A. hydrophila infection, hepcidin expression significantly increased in the liver, spleen, and head kidney. In vitro antibacterial assays showed that hepcidin exhibits strong broad spectrum antibacterial activity. Furthermore, we examined the regulatory effect of hepcidin on the intestinal flora and found that A. fasciatus hepcidin restored the reduced diversity and compositional changes in intestinal flora caused by A. hydrophila infection. Our results suggest that hepcidin could regulate the intestinal flora in fishes; however, the underlying mechanisms need to be explored in greater detail.
Assuntos
Cyprinidae , Doenças dos Peixes , Microbioma Gastrointestinal , Animais , Aeromonas hydrophila/fisiologia , Hepcidinas/genética , Hepcidinas/química , Peptídeos Antimicrobianos , Proteínas de Peixes/metabolismo , Filogenia , Doenças dos Peixes/microbiologia , Cyprinidae/metabolismo , Antibacterianos/farmacologiaRESUMO
The toxicity of triphenyl phosphate (TPhP) to aquatic organisms in surface waters has been demonstrated; However, an understanding of toxicity profiles of TPhP in amphibians is limited. Therefore, the adverse effects and threshold concentrations of TPhP on metamorphosis, growth, locomotion, and hepatic antioxidants of Gosner stage 25 Polypedates megacephalus tadpoles under long-term (35 d) exposure to six TPhP concentrations until complete metamorphosis were assessed. Additionally, the overall effect of using integrated multiple biomarkers were determined to demonstrate the potential ecological risks of waterborne TPhP at environmentally relevant concentrations in amphibian tadpoles. With increasing TPhP concentrations, physical parameters (snout-vent length, body mass, condition factor, and hepatic somatic index), jumping distance, hepatic catalase, and superoxide dismutase activities decreased, whereas metamorphosis time and malondialdehyde content increased. The threshold concentration of TPhP that affected the tadpole biomarker, except for metamorphosis rate and jumping distance, was 50-400 µg/L. Furthermore, the standardized scores of the examined integrated biomarkers in the six TPhP concentrations were visualized using radar plots and calculated as the integrated biomarker responses (IBRs). The varying TPhP concentrations had different scores in the radar plots, and the threshold for affecting the IBR value was 10 µg/L, which was close to the TPhP concentration in surface waters. Additionally, IBR values were strongly positively correlated with the TPhP concentrations. These findings indicate that environmentally relevant exposure to waterborne TPhP can pose an ecological risk to amphibian tadpoles. This study can serve as a reference and assist in the formulation of relevant policies and strategies to control TPhP pollution in water bodies.
Assuntos
Antioxidantes , Biomarcadores Ambientais , Animais , Catalase , Larva , Malondialdeído , Organofosfatos/toxicidade , Superóxido Dismutase , ÁguaRESUMO
Podocyte mitochondrial dysfunction plays a critical role in the pathogenesis of chronic kidney disease (CKD). Previous studies demonstrated that excessive mitochondrial fission could lead to the overproduction of reactive oxygen species (ROS) and promote podocyte apoptosis. Therefore, the maintenance of stable mitochondrial function is a newly identified way to protect podocytes and prevent the progression of CKD. As a mitochondria-targeted antioxidant, mitoquinone (MitoQ) has been proven to be a promising agent for the prevention of mitochondrial injury in cardiovascular disease and Parkinson's disease. The present study examined the effects of MitoQ on angiotensin II- (Ang II-) induced podocyte injury both in vivo and in vitro. Podocyte mitochondria in Ang II-infused mice exhibited morphological and functional alterations. The observed mitochondrial fragmentation and ROS production were alleviated with MitoQ treatment. In vitro, alterations in mitochondrial morphology and function in Ang II-stimulated podocytes, including mitochondrial membrane potential reduction, ROS overproduction, and adenosine triphosphate (ATP) deficiency, were significantly reversed by MitoQ. Moreover, MitoQ rescued the expression and translocation of Nrf2 (nuclear factor E2-related factor 2) and decreased the expression of Keap1 (Kelch-like ECH-associated protein 1) in Ang II-stimulated podocytes. Nrf2 knockdown partially blocked the protective effects of MitoQ on Ang II-induced mitochondrial fission and oxidative stress in podocytes. These results demonstrate that MitoQ exerts a protective effect in Ang II-induced mitochondrial injury in podocytes via the Keap1-Nrf2 signaling pathway.
Assuntos
Angiotensina II/efeitos adversos , Isoindóis/farmacologia , Isoquinolinas/farmacologia , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Mitocôndrias/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Podócitos/metabolismo , Quinonas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Angiotensina II/farmacologia , Animais , Humanos , Camundongos , Podócitos/patologiaRESUMO
Disturbed renal lipid metabolism, especially cholesterol dysregulation plays a crucial role in the pathogenesis of chronic kidney disease (CKD). We recently reported that angiotensin (Ang) II could induce cholesterol accumulation and injury in podocytes. However, the underlying mechanisms for these alterations remain unknown. Methods: Bioinformatics analysis of renal biopsy specimens from patients with hypertensive nephropathy (HN) suggests the involvement of Sirtuin 6 (Sirt6) in Ang II-induced dysregulation of glomerular cholesterol. Using a podocyte-specific Sirt6 knockout mouse model, the effects of Sirt6 on Ang II-induced cholesterol accumulation in podocytes and the therapeutic efficacies of cholesterol-lowering agents were evaluated. Results: Cholesterol accumulation was detected in the podocytes of Ang II-infused mice, whereas selective deletion of Sirt6 in podocytes not only increased cholesterol accumulation in these cells but also exacerbated Ang II-induced kidney injury. Deletion of Sirt6 also attenuated the protective effect of cyclodextrin (CD) on Ang II-induced urinary albumin excretion, glomerulosclerosis and podocyte injury. In addition, we demonstrated that Sirt6 affected cholesterol efflux in podocytes by regulating the expression of ATP-binding cassette transporter G1 (ABCG1). Conclusions: These findings provide evidence that Sirt6 is a potential target for renin-angiotensin system (RAS)-associated podocyte injury and provide a rationale for the application of cholesterol-lowering agents in patients with CKD.
Assuntos
Colesterol/metabolismo , Podócitos/patologia , Insuficiência Renal Crônica/patologia , Sirtuínas/deficiência , Sirtuínas/metabolismo , Adulto , Angiotensina II/administração & dosagem , Angiotensina II/toxicidade , Animais , Anticolesterolemiantes/farmacologia , Anticolesterolemiantes/uso terapêutico , Biópsia , Estudos de Casos e Controles , Linhagem Celular , Colesterol/sangue , Conjuntos de Dados como Assunto , Modelos Animais de Doenças , Feminino , Perfilação da Expressão Gênica , Humanos , Metabolismo dos Lipídeos/efeitos dos fármacos , Masculino , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos , Podócitos/efeitos dos fármacos , Insuficiência Renal Crônica/sangue , Insuficiência Renal Crônica/induzido quimicamente , Insuficiência Renal Crônica/tratamento farmacológico , Sirtuínas/genéticaRESUMO
Natural rubber is an important industrial material, which is obtained from the only commercially cultivated rubber tree, Hevea brasiliensis. In rubber latex production, ethylene has been extensively used as a stimulant. Recent research showed that post-translational modifications (PTMs) of latex proteins, such as phosphorylation, glycosylation and ubiquitination, are crucial in natural rubber biosynthesis. In this study, comparative proteomics was performed to identify the glycosylated proteins in rubber latex treated with ethylene for different days. Combined with Pro-Q Glycoprotein gel staining and mass spectrometry techniques, we provided the first visual profiling of glycoproteomics of rubber latex and finally identified 144 glycosylated protein species, including 65 differentially accumulated proteins (DAPs) after treating with ethylene for three and/or five days. Gene Ontology (GO) functional annotation showed that these ethylene-responsive glycoproteins are mainly involved in cell parts, membrane components and metabolism. Pathway analysis demonstrated that these glycosylated rubber latex proteins are mainly involved in carbohydrate metabolism, energy metabolism, degradation function and cellular processes in rubber latex metabolism. Protein-protein interaction analysis revealed that these DAPs are mainly centered on acetyl-CoA acetyltransferase and hydroxymethylglutaryl-CoA synthase (HMGS) in the mevalonate pathway for natural rubber biosynthesis. In our glycoproteomics, three protein isoforms of HMGS2 were identified from rubber latex, and only one HMGS2 isoform was sharply increased in rubber latex by ethylene treatment for five days. Furthermore, the HbHMGS2 gene was over-expressed in a model rubber-producing grass Taraxacum Kok-saghyz and rubber content in the roots of transgenic rubber grass was significantly increased over that in the wild type plant, indicating HMGS2 is the key component for natural rubber production.
Assuntos
Etilenos/farmacologia , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Glicoproteínas/biossíntese , Hevea/metabolismo , Proteínas de Plantas/biossíntese , Perfilação da Expressão GênicaRESUMO
Renal fibrosis is controlled by profibrotic and antifibrotic forces. Exploring anti-fibrosis factors and mechanisms is an attractive strategy to prevent organ failure. Here we identified the JNK-associated leucine zipper protein (JLP) as a potential endogenous antifibrotic factor. JLP, predominantly expressed in renal tubular epithelial cells (TECs) in normal human or mouse kidneys, was downregulated in fibrotic kidneys. Jlp deficiency resulted in more severe renal fibrosis in unilateral ureteral obstruction (UUO) mice, while renal fibrosis resistance was observed in TECs-specific transgenic Jlp mice. JLP executes its protective role in renal fibrosis via negatively regulating TGF-ß1 expression and autophagy, and the profibrotic effects of ECM production, epithelial-to-mesenchymal transition (EMT), apoptosis and cell cycle arrest in TECs. We further found that TGF-ß1 and FGF-2 could negatively regulate the expression of JLP. Our study suggests that JLP plays a central role in renal fibrosis via its negative crosstalk with the profibrotic factor, TGF-ß1.
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Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Células Epiteliais/patologia , Fibrose/patologia , Nefropatias/patologia , Fator de Crescimento Transformador beta1/metabolismo , Obstrução Ureteral/fisiopatologia , Animais , Autofagia , Células Epiteliais/metabolismo , Transição Epitelial-Mesenquimal , Retroalimentação Fisiológica , Feminino , Fibrose/genética , Fibrose/metabolismo , Nefropatias/genética , Nefropatias/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transdução de Sinais , Fator de Crescimento Transformador beta1/genéticaRESUMO
Increasing evidence suggests that mitochondrial dysfunction plays a critical role in the development of diabetic kidney disease (DKD), however, its specific pathomechanism remains unclear. A-kinase anchoring protein (AKAP) 1 is a scaffold protein in the AKAP family that is involved in mitochondrial fission and fusion. Here, we show that rats with streptozotocin (STZ)-induced diabetes developed podocyte damage accompanied by AKAP1 overexpression and that AKAP1 closely interacted with the mitochondrial fission enzyme dynamin-related protein 1 (Drp1). At the molecular level, high glucose (HG) promoted podocyte injury and Drp1 phosphorylation at Ser637 as proven by decreased mitochondrial membrane potential, elevated reactive oxygen species generation, reduced adenosine triphosphate synthesis, and increased podocyte apoptosis. Furthermore, the AKAP1 knockdown protected HG-induced podocyte injury and suppressed HG-induced Drp1 phosphorylation at Ser637. AKAP1 overexpression aggravated HG-induced mitochondrial fragmentation and podocyte apoptosis. The coimmunoprecipitation assay showed that HG-induced Drp1 interacted with AKAP1, revealing that AKAP1 could recruit Drp1 from the cytoplasm under HG stimulation. Subsequently, we detected the effect of drp1 phosphorylation on Ser637 by transferring several different Drp1 mutants. We demonstrated that activated AKAP1 promoted Drp1 phosphorylation at Ser637, which promoted the transposition of Drp1 to the surface of the mitochondria and accounts for mitochondrial dysfunction events. These findings indicate that AKAP1 is the main pathogenic factor in the development and progression of HG-induced podocyte injury through the destruction of mitochondrial dynamic homeostasis by regulating Drp1 phosphorylation in human podocytes.
Assuntos
Proteínas de Ancoragem à Quinase A/metabolismo , Dinaminas/metabolismo , Glucose/metabolismo , Mitocôndrias/metabolismo , Dinâmica Mitocondrial/fisiologia , Podócitos/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Apoptose/fisiologia , Células Cultivadas , Citoplasma/metabolismo , Homeostase/fisiologia , Humanos , Masculino , Potencial da Membrana Mitocondrial/fisiologia , Mitocôndrias/fisiologia , Fosforilação/fisiologia , Podócitos/fisiologia , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismoRESUMO
Acute kidney injury (AKI) is a condition that has a high incidence and death rate. Unfortunately, the kidney may not recover completely after AKI, which then develops to chronic kidney disease (CKD). Therefore, it is necessary to identify potential curative targets to avoid its development to CKD. As an NAD+ -dependent deacetylase, sirtuin 6 (Sirt6) has been linked to different types of biological processes. In the present work, our group investigated the role of Sirt6 in tubular epithelial cells (TECs) under hypoxic stress. Sirt6 expression was examined in mouse kidney following ischemia/reperfusion (IR) injury and hypoxia-challenged TECs. Using Sirt6 plasmid and small interfering RNA, we also investigated how, in regard to inflammation and epithelial-to-mesenchymal transition, Sirt6 affects hypoxia-triggered injury. In addition, cell cycle was detected in hypoxia-challenged TECs. Sirt6 was downregulated in the kidney of mice with IR injury and hypoxia-challenged TECs. Consequently, Sirt6 depletion aggravated hypoxia-induced injury and G2/M phase arrest. Sirt6 overexpression attenuated hypoxia-triggered damage and G2/M phase arrest in TECs. Sirt6 prevented hypoxia-triggered TEC damage via suppressing G2/M phase arrest. Thus, Sirt6 is a possible candidate for alleviating the effects of kidney injury.
Assuntos
Injúria Renal Aguda/genética , Túbulos Renais/lesões , Insuficiência Renal Crônica/genética , Sirtuínas/genética , Injúria Renal Aguda/patologia , Animais , Pontos de Checagem do Ciclo Celular/genética , Hipóxia Celular/genética , Modelos Animais de Doenças , Células Epiteliais/patologia , Pontos de Checagem da Fase G2 do Ciclo Celular/genética , Regulação da Expressão Gênica/genética , Humanos , Túbulos Renais/patologia , Camundongos , Insuficiência Renal Crônica/patologia , Traumatismo por Reperfusão/genética , Traumatismo por Reperfusão/patologiaRESUMO
Cadmium (Cd) exposure is harmful to amphibians in natural environments and the Cd concentration is a key parameter in water monitoring. Cd pollution has been a severe issue in the Yangtze River and its southern reaches in recent years. Acute toxicity assays were employed to determine the tolerance limits of Cd for Microhyla fissipes tadpoles and five different concentrations of Cd (0, 50, 100, 200 and 300 µg/L) were involved to detect its chronic effects on metamorphosis, growth, locomotion, genotoxicity and enzymatic activities of M. fissipes tadpoles. The results showed that the 24-h and 48-h LC50 values of Cd on M. fissipes tadpoles were 2591.3 µg/L and 1567.9 µg/L, respectively, and the presumable non-lethal concentration obtained was 172.2 µg/L. During the 70-day chronic toxicity assays, Cd showed negative impacts on survival, growth, metamorphosis and the frequency of erythrocytes nuclear abnormality of M. fissipes tadpoles. However, the Cd exposure caused the increased body size and condition of tadpoles at complete metamorphosis (GS46). The tadpoles exposed to 200 µg/L of Cd exhibited degraded locomotor performance at GS46. Weight increments of tadpoles were inhibited at Day 14 and massive deaths were observed over the next 14 days. The enzymatic activities of tadpoles experienced a shock response stage (GS30-GS35) and a complete recovery stage (GS36-GS41) in all treatments. However, the enzymatic activities (except alkaline phosphatase) of tadpoles at GS46 increased after Cd exposure, especially at high concentrations. In summary, Cd is a threat to M. fissipes tadpoles as that causes reduced fitness.
Assuntos
Anuros/fisiologia , Cádmio/toxicidade , Locomoção/efeitos dos fármacos , Metamorfose Biológica/efeitos dos fármacos , Poluentes Químicos da Água/toxicidade , Animais , Anuros/crescimento & desenvolvimento , Larva/efeitos dos fármacos , Larva/enzimologia , Dose Letal MedianaRESUMO
AIMS: Previous studies showed that abnormal mitochondrial structure and function were involved in the pathological process of diabetic nephropathy (DN). The dynamic mitochondrial processes, including fusion and fission, maintain the mass and quantity of mitochondria. Podocyte injury is a critical factor in the development and progression of DN. The present study evaluated the mitochondrial fission of podocytes in patients with DN. METHODS: We recruited 31 patients with biopsy-confirmed DN. A quantitative analysis of the mitochondrial morphology was conducted with electron microscopy using a computer-assisted morphometric analysis application to calculate the aspect ratio values. Immunofluorescence assays were used to evaluate protein colocalization in the glomeruli of patients. RESULTS: The urine protein level was significantly increased in DN patients compared to non-DN patients (P < 0.001), and the mitochondria in the podocytes from DN patients were more fragmentated than those from patients without DN. The mitochondrial aspect ratio values were negatively correlated with the proteinuria levels (r = -0.574, P = 0.01), and multiple regression analysis verified that the mitochondrial aspect ratio was significantly and independently associated with the urine protein level (ß = -0.519, P = 0.007). In addition, Drp1, a mitochondrial fission factor, preferentially combines with AKAP1, which is located in the mitochondrial membrane. CONCLUSIONS: In the podocytes of DN patients, mitochondrial fragmentation was increased, and mitochondrial aspect ratio values were correlated with the proteinuria levels. The AKAP1-Drp1 pathway may contribute to mitochondrial fission in the pathogenesis of DN.
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BACKGROUND: Two-dimensional electrophoresis (2-DE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) are widely used in plant proteomics research. However, these two techniques cannot be simultaneously satisfied by traditional protein extraction methods when investigate cotton leaf proteome. RESULTS: Here, we evaluated the efficiency of three different protein extraction methods for 2-DE and LC-MS/MS analyses of total proteins obtained from cotton leaves. The protein yield of the borax/PVPP/phenol (BPP) method (0.14%) was significantly lower than the yields of the trichloroacetic acid/acetone (TCA) precipitation method (1.42%) and optimized TCA combined with BPP (TCA-B) method (0.47%). The BPP method was failed to get a clear 2-DE electrophoretogram. Fifty pairs of protein spots were randomly selected from the 2-DE gels of TCA- and TCA-B-extracted proteins for identification by MALDI TOF/TOF, and the results of 42 pairs were consistent. High-throughput proteomic analysis showed that 6339, 9282 and 9697 unique proteins were identified from the total cotton leaf proteins extracted by the TCA, BPP and TCA-B methods, respectively. Gene Ontology (GO) analysis revealed that the proteins specifically identified by TCA method were primarily distributed in the plasma membrane, while BPP and TCA-B methods specific proteins distributed in the cytosol, indicating the sub-cellular preference of different protein extraction methods. Further, ATP-dependent zinc metalloprotease FTSH 8 could be observed in the 2-DE gels of TCA and TCA-B methods, and could only be detected in the LC-MS/MS results of the BPP and TCA-B methods, showing that TCA-B method might be the optimized choice for both 2-DE and LC-MS/MS. CONCLUSION: Our data provided an improved TCA-B method for protein extraction that is compatible with 2-DE and LC-MS/MS for cotton leaves and similar plant tissues which is rich in polysaccharides and polyphenols.
Assuntos
Fracionamento Químico/métodos , Gossypium/metabolismo , Folhas de Planta/metabolismo , Proteínas de Plantas/isolamento & purificação , Proteínas de Plantas/metabolismo , Sequência de Aminoácidos , Cromatografia Líquida , Eletroforese em Gel Bidimensional , Proteínas de Plantas/química , Proteômica , Espectrometria de Massas em TandemRESUMO
Percutaneous insertion of peritoneal dialysis catheters is theoretically most preferred by nephrologists because of the advantages of bedside performing, surgery independence, and minimal injury over other procedures of catheter placement such as open surgical dissection or laparoscopic operation. However, blindly placing catheters in the percutaneous procedure brings the risk of catheter malposition or bowel perforation; this largely retarded it's implementation. We had previously developed a novel technique termed "Wang's forceps-assisted catheter insertion and fixation," which had been successfully applied in the open surgical catheter insertion and displaced catheter reposition in our center. In this study, we further explored the possibility of applying the Wang's forceps in the procedure of percutaneous catheter insertion both in porcine model and patients with end stage renal disease (ESRD). A total of three miniature pigs successfully received percutaneous catheter insertion using Seldinger's technique with Wang's forceps assistance. The catheters were all placed in the right position and functioning well in dialysate drainage. This novel method of percutaneous catheter insertion was then performed on 20 ESRD patients. The procedure showed effective time-saving with the average operating time of 29.2 ± 3.53 min and was well tolerated by patients with minimal pain and injury. During a follow-up time of 6 months, no complications of catheter displacement, leakage, or blockade occurred. Our preliminary observation demonstrates that utilization of Wang's forceps in a percutaneous procedure conferred benefits of accurately placing and fixing catheters while preserving the merits of minimal invasion and simple performance.
Assuntos
Cateterismo/instrumentação , Cateteres de Demora , Falência Renal Crônica/terapia , Diálise Peritoneal/instrumentação , Instrumentos Cirúrgicos , Adulto , Idoso , Animais , Cateterismo/métodos , Modelos Animais de Doenças , Desenho de Equipamento , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Diálise Peritoneal/métodos , SuínosRESUMO
Our previous study showed that angiotensin II (Ang II) exposure diminished the interaction between nephrin and c-Abl, then c-Abl mediated SHIP2-Akt pathway in the process of podocyte injury in vivo and vitro. However, the relationship between nephrin and c-Abl was unknown. Recently, various studies showed that nephrin was required for cytoskeletal remodeling in glomerular podocytes. But its specific mechanisms remain incompletely understood. As a nonreceptor tyrosine kinase involved in cytoskeletal regulation, c-Abl may be a candidate of signaling proteins interacting with Src homology 2/3 (SH2/SH3) domains of nephrin. Therefore, it is proposed that c-Abl contributes to nephrin-dependent cytoskeletal remodeling of podocytes. Herein, we observed that nephrin-c-Abl colocalization were suppressed in glomeruli of patients with proteinuria. Next, CD16/7-nephrin and c-Abl vectors were constructed to investigate the nephrin-c-Abl signaling pathway in podocyte actin-cytoskeletal remodeling. The disorganized cytoskeleton stimulated by cytochalasin D in COS7 cells was dramatically restored by co-transfection with phosphorylated CD16/7-nephrin and c-Abl full-length constructs. Further, co-immunoprecipitation showed that phosphorylated CD16/7-nephrin interacted with wild-type c-Abl, but not with SH2/SH3-defective c-Abl. These findings suggest that phosphorylated nephrin is able to recruit c-Abl in a SH2/SH3-dependent manner and detached c-Abl from dephosphorylated nephrin contributes to cytoskeletal remodeling in podocytes.