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The increasing incidence of oropharyngeal squamous cell carcinoma (OPSCC) is primarily due to human papillomavirus, and understanding the tumor biology caused by the virus is crucial. Our goal was to investigate the proteins present in the serum of patients with OPSCC, which were not previously studied in OPSCC tissue. We examined the difference in expression of these proteins between HPV-positive and -negative tumors and their correlation with clinicopathological parameters and patient survival. The study included 157 formalin-fixed, paraffin-embedded tissue samples and clinicopathological data. Based on the protein levels in the sera of OPSCC patients, we selected 12 proteins and studied their expression in HPV-negative and HPV-positive OPSCC cell lines. LRG1, SDR16C5, PIP4K2C and MVD proteins were selected for immunohistochemical analysis in HPV-positive and -negative OPSCC tissue samples. These protein´s expression levels were compared with clinicopathological parameters and patient survival to investigate their clinical relevance. LRG1 expression was strong in HPV-negative whereas SDR16C5 expression was strong in HPV-positive tumors. Correlation was observed between LRG1, SDR16C5, and PIP4K2C expression and patient survival. High expression of PIP4K2C was found to be an independent prognostic factor for overall survival and expression correlated with HPV-positive tumor status. The data suggest the possible role of LRG1, SDR16C5 and PIP4K2C in OPSCC biology.
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Neoplasias Orofaríngeas , Infecções por Papillomavirus , Adulto , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Biomarcadores Tumorais/metabolismo , Carcinoma de Células Escamosas/virologia , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Glicoproteínas/metabolismo , Neoplasias Orofaríngeas/virologia , Neoplasias Orofaríngeas/metabolismo , Neoplasias Orofaríngeas/patologia , Papillomaviridae/genética , Infecções por Papillomavirus/virologia , Infecções por Papillomavirus/metabolismo , Infecções por Papillomavirus/complicações , Infecções por Papillomavirus/patologia , Prognóstico , Carcinoma de Células Escamosas de Cabeça e Pescoço/virologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologiaRESUMO
Changes in the dynamic architecture of podocytes, the glomerular epithelial cells, lead to kidney dysfunction. Previous studies on protein kinase C and casein kinase 2 substrates in neurons 2 (PACSIN2), a known regulator of endocytosis and cytoskeletal organization, reveal a connection between PACSIN2 and kidney pathogenesis. Here, we show that the phosphorylation of PACSIN2 at serine 313 (S313) is increased in the glomeruli of rats with diabetic kidney disease. We found that phosphorylation at S313 is associated with kidney dysfunction and increased free fatty acids rather than with high glucose and diabetes alone. Phosphorylation of PACSIN2 emerged as a dynamic process that fine-tunes cell morphology and cytoskeletal arrangement, in cooperation with the regulator of the actin cytoskeleton, Neural Wiskott-Aldrich syndrome protein (N-WASP). PACSIN2 phosphorylation decreased N-WASP degradation while N-WASP inhibition triggered PACSIN2 phosphorylation at S313. Functionally, pS313-PACSIN2 regulated actin cytoskeleton rearrangement depending on the type of cell injury and the signaling pathways involved. Collectively, this study indicates that N-WASP induces phosphorylation of PACSIN2 at S313, which serves as a mechanism whereby cells regulate active actin-related processes. The dynamic phosphorylation of S313 is needed to regulate cytoskeletal reorganization.
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Caseínas , Podócitos , Ratos , Animais , Fosforilação , Caseínas/metabolismo , Podócitos/metabolismo , Serina/metabolismo , Neurônios/metabolismoRESUMO
Consumers and manufacturers are exposed to nanosized zinc oxide (nZnO) and silver particles (nAg) via airways, but their biological effects are still not fully elucidated. To understand the immune effects, we exposed mice to 2, 10, or 50 µg of nZnO or nAg by oropharyngeal aspiration and analyzed the global gene expression profiles and immunopathological changes in the lungs after 1, 7, or 28 days. Our results show that the kinetics of responses varied in the lungs. Exposure to nZnO resulted in the highest accumulation of F4/80- and CD3-positive cells, and the largest number of differentially expressed genes (DEGs) were identified after day 1, while exposure to nAg caused peak responses at day 7. Additionally, nZnO mainly activated the innate immune responses leading to acute inflammation, whereas the nAg activated both innate and adaptive immune pathways, with long-lasting effects. This kinetic-profiling study provides an important data source to understand the cellular and molecular processes underlying nZnO- and nAg-induced transcriptomic changes, which lead to the characterization of the corresponding biological and toxicological effects of nZnO and nAg in the lungs. These findings could improve science-based hazard and risk assessment and the development of safe applications of engineered nanomaterials (ENMs), e.g., in biomedical applications.
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Nanopartículas Metálicas , Nanopartículas , Óxido de Zinco , Camundongos , Animais , Óxido de Zinco/toxicidade , Nanopartículas Metálicas/toxicidade , Prata/toxicidade , Transcriptoma , PulmãoRESUMO
Ebselen, a multifunctional organoselenium compound, has been recognized as a potential treatment for diabetes-related disorders. However, the underlying mechanisms whereby ebselen regulates metabolic pathways remain elusive. We discovered that ebselen inhibits lipid phosphatase SHIP2 (Src homology 2 domain-containing inositol-5-phosphatase 2), an emerging drug target to ameliorate insulin resistance in diabetes. We found that ebselen directly binds to and inhibits the catalytic activity of the recombinant SHIP2 phosphatase domain and SHIP2 in cultured cells, the skeletal muscle and liver of the diabetic db/db mice, and the liver of the SHIP2 overexpressing (SHIP2-Tg) mice. Ebselen increased insulin-induced Akt phosphorylation in cultured myotubes, enhanced insulin sensitivity and protected liver tissue from lipid peroxidation and inflammation in the db/db mice, and improved glucose tolerance more efficiently than metformin in the SHIP2-Tg mice. SHIP2 overexpression abrogated the ability of ebselen to induce glucose uptake and reduce ROS production in myotubes and blunted the effect of ebselen to inhibit SHIP2 in the skeletal muscle of the SHIP2-Tg mice. Our data reveal ebselen as a potent SHIP2 inhibitor and demonstrate that the ability of ebselen to ameliorate insulin resistance and act as an antioxidant is at least in part mediated by the reduction of SHIP2 activity.
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Diabetes Mellitus Experimental , Resistência à Insulina , Animais , Diabetes Mellitus Experimental/tratamento farmacológico , Inflamação/tratamento farmacológico , Insulina/metabolismo , Isoindóis , Camundongos , Compostos Organosselênicos , Estresse Oxidativo , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases , Monoéster Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/metabolismo , Fosforilação , Transdução de SinaisRESUMO
Old-world orthohantaviruses cause hemorrhagic fever with renal syndrome (HFRS), characterized by acute kidney injury (AKI) with transient proteinuria. It seems plausible that proteinuria during acute HFRS is mediated by the disruption of the glomerular filtration barrier (GFB) due to vascular leakage, a hallmark of orthohantavirus-caused diseases. However, direct infection of endothelial cells by orthohantaviruses does not result in increased endothelial permeability, and alternative explanations for vascular leakage and diminished GFB function are necessary. Vascular integrity is partly dependent on an intact endothelial glycocalyx, which is susceptible to cleavage by heparanase (HPSE). To understand the role of glycocalyx degradation in HFRS-associated proteinuria, we investigated the levels of HPSE in urine and plasma during acute, convalescent and recovery stages of HFRS caused by Puumala orthohantavirus. HPSE levels in urine during acute HFRS were significantly increased and strongly associated with the severity of AKI and other markers of disease severity. Furthermore, increased expression of HPSE was detected in vitro in orthohantavirus-infected podocytes, which line the outer surfaces of glomerular capillaries. Taken together, these findings suggest the local activation of HPSE in the kidneys of orthohantavirus-infected patients with the potential to disrupt the endothelial glycocalyx, leading to increased protein leakage through the GFB, resulting in high amounts of proteinuria.
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Injúria Renal Aguda , Febre Hemorrágica com Síndrome Renal , Virus Puumala , Células Endoteliais , Glucuronidase , Febre Hemorrágica com Síndrome Renal/complicações , Humanos , Proteinúria/etiologia , Índice de Gravidade de DoençaRESUMO
Protein kinase C and casein kinase substrate in neurons (PACSINs), or syndapins (synaptic dynamin-associated proteins), are a family of proteins involved in the regulation of cell cytoskeleton, intracellular trafficking and signalling. Over the last twenty years, PACSINs have been mostly studied in the in vitro and ex vivo settings, and only in the last decade reports on their function in vivo have emerged. We first summarize the identification, structure and cellular functions of PACSINs, and then focus on the relevance of PACSINs in vivo. During development in various model organisms, PACSINs participate in diverse processes, such as neural crest cell development, gastrulation, laterality development and neuromuscular junction formation. In mouse, PACSIN2 regulates angiogenesis during retinal development and in human, PACSIN2 associates with monosomy and embryonic implantation. In adulthood, PACSIN1 has been extensively studied in the brain and shown to regulate neuromorphogenesis, receptor trafficking and synaptic plasticity. Several genetic studies suggest a role for PACSIN1 in the development of schizophrenia, which is also supported by the phenotype of mice depleted of PACSIN1. PACSIN2 plays an essential role in the maintenance of intestinal homeostasis and participates in kidney repair processes after injury. PACSIN3 is abundant in muscle tissue and necessary for caveolar biogenesis to create membrane reservoirs, thus controlling muscle function, and has been linked to certain genetic muscular disorders. The above examples illustrate the importance of PACSINs in diverse physiological or tissue repair processes in various organs, and associations to diseases when their functions are disturbed.
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Proteínas Adaptadoras de Transdução de Sinal , Proteínas do Citoesqueleto , Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Animais , Proteínas de Transporte/fisiologia , Proteínas do Citoesqueleto/fisiologia , Citoesqueleto/fisiologia , Camundongos , Neurônios/fisiologiaRESUMO
AIMS/HYPOTHESIS: Chronic low-grade inflammation with local upregulation of proinflammatory molecules plays a role in the progression of obesity-related renal injury. Reduced serum concentration of anti-inflammatory adiponectin may promote chronic inflammation. Here, we investigated the potential anti-inflammatory and renoprotective effects and mechanisms of action of AdipoRon, an adiponectin receptor agonist. METHODS: Wild-type DBA/2J mice were fed with high-fat diet (HFD) supplemented or not with AdipoRon to model obesity-induced metabolic endotoxaemia and chronic low-grade inflammation and we assessed changes in the glomerular morphology and expression of proinflammatory markers. We also treated human glomeruli ex vivo and human podocytes in vitro with AdipoRon and bacterial lipopolysaccharide (LPS), an endotoxin upregulated in obesity and diabetes, and analysed the secretion of inflammatory cytokines, activation of inflammatory signal transduction pathways, apoptosis and migration. RESULTS: In HFD-fed mice, AdipoRon attenuated renal inflammation, as demonstrated by reduced expression of glomerular activated NF-κB p65 subunit (NF-κB-p65) (70%, p < 0.001), TNFα (48%, p < 0.01), IL-1ß (51%, p < 0.001) and TGFß (46%, p < 0.001), renal IL-6 and IL-4 (21% and 20%, p < 0.05), and lowered glomerular F4/80-positive macrophage infiltration (31%, p < 0.001). In addition, AdipoRon ameliorated HFD-induced glomerular hypertrophy (12%, p < 0.001), fibronectin accumulation (50%, p < 0.01) and podocyte loss (12%, p < 0.001), and reduced podocyte foot process effacement (15%, p < 0.001) and thickening of the glomerular basement membrane (18%, p < 0.001). In cultured podocytes, AdipoRon attenuated the LPS-induced activation of the central inflammatory signalling pathways NF-κB-p65, c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38-MAPK) (30%, 36% and 22%, respectively, p < 0.001), reduced the secretion of TNFα (32%, p < 0.01), and protected against podocyte apoptosis and migration. In human glomeruli ex vivo, AdipoRon reduced the LPS-induced secretion of inflammatory cytokines IL-1ß, IL-18, IL-6 and IL-10. CONCLUSIONS/INTERPRETATION: AdipoRon attenuated the renal expression of proinflammatory cytokines in HFD-fed mice and LPS-stimulated human glomeruli, which apparently contributed to the amelioration of glomerular inflammation and injury. Mechanistically, based on assays on cultured podocytes, AdipoRon reduced LPS-induced activation of the NF-κB-p65, JNK and p38-MAPK pathways, thereby impelling the decrease in apoptosis, migration and secretion of TNFα. We conclude that the activation of the adiponectin receptor by AdipoRon is a potent strategy to attenuate endotoxaemia-associated renal inflammation.
Assuntos
Dieta Hiperlipídica , Glomérulos Renais/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , Nefrite/tratamento farmacológico , Piperidinas/uso terapêutico , Receptores de Adiponectina/agonistas , Idoso , Idoso de 80 Anos ou mais , Animais , Proteínas de Ligação ao Cálcio/metabolismo , Citocinas/metabolismo , Modelos Animais de Doenças , Endotoxinas/farmacologia , Feminino , Humanos , Immunoblotting , Imuno-Histoquímica , Glomérulos Renais/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos DBA , Camundongos Knockout , Pessoa de Meia-Idade , Nefrite/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Fator de Transcrição RelA/metabolismoRESUMO
BACKGROUND: Organ protection for transplantation is perfusion with ice-cold preservation solutions, although saline is also used in animal experiments and living donor transplantations. However, ice-cold perfusion can contribute to initial graft injury. Our aim was to test if cytoskeletal damage of parenchymal cells is caused by saline itself or by the ice-cold solution. METHODS: F344 rat kidneys were flushed with cold (4 °C) saline, ischemic and sham kidneys were not perfused. In a separate set, F344 kidneys were flushed with saline or preservation solution at 4 or 15 °C. Ischemia time was 30 min. RESULTS: Renal injury was significantly more severe following cold ischemia (CI) than after ischemia-reperfusion without flushing (ischemia/reperfusion (I/R)). Functional and morphologic damage was accompanied by severe loss of ezrin from glomerular and tubular epithelial cells after CI. Moreover, saline caused serious injury independently from its temperature, while the perfusion solution was more beneficial, especially at 4 °C. CONCLUSIONS: Flushing the kidney with ice-cold saline can cause more severe injury than ischemia-reperfusion at body temperature even during a short (30 min) ischemia. Saline perfusion can prolong recovery from ischemia in kidney transplantation, which can be prevented by using preservation solutions.
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Intestinal permeability (IP) tests are used to assess intestinal damage in patients and research models. The probe iohexol has shown advantages compared to 51Cr-EDTA or absorbable/nonabsorbable sugars. During IP tests, animals are housed in metabolic cages (MCs) to collect urine. We examined the performance of an iohexol IP test in mice. Rag1-/- (C57BL/6) mice of both sexes were divided into controls or treatment groups, the latter receiving injections of effector/memory T cells to induce intestinal inflammation. After two, four and five weeks (W), a single dose of iohexol was orally administered. Urine was collected seven times over 24 h in MCs. Iohexol concentration was measured by ELISA. Intestinal histological damage was scored in duodenal sections. In control and treated mice of both sexes, urinary excretion of iohexol peaked at 4 h. From W2 to W4/W5, urinary iohexol excretion increased in treated mice of both sexes, consistent with development of duodenitis in this model. Positive correlations were observed between the urinary excretion of iohexol in W4/W5 and the histological severity of duodenitis in treated male mice. We conclude that a 6 h cumulative urine sample appears sufficient to evaluate small IP to iohexol in this mouse model, improving animal welfare by reducing cage periods.
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Metformin is the most commonly prescribed drug for treating type 2 diabetes mellitus (T2D). Its mechanisms of action have been under extensive investigation, revealing that it has multiple cellular targets, either direct or indirect ones, via which it regulates numerous cellular pathways. Diabetic kidney disease (DKD), the serious complication of T2D, develops in up to 50% of the individuals with T2D. Various mechanisms contribute to the development of DKD, including hyperglycaemia, dyslipidemia, oxidative stress, chronic low-grade inflammation, altered autophagic activity and insulin resistance, among others. Metformin has been shown to affect these pathways, and thus, it could slow down or prevent the progression of DKD. Despite several animal studies demonstrating the renoprotective effects of metformin, there is no concrete evidence in clinical settings. This review summarizes the renoprotective effects of metformin in experimental settings. Special emphasis is on the effects of metformin on podocytes, the glomerular epithelial cells that are central in maintaining the glomerular ultrafiltration function.
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OBJECTIVE: Human TNKS, encoding tankyrase 1 (TNKS1), localizes to a susceptibility locus for obesity and type 2 diabetes mellitus (T2DM). Here, we addressed the therapeutic potential of G007-LK, a TNKS-specific inhibitor, for obesity and T2DM. METHODS: We administered G007-LK to diabetic db/db mice and measured the impact on body weight, abdominal adiposity, and serum metabolites. Muscle, liver, and white adipose tissues were analyzed by quantitative RT-PCR and western blotting to determine TNKS inhibition, lipolysis, beiging, adiponectin level, mitochondrial oxidative metabolism and mass, and gluconeogenesis. Protein interaction and PARylation analyses were carried out by immunoprecipitation, pull-down and in situ proximity ligation assays. RESULTS: TNKS inhibition reduced body weight gain, abdominal fat content, serum cholesterol levels, steatosis, and proteins associated with lipolysis in diabetic db/db mice. We discovered that TNKS associates with PGC-1α and that TNKS inhibition attenuates PARylation of PGC-1α, contributing to increased PGC-1α level in WAT and muscle in db/db mice. PGC-1α upregulation apparently modulated transcriptional reprogramming to increase mitochondrial mass and fatty acid oxidative metabolism in muscle, beiging of WAT, and raised circulating adiponectin level in db/db mice. This was in sharp contrast to the liver, where TNKS inhibition in db/db mice had no effect on PGC-1α expression, lipid metabolism, or gluconeogenesis. CONCLUSION: Our study unravels a novel molecular mechanism whereby pharmacological inhibition of TNKS in obesity and diabetes enhances oxidative metabolism and ameliorates lipid disorder. This happens via tissue-specific PGC-1α-driven transcriptional reprogramming in muscle and WAT, without affecting liver. This highlights inhibition of TNKS as a potential pharmacotherapy for obesity and T2DM.
Assuntos
Diabetes Mellitus Tipo 2/metabolismo , Dislipidemias/tratamento farmacológico , Obesidade/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Tanquirases/antagonistas & inibidores , Gordura Abdominal , Tecido Adiposo Branco , Animais , Peso Corporal , Fígado , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos , Oxirredução , Poli ADP Ribosilação , Sulfonas/uso terapêutico , Tanquirases/metabolismo , Triazóis/uso terapêuticoRESUMO
In oropharyngeal squamous cell carcinoma (OPSCC), the expression pattern of toll-like receptors (TLRs), in comparison between human papillomavirus (HPV)-positive and -negative tumors differs. TLRs control innate immune responses by activating, among others, the nuclear factor-κΒ (NF-κΒ) signaling pathway. Elevated NF-κΒ activity is detectable in several cancers and regulates cancer development and progression. We studied TLR5 expression in 143 unselected consecutive OPSCC tumors, and its relation to HPV-DNA and p16 status, clinicopathological parameters, and patient outcome, and studied TLR5 stimulation and consecutive NF-κB cascade activation in vitro in two human OPSCC cell lines and immortalized human keratinocytes (HaCat). Clinicopathological data came from hospital registries, and TLR5 immunoexpression was evaluated by immunohistochemistry. Flagellin served to stimulate TLR5 in cultured cells, followed by analysis of the activity of the NF-κB signaling cascade with In-Cell Western for IκΒ and p-IκΒ. High TLR5 expression was associated with poor disease-specific survival in HPV-positive OPSCC, which typically shows low TLR5 immunoexpression. High TLR5 immunoexpression was more common in HPV-negative OPSCC, known for its less-favorable prognosis. In vitro, we detected NF-κΒ cascade activation in the HPV-positive OPSCC cell line and in HaCat cells, but not in the HPV-negative OPSCC cell line. Our results suggest that elevated TLR5 immunoexpression may be related to reduced NF-κΒ activity in HPV-negative OPSCC. The possible prognosis-worsening mechanisms among these high-risk OPSCC patients however, require further evaluation.
Assuntos
Carcinoma de Células Escamosas/genética , Neoplasias Orofaríngeas/genética , Receptor 5 Toll-Like/genética , Fator de Transcrição RelA/genética , Carcinoma de Células Escamosas/patologia , Carcinoma de Células Escamosas/virologia , Intervalo Livre de Doença , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , NF-kappa B/genética , Neoplasias Orofaríngeas/patologia , Neoplasias Orofaríngeas/virologia , Papillomaviridae/patogenicidade , Infecções por Papillomavirus/genética , Infecções por Papillomavirus/patologia , Infecções por Papillomavirus/virologia , PrognósticoRESUMO
A series of substituted sulfonanilide analogs were prepared and evaluated as novel potent inhibitors of SH2 domain-containing inositol polyphosphate 5'-phosphatase 2 (SHIP2). SHIP2 has been shown to be a new attractive target for the treatment of insulin resistance in type 2 diabetes mellitus (T2D), which can lead to life-threatening diabetic kidney disease (DKD). Amongst the synthesized compounds, the two most promising candidates, 10 and 11, inhibited SHIP2 significantly. Additionally, these compounds induced Akt activation in a dose-dependent manner, increased the presence of glucose transporter 4 at the plasma membrane, and enhanced glucose uptake in cultured myotubes in vitro at lower concentrations than metformin, the most widely used antidiabetic drug. These results show that the novel SHIP2 inhibitors have insulin sensitizing capacity and provide prototypes for further drug development for T2D and DKD.
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SHIP2 (Src homology 2 domain-containing inositol 5'-phosphatase 2) belongs to the family of 5'-phosphatases. It regulates the phosphoinositide 3-kinase (PI3K)-mediated insulin signalling cascade by dephosphorylating the 5'-position of PtdIns(3,4,5)P3 to generate PtdIns(3,4)P2, suppressing the activity of the pathway. SHIP2 mouse models and genetic studies in human propose that increased expression or activity of SHIP2 contributes to the pathogenesis of the metabolic syndrome, hypertension and type 2 diabetes. This has raised great interest to identify SHIP2 inhibitors that could be used to design new treatments for metabolic diseases. This review summarizes the central mechanisms associated with the development of diabetic kidney disease, including the role of insulin resistance, and then moves on to describe the function of SHIP2 as a regulator of metabolism in mouse models. Finally, the identification of SHIP2 inhibitors and their effects on metabolic processes in vitro and in vivo are outlined. One of the newly identified SHIP2 inhibitors is metformin, the first-line medication prescribed to patients with type 2 diabetes, further boosting the attraction of SHIP2 as a treatment target to ameliorate metabolic disorders.
Assuntos
Diabetes Mellitus Tipo 2/tratamento farmacológico , Hipoglicemiantes/uso terapêutico , Metformina/uso terapêutico , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/antagonistas & inibidores , Nefropatias Diabéticas , Barreira de Filtração Glomerular , Humanos , Metformina/farmacologiaRESUMO
Septins are a conserved family of GTP-binding proteins that assemble into cytoskeletal filaments to function in a highly sophisticated and physiologically regulated manner. Originally septins were discovered in the budding yeast as membrane-associated filaments that affect cell polarity and cytokinesis. In the last decades, much progress has been made in understanding the biochemical properties and cell biological functions of septins. In line with this, mammalian septins have been shown to be involved in various cellular processes, including regulation of cell polarity, cytoskeletal organization, vesicle trafficking, ciliogenesis, and cell-pathogen interactions. A growing number of studies have shown that septins play important roles in tissue and organ development and physiology; yet, little is known about their role in the kidney. In the following review, we discuss the structure and functions of septins in general and summarize the evidence for their presence and roles in the kidney.
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Rim/metabolismo , Septinas/metabolismo , Animais , Movimento Celular/genética , Movimento Celular/fisiologia , Polaridade Celular/genética , Polaridade Celular/fisiologia , Citocinese/genética , Citocinese/fisiologia , Septinas/genéticaRESUMO
Metformin, the first-line drug to treat type 2 diabetes (T2D), inhibits mitochondrial glycerolphosphate dehydrogenase in the liver to suppress gluconeogenesis. However, the direct target and the underlying mechanisms by which metformin increases glucose uptake in peripheral tissues remain uncharacterized. Lipid phosphatase Src homology 2 domain-containing inositol-5-phosphatase 2 (SHIP2) is upregulated in diabetic rodent models and suppresses insulin signaling by reducing Akt activation, leading to insulin resistance and diminished glucose uptake. Here, we demonstrate that metformin directly binds to and reduces the catalytic activity of the recombinant SHIP2 phosphatase domain in vitro. Metformin inhibits SHIP2 in cultured cells and in skeletal muscle and kidney of db/db mice. In SHIP2-overexpressing myotubes, metformin ameliorates reduced glucose uptake by slowing down glucose transporter 4 endocytosis. SHIP2 overexpression reduces Akt activity and enhances podocyte apoptosis, and both are restored to normal levels by metformin. SHIP2 activity is elevated in glomeruli of patients with T2D receiving nonmetformin medication, but not in patients receiving metformin, compared with people without diabetes. Furthermore, podocyte loss in kidneys of metformin-treated T2D patients is reduced compared with patients receiving nonmetformin medication. Our data unravel a novel molecular mechanism by which metformin enhances glucose uptake and acts renoprotectively by reducing SHIP2 activity.-Polianskyte-Prause, Z., Tolvanen, T. A., Lindfors, S., Dumont, V., Van, M., Wang, H., Dash, S. N., Berg, M., Naams, J.-B., Hautala, L. C., Nisen, H., Mirtti, T., Groop, P.-H., Wähälä, K., Tienari, J., Lehtonen, S. Metformin increases glucose uptake and acts renoprotectively by reducing SHIP2 activity.
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Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Tipo 2/tratamento farmacológico , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Nefropatias/prevenção & controle , Metformina/farmacologia , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/antagonistas & inibidores , Animais , Células Cultivadas , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patologia , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patologia , Humanos , Hipoglicemiantes/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fibras Musculares Esqueléticas/citologia , Fibras Musculares Esqueléticas/efeitos dos fármacos , Fibras Musculares Esqueléticas/metabolismo , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/metabolismo , Podócitos/citologia , Podócitos/efeitos dos fármacos , Podócitos/metabolismo , RatosRESUMO
Diabetic kidney disease (DKD) is a major microvascular complication of diabetes and a common cause of end-stage renal disease worldwide. DKD manifests as an increased urinary protein excretion (albuminuria). Multiple studies have shown that insulin resistance correlates with the development of albuminuria in non-diabetic and diabetic patients. There is also accumulating evidence that glomerular epithelial cells or podocytes are insulin sensitive and that insulin signaling in podocytes is essential for maintaining normal kidney function. At the cellular level, the mechanisms leading to the development of insulin resistance include mutations in the insulin receptor gene, impairments in the phosphoinositide 3-kinase (PI3K)/AKT signaling pathway, or perturbations in the trafficking of glucose transporters (GLUTs), which mediate the uptake of glucose into cells. Podocytes express several GLUTs, including GLUT1, GLUT2, GLUT3, GLUT4, and GLUT8. Of these, the most studied ones are GLUT1 and GLUT4, both shown to be insulin responsive in podocytes. In the basal state, GLUT4 is preferentially located in perinuclear and cytosolic vesicular structures and to a lesser extent at the plasma membrane. After insulin stimulation, GLUT4 is sorted into GLUT4-containing vesicles (GCVs) that translocate to the plasma membrane. GCV trafficking consists of several steps, including approaching of the GCVs to the plasma membrane, tethering, and docking, after which the lipid bilayers of the GCVs and the plasma membrane fuse, delivering GLUT4 to the cell surface for glucose uptake into the cell. Studies have revealed novel molecular regulators of the GLUT trafficking in podocytes and unraveled unexpected roles for GLUT1 and GLUT4 in the development of DKD, summarized in this review. These findings pave the way for better understanding of the mechanistic pathways associated with the development and progression of DKD and aid in the development of new treatments for this devastating disease.
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Lack of CD2-associated protein (CD2AP) in mice increases podocyte apoptosis and leads to glomerulosclerosis and renal failure. We showed previously that SHIP2, a negative regulator of the PI3K/AKT signalling pathway, interacts with CD2AP. Here, we found that the expression level and activity of SHIP2 and production of reactive oxygen species (ROS) are increased in cultured CD2AP knockout (CD2AP-/-) mouse podocytes. Oxidative stress was also increased in CD2AP-/- mouse glomeruli in vivo. We found that puromycin aminonucleoside (PA), known to increase ROS production and apoptosis, increases SHIP2 activity and reduces CD2AP expression in cultured human podocytes. PDK1 and CDK2, central regulators of AKT, were downregulated in CD2AP-/- or PA-treated podocytes. Downregulation of PDK1 and CDK2, ROS generation and apoptosis were prevented by CD2AP overexpression in both models. Notably, inhibition of SHIP2 activity with a small molecule inhibitor AS1949490 ameliorated ROS production in CD2AP-/- podocytes, but, surprisingly, further reduced PDK1 expression and aggravated apoptosis. AKT- and ERK-mediated signalling was diminished and remained reduced after AS1949490 treatment in the absence of CD2AP. The data suggest that inhibition of the catalytic activity of SHIP2 is beneficial in reducing oxidative stress, but leads to deleterious increase in apoptosis in podocytes with reduced expression of CD2AP.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/deficiência , Apoptose/genética , Proteínas do Citoesqueleto/deficiência , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/antagonistas & inibidores , Podócitos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Animais , Biomarcadores , Células Cultivadas , Imunofluorescência , Regulação da Expressão Gênica , Humanos , Glomérulos Renais/metabolismo , Glomérulos Renais/patologia , Camundongos , Fosfatidilinositol 3-Quinases/metabolismo , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/genética , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de SinaisRESUMO
Nephrin is a core component of podocyte (glomerular epithelial cell) slit diaphragm and is required for kidney ultrafiltration. Down-regulation or mislocalization of nephrin has been observed in diabetic kidney disease (DKD), characterized by albuminuria. Here, we investigate the role of protein kinase C and casein kinase 2 substrate in neurons 2 (PACSIN2), a regulator of endocytosis and recycling, in the trafficking of nephrin and development of DKD. We observe that PACSIN2 is up-regulated and nephrin mislocalized in podocytes of obese Zucker diabetic fatty (ZDF) rats that have altered renal function. In cultured podocytes, PACSIN2 and nephrin colocalize and interact. We show that nephrin is endocytosed in PACSIN2-positive membrane regions and that PACSIN2 overexpression increases both nephrin endocytosis and recycling. We identify rabenosyn-5, which is involved in early endosome maturation and endosomal sorting, as a novel interaction partner of PACSIN2. Interestingly, rabenosyn-5 expression is increased in podocytes in obese ZDF rats, and, in vitro, its overexpression enhances the association of PACSIN2 and nephrin. We also show that palmitate, which is elevated in diabetes, enhances this association. Collectively, PACSIN2 is up-regulated and nephrin is abnormally localized in podocytes of diabetic ZDF rats. In vitro, PACSIN2 enhances nephrin turnover apparently via a mechanism involving rabenosyn-5. The data suggest that elevated PACSIN2 expression accelerates nephrin trafficking and associates with albuminuria.-Dumont, V., Tolvanen, T. A., Kuusela, S., Wang, H., Nyman, T. A., Lindfors, S., Tienari, J., Nisen, H., Suetsugu, S., Plomann, M., Kawachi, H., Lehtonen, S. PACSIN2 accelerates nephrin trafficking and is up-regulated in diabetic kidney disease.