RESUMO
BACKGROUND: The nicotinamide adenine dinucleotide phosphate oxidase isoform 4 (Nox4) mediates reactive oxygen species (ROS) production and renal fibrosis in diabetic kidney disease (DKD) at the level of the podocyte. However, the mitochondrial localization of Nox4 and its role as a mitochondrial bioenergetic sensor has recently been reported. Whether Nox4 drives pathology in DKD within the proximal tubular compartment, which is densely packed with mitochondria, is not yet known. METHODS: We generated a proximal tubular-specific Nox4 knockout mouse model by breeding Nox4flox/flox mice with mice expressing Cre recombinase under the control of the sodium-glucose cotransporter-2 promoter. Subsets of Nox4ptKO mice and their Nox4flox/flox littermates were injected with streptozotocin (STZ) to induce diabetes. Mice were followed for 20 weeks and renal injury was assessed. RESULTS: Genetic ablation of proximal tubular Nox4 (Nox4ptKO) resulted in no change in renal function and histology. Nox4ptKO mice and Nox4flox/flox littermates injected with STZ exhibited the hallmarks of DKD, including hyperfiltration, albuminuria, renal fibrosis and glomerulosclerosis. Surprisingly, diabetes-induced renal injury was not improved in Nox4ptKO STZ mice compared with Nox4flox/flox STZ mice. Although diabetes conferred ROS overproduction and increased the mitochondrial oxygen consumption rate, proximal tubular deletion of Nox4 did not normalize oxidative stress or mitochondrial bioenergetics. CONCLUSIONS: Taken together, these results demonstrate that genetic deletion of Nox4 from the proximal tubules does not influence DKD development, indicating that Nox4 localization within this highly energetic compartment is dispensable for chronic kidney disease pathogenesis in the setting of diabetes.
Assuntos
Diabetes Mellitus Experimental , Nefropatias Diabéticas , Animais , Nefropatias Diabéticas/genética , Rim , Túbulos Renais , Túbulos Renais Proximais , Camundongos , NADP , NADPH Oxidase 4/genética , NADPH Oxidases/genética , Espécies Reativas de OxigênioRESUMO
Lesions issued from the ischemia/reperfusion (I/R) stress are a major challenge in human pathophysiology. Of human organs, the kidney is highly sensitive to I/R because of its high oxygen demand and poor regenerative capacity. Previous studies have shown that targeting the hypusination pathway of eIF5A through GC7 greatly improves ischemic tolerance and can be applied successfully to kidney transplants. The protection process correlates with a metabolic shift from oxidative phosphorylation to glycolysis. Because the protein kinase B Akt is involved in ischemic protective mechanisms and glucose metabolism, we looked for a link between the effects of GC7 and Akt in proximal kidney cells exposed to anoxia or the mitotoxic myxothiazol. We found that GC7 treatment resulted in impaired Akt phosphorylation at the Ser473 and Thr308 sites, so the effects of direct Akt inhibition as a preconditioning protocol on ischemic tolerance were investigated. We evidenced that Akt inhibitors provide huge protection for kidney cells against ischemia and myxothiazol. The pro-survival effect of Akt inhibitors, which is reversible, implied a decrease in mitochondrial ROS production but was not related to metabolic changes or an antioxidant defense increase. Therefore, the inhibition of Akt can be considered as a preconditioning treatment against ischemia.
Assuntos
Hipóxia/tratamento farmacológico , Rim/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Animais , Antioxidantes/farmacologia , Células Cultivadas , Hipóxia/metabolismo , Precondicionamento Isquêmico/métodos , Rim/metabolismo , Metacrilatos/farmacologia , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Fosforilação/efeitos dos fármacos , Substâncias Protetoras/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Traumatismo por Reperfusão/tratamento farmacológico , Traumatismo por Reperfusão/metabolismo , Tiazóis/farmacologiaRESUMO
The eIF5A hypusination inhibitor GC7 (N1-guanyl-1,7-diaminoheptane) was shown to protect from ischemic injuries. We hypothesized that GC7 could be useful for preconditioning kidneys from donors before transplantation. Using a preclinical porcine brain death (BD) donation model, we carried out in vivo evaluation of GC7 pretreatment (3 mg/kg iv, 5 minutes after BD) at the beginning of the 4h-donor management, after which kidneys were collected and cold-stored (18h in University of Wisconsin solution) and 1 was allotransplanted. Groups were defined as following (n = 6 per group): healthy (CTL), untreated BD (Vehicle), and GC7-treated BD (Vehicle + GC7). At the end of 4h-management, GC7 treatment decreased BD-induced markers, as radical oxygen species markers. In addition, GC7 increased expression of mitochondrial protective peroxisome proliferator-activated receptor-gamma coactivator-1-alpha (PGC1α) and antioxidant proteins (superoxyde-dismutase-2, heme oxygenase-1, nuclear factor [erythroid-derived 2]-like 2 [NRF2], and sirtuins). At the end of cold storage, GC7 treatment induced an increase of NRF2 and PGC1α mRNA and a better mitochondrial integrity/homeostasis with a decrease of dynamin- related protein-1 activation and increase of mitofusin-2. Moreover, GC7 treatment significantly improved kidney outcome during 90 days follow-up after transplantation (fewer creatininemia and fibrosis). Overall, GC7 treatment was shown to be protective for kidneys against BD-induced injuries during donor management and subsequently appeared to preserve antioxidant defenses and mitochondria homeostasis; these protective effects being accompanied by a better transplantation outcome.
Assuntos
Transplante de Rim , Traumatismo por Reperfusão , Adenosina , Alopurinol , Animais , Morte Encefálica , Glutationa , Insulina , Rim/metabolismo , Transplante de Rim/efeitos adversos , Soluções para Preservação de Órgãos , Fatores de Iniciação de Peptídeos/metabolismo , Proteínas de Ligação a RNA , Rafinose , Traumatismo por Reperfusão/etiologia , Traumatismo por Reperfusão/prevenção & controle , SuínosRESUMO
The eukaryotic initiation factor 5A (eIF5A), which is highly conserved throughout evolution, has the unique characteristic of post-translational activation through hypusination. This modification is catalyzed by two enzymatic steps involving deoxyhypusine synthase (DHPS) and deoxyhypusine hydroxylase (DOHH). Notably, eIF5A may be involved in regulating the lifespan of Drosophila during long-term hypoxia. Therefore, we investigated the possibility of a link between eIF5A hypusination and cellular resistance to hypoxia/anoxia. Pharmacologic targeting of DHPS by N1-guanyl-1,7-diaminoheptane (GC7) or RNA interference-mediated inhibition of DHPS or DOHH induced tolerance to anoxia in immortalized mouse renal proximal cells. Furthermore, GC7 treatment of cells reversibly induced a metabolic shift toward glycolysis as well as mitochondrial remodeling and led to downregulated expression and activity of respiratory chain complexes, features characteristic of mitochondrial silencing. GC7 treatment also attenuated anoxia-induced generation of reactive oxygen species in these cells and in normoxic conditions, decreased the mitochondrial oxygen consumption rate of cultured cells and mice. In rats, intraperitoneal injection of GC7 substantially reduced renal levels of hypusinated eIF5A and protected against ischemia-reperfusion-induced renal injury. Finally, in the preclinical pig kidney transplant model, intravenous injection of GC7 before kidney removal significantly improved graft function recovery and late graft function and reduced interstitial fibrosis after transplant. This unconventional signaling pathway offers an innovative therapeutic target for treating hypoxic-ischemic human diseases and organ transplantation.
Assuntos
Morte Celular/efeitos dos fármacos , Transplante de Rim , Lisina/análogos & derivados , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/fisiologia , Fatores de Iniciação de Peptídeos/efeitos dos fármacos , Proteínas de Ligação a RNA/efeitos dos fármacos , Animais , Hipóxia Celular/efeitos dos fármacos , Células Cultivadas , Feminino , Lisina/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Oxigenases de Função Mista , Ratos , Ratos Wistar , Suínos , Resultado do Tratamento , Fator de Iniciação de Tradução Eucariótico 5ARESUMO
Loss of E-cadherin marks a defect in epithelial integrity and polarity during tissue injury and fibrosis. Whether loss of E-cadherin plays a causal role in fibrosis is uncertain. α3ß1 Integrin has been identified to complex with E-cadherin in cell-cell adhesion, but little is known about the details of their cross talk. Herein, E-cadherin gene (Cdh1) was selectively deleted from proximal tubules of murine kidney by Sglt2Cre. Ablation of E-cadherin up-regulated α3ß1 integrin at cell-cell adhesion. E-cadherin-deficient proximal tubular epithelial cell displayed enhanced transforming growth factor-ß1-induced α-smooth muscle actin (α-SMA) and vimentin expression, which was suppressed by siRNA silencing of α3 integrin, but not ß1 integrin. Up-regulation of transforming growth factor-ß1-induced α-SMA was mediated by an α3 integrin-dependent increase in integrin-linked kinase (ILK). Src phosphorylation of ß-catenin and consequent p-ß-catenin-Y654/p-Smad2 transcriptional complex underlies the transcriptional up-regulation of ILK. Kidney fibrosis after unilateral ureteric obstruction or ischemia reperfusion was increased in proximal tubule E-cadherin-deficient mice in comparison to that of E-cadherin intact control mice. The exacerbation of fibrosis was explained by the α3 integrin-dependent increase of ILK, ß-catenin nuclear translocation, and α-SMA/proximal tubular-specific Cre double positive staining in proximal tubular epithelial cell. These studies delineate a nonconventional integrin/ILK signaling by α3 integrin-dependent Src/p-ß-catenin-Y654/p-Smad2-mediated up-regulation of ILK through which loss of E-cadherin leads to kidney fibrosis.
Assuntos
Caderinas/deficiência , Integrina alfa3beta1/metabolismo , Nefropatias/patologia , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Western Blotting , Adesão Celular , Imunoprecipitação da Cromatina , Modelos Animais de Doenças , Fibrose/metabolismo , Fibrose/patologia , Imuno-Histoquímica , Imunoprecipitação , Nefropatias/metabolismo , Túbulos Renais Proximais/metabolismo , Túbulos Renais Proximais/patologia , Camundongos , Camundongos Knockout , Microscopia Eletrônica de Transmissão , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais/fisiologiaRESUMO
The study of kidney cancer pathogenesis and its treatment has been limited by the scarcity of genetically defined animal models. The FLCN gene that codes for the protein folliculin, mutated in Birt-Hogg-Dubé syndrome, presents a new target for mouse modeling of kidney cancer. Here we developed a kidney-specific knockout model by disrupting the mouse Flcn in the proximal tubules, thus avoiding homozygous embryonic lethality or neonatal mortality, and eliminating the requirement of loss of heterozygosity for tumorigenesis. This knockout develops renal cysts and early onset (6 months) of multiple histological subtypes of renal neoplasms featuring high tumor penetrance. Although the majority of the tumors were chromophobe renal cell carcinomas in affected mice under 1 year of age, papillary renal cell carcinomas predominated in the kidneys of older knockout mice. This renal neoplasia from cystic hyperplasia at 4 months to high-grade renal tumors by 16 months represented the progression of tumorigenesis. The mTOR and TGF-ß signalings were upregulated in Flcn-deficient tumors, and these two activated pathways may synergetically cause renal tumorigenesis. Treatment of knockout mice with the mTOR inhibitor rapamycin for 10 months led to the suppression of tumor growth. Thus, our model recapitulates human Birt-Hogg-Dubé kidney tumorigenesis, provides a valuable tool for further study of Flcn-deficient renal tumorigenesis, and tests new drugs/approaches to their treatment.
Assuntos
Carcinoma de Células Renais/genética , Carcinoma de Células Renais/patologia , Cistos/patologia , Modelos Animais de Doenças , Neoplasias Renais/genética , Neoplasias Renais/patologia , Túbulos Renais Proximais/patologia , Proteínas Proto-Oncogênicas/genética , Proteínas Supressoras de Tumor/genética , Animais , Antibióticos Antineoplásicos/uso terapêutico , Carcinogênese/genética , Carcinoma de Células Renais/tratamento farmacológico , Carcinoma de Células Renais/metabolismo , Cistos/genética , Hiperplasia/patologia , Neoplasias Renais/tratamento farmacológico , Neoplasias Renais/metabolismo , Camundongos , Camundongos Knockout , Transdução de Sinais , Sirolimo/uso terapêutico , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/metabolismo , Fator de Crescimento Transformador beta/metabolismoRESUMO
Mechanical forces associated with fluid flow and/or circumferential stretch are sensed by renal epithelial cells and contribute to both adaptive or disease states. Non-selective stretch-activated ion channels (SACs), characterized by a lack of inactivation and a remarkably slow deactivation, are active at the basolateral side of renal proximal convoluted tubules. Knockdown of Piezo1 strongly reduces SAC activity in proximal convoluted tubule epithelial cells. Similarly, overexpression of Polycystin-2 (PC2) or, to a greater extent its pathogenic mutant PC2-740X, impairs native SACs. Moreover, PC2 inhibits exogenous Piezo1 SAC activity. PC2 coimmunoprecipitates with Piezo1 and deletion of its N-terminal domain prevents both this interaction and inhibition of SAC activity. These findings indicate that renal SACs depend on Piezo1, but are critically conditioned by PC2.
Assuntos
Células Epiteliais/metabolismo , Canais Iônicos/metabolismo , Mecanotransdução Celular , Canais de Cátion TRPP/metabolismo , Potenciais de Ação , Animais , Sítios de Ligação , Células COS , Células Cultivadas , Chlorocebus aethiops , Células Epiteliais/fisiologia , Túbulos Renais/citologia , Mutação , Ligação Proteica , Canais de Cátion TRPC/metabolismo , Canais de Cátion TRPP/química , Canais de Cátion TRPP/genéticaRESUMO
Suppressor of cytokine signaling 3 (SOCS-3) is an important intracellular negative regulator of several signaling pathways. We found that SOCS-3 is highly expressed in renal proximal tubules during acute kidney injury. To test the impact of this, conditional proximal tubular knockout mice (SOCS-3(sglt2Δ/sglt2Δ)) were created. These mice had better kidney function than their wild-type counterparts in aristolochic acid nephropathy and after ischemia/reperfusion injury. Kidneys of these knockout mice showed significantly more proximal tubular cell proliferation during the repair phase. A direct effect of SOCS-3 on tubular cell cycling was demonstrated by in vitro experiments showing a JAK/STAT pathway-dependent antimitotic effect of SOCS-3. Furthermore, acute damaged kidneys of the knockout mice contained increased numbers of F4/80(+) cells. Phenotypic analysis of these F4/80(+) cells indicated a polarization from classically activated to alternatively activated macrophages. In vitro, SOCS-3-overexpressing renal epithelial cells directly induced classical activation in cocultured macrophages, supporting the observed in vivo phenomenon. Thus, upregulation of SOCS-3 in stressed proximal tubules plays an important role during acute kidney injury by inhibition of reparative proliferation and by modulation of the macrophage phenotype. Antagonizing SOCS-3 could have therapeutic potential for acute kidney injury.
Assuntos
Injúria Renal Aguda/metabolismo , Túbulos Renais Proximais/metabolismo , Macrófagos/metabolismo , Proteínas Supressoras da Sinalização de Citocina/deficiência , Injúria Renal Aguda/genética , Injúria Renal Aguda/imunologia , Injúria Renal Aguda/patologia , Injúria Renal Aguda/fisiopatologia , Animais , Antígenos de Diferenciação/metabolismo , Proliferação de Células , Células Cultivadas , Técnicas de Cocultura , Modelos Animais de Doenças , Regulação da Expressão Gênica , Genótipo , Janus Quinases/metabolismo , Túbulos Renais Proximais/imunologia , Túbulos Renais Proximais/patologia , Túbulos Renais Proximais/fisiopatologia , Ativação de Macrófagos , Macrófagos/imunologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenótipo , Interferência de RNA , Fatores de Transcrição STAT/metabolismo , Transdução de Sinais , Proteína 3 Supressora da Sinalização de Citocinas , Proteínas Supressoras da Sinalização de Citocina/genética , Fatores de Tempo , TransfecçãoRESUMO
Adaptation to hypoxia is an essential physiological response to decrease in tissue oxygenation. This process is primarily under the control of transcriptional activator hypoxia-inducible factor (HIF1). A better understanding of the intracellular HIF1 stabilization pathway would help in management of various diseases characterized by anemia. Among human pathologies, cystic fibrosis disease is characterized by a chronic anemia that is inadequately compensated by the classical erythroid response mediated by the HIF pathway. Because the kidney expresses CFTR and is a master organ involved in the adaptation to hypoxia, we used renal cells to explore the relationship between CFTR and the HIF1-mediated pathway. To monitor the adaptive response to hypoxia, we engineered a hypoxia-induced fluorescent reporter system to determine whether CFTR modulates hypoxia-induced HIF1 stabilization. We show that CFTR is a regulator of HIF stabilization by controlling the intracellular reactive oxygen species (ROS) level through its ability to transport glutathione (a ROS scavenger) out of the cell. Moreover, we demonstrated in a mouse model that both the pharmacological inhibition and the ΔF508 mutation of CFTR lead to an impairment of the adaptive erythroid response to oxygen deprivation. We conclude that CFTR controls HIF stabilization through control of the level of intracellular ROS that act as signaling agents in the HIF-1 pathway.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Fibrose Cística/metabolismo , Fibrose Cística/fisiopatologia , Espaço Intracelular/metabolismo , Acetilcisteína/farmacologia , Animais , Anidrases Carbônicas/metabolismo , Hipóxia Celular/efeitos dos fármacos , Linhagem Celular , Canais de Cloreto/metabolismo , Fibrose Cística/urina , Regulador de Condutância Transmembrana em Fibrose Cística/antagonistas & inibidores , Modelos Animais de Doenças , Glutationa/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/química , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Espaço Intracelular/efeitos dos fármacos , Túbulos Renais Proximais/efeitos dos fármacos , Túbulos Renais Proximais/metabolismo , Túbulos Renais Proximais/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Modelos Biológicos , Mutação/genética , Concentração Osmolar , Oxirredução/efeitos dos fármacos , Estabilidade Proteica/efeitos dos fármacos , Estrutura Terciária de Proteína , Espécies Reativas de Oxigênio/metabolismoRESUMO
Through kidney transplantation, ischemia/reperfusion is known to induce tissular injury due to cell energy shortage, oxidative stress, and endoplasmic reticulum (ER) stress. ER stress stems from an accumulation of unfolded or misfolded proteins in the lumen of ER, resulting in the unfolded protein response (UPR). Adaptive UPR pathways can either restore protein homeostasis or can turn into a stress pathway leading to apoptosis. We have demonstrated that N1-guanyl-1,7-diamineoheptane (GC7), a specific inhibitor of eukaryotic Initiation Factor 5A (eIF5A) hypusination, confers an ischemic protection of kidney cells by tuning their metabolism and decreasing oxidative stress, but its role on ER stress was unknown. To explore this, we used kidney cells pretreated with GC7 and submitted to either warm or cold anoxia. GC7 pretreatment promoted cell survival in an anoxic environment concomitantly to an increase in xbp1 splicing and BiP level while eiF2α phosphorylation and ATF6 nuclear level decreased. These demonstrated a specific modulation of UPR pathways. Interestingly, the pharmacological inhibition of xbp1 splicing reversed the protective effect of GC7 against anoxia. Our results demonstrated that eIF5A hypusination inhibition modulates distinctive UPR pathways, a crucial mechanism for the protection against anoxia/reoxygenation.
Assuntos
Estresse do Retículo Endoplasmático , Isquemia , Rim , Fatores de Iniciação de Peptídeos , Traumatismo por Reperfusão , Humanos , Estresse do Retículo Endoplasmático/genética , Estresse do Retículo Endoplasmático/fisiologia , Hipóxia/genética , Hipóxia/metabolismo , Isquemia/genética , Isquemia/metabolismo , Rim/irrigação sanguínea , Rim/metabolismo , Fatores de Iniciação de Peptídeos/genética , Fatores de Iniciação de Peptídeos/metabolismo , Traumatismo por Reperfusão/genética , Traumatismo por Reperfusão/metabolismo , Resposta a Proteínas não Dobradas , Fator de Iniciação de Tradução Eucariótico 5ARESUMO
Small bowel transplantation has become an established procedure for treatment of irreversible intestinal failure. In this procedure, primary ischemia and reperfusion is inevitable and will lead to some level of tissue injury. Both clinical and experimental data demonstrate that events occurring at the time of transplantation, called ischemia reperfusion injury (IRI), may have deleterious short- and long-term effects, manifesting as increased episodes of acute rejection and chronic allograft dysfunction. Recently, the acute phase of IRI has been increasingly viewed as part of the innate immune response to the lack of vascular perfusion and oxygen. Research on intestinal IRI that aims to understand its mechanisms and the means to reduce its impact on morbidity and mortality related to intestinal transplantations is considered important because a link has been suggested between innate immunity, adaptive immune responses and organ regeneration, and thus long-term graft function. This article provides an overview of porcine models commonly used to study intestinal reperfusion injury and to evaluate intestinal transplant protocols. It also updates the current knowledge obtained from this model, establishing the pig as a reference standard in intestinal transplantation research.
Assuntos
Intestinos/irrigação sanguínea , Intestinos/transplante , Modelos Animais , Traumatismo por Reperfusão/etiologia , Animais , Heme Oxigenase-1/fisiologia , Sistema Imunitário/anatomia & histologia , Intestinos/anatomia & histologia , Óxido Nítrico/fisiologia , Traumatismo por Reperfusão/imunologia , Suínos , Transplante HomólogoRESUMO
OBJECTIVES: We evaluated whether IGL-1, a graft preservation solution containing polyethylene glycol, improves the outcome of small bowel grafts in comparison to the University of Wisconsin (UW) solution in a pig allotransplantation model. MATERIALS AND METHODS: Seventeen pigs were randomly allocated to group 1 (n = 10; intestinal allotransplantation with IGL-1) and group 2 (n = 7; allotransplantation with UW). Pigs received no immunosuppression and were sacrificed on postoperative d (POD) 8. Intestinal specimens were obtained from the animal immediately before cold flushing (T0), 2 h after graft reperfusion (T1), and at sacrifice (T2). RESULTS: Survival rate to POD 8 was 50% in group 1 compared with 16% in group 2 (P < 0.05); 62% of pigs in group 1 did not present any acute cellular rejection (ACR) compared to 16% in group 2 (P < 0.05). Severe ACR rate was 25% in group 1 and 66% in group 2 (P < 0.05). iNOS activity and intestinal caspase 3 levels increased significantly between T0 and T1 in group 1 compared to group 2 (P < 0.05). Cell necrosis increased significantly between TO and T1 in group 2 compared with group 1 (P < 0.05) whereas cell apoptosis was significantly higher at T1 compared with T0 in group 1 in comparison to group 2. CONCLUSIONS: Our results show that IGL-1 improves intestinal graft viability as compared to UW solution, possibly by reducing graft immunogenicity and by favoring intestinal epithelial repair.
Assuntos
Rejeição de Enxerto/prevenção & controle , Sobrevivência de Enxerto/efeitos dos fármacos , Intestino Delgado/transplante , Soluções para Preservação de Órgãos/farmacologia , Polietilenoglicóis/farmacologia , Doença Aguda , Adenosina/farmacologia , Alopurinol/farmacologia , Animais , Apoptose/imunologia , Caspase 3/metabolismo , Feminino , Glutationa/farmacologia , Rejeição de Enxerto/imunologia , Rejeição de Enxerto/mortalidade , Sobrevivência de Enxerto/imunologia , Terapia de Imunossupressão , Insulina/farmacologia , Mucosa Intestinal/imunologia , Mucosa Intestinal/patologia , Intestino Delgado/imunologia , Intestino Delgado/patologia , Rafinose/farmacologia , Traumatismo por Reperfusão/imunologia , Traumatismo por Reperfusão/mortalidade , Traumatismo por Reperfusão/prevenção & controle , Sus scrofa , Transplante HomólogoRESUMO
Intracellular pH is a vital parameter that is maintained close to neutrality in all mammalian cells and tissues and acidic in most intracellular compartments. After presenting the main techniques used for intracellular an vesicular pH measurements we will briefly recall the main molecular mechanisms that affect and regulate intracellular pH. Following this we will discuss the large functional redundancy found in the transporters of H+ or acid-base equivalents. For this purpose, we will use mathematical modeling to simulate cellular response to persistent and/or transient acidification, in the presence of different transporters, single or in combination. We will also test the presence or absence of intracellular buffering. This latter section will highlight how modeling can yield fundamental insight into deep biological questions such as the utility of functional redundancy in natural selection.
RESUMO
The present study tested the hypothesis that intrarenal adenoviral transfer of an intracellular cyan fluorescent fusion of angiotensin II (ECFP/ANG II) selectively in proximal tubules of the kidney increases blood pressure by activating AT(1) (AT(1a)) receptors. Intrarenal transfer of ECFP/ANG II was induced in the superficial cortex of rat and mouse kidneys, and the sodium and glucose cotransporter 2 (sglt2) promoter was used to drive ECFP/ANG II expression selectively in proximal tubules. Intrarenal transfer of ECFP/ANG II induced a time-dependent, proximal tubule-selective expression of ECFP/ANG II in the cortex, which peaked at 2 wk and was sustained for 4 wk. ECFP/ANG II expression was low in the glomeruli and the entire medulla and was absent in the contralateral kidney or extrarenal tissues. At its peak of expression in proximal tubules at day 14, ANG II was increased by twofold in the kidney (P < 0.01) and more than threefold in proximal tubules (P < 0.01), but remained unchanged in plasma or urine. Systolic blood pressure was increased in ECFP/ANG II-transferred rats by 28 ± 6 mmHg (P < 0.01), whereas fractional sodium excretion was decreased by 20% (P < 0.01) and fractional lithium excretion was reduced by 24% (P < 0.01). These effects were blocked by losartan and prevented in AT(1a) knockout mice. Transfer of a scrambled ECFP/ANG IIc had no effects on blood pressure, kidney, and proximal tubule ANG II, or sodium excretion. These results provide evidence that proximal tubule-selective transfer of an intracellular ANG II fusion protein increases blood pressure by activating AT(1a) receptors and increasing sodium reabsorption in proximal tubules.
Assuntos
Angiotensina II/biossíntese , Pressão Sanguínea , Técnicas de Transferência de Genes , Proteínas de Fluorescência Verde/biossíntese , Hipertensão/metabolismo , Túbulos Renais Proximais/metabolismo , Adenoviridae/genética , Angiotensina II/sangue , Angiotensina II/genética , Angiotensina II/urina , Bloqueadores do Receptor Tipo 1 de Angiotensina II/farmacologia , Animais , Pressão Sanguínea/efeitos dos fármacos , Pressão Sanguínea/genética , Células Cultivadas , Modelos Animais de Doenças , Vetores Genéticos , Proteínas de Fluorescência Verde/sangue , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/urina , Hipertensão/genética , Hipertensão/fisiopatologia , Hipertensão/prevenção & controle , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Natriurese , Ratos , Ratos Sprague-Dawley , Receptor Tipo 1 de Angiotensina/deficiência , Receptor Tipo 1 de Angiotensina/efeitos dos fármacos , Receptor Tipo 1 de Angiotensina/genética , Receptor Tipo 1 de Angiotensina/metabolismo , Proteínas Recombinantes de Fusão/biossíntese , Fatores de Tempo , Transdução Genética , Transfecção , Regulação para Cima , MicçãoRESUMO
In eukaryotes, the polyamine pathway generates spermidine that activates the hypusination of the translation factor eukaryotic initiation factor 5A (eIF5A). Hypusinated-eIF5A modulates translation, elongation, termination and mitochondrial function. Evidence in model organisms like drosophila suggests that targeting polyamines synthesis might be of interest against ischemia. However, the potential of targeting eIF5A hypusination in stroke, the major therapeutic challenge specific to ischemia, is currently unknown. Using in vitro models of ischemic-related stress, we documented that GC7, a specific inhibitor of a key enzyme in the eIF5A activation pathway, affords neuronal protection. We identified the preservation of mitochondrial function and thereby the prevention of toxic ROS generation as major processes of GC7 protection. To represent a thoughtful opportunity of clinical translation, we explored whether GC7 administration reduces the infarct volume and functional deficits in an in vivo transient focal cerebral ischemia (tFCI) model in mice. A single GC7 pre- or post-treatment significantly reduces the infarct volume post-stroke. Moreover, GC7-post-treatment significantly improves mouse performance in the rotarod and Morris water-maze, highlighting beneficial effects on motor and cognitive post-stroke deficits. Our results identify the targeting of the polyamine-eIF5A-hypusine axis as a new therapeutic opportunity and new paradigm of research in stroke and ischemic diseases.
Assuntos
Guanina/análogos & derivados , Lisina/análogos & derivados , Mitocôndrias/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/antagonistas & inibidores , Fatores de Iniciação de Peptídeos/metabolismo , Proteínas de Ligação a RNA/metabolismo , Acidente Vascular Cerebral/terapia , Animais , Comportamento Animal/efeitos dos fármacos , Cognição/efeitos dos fármacos , Guanina/administração & dosagem , Guanina/farmacologia , Guanina/uso terapêutico , Injeções Intraperitoneais , Ataque Isquêmico Transitório/tratamento farmacológico , Ataque Isquêmico Transitório/prevenção & controle , Lisina/antagonistas & inibidores , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/ultraestrutura , Modelos Animais , Neuroproteção/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Fatores de Iniciação de Peptídeos/efeitos dos fármacos , Poliaminas/metabolismo , Proteínas de Ligação a RNA/efeitos dos fármacos , Espécies Reativas de Oxigênio/toxicidade , Acidente Vascular Cerebral/metabolismo , Fator de Iniciação de Tradução Eucariótico 5ARESUMO
Disorders characterized by ischemia/reperfusion (I/R) are the most common causes of debilitating diseases and death in stroke, cardiovascular ischemia, acute kidney injury or organ transplantation. In the latter example the I/R step defines both the amplitude of the damages to the graft and the functional recovery outcome. During transplantation the kidney is subjected to blood flow arrest followed by a sudden increase in oxygen supply at the time of reperfusion. This essential clinical protocol causes massive oxidative stress which is at the basis of cell death and tissue damage. The involvement of both reactive oxygen species (ROS) and nitric oxides (NO) has been shown to be a major cause of these cellular damages. In fact, in non-physiological situations, these species escape endogenous antioxidant control and dangerously accumulate in cells. In recent years, the objective has been to find clinical and pharmacological treatments to reduce or prevent the appearance of oxidative stress in ischemic pathologies. This is very relevant because, due to the increasing success of organ transplantation, clinicians are required to use limit organs, the preservation of which against oxidative stress is crucial for a better outcome. This review highlights the key actors in oxidative stress which could represent new pharmacological targets.
Assuntos
Transplante de Rim , Traumatismo por Reperfusão , Antioxidantes/uso terapêutico , Humanos , Estresse Oxidativo , Espécies Reativas de OxigênioRESUMO
Since the demonstration of its involvement in cell proliferation, the eukaryotic initiation factor 5A (eIF5A) has been studied principally in relation to the development and progression of cancers in which the isoform A2 is mainly expressed. However, an increasing number of studies report that the isoform A1, which is ubiquitously expressed in normal cells, exhibits novel molecular features that reveal its new relationships between cellular functions and organ homeostasis. At a first glance, eIF5A can be regarded, among other things, as a factor implicated in the initiation of translation. Nevertheless, at least three specificities: (1) its extreme conservation between species, including plants, throughout evolution, (2) its very special and unique post-translational modification through the activating-hypusination process, and finally (3) its close relationship with the polyamine pathway, suggest that the role of eIF5A in living beings remains to be uncovered. In fact, and beyond its involvement in facilitating the translation of proteins containing polyproline residues, eIF5A is implicated in various physiological processes including ischemic tolerance, metabolic adaptation, aging, development, and immune cell differentiation. These newly discovered physiological properties open up huge opportunities in the clinic for pathologies such as, for example, the ones in which the oxygen supply is disrupted. In this latter case, organ transplantation, myocardial infarction or stroke are concerned, and the current literature defines eIF5A as a new drug target with a high level of potential benefit for patients with these diseases or injuries. Moreover, the recent use of genomic and transcriptomic association along with metadata studies also revealed the implication of eIF5A in genetic diseases. Thus, this review provides an overview of eIF5A from its molecular mechanism of action to its physiological roles and the clinical possibilities that have been recently reported in the literature.
RESUMO
Signaling, proliferation, and inflammation are dependent on K63-linked ubiquitination-conjugation of a chain of ubiquitin molecules linked via lysine 63. However, very little information is currently available about how K63-linked ubiquitination is subverted in cancer. The present study provides, for the first time, evidence that cadmium (Cd), a widespread environmental carcinogen, is a potent activator of K63-linked ubiquitination, independently of oxidative damage, activation of ubiquitin ligase, or proteasome impairment. We show that Cd induces the formation of protein aggregates that sequester and inactivate cylindromatosis (CYLD) and selective autophagy, two tumor suppressors that deubiquitinate and degrade K63-ubiquitinated proteins, respectively. The aggregates are constituted of substrates of selective autophagy-SQSTM1, K63-ubiquitinated proteins, and mitochondria. These protein aggregates also cluster double-membrane remnants, which suggests an impairment in autophagosome maturation. However, failure to eliminate these selective cargos is not due to alterations in the general autophagy process, as degradation of long-lived proteins occurs normally. We propose that the simultaneous disruption of CYLD and selective autophagy by Cd feeds a vicious cycle that further amplifies K63-linked ubiquitination and downstream activation of the NF-κB pathway, processes that support cancer progression. These novel findings link together impairment of selective autophagy, K63-linked ubiquitination, and carcinogenesis.
RESUMO
Inhibition of the eukaryotic initiation factor 5A activation by the spermidine analogue GC7 has been shown to protect proximal cells and whole kidneys against an acute episode of ischaemia. The highlighted mechanism involves a metabolic switch from oxidative phosphorylation toward glycolysis allowing cells to be transiently independent of oxygen supply. Here we show that GC7 decreases protein expression of the renal GLUT1 glucose transporter leading to a decrease in transcellular glucose flux. At the same time, GC7 modifies the native energy source of the proximal cells from glutamine toward glucose use. Thus, GC7 acutely and reversibly reprogrammes function and metabolism of kidney cells to make glucose its single substrate, and thus allowing cells to be oxygen independent through anaerobic glycolysis. The physiological consequences are an increase in the renal excretion of glucose and lactate reflecting a decrease in glucose reabsorption and an increased glycolysis. Such a reversible reprogramming of glucose handling and oxygen dependence of kidney cells by GC7 represents a pharmacological opportunity in ischaemic as well as hyperglycaemia-associated pathologies from renal origin.
Assuntos
Glucose/metabolismo , Rim/metabolismo , Fatores de Iniciação de Peptídeos/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Masculino , Camundongos , Fator de Iniciação de Tradução Eucariótico 5ARESUMO
[Figure: see text].