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It is well-established that dysfunction of megalin-mediated albumin endocytosis by proximal tubule epithelial cells (PTECs) and the activation of the Renin-Angiotensin System (RAS) play significant roles in the development of Diabetic Kidney Disease (DKD). However, the precise correlation between these factors still requires further investigation. In this study, we aimed to elucidate the potential role of angiotensin II (Ang II), a known effector of RAS, as the mediator of albumin endocytosis dysfunction induced by high glucose (HG) in PTECs. To achieve this, we utilized LLC-PK1 and HK-2 cells, which are well-established in vitro models of PTECs. Using albumin-FITC or DQ-albumin as tracers, we observed that incubation of LLC-PK1 and HK-2 cells with HG (25 mM for 48 h) significantly reduced canonical receptor-mediated albumin endocytosis, primarily due to the decrease in megalin expression. HG increased the concentration of Ang II in the LLC-PK1 cell supernatant, a phenomenon associated with an increase in angiotensin-converting enzyme (ACE) expression and a decrease in prolyl carboxypeptidase (PRCP) expression. ACE type 2 (ACE2) expression remained unchanged. To investigate the potential impact of Ang II on HG effects, the cells were co-incubated with angiotensin receptor inhibitors. Only co-incubation with 10-7 M losartan (an antagonist for type 1 angiotensin receptor, AT1R) attenuated the inhibitory effect of HG on albumin endocytosis, as well as megalin expression. Our findings contribute to understanding the genesis of tubular albuminuria observed in the early stages of DKD, which involves the activation of the Ang II/AT1R axis by HG.
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Tubular proteinuria is a common feature in COVID-19 patients, even in the absence of established acute kidney injury. SARS-CoV-2 spike protein (S protein) was shown to inhibit megalin-mediated albumin endocytosis in proximal tubule epithelial cells (PTECs). Angiotensin-converting enzyme type 2 (ACE2) was not directly involved. Since Toll-like receptor 4 (TLR4) mediates S protein effects in various cell types, we hypothesized that TLR4 could be participating in the inhibition of PTECs albumin endocytosis elicited by S protein. Two different models of PTECs were used: porcine proximal tubule cells (LLC-PK1) and human embryonic kidney cells (HEK-293). S protein reduced Akt activity by specifically inhibiting of threonine 308 (Thr308) phosphorylation, a process mediated by phosphoinositide-dependent kinase 1 (PDK1). GSK2334470, a PDK1 inhibitor, decreased albumin endocytosis and megalin expression mimicking S protein effect. S protein did not change total TLR4 expression but decreased its surface expression. LPS-RS, a TLR4 antagonist, also counteracted the effects of the S protein on Akt phosphorylation at Thr308, albumin endocytosis, and megalin expression. Conversely, these effects of the S protein were replicated by LPS, an agonist of TLR4. Incubation of PTECs with a pseudovirus containing S protein inhibited albumin endocytosis. Null or VSV-G pseudovirus, used as control, had no effect. LPS-RS prevented the inhibitory impact of pseudovirus containing the S protein on albumin endocytosis but had no influence on virus internalization. Our findings demonstrate that the inhibitory effect of the S protein on albumin endocytosis in PTECs is mediated through TLR4, resulting from a reduction in megalin expression.
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Endocitose , Túbulos Renais Proximais , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus , Receptor 4 Toll-Like , Receptor 4 Toll-Like/metabolismo , Endocitose/efeitos dos fármacos , Humanos , Túbulos Renais Proximais/metabolismo , Túbulos Renais Proximais/virologia , Animais , Glicoproteína da Espícula de Coronavírus/metabolismo , SARS-CoV-2/metabolismo , Células HEK293 , Suínos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fosforilação , COVID-19/metabolismo , COVID-19/virologia , COVID-19/patologia , Albuminas/metabolismo , Células LLC-PK1 , Células Epiteliais/metabolismo , Células Epiteliais/virologiaRESUMO
Glioblastoma (GB) is the most aggressive primary malignant brain tumor and is associated with short survival. O-GlcNAcylation is an intracellular glycosylation that regulates protein function, enzymatic activity, protein stability, and subcellular localization. Aberrant O-GlcNAcylation is related to the tumorigenesis of different tumors, and mounting evidence supports O-GlcNAc transferase (OGT) as a potential therapeutic target. Here, we used two human GB cell lines alongside primary human astrocytes as a non-tumoral control to investigate the role of O-GlcNAcylation in cell proliferation, cell cycle, autophagy, and cell death. We observed that hyper O-GlcNAcylation promoted increased cellular proliferation, independent of alterations in the cell cycle, through the activation of autophagy. On the other hand, hypo O-GlcNAcylation inhibited autophagy, promoted cell death by apoptosis, and reduced cell proliferation. In addition, the decrease in O-GlcNAcylation sensitized GB cells to the chemotherapeutic temozolomide (TMZ) without affecting human astrocytes. Combined, these results indicated a role for O-GlcNAcylation in governing cell proliferation, autophagy, cell death, and TMZ response, thereby indicating possible therapeutic implications for treating GB. These findings pave the way for further research and the development of novel treatment approaches which may contribute to improved outcomes and increased survival rates for patients facing this challenging disease.
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BACKGROUND: Diabetic kidney disease (DKD) is a severe complication of diabetes mellitus (DM). It has been proposed that modifications in the function of proximal tubule epithelial cells (PTECs) precede glomerular damage during the onset of DKD. This study aimed to identify modifications in renal sodium handling in the early stage of DM and its molecular mechanism. METHODS: Streptozotocin (STZ)-induced diabetic BALB/c mice (STZ group) and LLC-PK1 cells, a model of PTECs, were used. All parameters were assessed in the 4th week after an initial injection of STZ. RESULTS: Early stage of DKD was characterized by hyperfiltration and PTEC dysfunction. STZ group exhibited increased urinary sodium excretion due to impairment of tubular sodium reabsorption. This was correlated to a decrease in cortical (Na++K+)ATPase (NKA) α1 subunit expression and enzyme activity and an increase in O-GlcNAcylation. RNAseq analysis of patients with DKD revealed an increase in expression of the glutamine-fructose aminotransferase (GFAT) gene, a rate-limiting step of hexosamine biosynthetic pathway, and a decrease in NKA expression. Incubation of LLC-PK1 cells with 10 µM thiamet G, an inhibitor of O-GlcNAcase, reduced the expression and activity of NKA and increased O-GlcNAcylation. Furthermore, 6-diazo-5-oxo-L-norleucine (DON), a GFAT inhibitor, or dapagliflozin, an SGLT2 inhibitor, avoided the inhibitory effect of HG on expression and activity of NKA associated with the decrease in O-GlcNAcylation. CONCLUSION: Our results show that the impairment of tubular sodium reabsorption, in the early stage of DM, is due to SGLT2-mediated HG influx in PTECs, increase in O-GlcNAcylation and reduction in NKA expression and activity.
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Diabetes Mellitus , Nefropatias Diabéticas , Camundongos , Suínos , Animais , Humanos , Túbulos Renais Proximais/metabolismo , Rim/metabolismo , Nefropatias Diabéticas/metabolismo , Sódio/metabolismo , Adenosina Trifosfatases/metabolismo , Diabetes Mellitus/metabolismoRESUMO
Introduction: Rapamycin is an immunosuppressor that acts by inhibiting the serine/threonine kinase mechanistic target of rapamycin complex 1. Therapeutic use of rapamycin is limited by its adverse effects. Proteinuria is an important marker of kidney damage and a risk factor for kidney diseases progression and has been reported in patients and animal models treated with rapamycin. However, the mechanism underlying proteinuria induced by rapamycin is still an open matter. In this work, we investigated the effects of rapamycin on parameters of renal function and structure and on protein handling by proximal tubule epithelial cells (PTECs). Methods: Healthy BALB/c mice were treated with 1.5 mg/kg rapamycin by oral gavage for 1, 3, or 7 days. At the end of each treatment, the animals were kept in metabolic cages and renal function and structural parameters were analyzed. LLC-PK1 cell line was used as a model of PTECs to test specific effect of rapamycin. Results: Rapamycin treatment did not change parameters of glomerular structure and function. Conversely, there was a transient increase in 24-h proteinuria, urinary protein to creatinine ratio (UPCr), and albuminuria in the groups treated with rapamycin. In accordance with these findings, rapamycin treatment decreased albumin-fluorescein isothiocyanate uptake in the renal cortex. This effect was associated with reduced brush border expression and impaired subcellular distribution of megalin in PTECs. The effect of rapamycin seems to be specific for albumin endocytosis machinery because it did not modify renal sodium handling or (Na++K+)ATPase activity in BALB/c mice and in the LLC-PK1 cell line. A positive Pearson correlation was found between megalin expression and albumin uptake while an inverse correlation was shown between albumin uptake and UPCr or 24-h proteinuria. Despite its effect on albumin handling in PTECs, rapamycin treatment did not induce tubular injury measured by interstitial space and collagen deposition. Conclusion: These findings suggest that proteinuria induced by rapamycin could have a tubular rather than a glomerular origin. This effect involves a specific change in protein endocytosis machinery. Our results open new perspectives on understanding the undesired effect of proteinuria generated by rapamycin.
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Subclinical acute kidney injury (subAKI) is characterized by tubule-interstitial injury without significant changes in glomerular function. SubAKI is associated with the pathogenesis and progression of acute and chronic kidney diseases. Currently, therapeutic strategies to treat subAKI are limited. The use of gold nanoparticles (AuNPs) has shown promising benefits in different models of diseases. However, their possible effects on subAKI are still unknown. Here, we investigated the effects of AuNPs on a mouse model of subAKI. Animals with subAKI showed increased functional and histopathologic markers of tubular injury. There were no changes in glomerular function and structure. The animals with subAKI also presented an inflammatory profile demonstrated by activation of Th1 and Th17 cells in the renal cortex. This phenotype was associated with decreased megalin-mediated albumin endocytosis and expression of proximal tubular megalin. AuNP treatment prevented tubule-interstitial injury induced by subAKI. This effect was associated with a shift to an anti-inflammatory Th2 response. Furthermore, AuNP treatment preserved megalin-mediated albumin endocytosis in vivo and in vitro. AuNPs were not nephrotoxic in healthy mice. These results suggest that AuNPs have a protective effect in the tubule-interstitial injury observed in subAKI, highlighting a promising strategy as a future antiproteinuric treatment.
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Injúria Renal Aguda , Nanopartículas Metálicas , Camundongos , Animais , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Ouro/farmacologia , Túbulos Renais Proximais , Modelos Animais de Doenças , Proteinúria/metabolismo , Proteinúria/patologia , Albuminas/metabolismo , Injúria Renal Aguda/metabolismoRESUMO
Diabetic kidney disease (DKD) is characterized by progressive impairment of kidney function. It has been postulated that tubule-interstitial injury, associated with tubular albuminuria, precedes glomerular damage in the early stage of DKD. Here, we wanted to determine if the development of tubule-interstitial injury at the early stage of DKD implies modulation of megalin-mediated protein reabsorption in proximal tubule epithelial cells (PTECs) by SGLT2-dependent high glucose influx. Rats with streptozotocin (STZ)-induced diabetes were treated or not with dapagliflozin (DAPA) for 8 weeks. Four experimental groups were generated: (1) CONT, control; (2) DAPA, rats treated with DAPA; (3) STZ, diabetic rats; (4) STZ + DAPA, diabetic rats treated with DAPA. No changes in glomerular structure and function were observed. The STZ group presented proteinuria and albuminuria associated with an increase in the fractional excretion of proteins. A positive correlation between glycemia and proteinuria was found. These phenomena were linked to a decrease in luminal and total megalin expression and, consequently, in albumin reabsorption in PTECs. We also observed tubule-interstitial injury characterized by an increase in urinary tubular injury biomarkers and changes in tubular histomorphometry parameters. In addition, inverse correlations were found between cortical albumin uptake and tubule-interstitial injury or glycemia. All these modifications were attenuated in the STZ + DAPA group. These results suggest that SGLT2-dependent high glucose influx into PTECs promotes a harmful effect on the PTECs, leading to the development of tubular albuminuria and tubule-interstitial injury preceding glomerular damage. These results expand current knowledge on the renoprotective effects of gliflozins.
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Diabetes Mellitus Experimental , Nefropatias Diabéticas , Ratos , Animais , Nefropatias Diabéticas/metabolismo , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Albuminúria , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Experimental/induzido quimicamente , Transportador 2 de Glucose-Sódio/metabolismo , Proteínas/metabolismo , Albuminas/metabolismo , Glucose/efeitos adversosRESUMO
Since the outbreak of COVID-19 disease, a bidirectional interaction between kidney disease and the progression of COVID-19 has been demonstrated. Kidney disease is an independent risk factor for mortality of patients with COVID-19 as well as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection leading to the development of acute kidney injury (AKI) and chronic kidney disease (CKD) in patients with COVID-19. However, the detection of kidney damage in patients with COVID-19 may not occur until an advanced stage based on the current clinical blood and urinary examinations. Some studies have pointed out the development of subclinical acute kidney injury (subAKI) syndrome with COVID-19. This syndrome is characterized by significant tubule interstitial injury without changes in the estimated glomerular filtration rate. Despite the complexity of the mechanism(s) underlying the development of subAKI, the involvement of changes in the protein endocytosis machinery in proximal tubule (PT) epithelial cells (PTECs) has been proposed. This paper focuses on the data relating to subAKI and COVID-19 and the role of PTECs and their protein endocytosis machinery in its pathogenesis.
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Injúria Renal Aguda , COVID-19 , Insuficiência Renal Crônica , Humanos , COVID-19/complicações , SARS-CoV-2 , Injúria Renal Aguda/metabolismo , Insuficiência Renal Crônica/metabolismo , Túbulos Renais Proximais/metabolismoRESUMO
Kidneys maintain internal milieu homeostasis through a well-regulated manipulation of body fluid composition. This task is performed by the correlation between structure and function in the nephron. Kidney diseases are chronic conditions impacting healthcare programs globally, and despite efforts, therapeutic options for its treatment are limited. The development of chronic degenerative diseases is associated with changes in protein O-GlcNAcylation, a post-translation modification involved in the regulation of diverse cell function. O-GlcNAcylation is regulated by the enzymatic balance between O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) which add and remove GlcNAc residues on target proteins, respectively. Furthermore, the hexosamine biosynthetic pathway provides the substrate for protein O-GlcNAcylation. Beyond its physiological role, several reports indicate the participation of protein O-GlcNAcylation in cardiovascular, neurodegenerative, and metabolic diseases. In this review, we discuss the impact of protein O-GlcNAcylation on physiological renal function, disease conditions, and possible future directions in the field.
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Acetilglucosamina , N-Acetilglucosaminiltransferases , Acetilglucosamina/metabolismo , Hexosaminas/metabolismo , Homeostase , Rim/metabolismo , N-Acetilglucosaminiltransferases/metabolismo , Processamento de Proteína Pós-TraducionalRESUMO
Patients with COVID-19 have high prevalence of albuminuria which is used as a marker of progression of renal disease and is associated with severe COVID-19. We hypothesized that SARS-CoV-2 spike protein (S protein) could modulate albumin handling in proximal tubule epithelial cells (PTECs) and, consequently contribute to the albuminuria observed in patients with COVID-19. In this context, the possible effect of S protein on albumin endocytosis in PTECs was investigated. Two PTEC lines were used: HEK-293A and LLC-PK1. Incubation of both cell types with S protein for 16 h inhibited albumin uptake at the same magnitude. This effect was associated with canonical megalin-mediated albumin endocytosis because: (1) DQ-albumin uptake, a marker of the lysosomal degradation pathway, was reduced at a similar level compared with fluorescein isothiocyanate (FITC)-albumin uptake; (2) dextran-FITC uptake, a marker of fluid-phase endocytosis, was not changed; (3) cell viability and proliferation were not changed. The inhibitory effect of S protein on albumin uptake was only observed when it was added at the luminal membrane, and it did not involve the ACE2/Ang II/AT1R axis. Although both cells uptake S protein, it does not seem to be required for modulation of albumin endocytosis. The mechanism underlying the inhibition of albumin uptake by S protein encompasses a decrease in megalin expression without changes in megalin trafficking and stability. These results reveal a possible mechanism to explain the albuminuria observed in patients with COVID-19.
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COVID-19 , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade , Albuminas/metabolismo , Albuminas/farmacologia , Albuminúria/metabolismo , Enzima de Conversão de Angiotensina 2 , Células Cultivadas , Dextranos/farmacologia , Endocitose/fisiologia , Células Epiteliais/metabolismo , Fluoresceína-5-Isotiocianato/metabolismo , Fluoresceína-5-Isotiocianato/farmacologia , Humanos , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , SARS-CoV-2 , Glicoproteína da Espícula de CoronavírusRESUMO
1,8-Cineole is a naturally occurring compound found in essential oils of different plants and has well-known anti-inflammatory and antimicrobial activities. In the present work, we aimed to investigate its potential antimalarial effect, using the following experimental models: (1) the erythrocytic cycle of Plasmodium falciparum; (2) an adhesion assay using brain microvascular endothelial cells; and (3) an experimental cerebral malaria animal model induced by Plasmodium berghei ANKA infection in susceptible mice. Using the erythrocytic cycle of Plasmodium falciparum, we characterized the schizonticidal effect of 1,8-cineole. This compound decreased parasitemia in a dose-dependent manner with a half maximal inhibitory concentration of 1045.53 ± 63.30 µM. The inhibitory effect of 972 µM 1,8-cineole was irreversible and independent of parasitemia. Moreover, 1,8-cineole reduced the progression of intracellular development of the parasite over 2 cycles, inducing important morphological changes. Ultrastructure analysis revealed a massive loss of integrity of endomembranes and hemozoin crystals in infected erythrocytes treated with 1,8-cineole. The monoterpene reduced the adhesion index of infected erythrocytes to brain microvascular endothelial cells by 60%. Using the experimental cerebral malaria model, treatment of infected mice for 6 consecutive days with 100 mg/kg/day 1,8-cineole reduced cerebral edema with a 50% reduction in parasitemia. Our data suggest a potential antimalarial effect of 1,8-cineole with an impact on the parasite erythrocytic cycle and severe disease.
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Antimaláricos , Edema Encefálico , Malária Cerebral , Animais , Antimaláricos/química , Antimaláricos/farmacologia , Antimaláricos/uso terapêutico , Modelos Animais de Doenças , Células Endoteliais , Eucaliptol/farmacologia , Malária Cerebral/tratamento farmacológico , Malária Cerebral/parasitologia , Malária Cerebral/prevenção & controle , Camundongos , Camundongos Endogâmicos C57BL , Monoterpenos/farmacologia , Monoterpenos/uso terapêutico , Parasitemia/tratamento farmacológico , Parasitemia/parasitologia , Plasmodium berghei , Plasmodium falciparumRESUMO
Renal proximal tubule cells (PTECs) act as urine gatekeepers, constantly and efficiently avoiding urinary protein waste through receptor-mediated endocytosis. Despite its importance, little is known about how this process is modulated in physiologic conditions. Data suggest that the phosphoinositide-3-kinase (PI3K)/protein kinase B (AKT) pathway regulates PTEC protein reabsorption. Here, we worked on the hypothesis that the physiologic albumin concentration and PI3K/AKT pathway form a positive feedback loop to expand endocytic capacity. Using LLC-PK1 cells, a model of PTECs, we showed that the PI3K/AKT pathway is required for megalin recycling and surface expression, affecting albumin uptake. Inhibition of this pathway stalls megalin at EEA1+ endosomes. Physiologic albumin concentration (0.01 mg/mL) activated AKT; this depends on megalin-mediated albumin endocytosis and requires previous activation of PI3K/mTORC2. This effect is correlated to the increase in albumin endocytosis, a phenomenon that we refer to as "albumin-induced albumin endocytosis". Mice treated with L-lysine present decreased albumin endocytosis leading to proteinuria and albuminuria associated with inhibition of AKT activity. Renal cortex explants obtained from control mice treated with MK-2206 decreased albumin uptake and promoted megalin internalization. Our data highlight the mechanism behind the capacity of PTECs to adapt albumin reabsorption to physiologic fluctuations in its filtration, avoiding urinary excretion.
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Células Epiteliais/metabolismo , Retroalimentação Fisiológica , Túbulos Renais Proximais/metabolismo , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Albuminas/metabolismo , Animais , Biomarcadores , Endocitose , Células Epiteliais/efeitos dos fármacos , Imunofluorescência , Expressão Gênica , Túbulos Renais Proximais/citologia , Masculino , Camundongos , Fosfatidilinositol 3-Quinases/metabolismo , Transporte Proteico , Transdução de Sinais/efeitos dos fármacosRESUMO
Megalin-mediated albumin endocytosis plays a critical role in albumin reabsorption in proximal tubule (PT) epithelial cells (PTECs). Some studies have pointed out the modulatory effect of bradykinin (BK) on urinary protein excretion, but its role in PT protein endocytosis has not yet been determined. Here, we studied the possible correlation between BK and albumin endocytosis in PT. Using LLC-PK1 cells, a model of PTECs, we showed that BK specifically inhibited megalin-mediated albumin endocytosis. This inhibitory effect of BK was mediated by B2 receptor (B2R) because it was abolished by HOE140, an antagonist of B2R, but it was not affected by Lys-des-Arg9-BK, an antagonist of B1. BK induced the stall of megalin in EEA1+ endosomes, but not in LAMP1+ lysosomes, leading to a decrease in surface megalin expression. In addition, we showed that BK, through B2R, activated calphostin C-sensitive protein kinase C, which mediated its effect on the surface megalin expression and albumin endocytosis. These results reveal an important modulatory mechanism of PT albumin endocytosis by BK, which opens new possibilities to understanding the effect of BK on urinary albumin excretion.
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Albuminas/metabolismo , Bradicinina/farmacologia , Endocitose/efeitos dos fármacos , Túbulos Renais Proximais/efeitos dos fármacos , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Animais , Linhagem Celular , Ativação Enzimática , Túbulos Renais Proximais/metabolismo , Células LLC-PK1 , Proteína Quinase C/metabolismo , Receptor B2 da Bradicinina/metabolismo , SuínosRESUMO
Although bone marrow-derived mesenchymal stromal cells (BM-MSCs) from patients with chronic obstructive pulmonary disease (COPD) appear to be phenotypically and functionally similar to BM-MSCs from healthy sources in vitro, the impact of COPD on MSC metabolism and mitochondrial function has not been evaluated. In this study, we aimed to comparatively characterize MSCs from healthy and emphysematous donors (H-MSCs and E-MSCs) in vitro and to assess the therapeutic potential of these MSCs and their extracellular vesicles (H-EVs and E-EVs) in an in vivo model of severe emphysema. For this purpose, C57BL/6 mice received intratracheal porcine pancreatic elastase once weekly for 4 weeks to induce emphysema; control animals received saline under the same protocol. Twenty-four hours after the last instillation, animals received saline, H-MSCs, E-MSCs, H-EVs, or E-EVs intravenously. In vitro characterization demonstrated that E-MSCs present downregulation of anti-inflammatory (TSG-6, VEGF, TGF-ß, and HGF) and anti-oxidant (CAT, SOD, Nrf2, and GSH) genes, and their EVs had larger median diameter and lower average concentration. Compared with H-MSC, E-MSC mitochondria also exhibited a higher respiration rate, were morphologically elongated, expressed less dynamin-related protein-1, and produced more superoxide. When co-cultured with alveolar macrophages, both H-MSCs and E-MSCs induced an increase in iNOS and arginase-1 levels, but only H-MSCs and their EVs were able to enhance IL-10 levels. In vivo, emphysematous mice treated with E-MSCs or E-EVs demonstrated no amelioration in cardiorespiratory dysfunction. On the other hand, H-EVs, but not H-MSCs, were able to reduce the neutrophil count, the mean linear intercept, and IL-1ß and TGF-ß levels in lung tissue, as well as reduce pulmonary arterial hypertension and increase the right ventricular area in a murine model of elastase-induced severe emphysema. In conclusion, E-MSCs and E-EVs were unable to reverse cardiorespiratory dysfunction, whereas H-EVs administration was associated with a reduction in cardiovascular and respiratory damage in experimental severe emphysema.
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BACKGROUND: Tubule-interstitial injury (TII) is one of the mechanisms involved in the progression of renal diseases with progressive proteinuria. Angiotensin II (Ang II) type 1 receptor blockers (ARBs) have been successfully used to treat renal diseases. However, the mechanism correlating treatment with ARBs and proteinuria is not completely understood. The hypothesis that the anti-proteinuric effect of losartan is associated with the modulation of albumin endocytosis in PT epithelial cells (PTECs) was assessed. METHODS: We used a subclinical acute kidney injury animal model (subAKI) and LLC-PK1 cells, a model of PTECs. RESULTS: In subAKI, PT albumin overload induced TII development, measured by: (1) increase in urinary lactate dehydrogenase and γ-glutamyltranspeptidase activity; (2) proteinuria associated with impairment in megalin-mediated albumin reabsorption; (3) increase in luminal and interstitial space in tubular cortical segments. These effects were avoided by treating the animals with losartan, an ARB. Using LLC-PK1 cells, we observed that: (1) 20 mg/mL albumin increased the secretion of Ang II and decreased megalin-mediated albumin endocytosis; (2) the effects of Ang II and albumin were abolished by 10-8 M losartan; (3) MEK/ERK pathway is the molecular mechanism underlying the Ang II-mediated inhibitory effect of albumin on PT albumin endocytosis. CONCLUSION: Our results show that PT megalin-mediated albumin endocytosis is a possible target during the treatment of renal diseases patients with ARB. GENERAL SIGNIFICANCE: The findings obtained in the present work represents a step forward to the current knowledge on about the role of ARBs in the treatment of renal disease.
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Injúria Renal Aguda/tratamento farmacológico , Albuminas/antagonistas & inibidores , Bloqueadores do Receptor Tipo 1 de Angiotensina II/farmacologia , Túbulos Renais Proximais/efeitos dos fármacos , Losartan/farmacologia , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade/antagonistas & inibidores , Injúria Renal Aguda/metabolismo , Albuminas/metabolismo , Angiotensina II/metabolismo , Animais , Células Cultivadas , Modelos Animais de Doenças , Endocitose/efeitos dos fármacos , Túbulos Renais Proximais/metabolismo , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
BACKGROUND: Malaria is a parasitic disease that compromises the human host. Currently, control of the Plasmodium falciparum burden is centered on artemisinin-based combination therapies. However, decreased sensitivity to artemisinin and derivatives has been reported, therefore it is important to identify new therapeutic strategies. METHOD: We used human erythrocytes infected with P. falciparum and experimental cerebral malaria (ECM) animal model to assess the potential antimalarial effect of eugenol, a component of clove bud essential oil. RESULTS: Plasmodium falciparum cultures treated with increasing concentrations of eugenol reduced parasitemia in a dose-dependent manner, with IC50 of 532.42 ± 29.55 µM. This effect seems to be irreversible and maintained even in the presence of high parasitemia. The prominent effect of eugenol was detected in the evolution from schizont to ring forms, inducing important morphological changes, indicating a disruption in the development of the erythrocytic cycle. Aberrant structural modification was observed by electron microscopy, showing the separation of the two nuclear membrane leaflets as well as other subcellular membranes, such as from the digestive vacuole. Importantly, in vivo studies using ECM revealed a reduction in blood parasitemia and cerebral edema when mice were treated for 6 consecutive days upon infection. CONCLUSIONS: These data suggest a potential effect of eugenol against Plasmodium sp. with an impact on cerebral malaria. GENERAL SIGNIFICANCE: Our results provide a rational basis for the use of eugenol in therapeutic strategies to the treatment of malaria.
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Antimaláricos/farmacologia , Edema Encefálico/tratamento farmacológico , Eugenol/farmacologia , Estágios do Ciclo de Vida/efeitos dos fármacos , Malária Cerebral/tratamento farmacológico , Malária Falciparum/tratamento farmacológico , Plasmodium falciparum/efeitos dos fármacos , Animais , Barreira Hematoencefálica/efeitos dos fármacos , Barreira Hematoencefálica/metabolismo , Barreira Hematoencefálica/parasitologia , Edema Encefálico/parasitologia , Modelos Animais de Doenças , Eritrócitos/efeitos dos fármacos , Eritrócitos/parasitologia , Humanos , Concentração Inibidora 50 , Estágios do Ciclo de Vida/fisiologia , Malária Cerebral/parasitologia , Malária Falciparum/parasitologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Plasmodium berghei/efeitos dos fármacos , Plasmodium berghei/crescimento & desenvolvimento , Plasmodium berghei/parasitologia , Plasmodium falciparum/crescimento & desenvolvimento , Plasmodium falciparum/patogenicidadeRESUMO
Coronavirus disease 2019 (COVID-19), caused by the SARS-CoV-2 novel coronavirus, has spread worldwide causing high fatality rates. Neither a vaccine nor specific therapeutic approaches are available, hindering the fight against this disease and making better understanding of its pathogenesis essential. Despite similarities between SARS-CoV-2 and SARS-CoV, the former has unique characteristics which represent a great challenge to physicians. The mechanism of COVID-19 infection and pathogenesis is still poorly understood. In the present review, we highlight possible pathways involved in the pathogenesis of COVID-19 and potential therapeutic targets, focusing on the role of the renin-angiotensin-aldosterone system.
Assuntos
Betacoronavirus/patogenicidade , Infecções por Coronavirus/virologia , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/virologia , Sistema Renina-Angiotensina , Antagonistas de Receptores de Angiotensina/uso terapêutico , Enzima de Conversão de Angiotensina 2 , Inibidores da Enzima Conversora de Angiotensina/uso terapêutico , Animais , Antivirais/uso terapêutico , Betacoronavirus/efeitos dos fármacos , COVID-19 , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/tratamento farmacológico , Medicina Baseada em Evidências , Interações Hospedeiro-Patógeno , Humanos , Pandemias , Peptidil Dipeptidase A/uso terapêutico , Pneumonia Viral/diagnóstico , Pneumonia Viral/tratamento farmacológico , Sistema Renina-Angiotensina/efeitos dos fármacos , SARS-CoV-2 , Tratamento Farmacológico da COVID-19RESUMO
Increasing evidence has highlighted the role of tubule-interstitial injury (TII) as a vital step in the pathogenesis of acute kidney injury (AKI). Incomplete repair of TII during AKI could lead to the development of chronic kidney disease. Changes in albumin endocytosis in proximal tubule epithelial cells (PTECs) is linked to the development of TII. In this context, interleukin (IL)-4 has been shown to be an important factor in modulating recovery of TII. We have studied the possible role of IL-4 in TII induced by albumin overload. A subclinical AKI model characterized by albumin overload in the proximal tubule was used, without changing glomerular function. Four groups were generated: (1) CONT, wild-type mice treated with saline; (2) BSA, wild-type mice treated with 10 g/kg/day bovine serum albumin (BSA); (3) KO, IL4Rα-/- mice treated with saline; and (4) KO + BSA, IL4Rα-/- mice treated with BSA. As reported previously, mice in the BSA group developed TII without changes in glomerular function. The following parameters were increased in the KO + BSA group compared with the BSA group: (1) tubular injury score; (2) urinary γ-glutamyltransferase; (3) CD4+ T cells, dendritic cells, macrophages, and neutrophils are associated with increases in renal IL-6, IL-17, and transforming growth factor ß. A decrease in M2-subtype macrophages associated with a decrease in collagen deposition was observed. Using LLC-PK1 cells, a model of PTECs, we observed that (1) these cells express IL-4 receptor α chain associated with activation of the JAK3/STAT6 pathway; (2) IL-4 alone did not change albumin endocytosis but did reverse the inhibitory effect of higher albumin concentration. This effect was abolished by JAK3 inhibitor. A further increase in urinary protein and creatinine levels was observed in the KO + BSA group compared with the BSA group, but not compared with the CONT group. These observations indicate that IL-4 has a protective role in the development of TII induced by albumin overload that is correlated with modulation of the pro-inflammatory response. We propose that megalin-mediated albumin endocytosis in PTECs could work as a sensor, transducer, and target during the genesis of TII.
RESUMO
Background: Plasmodium falciparum, the etiologic agent of malaria, is a major cause of infant death in Africa. Although research on the contact system has been revitalized by recent discoveries in the field of thrombosis, limited efforts were done to investigate the role of its proinflammatory arm, the kallikrein kinin system (KKS), in the pathogenesis of neglected parasitic diseases, such as malaria. Owing to the lack of animal models, the dynamics of central nervous system (CNS) pathology caused by the sequestration of erythrocytic stages of P. falciparum is not fully understood. Given the precedent that kinins destabilize the blood brain barrier (BBB) in ischemic stroke, here we sought to determine whether Plasmodium falciparum infected erythrocytes (Pf-iRBC) conditioned medium enhances parasite sequestration and impairs BBB integrity via activation of the kallikrein kinin system (KKS). Methods: Monolayers of human brain endothelial cell line (BMECs) are preincubated with the conditioned medium from Pf-iRBCs or RBCs (controls) in the presence or absence of HOE-140 or DALBK, antagonists of bradykinin receptor B2 (B2R) and bradykinin receptor B1 (B1R), respectively. Following washing, the treated monolayers are incubated with erythrocytes, infected or not with P. falciparum mature forms, to examine whether the above treatment (i) has impact on the adhesion of Pf-iRBC to BMEC monolayer, (ii) increases the macromolecular permeability of the tracer BSA-FITC, and (iii) modifies the staining pattern of junctional proteins (ZO-1 and ß-catenin). Results: We found that kinins generated in the parasite conditioned medium, acting via bradykinin B2 and/or B1 receptors (i) enhanced Pf-iRBC adhesion to the endothelium monolayer and (ii) impaired the endothelial junctions formed by ZO-1 and ß-catenin, consequently disrupting the integrity of the BBB. Conclusions: Our studies raise the possibility that therapeutic targeting of kinin forming enzymes and/or endothelial bradykinin receptors might reduce extent of Pf-iRBC sequestration and help to preserve BBB integrity in cerebral malaria (CM).
RESUMO
Tubule-interstitial injury (TII) is a critical step in the progression of renal disease. It has been proposed that changes in proximal tubule (PT) albumin endocytosis plays an important role in the development of TII. Some reports have shown protective effects of lithium on kidney injury animal models that was correlated to proteinuria. We tested the hypothesis that lithium treatment ameliorates the development of TII due to changes in albumin endocytosis. Two experimental models were used: (1) TII induced by albumin overload in an animal model; (2) LLC-PK1 cells, a PT cell line. Lithium treatment ameliorates TII induced by albumin overload measured by (1) proteinuria; (2) collagen deposition; (3) area of tubule-interstitial space, and (4) macrophage infiltration. Lithium treatment increased mTORC2 activity leading to the phosphorylation of protein kinase B (PKB) at Ser473 and its activation. This mechanism enhanced albumin endocytosis in PT cells, which decreased the proteinuria observed in TII induced by albumin overload. This effect did not involve changes in the expression of megalin, a PT albumin receptor. In addition, activation of this pathway decreased apoptosis in LLC-PK1 cells, a PT cell line, induced by higher albumin concentration, similar to that found in pathophysiologic conditions. Our results indicate that the protective role of lithium treatment on TII induced by albumin overload involves an increase in PT albumin endocytosis due to activation of the mTORC2/PKB pathway. These results open new possibilities in understanding the effects of lithium on the progression of renal disease.