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Mammalian cell membranes are very dynamic where they respond to several environmental stimuli by rearranging the membrane composition by basic biological processes, including endocytosis. In this context, receptor-mediated endocytosis, either clathrin-dependent or caveolae-dependent, is involved in different physiological and pathological conditions. In the last years, an important amount of evidence has been reported that kidney function involves the modulation of different types of endocytosis, including renal protein handling. In addition, the dysfunction of the endocytic machinery is involved with the development of proteinuria as well as glomerular and tubular injuries observed in kidney diseases associated with hypertension, diabetes, and others. In this present review, we will discuss the mechanisms underlying the receptor-mediated endocytosis in different glomerular cells and proximal tubule epithelial cells as well as their modulation by different factors during physiological and pathological conditions. These findings could help to expand the current understanding regarding renal protein handling as well as identify possible new therapeutic targets to halt the progression of kidney disease.
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Endocitosis , Humanos , Animales , Enfermedades Renales/metabolismo , Enfermedades Renales/patología , Riñón/metabolismo , Riñón/patología , Receptores de Superficie Celular/metabolismoRESUMEN
IgA Nephropathy (IgAN) is the commonest primary glomerular disease around the world and represents a significant cause of end-stage renal disease. IgAN is characterized by mesangial deposition of IgA-immune complexes and mesangial expansion. The pathophysiological process includes an abnormally glycosylated IgA1, which is an antigenic target. Autoantibodies specifically recognize galactose-deficient IgA1 forming immune complexes that are amplified in size by the soluble IgA Fc receptor CD89 leading to deposition in the mesangium through interaction with non-classical IgA receptors. The local production of cytokines promotes local inflammation and complement system activation, besides the stimulation of mesangial proliferation. The spectrum of clinical manifestations is quite variable from asymptomatic microscopic hematuria to rapidly progressive glomerulonephritis. Despite all the advances, the pathophysiology of the disease is still not fully elucidated. The mucosal immune system is quoted to be a factor in triggering IgAN and a "gut-kidney axis" is proposed in its development. Furthermore, many recent studies have demonstrated that food intake interferes directly with disease prognosis. In this review, we will discuss how mucosal immunity, microbiota, and nutritional status could be interfering directly with the activation of intrinsic pathways of the mesangial cells, directly resulting in changes in their function, inflammation and development of IgAN.
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Mesangial cells (MC) maintain the architecture and cellular communication and indirectly join in the glomerular filtration rate for the correct functioning of the glomerulus. Consequently, these cells are activated constantly in response to changes in the intraglomerular environment due to a metabolic imbalance or infection. IL-36, a member of the IL-1 family, is a cytokine that initiates and maintains inflammation in different tissues in acute and chronic pathologies, including the skin, lungs, and intestines. In the kidney, IL-36 has been described in the development of tubulointerstitial lesions, the production of an inflammatory environment, and is associated with metabolic and mesangioproliferative disorders. The participation of IL-36 in functional dysregulation and the consequent generation of the inflammatory environment by MCs in the presence of microbial stimulation is not yet elucidated. In this work, the MES SV40 cell cultures were stimulated with classical pathogen-associated molecular patterns (PAMPs), mimicking an infection by negative and positive bacteria as well as a viral infection. Lipopolysaccharide (LPS), peptidoglycan (PGN) microbial wall components, and a viral mimic poly I:C were used, and the mRNA and protein expression of the IL-36 members were assessed. We observed a differential and dose-dependent IL-36 mRNA and protein expression under LPS, PGN, and poly I:C stimulation. IL-36ß was only found when the cells were treated with LPS, while IL-36α and IL-36γ were favored by PGN and poly I:C stimulation. We suggest that the microbial components participate in the activation of MCs, leading them to the production of IL-36, in which a specific member may participate in the origin and maintenance of inflammation in the glomerular environment that is associated with infections.
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Citocinas , Lipopolisacáridos , Citocinas/metabolismo , Humanos , Inflamación , Interleucina-1/genética , Interleucina-1/metabolismo , Lipopolisacáridos/farmacología , Moléculas de Patrón Molecular Asociado a Patógenos , Peptidoglicano/farmacología , Poli I-C , ARN Mensajero/genéticaRESUMEN
Abstract Introduction: Diabetes mellitus (DM) is a chronic disease characterized by hyperglycemia that leads to diabetic nephropathy (DN). We showed that P2X7, a purinergic receptor, was highly expressed in DM; however, when oxidative stress was controlled, renal NO recovered, and the activation of this receptor remained significantly reduced. The aim of this study was to assess the influence of NO on the P2X7 and apoptosis in mouse immortalized mesangial cells (MiMC) cultured in high glucose (HG) medium. Methods: MiMCs were cultured with DMEM and exposed to normal glucose (NG), mannitol (MA), or HG. Cell viability was assessed by an automated counter. Supernatants were collected for NO quantification, and proteins were extracted for analysis of NO synthases (iNOS and eNOS), caspase-3, and P2X7. Results: Cell viability remained above 90% in all groups. There was a significant increase in the proliferation of cells in HG compared to MA and NG. NO, iNOS, caspase-3, and P2X7 were significantly increased in HG compared to NG and MA, with no changes in eNOS. We observed that there was a strong and significant correlation between P2X7 and NO. Discussion: The main finding was that the production of NO by iNOS was positively correlated with the increase of P2X7 in MCs under HG conditions, showing that there is a common stimulus between them and that NO interacts with the P2X7 pathway, contributing to apoptosis in experimental DM. These findings could be relevant to studies of therapeutic targets for the prevention and/or treatment of hyperglycemia-induced kidney damage to delay DN progression.
Resumo Introdução: Diabetes mellitus (DM) é uma doença crônica caracterizada por hiperglicemia levando à nefropatia diabética (ND). Mostramos que P2X7, um receptor purinérgico, foi altamente expresso na DM; entretanto, quando o estresse oxidativo foi controlado, o NO renal recuperou-se, e a ativação deste receptor permaneceu significativamente reduzida. Este estudo objetivou avaliar a influência do NO no P2X7 e a apoptose em células mesangiais imortalizadas de camundongos (CMiC) cultivadas em meio de glicose elevada (GE). Métodos: CMiCs foram cultivadas em meio DMEM e expostas à glicose normal (GN), manitol (MA), ou GE. A viabilidade celular foi avaliada por contador automático. Sobrenadantes foram coletados para quantificação de NO, e foram extraídas proteínas para análise de NO sintases (iNOS e eNOS), caspase-3, e P2X7. Resultados: A viabilidade celular permaneceu acima de 90% em todos os grupos. Houve aumento significativo na proliferação de células na GE comparado com MA e GN. NO, iNOS, caspase-3 e P2X7 foram significativamente aumentados na GE comparados com GN e MA, sem alterações na eNOS. Observamos que houve correlação forte e significativa entre P2X7 e NO. Discussão: O principal achado foi que a produção de NO pela iNOS foi positivamente correlacionada com aumento de P2X7 em CMs sob condições de GE, mostrando que existe um estímulo comum entre eles e que o NO interage com a via do P2X7, contribuindo para apoptose na DM experimental. Estes achados podem ser relevantes para estudos de alvos terapêuticos para a prevenção e/ou tratamento de danos renais induzidos por hiperglicemia para retardar a progressão da ND.
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Nanosized copper particles (nano Cu) have been incorporated into products in multiple industries, although studies have demonstrated that these particles are nephrotoxic. We investigated the cytotoxicity of nanosized copper particles on rat mesangial cells and measured rates of apoptosis, the expression of caspase-3, and generation of reactive oxygen species. We also measured autophagy through the acridine orange (AO) staining and expression of Beclin-1, microtubule-associated protein 1 light chain 3, and p62 to screen the underlying mechanism of toxicity. Nanosized copper particles inhibited mesangial cell viability, up-regulated the activity of caspase-3, and increased the rates of apoptosis and the generation of reactive oxygen species in a concentration-dependent manner. Exposure to nano Cu increased the formation of acidic vesicular organelles and the expression of Beclin-1, microtubule-associated protein 1 light chain 3, and p62, and treatment with an autophagy inhibitor reduced nephrotoxicity. This indicated that the autophagy pathway is involved in the toxicity induced by nanosized copper particles to mesangial cells. This finding can contribute to the development of safety guidelines for the evaluation of nanomaterials in the future.
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Chronic kidney disease (CKD) is a major public health concern and its prevalence and incidence are rising quickly. It is a non-communicable disease primarily caused by diabetes and/or hypertension and is associated with high morbidity and mortality. Despite decades of research efforts, the pathogenesis of CKD remains a puzzle with missing pieces. Understanding the cellular and molecular mechanisms that govern the loss of kidney function is crucial. Abrupt regulation of gene expression in kidney cells is apparent in CKD and shown to be responsible for disease onset and progression. Gene expression regulation extends beyond DNA sequence and involves epigenetic mechanisms including changes in DNA methylation and post-translational modifications of histones, driven by the activity of specific enzymes. Recent advances demonstrate the essential participation of epigenetics in kidney (patho)physiology, as its actions regulate both the integrity of cells but also triggers deleterious signaling pathways. Here, we review the known epigenetic processes regulating the complex filtration unit of the kidney, the glomeruli. The review will elaborate on novel insights into how epigenetics contributes to cell injury in the CKD setting majorly focusing on kidney glomerular cells: the glomerular endothelial cells, the mesangial cells, and the specialized and terminally differentiated podocyte cells.
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Susceptibilidad a Enfermedades , Epigénesis Genética , Regulación de la Expresión Génica , Enfermedades Renales/etiología , Enfermedades Renales/metabolismo , Glomérulos Renales/citología , Glomérulos Renales/metabolismo , Animales , Biomarcadores , Metilación de ADN , Células Endoteliales/metabolismo , Histonas/metabolismo , Humanos , Enfermedades Renales/patología , Glomérulos Renales/patología , Células Mesangiales/metabolismo , Podocitos/metabolismo , Procesamiento Proteico-PostraduccionalRESUMEN
ABSTRACT Objective To analyze whether passive inhalation of cigarette smoke causes morphological, structural, and functional changes in kidneys of rats. Methods Wistar rats, aged eight weeks, weighing on average 260g, were divided into Control Group and Smoking Group. Each group was subdivided into four groups of ten animals for morphofunctional analysis, in a period of seven and 28 days. The Smoking Group was exposed to smoke of 40 cigarettes per day, at certain times and in automated equipment for cigarette burning, called smoking machine (SM-MC-01). After the exposure period, urine and blood samples were collected for the functional analyses, and the kidneys were dissected and submitted to histological procedures for morphoquantitative analyses. Results After exposure of animals of the Smoking Group, the following were observed: lower weight gain; lower water and feed intake; decreased renal weight, diameter, and volume; reduction in cortical thickness and glomerular volume density; decrease in glomerular and capsular diameter; increase in mesangial density; decreased urine volume; increased levels of glucose, serum creatinine and microalbuminuria; decreased urinary creatinine levels and creatinine clearance rate. Conclusion Passive smoking negatively influences renal morphology and glomerular filtration rate, with effects similar to those described in the literature regarding active smoking.
RESUMO Objetivo Analisar se a inalação passiva da fumaça do cigarro proporciona alterações morfológicas, estruturais e funcionais nos rins de ratos. Métodos Ratos Wistar, com oito semanas de idade, pesando, em média, 260g, foram divididos em Grupo Controle e Grupo Tabagista. Cada grupo foi subdividido em quatro grupos de dez animais para análise morfofuncional, em um período de sete e 28 dias. O Grupo Tabagista foi exposto à fumaça de 40 cigarros por dia, em horários determinados e equipamento automatizado de queima de cigarros, denominado smoking machine (SM-MC-01). Após o período de exposição, foram coletadas amostras de urina e sangue para as análises funcionais, e os rins foram dissecados e submetidos a procedimentos histológicos para análises morfoquantitativas. Resultados Após a exposição dos animais do Grupo Tabagista, observou-se menor ganho de peso; menor consumo de água e ração; menor peso, diâmetro e volume renal; redução em espessura cortical e densidade de volume glomerular; diminuição no diâmetro glomerular e capsular; aumento na densidade mesangial; volume urinário diminuído; níveis aumentados de glicose, creatinina sérica e microalbuminúria; níveis reduzidos de creatinina urinária e redução da taxa de depuração da creatinina. Conclusão O tabagismo passivo influencia negativamente na morfologia renal e na taxa de filtração glomerular, com efeitos semelhantes aos descritos na literatura em relação ao tabagismo ativo.
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Animales , Ratas , Contaminación por Humo de Tabaco/efectos adversos , Fumar/efectos adversos , Ratas Wistar , Tasa de Filtración Glomerular , RiñónRESUMEN
The kidney is an important target of the renin-ANG-aldosterone system (RAAS). To date, several studies have demonstrated the existence of a local RAAS in various tissues, including the renal tissue. The mineralocorticoid aldosterone is known to play a critical role in the classical RAAS; however, its effect on mesangial cells (MCs) remains to be elucidated. Based on this, our aim was to investigate whether aldosterone stimulation can modulate the intracellular RAAS of immortalized human MCs by evaluating ANG-converting enzyme (ACE)/ANG II/ANG II receptor type 1 (AT1) and ANG-converting enzyme 2 (ACE2)/ANG (1-7)/MAS receptor axes. To realise this, protein expression, enzyme activity, and immunofluorescence were performed under aldosterone stimulation and in the presence of the mineralocorticoid receptor (MR) antagonist spironolactone (SPI). We observed that high doses of aldosterone increase ACE activity. The effect of aldosterone on the catalytic activity of ACE was completely abolished with the pretreatment of SPI suggesting that the aldosterone-induced cell injuries through ANG II release were attenuated. Aldosterone treatment also decreased the expression of MAS receptor, but did not alter the expression or the catalytic activity of ACE 2 and ANG (1-7) levels. Spironolactone modulated the localization of ANG II and AT1 receptor and decreased ANG (1-7) and MAS receptor levels. Our data suggest that both aldosterone and the MR receptor antagonist can modulate both of these axes and that spironolactone can protect MCs from the damage induced by aldosterone.
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Aldosterona/farmacología , Células Mesangiales/efectos de los fármacos , Células Mesangiales/metabolismo , Espironolactona/farmacología , Angiotensina I/genética , Angiotensina I/metabolismo , Enzima Convertidora de Angiotensina 2 , Células Cultivadas , Glicosilación/efectos de los fármacos , Humanos , Células Mesangiales/citología , Antagonistas de Receptores de Mineralocorticoides/farmacología , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Peptidil-Dipeptidasa A/genética , Peptidil-Dipeptidasa A/metabolismo , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Receptor de Angiotensina Tipo 1/genética , Receptor de Angiotensina Tipo 1/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismoRESUMEN
BACKGROUND: Activated cardiac fibroblasts (CFs), preglomerular vascular smooth muscle cells (PGVSMCs), and glomerular mesangial cells (GMCs) proliferate, cause hypertrophy, and produce collagen; in this way, activated CFs contribute to cardiac fibrosis, and activated PGVSMCs and GMCs promote renal fibrosis. In heart and kidney diseases, SDF-1α (stromal cell-derived factor 1α; endogenous CXCR4 [C-X-C motif chemokine receptor 4] receptor agonist) levels are often elevated; therefore, it is important to know whether and how the SDF-1α/CXCR4 axis activates CFs, PGVSMCs, or GMCs. METHODS AND RESULTS: Here we investigated whether SDF-1α activates CFs, PGVSMCs, and GMCs to proliferate, hypertrophy, or produce collagen. DPP4 (dipeptidyl peptidase 4) inactivates SDF-1α and previous experiments show that growth-promoting peptides have greater effects in cells from genetically-hypertensive animals. Therefore, we performed experiments in the absence and presence of sitagliptin (DPP4 inhibitor) and in cells from normotensive Wistar-Kyoto rats and spontaneously hypertensive rats. Our studies show (1) that spontaneously hypertensive and Wistar-Kyoto rat CFs, PGVSMCs, and GMCs express CXCR4 receptors and DPP4 activity; (2) that chronic treatment with physiologically relevant concentrations of SDF-1α causes concentration-dependent increases in the proliferation (cell number) and hypertrophy (3H-leucine incorporation) of and collagen production (3H-proline incorporation) by CFs, PGVSMCs, and GMCs; (3) that sitagliptin augments these effects of SDF-1α; (4) that interactions between SDF-1α and sitagliptin are greater in spontaneously hypertensive rat cells; (5) that CXCR4 antagonism (AMD3100) blocks all effects of SDF-1α; and (6) that SDF-1α/CXCR4 signal transduction likely involves the RACK1 (receptor for activated C kinase 1)/Gßγ/PLC (phospholipase C)/PKC (protein kinase C) signaling complex. CONCLUSIONS: The SDF-1α/CXCR4 axis drives proliferation and hypertrophy of and collagen production by CFs, PGVSMCs, and GMCs, particularly in cells from genetically hypertensive animals and when DPP4 is inhibited.
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Cardiomiopatía Hipertrófica/genética , Quimiocina CXCL12/genética , Colágeno/biosíntesis , Regulación de la Expresión Génica , Células Mesangiales/patología , Músculo Liso Vascular/patología , Miocardio/patología , Animales , Western Blotting , Cardiomiopatía Hipertrófica/metabolismo , Cardiomiopatía Hipertrófica/patología , Proliferación Celular , Células Cultivadas , Quimiocina CXCL12/biosíntesis , Modelos Animales de Enfermedad , Fibroblastos/metabolismo , Fibroblastos/patología , Glomérulos Renales/metabolismo , Glomérulos Renales/patología , Masculino , Células Mesangiales/metabolismo , Microvasos/metabolismo , Microvasos/patología , Músculo Liso Vascular/metabolismo , Miocardio/metabolismo , ARN/genética , Ratas , Ratas Endogámicas SHR , Ratas Endogámicas WKY , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores CXCR4/biosíntesis , Receptores CXCR4/genética , Arteria Renal/metabolismo , Arteria Renal/patologíaRESUMEN
Resveratrol (Resv) is natural polyphenol found in grapes. This study evaluated the protective effect of Resv against the effects of uric acid (UA) in immortalized human mesangial cells (ihMCs). ihMCs were preincubated with Resv (12.5 µM) for 1 h and treated with UA (10 mg/dL) for 6 or 12 h. The intracellular calcium concentration [Ca2+]i was quantified by fluorescence using flow cytometry. Angiotensinogen (AGT) and pre-pro endothelin-1 (ppET-1) mRNA were assayed by quantitative real-time RT-PCR. Angiotensin II (AII) and endothelin-1 (ET-1) were assayed by ELISA. UA significantly increased [Ca2+]i. Pre-incubation with Resv significantly reduced the change in [Ca2+]i induced by UA. Incubation with UA for 6 or 12 h also increased AGT mRNA expression and AII protein synthesis. Resv blunted these increases in AGT mRNA expression and AII protein. Incubation with UA in the ihMCs increased ppET-1 expression and ET-1 protein synthesis at 6 and 12 h. When ihMCs were pre-incubated with Resv, UA had a significantly diminished effect on ppET-1 mRNA expression and ET-1 protein synthesis at 6 and 12 h, respectively. Our results suggested that UA triggers reactions including AII and ET-1 production in mesangial cells. The renin-angiotensin system may contribute to the pathogenesis of renal function and chronic kidney disease. Resv can minimize the impact of UA on AII, ET-1 and the increase of [Ca2+]i in mesangial cells, suggesting that, at least in part, Resv can prevent the effects of soluble UA in mesangial cells.
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Somatic angiotensin-I converting enzyme (sACE) is a broadly distributed peptidase which plays a role in blood pressure and electrolyte homeostasis by the conversion of angiotensin I into angiotensin II. N-domain isoforms (nACE) with 65 and 90 kDa have been described in body fluids, tissues and mesangial cells (MC), and a 90 kDa nACE has been described only in spontaneously hypertensive rats. The aim of this study was to investigate the existence of proteolytic enzymes that may act in the hydrolysis of sACE generating nACEs in MC. After the confirmation of the presence of ACE sheddases in Immortalized MC (IMC), we purified and characterized these enzymes using fluorogenic substrates specifically designed for ACE sheddases. Purified enzyme identified as a serine protease by N-terminal sequence was able to generate nACE. In the present study, we described for the first time the presence of ACE sheddases in IMC, identified as serine proteases able to hydrolyze sACE in vitro. Further investigations are necessary to elucidate the mechanisms responsible for the expression and regulation of ACE sheddases in MC and their roles in the generation of nACEs, especially the 90 kDa form possibly related to hypertension.
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Hipertensión/enzimología , Peptidil-Dipeptidasa A/metabolismo , Proteolisis , Serina Proteasas/metabolismo , Angiotensina I/metabolismo , Angiotensina II/metabolismo , Animales , Presión Sanguínea , Humanos , Hipertensión/metabolismo , Células Mesangiales/enzimología , Células Mesangiales/metabolismo , Peptidil-Dipeptidasa A/biosíntesis , Isoformas de Proteínas/biosíntesis , Isoformas de Proteínas/metabolismo , Ratas , Serina Proteasas/biosíntesisRESUMEN
Chronic kidney disease (CKD) is an increasingly common condition characterized by progressive loss of functional nephrons leading to renal failure. TGF-ß1-induced mesangial cell (MC) phenotype alterations have been linked to the genesis of CKD. Here we show that TGF-ß1 regulates TBX3 gene expression in MC. This gene encodes for two main isoforms, TBX3.1 and TBX3+2α. TBX3.1 has been implicated in cell immortalization, proliferation and apoptosis by inhibiting p14(ARF)-Mdm2-p53 pathway, while TBX3+2α role has not been defined. We demonstrated that TBX3 overexpression abrogated MC apoptosis induced by serum deprivation. Moreover, we observed an enhancement in TBX3 protein expression both in glomerular and tubular regions in the model of 5/6 nephrectomy, temporally related to increased expression of TGF-ß1, type IV collagen and fibronectin. Our results indicate that TBX3 acts as an anti-apoptotic factor in MC in vitro and may be involved in the mechanism by which TGF-ß1 induces glomerulosclerosis and tubular fibrosis during the progression of nephropathies.