RESUMEN
The strong predictive value of proteinuria in chronic glomerulopathies is firmly established as well as the pathogenic role of angiotensin II promoting progression of glomerular disease with an altered glomerular filtration barrier, podocyte injury and scarring of glomeruli. Here we found that chronic angiotensin II-induced hypertension inhibited autophagy flux in mouse glomeruli. Deletion of Atg5 (a gene encoding a protein involved autophagy) specifically in the podocyte resulted in accelerated angiotensin II-induced podocytopathy, accentuated albuminuria and glomerulosclerosis. This indicates that autophagy is a key protective mechanism in the podocyte in this condition. Angiotensin-II induced calpain activity in podocytes inhibits autophagy flux. Podocytes from mice with transgenic expression of the endogenous calpain inhibitor calpastatin displayed higher podocyte autophagy at baseline that was resistant to angiotensin II-dependent inhibition. Also, sustained autophagy with calpastatin limited podocyte damage and albuminuria. These findings suggest that hypertension has pathogenic effects on the glomerular structure and function, in part through activation of calpains leading to blockade of podocyte autophagy. These findings uncover an original mechanism whereby angiotensin II-mediated hypertension inhibits autophagy via calcium-induced recruitment of calpain with pathogenic consequences in case of imbalance by calpastatin activity. Thus, preventing a calpain-mediated decrease in autophagy may be a promising new therapeutic strategy for nephropathies associated with high renin-angiotensin system activity.
Asunto(s)
Podocitos , Angiotensina II/toxicidad , Animales , Autofagia , Proteínas de Unión al Calcio , Glomérulos Renales , RatonesRESUMEN
Most kidney stones are made of calcium oxalate crystals. Randall's plaque, an apatite deposit at the tip of the renal papilla, is considered to at the origin of these stones. Hypercalciuria may promote Randall's plaque formation and growth. We analyzed whether long-term exposure of Abcc6-/- mice (a murine model of Randall's plaque) to vitamin D supplementation, with or without a calcium-rich diet, would accelerate the formation of Randall's plaque. Eight groups of mice (including Abcc6-/- and wild type) received vitamin D alone (100,000 UI/kg every 2 weeks), a calcium-enriched diet alone (calcium gluconate 2 g/L in drinking water), both vitamin D supplementation and a calcium-rich diet, or a standard diet (controls) for 6 months. Kidney calcifications were assessed by 3-dimensional microcomputed tomography, µ-Fourier transform infrared spectroscopy, field emission-scanning electron microscopy, transmission electron microscopy, and Yasue staining. At 6 months, Abcc6-/- mice exposed to vitamin D and calcium supplementation developed massive Randall's plaque when compared with control Abcc6-/- mice (P < 0.01). Wild-type animals did not develop significant calcifications when exposed to vitamin D. Combined administration of vitamin D and calcium significantly accelerates Randall's plaque formation in a murine model. This original model raises concerns about the cumulative risk of vitamin D supplementation and calcium intakes in Randall's plaque formation.
Asunto(s)
Calcio de la Dieta/efectos adversos , Suplementos Dietéticos/efectos adversos , Cálculos Renales/inducido químicamente , Médula Renal/metabolismo , Vitamina D/efectos adversos , Animales , Calcinosis/inducido químicamente , Calcinosis/metabolismo , Calcinosis/patología , Calcio de la Dieta/administración & dosificación , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Femenino , Cálculos Renales/metabolismo , Cálculos Renales/patología , Médula Renal/patología , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Factores de Tiempo , Vitamina D/administración & dosificaciónRESUMEN
BACKGROUND: Pseudoxanthoma elasticum (PXE) is a genetic disease caused by mutations in the ABCC6 gene that result in low pyrophosphate levels and subsequent progressive soft tissue calcifications. PXE mainly affects the skin, retina, and arteries. However, many patients with PXE experience kidney stones. We determined the prevalence of this pathology in patients with PXE and examined the possible underlying mechanisms in murine models. METHODS: We conducted a retrospective study in a large cohort of patients with PXE and analyzed urine samples and kidneys from Abcc6-/- mice at various ages. We used Yasue staining, scanning electron microscopy, electron microscopy coupled to electron energy loss spectroscopy, and Fourier transform infrared microspectroscopy to characterize kidney calcifications. RESULTS: Among 113 patients with PXE, 45 (40%) had a past medical history of kidney stones. Five of six computed tomography scans performed showed evidence of massive papillary calcifications (Randall plaques). Abcc6-/- mice spontaneously developed kidney interstitial apatite calcifications with aging. These calcifications appeared specifically at the tip of the papilla and formed Randall plaques similar to those observed in human kidneys. Compared with controls, Abcc6-/- mice had low urinary excretion of pyrophosphate. CONCLUSIONS: The frequency of kidney stones and probably, Randall plaque is extremely high in patients with PXE, and Abcc6-/- mice provide a new and useful model in which to study Randall plaque formation. Our findings also suggest that pyrophosphate administration should be evaluated for the prevention of Randall plaque and kidney stones.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/genética , Cálculos Renales/etiología , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Seudoxantoma Elástico/genética , Seudoxantoma Elástico/patología , Animales , Biopsia con Aguja , Calcinosis/genética , Calcinosis/patología , Estudios de Cohortes , Modelos Animales de Enfermedad , Femenino , Humanos , Inmunohistoquímica , Incidencia , Cálculos Renales/epidemiología , Cálculos Renales/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Pronóstico , Seudoxantoma Elástico/complicaciones , Seudoxantoma Elástico/diagnóstico por imagen , Estudios Retrospectivos , Medición de Riesgo , Tomografía Computarizada por Rayos X/métodos , UrinálisisRESUMEN
Calpains are intracellular proteases that play a key role in inflammation/immunity. Rare studies show that they are partially externalized. However, the mechanism of this secretion and the functions of exteriorized calpains remain poorly understood. In this study, we found that mouse and human lymphocytes secreted calpains through an ABCA1-driven process. In turn, extracellular calpains inhibited IL-17A expression. We were able to attribute this function to a cleavage of the TLR2 extracellular domain, which prevented TLR2-induced transcription of molecules essential for IL-17A induction. Calpain exteriorization and TLR2 cleavage were critical for the control of IL-17A expression by low doses of IL-2. By using newly developed transgenic mice in which extracellular calpains are specifically inactivated, we provide evidence for the relevance of calpain externalization in vivo in regulating IL-17A expression and function in experimental sterile peritonitis and autoimmune arthritis, respectively. Thus, this study identifies calpain exteriorization as a potential target for immune modulation.
Asunto(s)
Transportador 1 de Casete de Unión a ATP/biosíntesis , Calpaína/metabolismo , Interleucina-17/biosíntesis , Linfocitos T/inmunología , Receptor Toll-Like 2/metabolismo , Transportador 1 de Casete de Unión a ATP/genética , Animales , Artritis Experimental , Línea Celular , Proliferación Celular , Regulación de la Expresión Génica , Células HEK293 , Humanos , Inflamación/inmunología , Mediadores de Inflamación/inmunología , Interleucina-17/genética , Interleucina-2/uso terapéutico , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Neutrófilos/inmunología , Interferencia de ARN , ARN Interferente Pequeño , Bazo/citologíaRESUMEN
Vitamin D supplementation in humans should be accompanied by calcium administration to avoid bone demineralization through vitamin D receptor signaling. Here we analyzed whether long-term exposure of rats to vitamin D supplementation, with or without a calcium-rich diet, would promote kidney stone formation. Four groups of rats received vitamin D alone (100,000 UI/kg/3 weeks), a calcium-enriched diet alone, both vitamin D supplementation and calcium-enriched diet, or a standard diet (controls) for 6 months. Serum and urine parameters and crystalluria were monitored. Kidney stones were assessed by 3-dimensional micro-computed tomography, infrared spectroscopy, von Kossa/Yasue staining, and field emission scanning electron microscopy. Although serum calcium levels were similar in the 4 groups, rats receiving vitamin D had a progressive increase in urinary calcium excretion over time, especially those receiving both calcium and vitamin D. However, oral calcium supplementation alone did not increase urinary calcium excretion. At 6 months, rats exposed to both calcium and vitamin D, but not rats exposed to calcium or vitamin D alone, developed significant apatite kidney calcifications (mean volume, 0.121 mm(3)). Thus, coadministration of vitamin D and increased calcium intake had a synergistic role in tubular calcifications or kidney stone formation in this rat model. Hence, one should be cautious about the cumulative risk of kidney stone formation in humans when exposed to both vitamin D supplementation and high calcium intake.
Asunto(s)
Calcio de la Dieta/farmacología , Suplementos Dietéticos/efectos adversos , Cálculos Renales/etiología , Vitamina D/farmacología , Animales , Apatitas/metabolismo , Desmineralización Ósea Patológica/etiología , Calcio de la Dieta/sangre , Calcio de la Dieta/orina , Modelos Animales de Enfermedad , Sinergismo Farmacológico , Cálculos Renales/sangre , Cálculos Renales/química , Cálculos Renales/orina , Masculino , Microscopía Electrónica de Rastreo , Ratas , Ratas Sprague-Dawley , Receptores de Calcitriol/metabolismo , Eliminación Renal , Espectroscopía Infrarroja por Transformada de Fourier , Microtomografía por Rayos XRESUMEN
OBJECTIVE: Calpains, calcium-activated proteases, mediate the angiogenic signals of vascular endothelial growth factor. However, their involvement in vascular repair has not been investigated and the underlying mechanisms remain to be fully elucidated. METHODS AND RESULTS: A rapidly progressive form of glomerulonephritis in wild type and transgenic mice expressing high levels of calpastatin, a calpain-specific inhibitor, was studied. Calpastatin transgene expression prevented the repair of peritubular capillaries and the recovery of renal function, limiting mouse survival. In vitro analysis detected a significant reduction of both intracellular and extracellular calpain activities in transgene expressing cells, whereas Western blotting revealed that proangiogenic factors vascular endothelial growth factor and norepinephrine increased calpain exteriorization. In vitro, extracellular calpains increased endothelial cell proliferation, migration and capillary tube formation. In vivo, delivery of nonpermeable extracellular calpastatin was sufficient to blunt angiogenesis and vascular repair. Endothelial cell response to extracellular calpains was associated with fibronectin cleavage, generating fibronectin fragments with proangiogenic capacity. In vivo, fibronectin cleavage was limited in the kidney of calpastatin transgenic mice with nephritis. CONCLUSIONS: This study demonstrates that externalized calpains participate in angiogenesis and vascular repair, partly by promoting fibronectin cleavage and thereby amplifying vascular endothelial growth factor efficiency. Thus, manipulation of calpain externalization may have therapeutic implications to control angiogenesis.
Asunto(s)
Calpaína/fisiología , Progresión de la Enfermedad , Glomerulonefritis/fisiopatología , Neovascularización Fisiológica/fisiología , Animales , Vasos Sanguíneos/crecimiento & desarrollo , Calpaína/genética , Calpaína/farmacología , Movimiento Celular/efectos de los fármacos , Movimiento Celular/fisiología , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Modelos Animales de Enfermedad , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Fibronectinas/metabolismo , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones TransgénicosRESUMEN
RATIONALE: Sepsis, a leading cause of death worldwide, involves widespread activation of inflammation, massive activation of coagulation, and lymphocyte apoptosis. Calpains, calcium-activated cysteine proteases, have been shown to increase inflammatory reactions and lymphocyte apoptosis. Moreover, calpain plays an essential role in microparticle release. OBJECTIVES: We investigated the contribution of calpain in eliciting tissue damage during sepsis. METHODS: To test our hypothesis, we induced polymicrobial sepsis by cecal ligation and puncture in wild-type (WT) mice and transgenic mice expressing high levels of calpastatin, a calpain-specific inhibitor. MEASUREMENTS AND MAIN RESULTS: In WT mice, calpain activity increased transiently peaking at 6 hours after cecal ligation and puncture surgery. Calpastatin overexpression improved survival, organ dysfunction (including lung, kidney, and liver damage), and lymphocyte apoptosis. It decreased the sepsis-induced systemic proinflammatory response and disseminated intravascular coagulation, by reducing the number of procoagulant circulating microparticles and therefore delaying thrombin generation. The deleterious effect of microparticles in this model was confirmed by transferring microparticles from septic WT to septic transgenic mice, worsening their survival and coagulopathy. CONCLUSIONS: These results demonstrate an important role of the calpain/calpastatin system in coagulation/inflammation pathways during sepsis, because calpain inhibition is associated with less severe disseminated intravascular coagulation and better overall outcomes in sepsis.
Asunto(s)
Proteínas de Unión al Calcio/fisiología , Sepsis/fisiopatología , Animales , Apoptosis/fisiología , Calpaína/fisiología , Micropartículas Derivadas de Células/fisiología , Citocinas/fisiología , Modelos Animales de Enfermedad , Coagulación Intravascular Diseminada/fisiopatología , Linfocitos/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Insuficiencia Multiorgánica/fisiopatología , FN-kappa B/fisiología , Sepsis/mortalidad , Tromboplastina/fisiologíaRESUMEN
The renal urothelium, the monolayered epithelium that covers the papilla, is the direct target of increased pressure during obstruction, yet most studies have mainly focused on tubules, fibroblasts, and inflammatory cells. We studied this epithelium in a unilateral ureteral obstruction mouse mode land found that it was disrupted and had broken tight junctions, enlarged intercellular space, with loss of apicaluroplakins, and marginal lumen desquamation. Shortly after obstruction these urothelial cells proliferated, peaking at day 2. By day 14, the renal urothelium was transformed into a multilayered barrier with newly synthesized uroplakins including the de novo induction of uroplakin II. This proliferation was found to be fibroblast growth factor (FGF)dependent. Renal urothelial cells constitutively express the FGF receptor 2, and obstruction activated the receptor by phosphorylation. Treatment with FGF receptor 2-antisense or vitamin A (an inhibitor of the MAP kinase in the FGFR2 pathway) decreased renal urothelial cell proliferation. Among known FGF receptor 2 ligands, only FGF7 was upregulated.Infusion of FGF7 into control mice caused the formation of a multilayered structure at 7 days, resembling the urothelium 14 days following obstruction. Thus, the pressure/stretching of renal monolayered urothelial cells is a very efficient trigger for proliferation, causing the formation of a bladder-like multistratified barrier with enhanced apical uroplakin plaques. Presumably, this ensures efficient barrier protection and repair.
Asunto(s)
Diferenciación Celular , Proliferación Celular , Transdiferenciación Celular , Riñón/patología , Obstrucción Ureteral/patología , Vejiga Urinaria/patología , Urotelio/patología , Animales , Biomarcadores/metabolismo , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Transdiferenciación Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Femenino , Factor 7 de Crecimiento de Fibroblastos/administración & dosificación , Factor 7 de Crecimiento de Fibroblastos/metabolismo , Factores de Crecimiento de Fibroblastos/genética , Factores de Crecimiento de Fibroblastos/metabolismo , Riñón/efectos de los fármacos , Riñón/metabolismo , Mecanotransducción Celular , Ratones , Ratones Endogámicos C57BL , Oligonucleótidos Antisentido/metabolismo , Fenotipo , Fosforilación , Presión , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/metabolismo , Estrés Mecánico , Factores de Tiempo , Obstrucción Ureteral/genética , Obstrucción Ureteral/metabolismo , Vejiga Urinaria/efectos de los fármacos , Vejiga Urinaria/metabolismo , Uroplaquinas/metabolismo , Urotelio/efectos de los fármacos , Urotelio/metabolismo , Vitamina A/farmacologíaRESUMEN
Rejection of solid organ allograft involves alloreactive T-cell expansion. The importance of NF-κB and NFAT in this process is underscored by the therapeutic efficacy of immunosuppressive agents, which target the two transcription factors. Since calpains, calcium-activated proteases, are involved in the activation of NF-κB and NFAT, we investigated the role of calpains in allograft rejection. In human transplant kidneys undergoing acute or chronic rejection, we show an increased expression of CAPN 1 gene encoding µ-calpain, associated with a marked expression of µ-calpain, mainly in infiltrating T cells. To address the role of calpain in rejection, we used a skin transplant model in transgenic mice expressing high levels of calpastatin, a calpain-specific inhibitor. We show that calpain inhibition extended skin allograft survival, from 11 to 20 days. This delay was associated with a limitation in allograft infiltration by T cells. In vitro, calpain inhibition by calpastatin transgene expression limited dramatically T-cell migration but, unexpectedly, increased slightly T-cell proliferation. Amplification of IL-2 signaling via the stabilization of IL-2R common γ-chain provided an explanation for the proliferation response. This is the first study establishing that calpain inhibition delays allograft rejection by slowing down T-cell migration rather than proliferation.
Asunto(s)
Proteínas de Unión al Calcio/metabolismo , Calpaína/metabolismo , Rechazo de Injerto/metabolismo , Acrilatos/uso terapéutico , Traslado Adoptivo , Animales , Proteínas de Unión al Calcio/genética , Calpaína/antagonistas & inhibidores , Calpaína/genética , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Movimiento Celular/genética , Movimiento Celular/inmunología , Proliferación Celular/efectos de los fármacos , Expresión Génica/genética , Expresión Génica/inmunología , Rechazo de Injerto/inmunología , Rechazo de Injerto/patología , Rechazo de Injerto/prevención & control , Proteínas de Homeodominio/genética , Humanos , Interleucina-2/metabolismo , Riñón/inmunología , Riñón/metabolismo , Trasplante de Riñón/inmunología , Activación de Linfocitos/genética , Activación de Linfocitos/inmunología , Prueba de Cultivo Mixto de Linfocitos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , FN-kappa B/metabolismo , Factores de Transcripción NFATC/metabolismo , Trasplante de Piel/inmunología , Trasplante de Piel/patología , Linfocitos T/inmunología , Linfocitos T/metabolismo , Linfocitos T/patología , Linfocitos T/trasplante , Linfocitos T Citotóxicos/inmunología , Células Th17/inmunología , Células Th17/metabolismo , Trasplante HomólogoRESUMEN
In hypertension, angiotensin (Ang) II is a critical mediator of cardiovascular remodeling, whose prominent features include myocardial and vascular media hypertrophy, perivascular inflammation, and fibrosis. The signaling pathways responsible for these alterations are not completely understood. Here, we investigated the importance of calpains, calcium-dependent cysteine proteases. We generated transgenic mice constitutively expressing high levels of calpastatin, a calpain-specific inhibitor. Chronic infusion of Ang II led to similar increases in systolic blood pressure in wild-type and transgenic mice. In contrast, compared with wild-type mice, transgenic mice displayed a marked blunting of Ang II-induced hypertrophy of left ventricle. Ang II-dependent vascular remodeling, ie, media hypertrophy and perivascular inflammation and fibrosis, was also limited in both large arteries (aorta) and small kidney arteries from transgenic mice as compared with wild type. In vitro experiments using vascular smooth muscle cells showed that calpastatin transgene expression blunted calpain activation by Ang II through epidermal growth factor receptor transactivation. In vivo and in vitro models of inflammation showed that impaired recruitment of mononuclear cells in transgenic mice was attributable to a decrease in both the release of and the chemotactic response to monocyte chemoattractant protein-1. Finally, results from collagen synthesis assay and zymography suggested that limited fibrogenesis was attributable to a decrease in collagen deposition rather than an increase in collagen degradation. These results indicate a critical role for calpains as downstream mediators in Ang II-induced cardiovascular remodeling and, thus, highlight an attractive therapeutic target.
Asunto(s)
Proteínas de Unión al Calcio/metabolismo , Calpaína/metabolismo , Inhibidores de Cisteína Proteinasa/metabolismo , Terapia Genética , Hipertensión/complicaciones , Hipertrofia Ventricular Izquierda/prevención & control , Remodelación Ventricular , Angiotensina II/administración & dosificación , Animales , Aorta/enzimología , Aorta/patología , Presión Sanguínea , Proteínas de Unión al Calcio/genética , Calpaína/antagonistas & inhibidores , Inhibidores de Cisteína Proteinasa/genética , Modelos Animales de Enfermedad , Fibrosis , Terapia Genética/métodos , Hipertensión/inducido químicamente , Hipertensión/metabolismo , Hipertensión/fisiopatología , Hipertensión/terapia , Hipertrofia , Hipertrofia Ventricular Izquierda/etiología , Hipertrofia Ventricular Izquierda/metabolismo , Hipertrofia Ventricular Izquierda/fisiopatología , Inflamación/etiología , Inflamación/metabolismo , Inflamación/fisiopatología , Bombas de Infusión Implantables , Ratones , Ratones Transgénicos , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patología , Miocardio/enzimología , Miocardio/patología , FN-kappa B/metabolismo , Factores de Transcripción NFATC/metabolismo , Arteria Renal/enzimología , Arteria Renal/patología , Factores de TiempoRESUMEN
Calpains, intracellular proteases specifically inhibited by calpastatin, play a major role in neoangiogenesis involved in tumor invasiveness and metastasis. They are partly exteriorized via the ATP-binding cassette transporter A1(ABCA1) transporter, but the importance of this process in tumor growth is still unknown. The aim of our study was to investigate the role of extracellular calpains in a model of melanoma by blocking their extracellular activity or exteriorization. In the first approach, a B16-F10 model of melanoma was developed in transgenic mice expressing high extracellular levels of calpastatin. In these mice, tumor growth was inhibited by â¼ 3-fold compared with wild-type animals. In vitro cytotoxicity assays and in vivo tumor studies have demonstrated that this protection was associated with a defect in tumor neoangiogenesis. Similarly, in wild-type animals given probenecid to blunt ABCA1 activity, melanoma tumor growth was inhibited by â¼ 3-fold. Again, this response was associated with a defect in neoangiogenesis. In vitro studies confirmed that probenecid limited endothelial cell migration and capillary formation from vascular explants. The observed reduction in fibronectin cleavage under these conditions is potentially involved in the response. Collectively, these studies demonstrate that probenecid, by blunting ABCA1 activity and thereby calpain exteriorization, limits melanoma tumor neoangiogenesis and invasiveness.
Asunto(s)
Transportador 1 de Casete de Unión a ATP/antagonistas & inhibidores , Calpaína/metabolismo , Melanoma Experimental/tratamiento farmacológico , Neovascularización Patológica/tratamiento farmacológico , Probenecid/farmacología , Neoplasias Cutáneas/tratamiento farmacológico , Transportador 1 de Casete de Unión a ATP/genética , Transportador 1 de Casete de Unión a ATP/metabolismo , Animales , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Línea Celular Tumoral/trasplante , Proliferación Celular/efectos de los fármacos , Humanos , Masculino , Melanoma Experimental/irrigación sanguínea , Melanoma Experimental/patología , Ratones , Ratones Transgénicos , Neovascularización Patológica/patología , Probenecid/uso terapéutico , Neoplasias Cutáneas/irrigación sanguínea , Neoplasias Cutáneas/patologíaRESUMEN
BACKGROUND: The specific mTor inhibitor sirolimus has been implicated in the pathogenesis of renal glomerular lesions and nephrotic syndrome appearance after transplantation. Podocyte injury and focal segmental glomerulosclerosis have been related to sirolimus therapy in some patients but the pathways underlying these lesions remain hypothetical. METHODS: To go further in the comprehension of these mechanisms, primary cultures of human podocytes were exposed to therapeutic-range concentrations of sirolimus. RESULTS: Cell viability was not affected after 2 days' exposure to the drug but changes in cell phenotype and cytoskeleton reorganization were observed. We also evidenced that vascular endothelial growth factor (VEGF) synthesis and Akt phosphorylation were decreased by sirolimus addition. We did not observe any loss of podocyte differentiation markers with the notable exception of WT1, a transcription factor essential for maintaining podocyte integrity. WT1 gene and protein expression in podocytes were decreased in a dose-dependent manner after incubation with sirolimus. CONCLUSION: Taken together, these data suggest that sirolimus could impair pathways essential for podocyte integrity and therefore predisposes to glomerular injury.
Asunto(s)
Podocitos/efectos de los fármacos , Podocitos/metabolismo , Sirolimus/efectos adversos , Secuencia de Bases , Diferenciación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Citoesqueleto/efectos de los fármacos , Cartilla de ADN/genética , Genes del Tumor de Wilms/efectos de los fármacos , Humanos , Inmunosupresores/efectos adversos , Modelos Biológicos , Fenotipo , Podocitos/patología , Reacción en Cadena de la Polimerasa , Proteínas Quinasas/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal/efectos de los fármacos , Serina-Treonina Quinasas TOR , Factor A de Crecimiento Endotelial Vascular/biosíntesis , Proteínas WT1/genética , Proteínas WT1/metabolismoRESUMEN
Calpain 1 is a proinflammatory calcium-activated cysteine protease, which can be partly externalized. Extracellular calpains limit inflammatory processes and promote tissue repair, through cell proliferation and migration. Toll like receptor (TLR) 2 has been identified as a target of extracellular calpains in lymphocytes. The aim was to investigate the externalization of calpain 1 and the release of soluble TLR2 during tumor progression of pulmonary lepidic predominant adenocarcinoma (LPA). Extracellular calpain 1, soluble fragment of TLR2 and cytokines were analyzed by ELISA in bronchoalveolar lavage fluid (BALF) supernatants from patients with LPA (n=68). Source of calpain was analyzed by immunohistochemistry and soluble TLR2 by flow cytometry on polymorphonuclear neutrophils (PMN) and human lung cancer cell lines. Extracellular calpain 1, secreted by tumor cells, was associated to tumor progression, neutrophilic inflammation, with a poor prognostic factor on survival (P=0.003). TLR2 was expressed on PMN and tumor cells and decreased after calpain exposure. Soluble fragment of TLR2 in BALF supernatants was correlated to the extracellular calpain 1 concentration (r=0.624; P<0.001), and its high level was associated with tumor progression and a pro-inflammatory environment. Extracellular calpain 1 secreted by tumor cells, could participate in inflammatory microenvironment and tumor progression through TLR2 in LPA.
Asunto(s)
Adenocarcinoma/metabolismo , Líquido del Lavado Bronquioalveolar/química , Calpaína/análisis , Neoplasias Pulmonares/metabolismo , Proteínas de Neoplasias/metabolismo , Receptor Toll-Like 2/metabolismo , Adenocarcinoma/mortalidad , Adenocarcinoma/patología , Anciano , Calpaína/metabolismo , Línea Celular Tumoral , Progresión de la Enfermedad , Femenino , Humanos , Inflamación/metabolismo , Neoplasias Pulmonares/mortalidad , Neoplasias Pulmonares/patología , Masculino , Proteínas de Neoplasias/análisis , Neutrófilos/metabolismo , PronósticoRESUMEN
Thyroid calcification is frequent in thyroid nodules. The aim of our study was to evaluate the prevalence of calcifications in thyroid tissue samples of patients with various thyroid diseases, and to identify their composition according to their localization. Among 50 thyroid samples included, 56% were malignant (papillary carcinoma) and 44% were benign (adenoma, multinodular goiter, Graves' disease, sarcoidosis). Calcifications were found in 95% of samples using polarised light microscopy, whereas only 12% were described in initial pathological reports. Three types were individualised and analyzed by infrared spectrometry (µFTIR): colloid calcifications composed of calcium oxalate, capsular calcifications and psammoma bodies, both composed of calcium phosphate. Of notice, psammoma bodies characterized by FE-SEM were composed of concentric structure suggesting a slow process for crystal deposition. Calcium phosphates were found only in malignant samples whereas calcium oxalate was not associated with a define pathology. Proliferation assessed by KI67 staining was high (33% of positive follicles), and RUNX2, OPN, and CD44 positive staining were detected in thyrocytes with a broad variation between samples. However, thyrocyte proliferation and differentiation markers were not associated with the number of crystals. TRPV5 and CaSR expression was also detected in thyrocytes. mRNA transcripts expression was confirmed in a subgroup of 10 patients, altogether with other calcium transporters such as PMCA1 or Cav1.3. Interestingly, TRPV5 mRNA expression was significantly associated with number of colloid calcifications (rho = -0.72; p = 0.02). The high prevalence of calcium oxalate crystals within colloid gel raises intriguing issues upon follicle physiology for calcium and oxalate transport.
Asunto(s)
Calcinosis/epidemiología , Calcinosis/patología , Carcinoma Papilar/fisiopatología , Neoplasias de la Tiroides/fisiopatología , Nódulo Tiroideo/fisiopatología , Adulto , Estudios de Casos y Controles , Femenino , Francia/epidemiología , Humanos , Incidencia , Masculino , Persona de Mediana EdadRESUMEN
Increased urinary oxalate excretion (hyperoxaluria) promotes the formation of calcium oxalate crystals. Monogenic diseases due to hepatic enzymes deficiency result in chronic hyperoxaluria, promoting end-stage renal disease in children and young adults. Ethylene glycol poisoning also results in hyperoxaluria promoting acute renal failure and frequently death. Stiripentol is an antiepileptic drug used to treat children affected by Dravet syndrome, possibly by inhibiting neuronal lactate dehydrogenase 5 isoenzyme. As this isoenzyme is also the last step of hepatic oxalate production, we hypothesized that Stiripentol would potentially reduce hepatic oxalate production and urine oxalate excretion. In vitro, Stiripentol decreased in a dose-dependent manner the synthesis of oxalate by hepatocytes. In vivo, Stiripentol oral administration reduced significantly urine oxalate excretion in rats. Stiripentol protected kidneys against calcium oxalate crystal deposits in acute ethylene glycol intoxication and chronic calcium oxalate nephropathy models. In both models, Stiripentol improved significantly renal function. Patients affected by Dravet syndrome and treated with Stiripentol had a lower urine oxalate excretion than control patients. A young girl affected by severe type I hyperoxaluria received Stiripentol for several weeks: urine oxalate excretion decreased by two-thirds. Stiripentol is a promising potential therapy against genetic hyperoxaluria and ethylene glycol poisoning.
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Dioxolanos/farmacología , Glicol de Etileno/envenenamiento , Hiperoxaluria Primaria/prevención & control , Nefrolitiasis/prevención & control , Animales , Oxalato de Calcio/metabolismo , Epilepsias Mioclónicas/tratamiento farmacológico , Epilepsias Mioclónicas/metabolismo , Epilepsias Mioclónicas/patología , Femenino , Hepatocitos/metabolismo , Hepatocitos/patología , Humanos , Hiperoxaluria Primaria/metabolismo , Hiperoxaluria Primaria/patología , Riñón/metabolismo , Riñón/patología , Masculino , Nefrolitiasis/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
Most mouse kidney stone models induce nephrocalcinosis rather than urolithiasis. The aim of our study was to find an accelerated experimental model in order to study the early events of stone formation, that is, at the time of crystal binding to intrarenal urothelium. C57B6 mice exposed to vitamin D supplements and water containing hydroxyl-L-proline, ammonium chloride and calcium chloride were studied for 42 days. A group receiving urothelial cell mitogen Fibroblast Growth Factor 7 (FGF7) was compared to control group receiving saline. Calcium oxalate monohydrate (COM) crystals were detected in urines by day 2 and within urinary spaces in specialized fornix areas in both groups as soon as day 14 with enhanced deposits in FGF7 group compared to controls at day 21. Urothelial cells proliferation, uroplakin III downregulation and de novo expression of osteopontin receptor CD44 detected in FGF7 group, were delayed in the control group (day 42). Crystal aggregates within specialized fornix areas by day 42 were located in urinary spaces but also within and under a multilayered metaplastic urothelium, simultaneous to macrophages influx. Point of note, administration of a normal diet by day 21 was responsible for a spontaneous crystal clearance. Our data show that under supersaturation conditions, urothelial cell proliferation and calcium oxalate crystal retention occur within specialized fornix areas. Enhanced crystal deposits following FGF7 administration suggest that urothelium proliferation would be a relevant trigger for renal stone formation.
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Cálculos Renales/patología , Urotelio/patología , Animales , Proliferación Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Femenino , Factor 7 de Crecimiento de Fibroblastos/farmacología , Riñón/efectos de los fármacos , Riñón/patología , Ratones , Ratones Endogámicos C57BL , Fenotipo , Urotelio/efectos de los fármacosRESUMEN
Most of kidney stones are supposed to originate from Randall's plaque at the tip of the papilla or from papillary tubular plugs. Nevertheless, the frequency and the composition of crystalline plugs remain only partly described. The objective was to assess the frequency, the composition and the topography of papillary plugs in human kidneys. A total of 76 papillae from 25 kidneys removed for cancer and without stones were analysed by immunohistochemistry combined with Yasue staining, field emission-scanning electron microscopy and Fourier transformed infrared micro-spectroscopy. Papillary tubular plugs have been observed by Yasue staining in 23/25 patients (92%) and 52/76 papillae (68%). Most of these plugs were made of calcium phosphate, mainly carbonated apatite and amorphous calcium phosphate, and rarely octacalcium phosphate pentahydrate. Calcium and magnesium phosphate (whitlockite) have also been observed. Based upon immunostaining coupled to Yasue coloration, most of calcium phosphate plugs were located in the deepest part of the loop of Henle. Calcium oxalate monohydrate and dihydrate tubular plugs were less frequent and stood in collecting ducts. At last, we observed calcium phosphate plugs deforming and sometimes breaking adjacent collecting ducts. Papillary tubular plugging, which may be considered as a potential first step toward kidney stone formation, is a very frequent setting, even in kidneys of non-stone formers. The variety in their composition and the distal precipitation of calcium oxalate suggest that plugs may occur in various conditions of urine supersaturation. Plugs were sometimes associated with collecting duct deformation.
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Cálculos Renales/etiología , Túbulos Renales Colectores/patología , Asa de la Nefrona/patología , Anciano , Fosfatos de Calcio/análisis , Humanos , Cálculos Renales/química , Cálculos Renales/epidemiología , Cálculos Renales/ultraestructura , Túbulos Renales Colectores/química , Túbulos Renales Colectores/diagnóstico por imagen , Asa de la Nefrona/química , Asa de la Nefrona/diagnóstico por imagen , Microscopía Electrónica de Rastreo , Persona de Mediana Edad , Espectroscopía Infrarroja por Transformada de FourierRESUMEN
Severe hypertension can lead to malignant hypertension (MH) with renal thrombotic microangiopathy and hemolysis. The role of plasma heme release in this setting is unknown. We aimed at evaluating the effect of a mild plasma heme increase by hemin administration in angiotensin II (AngII)-mediated hypertensive rats. Prevalence of MH and blood pressure values were similar in AngII and AngII + hemin groups. MH rats displayed a decreased renal blood flow (RBF), increased renal vascular resistances (RVR), and increased aorta and interlobar arteries remodeling with a severe renal microcirculation assessed by peritubular capillaries (PTC) rarefaction. Hemin-treated rats with or without AngII displayed also a decreased RBF and increased RVR explained only by PCT rarefaction. In AngII rats, RBF was similar to controls (with increased RVR). PTC density appeared strongly correlated to tubular damage score (rho = -0.65, p < 0.0001) and also renal Heme Oygenase-1 (HO-1) mRNA (rho = -0.67, p < 0.0001). HO-1 was expressed in PTC and renal tubules in MH rats, but only in PTC in other groups. In conclusion, though increased plasma heme does not play a role in triggering or aggravating MH, heme release appears as a relevant toxic mediator leading to renal impairment, primarily through PTC endothelial dysfunction rather than direct tubular toxicity.
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Hemo/toxicidad , Hipertensión/complicaciones , Enfermedades Renales/inducido químicamente , Enfermedades Renales/patología , Rarefacción Microvascular/patología , Plasma/química , Animales , Hemina/administración & dosificación , Ratas Sprague-Dawley , Circulación RenalRESUMEN
Calpains are ubiquitous pro-inflammatory proteases, whose activity is controlled by calpastatin, their specific inhibitor. Transgenic mice over-expressing rabbit calpastatin (CalpTG) are protected against vascular remodelling and angiotensin II-dependent inflammation. We hypothesized that specific calpain inhibition would protect against aging-related lesions in arteries and kidneys. We analysed tissues from 2-months and 2-years-old CalpTG and wild-type mice and performed high throughput RNA-Sequencing of kidney tissue in aged mice. In addition, we analysed inflammatory response in the kidney of aged CalpTG and wild-type mice, and in both in vivo (monosodium urate peritonitis) and in vitro models of inflammation. At two years, CalpTG mice had preserved kidney tissue, less vascular remodelling and less markers of senescence than wild-type mice. Nevertheless, CalpTG mice lifespan was not extended, due to the development of lethal spleen tumors. Inflammatory pathways were less expressed in aged CalpTG mice, especially cytokines related to NF-κB and NLRP3 inflammasome activation. CalpTG mice had reduced macrophage infiltration with aging and CalpTG mice produced less IL-1α and IL-1ß in vivo in response to inflammasome activators. In vitro, macrophages from CalpTG mice produced less IL-1α in response to particulate activators of inflammasome. Calpains inhibition protects against inflammaging, limiting kidney and vascular lesions related to aging.
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Envejecimiento/efectos de los fármacos , Proteínas de Unión al Calcio/farmacología , Calpaína/antagonistas & inhibidores , Inhibidores de Cisteína Proteinasa/farmacología , Peritonitis/tratamiento farmacológico , Animales , Arterias/efectos de los fármacos , Arterias/crecimiento & desarrollo , Proteínas de Unión al Calcio/uso terapéutico , Calpaína/metabolismo , Células Cultivadas , Inhibidores de Cisteína Proteinasa/uso terapéutico , Citocinas/metabolismo , Inflamasomas/metabolismo , Riñón/efectos de los fármacos , Riñón/crecimiento & desarrollo , Ratones , Ratones Endogámicos C57BL , FN-kappa B/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , ConejosRESUMEN
Somatostatin (SRIF) exerts anti-inflammatory effects, in part by deactivating monocytes/macrophages. Thus, the objective of this study was to characterize specific receptors for SRIF on these cells. Macrophages isolated from mouse peritoneal cells bound [125I]Tyr(0), D-Trp(8) SRIF(14) specifically. Scatchard analysis of saturation binding data revealed two classes of binding sites with an affinity of 0.44+/-0.13 and 2.58+/-0.56 nM, respectively. By sensitive and specific RT-PCR, the mRNAs for the five SRIF receptors (SSTR1 to SSTR5) could be detected. Evidence for the involvement of SSTR1 and SSTR2 in the binding of SRIF to the high and low affinity sites, respectively, was obtained by the demonstration that (1) only SSTR1 and SSTR2 subtype-specific agonists were active in competing for [125I]Tyr(0), D-Trp(8) SRIF(14) binding to high and low affinity sites, respectively, and (2) [125I]Tyr(0), D-Trp(8) SRIF(14) bound to high but not low affinity sites on macrophages isolated from SSTR2 knock-out mice. In conclusion, we have identified and characterized two different SRIF receptor subtypes in murine macrophages.