RESUMEN
The mechanisms driving the development of extracapillary lesions in focal segmental glomerulosclerosis (FSGS) and crescentic glomerulonephritis (CGN) remain poorly understood. A key question is how parietal epithelial cells (PECs) invade glomerular capillaries, thereby promoting injury and kidney failure. Here we show that expression of the tetraspanin CD9 increases markedly in PECs in mouse models of CGN and FSGS, and in kidneys from individuals diagnosed with these diseases. Cd9 gene targeting in PECs prevents glomerular damage in CGN and FSGS mouse models. Mechanistically, CD9 deficiency prevents the oriented migration of PECs into the glomerular tuft and their acquisition of CD44 and ß1 integrin expression. These findings highlight a critical role for de novo expression of CD9 as a common pathogenic switch driving the PEC phenotype in CGN and FSGS, while offering a potential therapeutic avenue to treat these conditions.
Asunto(s)
Enfermedades Renales/patología , Tetraspanina 29/fisiología , Animales , Movimiento Celular/genética , Proliferación Celular/genética , Progresión de la Enfermedad , Femenino , Glomerulonefritis/genética , Glomerulonefritis/metabolismo , Glomerulonefritis/patología , Glomeruloesclerosis Focal y Segmentaria/genética , Glomeruloesclerosis Focal y Segmentaria/metabolismo , Glomeruloesclerosis Focal y Segmentaria/patología , Humanos , Enfermedades Renales/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Tetraspanina 29/genética , Tetraspanina 29/metabolismoRESUMEN
Necrotizing and crescentic rapidly progressive GN (RPGN) is a life-threatening syndrome characterized by a rapid loss of renal function. Evidence suggests that podocyte expression of the transcription factor peroxisome proliferator-activated receptor γ (PPARγ) may prevent podocyte injury, but the function of glomerular PPARγ in acute, severe inflammatory GN is unknown. Here, we observed marked loss of PPARγ abundance and transcriptional activity in glomerular podocytes in experimental RPGN. Blunted expression of PPARγ in podocyte nuclei was also found in kidneys from patients diagnosed with crescentic GN. Podocyte-specific Pparγ gene targeting accentuated glomerular damage, with increased urinary loss of albumin and severe kidney failure. Furthermore, a PPARγ gain-of-function approach achieved by systemic administration of thiazolidinedione (TZD) failed to prevent severe RPGN in mice with podocyte-specific Pparγ gene deficiency. In nuclear factor erythroid 2-related factor 2 (NRF2)-deficient mice, loss of podocyte PPARγ was observed at baseline. NRF2 deficiency markedly aggravated the course of RPGN, an effect that was partially prevented by TZD administration. Furthermore, delayed administration of TZD, initiated after the onset of RPGN, still alleviated the severity of experimental RPGN. These findings establish a requirement for the NRF2-PPARγ cascade in podocytes, and we suggest that these transcription factors have a role in augmenting the tolerance of glomeruli to severe immune-complex mediated injury. The NRF2-PPARγ pathway may be a therapeutic target for RPGN.
Asunto(s)
Glomerulonefritis/etiología , Factor 2 Relacionado con NF-E2/fisiología , PPAR gamma/biosíntesis , Podocitos/metabolismo , Animales , Masculino , RatonesRESUMEN
BACKGROUND: Cardiac-committed cells and biomimetic scaffolds independently improve the therapeutic efficacy of stem cells. In this study we tested the long-term effects of their combination. METHODS: Eighty immune-deficient rats underwent permanent coronary artery ligation. Five to 7 weeks later, those with an echocardiographically measured ejection fraction (EF) ≤55% were re-operated on and randomly allocated to receive a cell-free fibrin patch (n = 25), a fibrin patch loaded with 700,000 human embryonic stem cells (ESC) pre-treated to promote early cardiac differentiation (SSEA-1(+) progenitors [n = 30]), or to serve as sham-operated animals (n = 25). Left ventricular function was assessed by echocardiography at baseline and every month thereafter until 4 months. Hearts were then processed for assessment of fibrosis and angiogenesis and a 5-component heart failure score was constructed by integrating the absolute change in left ventricular end-systolic volume (LVESV) between 4 months and baseline, and the quantitative polymerase chain reaction (qPCR)-based expression of natriuretic peptides A and B, myosin heavy chain 7 and periostin. All data were recorded and analyzed in a blinded manner. RESULTS: The cell-treated group consistently yielded better functional outcomes than the sham-operated group (p = 0.002 for EF; p = 0.01 for LVESV). Angiogenesis in the border zone was also significantly greater in the cell-fibrin group (p = 0.006), which yielded the lowest heart failure score (p = 0.04 vs sham). Engrafted progenitors were only detected shortly after transplantation; no grafted cells were identified after 4 months. There was no teratoma identified. CONCLUSIONS: A fibrin scaffold loaded with ESC-derived cardiac progenitors resulted in sustained improvement in contractility and attenuation of remodeling without sustained donor cell engraftment. A paracrine effect, possibly on innate reparative responses, is a possible mechanism for this enduring effect.
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Células Madre Embrionarias , Fibrina , Miocardio/citología , Células Madre , Andamios del Tejido , Inductores de la Angiogénesis , Animales , Moléculas de Adhesión Celular/análisis , Vasos Coronarios , Ecocardiografía , Femenino , Insuficiencia Cardíaca , Humanos , Inmunohistoquímica , Ligadura , Ratones Desnudos , Cadenas Pesadas de Miosina , Péptidos Natriuréticos/análisis , Reacción en Cadena de la Polimerasa , Ratas , Volumen Sistólico , Función Ventricular IzquierdaRESUMEN
Both enzymatic dissociation of cells prior to needle-based injections and poor vascularization of myocardial infarct areas are two important contributors to cell death and impede the efficacy of cardiac cell therapy. Because these limitations could be overcome by scaffolds ensuring cell cohesiveness and codelivery of angiogenic cells, we used a chronic rat model of myocardial infarction to assess the long-term (6 months) effects of the epicardial delivery of a composite collagen-based patch harboring both cardiomyogenesis-targeted human embryonic SSEA-1(+) (stem cell-derived stage-specific embryonic antigen-1 positive) cardiovascular progenitors and autologous (rat) adipose tissue-derived angiogenesis-targeted stromal cells (n = 27). Cell-free patches served as controls (n = 28). Serial follow-up echocardiographic measurements of left ventricular ejection fraction (LVEF) showed that the composite patch group yielded a significantly better preservation of left ventricular function that was sustained over time as compared with controls, and this pattern persisted when the assessment was restricted to the subgroup of rats with initial LVEFs below 50%. The composite patch group was also associated with significantly less fibrosis and more vessels in the infarct area. However, although human progenitors expressing cardiac markers were present in the patches before implantation, none of them could be subsequently identified in the grafted tissue. These data confirm the efficacy of epicardial scaffolds as cell carriers for ensuring long-term functional benefits and suggest that these effects are likely related to paracrine effects and call for optimizing cross-talks between codelivered cell populations to achieve the ultimate goal of myocardial regeneration.
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Tratamiento Basado en Trasplante de Células y Tejidos/métodos , Infarto del Miocardio/terapia , Pericardio/citología , Tejido Adiposo/citología , Animales , Colágeno/administración & dosificación , Desfibriladores Implantables , Modelos Animales de Enfermedad , Femenino , Humanos , Miocardio/citología , Ratas , Ratas Wistar , Andamios del TejidoRESUMEN
RATIONALE: Purpura fulminans in adults is a rare but devastating disease. Its pathophysiology is not well known. OBJECTIVES: To understand the pathophysiology of skin lesions in purpura fulminans, the interplay between circulating blood and vascular alterations was assessed. METHODS: Prospective multicenter study in four intensive care units. Patients with severe sepsis without skin lesions were recruited as control subjects. MEASUREMENTS AND MAIN RESULTS: Twenty patients with severe sepsis and purpura fulminans were recruited for blood sampling, and skin biopsy was performed in deceased patients. High severity of disease and mortality rates (80%) was observed. Skin biopsies in purpura fulminans lesions revealed thrombosis and extensive vascular damage: vascular congestion and dilation, endothelial necrosis, alteration of markers of endothelial integrity (CD31) and of the protein C pathway receptors (endothelial protein C receptor, thrombomodulin). Elevated plasminogen activating inhibitor-1 mRNA was also observed. Comparison with control patients showed that these lesions were specific to purpura fulminans. By contrast, no difference was observed for blood hemostasis parameters, including soluble thrombomodulin, activated protein C, and disseminated intravascular coagulation markers. Bacterial presence at the vascular wall was observed specifically in areas of vascular damage in eight of nine patients tested (including patients with Streptococcus pneumoniae, Neisseria meningitidis, Escherichia coli, and Pseudomonas aeruginosa infection). CONCLUSIONS: Thrombi and extensive vascular damage with multifaceted prothrombotic local imbalance are characteristics of purpura fulminans. A "vascular wall infection" hypothesis, responsible for endothelial damage and subsequent skin lesions, can be put forward.
Asunto(s)
Endotelio Vascular/patología , Púrpura Fulminante/patología , Trombosis/complicaciones , Malformaciones Vasculares/complicaciones , Anciano , Anciano de 80 o más Años , Biomarcadores/metabolismo , Biopsia , Endotelio Vascular/metabolismo , Femenino , Humanos , Masculino , Persona de Mediana Edad , Inhibidor 1 de Activador Plasminogénico/metabolismo , Estudios Prospectivos , Púrpura Fulminante/complicaciones , Púrpura Fulminante/metabolismo , Sepsis/metabolismo , Piel/irrigación sanguínea , Trombomodulina/metabolismo , Trombosis/patología , Malformaciones Vasculares/metabolismo , Malformaciones Vasculares/patologíaRESUMEN
Nephrogenic systemic fibrosis (NSF) is a scleroderma-like disease associated with prior administration of certain gadolinium chelates (GCs). NSF occurs in patients with severe renal failure. The purpose of this study was to set up a rat model of GC-associated NSF to elucidate the mechanism of this devastating disease. Firstly, after characterization of the model, male Wistar rats received a 0.75% adenine-enriched diet for 8, 14, or 16 days to obtain various degrees of renal failure. Rats received five consecutive daily iv injections of saline or gadodiamide (2.5 mmol/kg/day). Secondly, the safety profile and in vivo propensity to dissociate of all categories of marketed GCs (gadoterate, gadobutrol, gadobenate, gadopentetate, and gadodiamide) were compared in rats receiving adenine-enriched diet for 16 days. Serial skin biopsies were performed for blinded histopathological study. Total Gd concentration in tissues was measured by Inductively Coupled Plasma Mass Spectrometry. Relaxometry was used to evaluate the presence of dissociated Gd in skin and bone. Gadodiamide-induced high mortality and skin lesions (dermal fibrosis, calcification, and inflammation) were related to adenine diet duration. No skin lesions were observed with other molecules. Unlike macrocyclic GCs, gadodiamide, gadopentetate, and gadobenate gradually increased the r(1) relaxivity value, consistent with in vivo dissociation and release of soluble Gd (or, in the case of gadobenate, the consequence of protein binding). Gadodiamide-induced cutaneous and systemic toxicity depended on baseline renal function. We demonstrate in vivo dissociation of linear GCs, gadodiamide, and gadopentetate, whereas macrocyclic agents remained stable over the study period.
Asunto(s)
Adenina/administración & dosificación , Medios de Contraste/toxicidad , Gadolinio/toxicidad , Dermopatía Fibrosante Nefrogénica/inducido químicamente , Compuestos Organometálicos/toxicidad , Insuficiencia Renal/complicaciones , Animales , Huesos/efectos de los fármacos , Huesos/metabolismo , Huesos/patología , Medios de Contraste/química , Medios de Contraste/farmacocinética , Dieta , Modelos Animales de Enfermedad , Gadolinio/química , Gadolinio/farmacocinética , Riñón/efectos de los fármacos , Riñón/metabolismo , Riñón/patología , Pruebas de Función Renal , Masculino , Dermopatía Fibrosante Nefrogénica/etiología , Dermopatía Fibrosante Nefrogénica/metabolismo , Dermopatía Fibrosante Nefrogénica/patología , Compuestos Organometálicos/química , Compuestos Organometálicos/farmacocinética , Ratas , Ratas Wistar , Insuficiencia Renal/inducido químicamente , Insuficiencia Renal/metabolismo , Piel/efectos de los fármacos , Piel/metabolismo , Piel/patología , Espectrofotometría Atómica , Distribución TisularRESUMEN
Tight regulation of calcium levels is required for many critical biological functions. The Ca2+-sensing receptor (CaSR) expressed by parathyroid cells controls blood calcium concentration by regulating parathyroid hormone (PTH) secretion. However, CaSR is also expressed in other organs, such as the kidney, but the importance of extraparathyroid CaSR in calcium metabolism remains unknown. Here, we investigated the role of extraparathyroid CaSR using thyroparathyroidectomized, PTH-supplemented rats. Chronic inhibition of CaSR selectively increased renal tubular calcium absorption and blood calcium concentration independent of PTH secretion change and without altering intestinal calcium absorption. CaSR inhibition increased blood calcium concentration in animals pretreated with a bisphosphonate, indicating that the increase did not result from release of bone calcium. Kidney CaSR was expressed primarily in the thick ascending limb of the loop of Henle (TAL). As measured by in vitro microperfusion of cortical TAL, CaSR inhibitors increased calcium reabsorption and paracellular pathway permeability but did not change NaCl reabsorption. We conclude that CaSR is a direct determinant of blood calcium concentration, independent of PTH, and modulates renal tubular calcium transport in the TAL via the permeability of the paracellular pathway. These findings suggest that CaSR inhibitors may provide a new specific treatment for disorders related to impaired PTH secretion, such as primary hypoparathyroidism.
Asunto(s)
Calcio/sangre , Hormona Paratiroidea/metabolismo , Receptores Sensibles al Calcio/fisiología , Aminoácidos/orina , Animales , Conservadores de la Densidad Ósea/farmacología , Conservadores de la Densidad Ósea/uso terapéutico , Calcio/metabolismo , Calcio/orina , Creatinina/orina , Difosfonatos/farmacología , Difosfonatos/uso terapéutico , Hipoparatiroidismo/sangre , Hipoparatiroidismo/tratamiento farmacológico , Asa de la Nefrona/metabolismo , Masculino , Naftalenos/farmacología , Naftalenos/uso terapéutico , Osteocalcina/sangre , Pamidronato , Paratiroidectomía , Permeabilidad/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Receptores Sensibles al Calcio/antagonistas & inhibidores , Receptores Sensibles al Calcio/metabolismo , Simportadores de Cloruro de Sodio-Potasio/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Miembro 1 de la Familia de Transportadores de Soluto 12RESUMEN
Familial hyperkalemic hypertension (FHHt) is a Mendelian form of arterial hypertension that is partially explained by mutations in WNK1 and WNK4 that lead to increased activity of the Na(+)-Cl(-) cotransporter (NCC) in the distal nephron. Using combined linkage analysis and whole-exome sequencing in two families, we identified KLHL3 as a third gene responsible for FHHt. Direct sequencing of 43 other affected individuals revealed 11 additional missense mutations that were associated with heterogeneous phenotypes and diverse modes of inheritance. Polymorphisms at KLHL3 were not associated with blood pressure. The KLHL3 protein belongs to the BTB-BACK-kelch family of actin-binding proteins that recruit substrates for Cullin3-based ubiquitin ligase complexes. KLHL3 is coexpressed with NCC and downregulates NCC expression at the cell surface. Our study establishes a role for KLHL3 as a new member of the complex signaling pathway regulating ion homeostasis in the distal nephron and indirectly blood pressure.
Asunto(s)
Proteínas Portadoras/genética , Transporte Iónico/genética , Nefronas/metabolismo , Seudohipoaldosteronismo/genética , Simportadores del Cloruro de Sodio/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Secuencia de Aminoácidos , Secuencia de Bases , Presión Sanguínea/genética , Niño , Femenino , Humanos , Riñón/metabolismo , Masculino , Proteínas de Microfilamentos , Persona de Mediana Edad , Datos de Secuencia Molecular , Polimorfismo de Nucleótido Simple , Seudohipoaldosteronismo/metabolismo , Seudohipoaldosteronismo/fisiopatología , Análisis de Secuencia de ADN , Transducción de Señal , Simportadores del Cloruro de Sodio/genética , Adulto JovenRESUMEN
IgA nephropathy (IgAN) often shows lesions morphologically identical with those of focal segmental glomerulosclerosis (FSGS). In order to determine the possible role of FSGS in IgAN lesions, we measured glomerular capsular adhesions, often the first step toward FSGS, in biopsies from 127 patients with IgAN, 100 with lupus nephritis, and 26 with primary FSGS. Capsular adhesions with no lesions in the underlying tuft, consistent with podocyte abnormality or loss, were found regularly in FSGS and IgAN, but infrequently in lupus. Fifteen biopsies of patients with IgAN were studied immunohistochemically using markers for podocytes, Bowman's parietal epithelial cells, proliferating cells, and macrophages. Cytokeratins CK-8 and C2562 differentiated normal podocytes (negative) from parietal epithelial cells (variably positive). There was focal loss of the podocyte markers synaptopodin, glomerular epithelial protein 1 (GLEPP-1), nephrin, and vascular endothelial growth factor (VEGF), particularly at sites of capsular adhesions in otherwise histologically normal glomeruli. Cells displaying the parietal epithelial cell markers PAX2 (paired box gene 2) and the cytokeratins were also positive for the proliferating cell marker, proliferating cell nuclear antigen. These cells gathered at sites of adhesion, and in response to active lesions in the tuft, grew inward along the adhesion onto the tuft, forming a monolayer positive for parietal markers and the podocyte marker Wilms tumor protein-1 (WT-1). These cells deposited a layer of collagen over the sclerosing tuft. Thus, all biopsies of patients with IgAN had changes basically identical to those classically described in FSGS. Hence, our study strongly suggests that podocytopathy of a type similar to that in primary FSGS occurs frequently in IgAN.
Asunto(s)
Glomerulonefritis por IGA/metabolismo , Glomeruloesclerosis Focal y Segmentaria/metabolismo , Inmunohistoquímica , Glomérulos Renales/química , Nefritis Lúpica/metabolismo , Antígenos CD/análisis , Antígenos de Diferenciación Mielomonocítica/análisis , Biomarcadores/análisis , Biopsia , Distribución de Chi-Cuadrado , Progresión de la Enfermedad , Células Epiteliales/química , Glomerulonefritis por IGA/inmunología , Glomerulonefritis por IGA/patología , Glomeruloesclerosis Focal y Segmentaria/inmunología , Glomeruloesclerosis Focal y Segmentaria/patología , Humanos , Queratina-8/análisis , Glomérulos Renales/inmunología , Glomérulos Renales/patología , Nefritis Lúpica/inmunología , Nefritis Lúpica/patología , Factor de Transcripción PAX2/análisis , Paris , Podocitos/química , Pronóstico , Antígeno Nuclear de Célula en Proliferación/análisis , Proteínas WT1/análisisRESUMEN
Angiotensin I-converting enzyme (ACE; kininase II) levels in humans are genetically determined. ACE levels have been linked to risk of myocardial infarction, but the association has been inconsistent, and the causality underlying it remains undocumented. We tested the hypothesis that genetic variation in ACE levels influences myocardial tolerance to ischemia. We studied ischemia-reperfusion injury in mice bearing 1 (ACE1c), 2 (ACE2c, wild type), or 3 (ACE3c) functional copies of the ACE gene and displaying an ACE level range similar to humans. Infarct size in ACE1c was 29% lower than in ACE2c (P<0.05). Pretreatment with a kinin B2 receptor antagonist suppressed this reduction. In ACE3c, infarct size was the same as in ACE2c. But ischemic preconditioning, which reduced infarct size in ACE2c (-63%, P<0.001) and ACE1c (-52%, P<0.05), was not efficient in ACE3c (-2%, NS, P<0.01 vs. ACE2c). In ACE3c, ischemic preconditioning did not decrease myocardial inflammation or cardiomyocyte apoptosis. Pretreatment with a renin inhibitor had no cardioprotective effect in ACE2c, but in ACE3c partially restored (38%) the cardioprotection of ischemic preconditioning. Thus, a modest genetic increase in ACE impairs myocardial tolerance to ischemia. ACE level plays a critical role in cardiac ischemia, through both kinin and angiotensin mediated mechanisms.
Asunto(s)
Corazón/efectos de los fármacos , Infarto del Miocardio/enzimología , Isquemia Miocárdica/enzimología , Miocardio/enzimología , Peptidil-Dipeptidasa A/metabolismo , Daño por Reperfusión/genética , Amidas/farmacología , Angiotensina I/farmacología , Angiotensina II/farmacología , Animales , Apoptosis/efectos de los fármacos , Presión Sanguínea/efectos de los fármacos , Bradiquinina/análogos & derivados , Bradiquinina/farmacología , Antagonistas de los Receptores de Bradiquinina , Fumaratos/farmacología , Cininas/farmacología , Pulmón/enzimología , Ratones , Ratones Mutantes , Infarto del Miocardio/genética , Isquemia Miocárdica/genética , Peptidil-Dipeptidasa A/genética , Renina/antagonistas & inhibidoresRESUMEN
The present work identifies a new mouse model of inductible acute glomerular injury leading to focal segmental glomerulonephritis. We take advantage of the suicide gene/prodrug nitroreductase/CB1954 combination, in which nitroreductase converts CB1954, a monofunctional alkylating agent, into its toxic form. We generate two lines of transgenic mice in which the nitroreductase gene was placed under the control of the podocyte-specific gene podocin. The functional analysis of transgenic mice lines showed that CB1954 treatment induced a severe but transitory proteinuria. Sequential histopathological analysis was performed on serial kidney biopsies. Injured glomeruli showed acute lesions with early podocyte vacuolization and detachment, podocyte apoptosis, and cellular proliferation leading to a marked hypercellularity of the urinary space that was associated with collapsing of the glomerular tuft. After 1 month, progressive scarring lead to focal segmental glomerulosclerosis with fibrous capsular adhesion, hyalinosis, and podocytosis associated with interstitial fibrosis. The phenotype of podocytes was changed exhibiting dedifferentiation characterized by the loss of podocyte specific proteins/transcription factor and the expression of injury markers. Bowman's capsule cells were also involved in the cellular changes in a manner suggesting epithelial to mesenchymal transition. This model of podocyte injury in transgenic mice provides new insights into the cellular mechanisms of podocytopathies and their progression to scarring.
Asunto(s)
Glomeruloesclerosis Focal y Segmentaria/fisiopatología , Péptidos y Proteínas de Señalización Intracelular/genética , Glomérulos Renales/metabolismo , Proteínas de la Membrana/genética , Nitrorreductasas/genética , Animales , Aziridinas/metabolismo , Modelos Animales de Enfermedad , Femenino , Glomeruloesclerosis Focal y Segmentaria/inducido químicamente , Glomeruloesclerosis Focal y Segmentaria/patología , Ratones , Ratones Transgénicos , Podocitos/patología , Regiones Promotoras Genéticas/fisiologíaRESUMEN
AIMS: To investigate the adaptations of left ventricular function and calcium handling to chronic heart rate reduction with ivabradine in the reperfused heart. METHODS AND RESULTS: Rabbits underwent 20 min coronary artery occlusion followed by 3 weeks of reperfusion. Throughout reperfusion, rabbits received ivabradine (10 mg/kg/day) or vehicle (control). Ivabradine reduced heart rate by about 20% and improved both ejection fraction (+35%) and systolic displacement (+26%) after 3 weeks of treatment. Interestingly, this was associated with a two-fold increase expression of FKBP12/12.6. There was no difference in the expressions of phospholamban, SERCA2a, calsequestrin, ryanodine, phospho-ryanodine, and Na(2+)/Ca(2+) exchanger in the two groups. Infarct scar and vascular density were similar in both groups. Administration of a single intravenous bolus of ivabradine (1 mg/kg) in control rabbits at 3 weeks of reperfusion also significantly improved acutely ejection fraction and systolic displacement. CONCLUSION: Chronic heart rate reduction protects the myocardium against ventricular dysfunction induced by myocardial ischaemia followed by 3 weeks of reperfusion. Beyond pure heart rate reduction, ivabradine improves global and regional systolic function of the reperfused heart through a dual mechanism involving a direct mechanical effect and a long-term adaptation in calcium handling, as supported by the increase in FKBP12/12.6 expression.
Asunto(s)
Antiarrítmicos/farmacología , Benzazepinas/farmacología , Frecuencia Cardíaca/efectos de los fármacos , Proteína 1A de Unión a Tacrolimus/metabolismo , Animales , Western Blotting , Proteínas de Unión al Calcio/metabolismo , Oclusión Coronaria/fisiopatología , Ivabradina , Infarto del Miocardio/fisiopatología , Reperfusión Miocárdica/métodos , Conejos , Volumen Sistólico/efectos de los fármacos , Disfunción Ventricular Izquierda/tratamiento farmacológico , Disfunción Ventricular Izquierda/fisiopatologíaRESUMEN
AIMS: We investigated whether rapid cooling instituted by total liquid ventilation (TLV) improves cardiac and mitochondrial function in rabbits submitted to ischaemia-reperfusion. METHODS AND RESULTS: Rabbits were chronically instrumented with a coronary artery occluder and myocardial ultrasonic crystals for assessment of segment length-shortening. Two weeks later they were re-anaesthetized and underwent either a normothermic 30-min coronary artery occlusion (CAO) (Control group, n = 7) or a comparable CAO with cooling initiated by a 10-min hypothermic TLV and maintained by a cold blanket placed on the skin. Cooling was initiated after 5 or 15 min of CAO (Hypo-TLV and Hypo-TLV(15') groups, n = 6 and 5, respectively). A last group underwent normothermic TLV during CAO (Normo-TLV group, n = 6). Wall motion was measured in the conscious state over three days of reperfusion before infarct size evaluation and histology. Additional experiments were done for myocardial sampling in anaesthetized rabbits for mitochondrial studies. The Hypo-TLV procedure induced a rapid decrease in myocardial temperature to 32-34 degrees C. Throughout reperfusion, segment length-shortening was significantly increased in Hypo-TLV and Hypo-TLV(15') vs. Control and Normo-TLV (15.1 +/- 3.3%, 16.4 +/- 2.3%, 1.8 +/- 0.6%, and 1.1 +/- 0.8% at 72 h, respectively). Infarct sizes were also considerably attenuated in Hypo-TLV and Hypo-TLV(15') vs. Control and Normo-TLV (4 +/- 1%, 11 +/- 5%, 39 +/- 2%, and 42 +/- 5% infarction of risk zones, respectively). Mitochondrial function in myocardial samples obtained at the end of ischaemia or after 10 min of reperfusion was improved by Hypo-TLV with respect to ADP-stimulated respiration and calcium-induced opening of mitochondrial permeability transition pores (mPTP). Calcium concentration opening mPTP was, e.g., increased at the end of ischaemia in the risk zone in Hypo-TLV vs. Control (157 +/- 12 vs. 86 +/- 12 microM). Histology and electron microscopy also revealed better preservation of lungs and of cardiomyocyte ultrastructure in Hypo-TLV when compared with Control. CONCLUSION: Institution of rapid cooling by TLV during ischaemia reduces infarct size as well as other sequelae of ischaemia, such as post-ischaemic contractile and mitochondrial dysfunction.
Asunto(s)
Hipotermia Inducida , Mitocondrias Cardíacas/patología , Isquemia Miocárdica/terapia , Miocardio/patología , Daño por Reperfusión/prevención & control , Disfunción Ventricular Izquierda/prevención & control , Adenosina Difosfato/metabolismo , Animales , Ropa de Cama y Ropa Blanca , Calcio/metabolismo , Modelos Animales de Enfermedad , Hemodinámica , Hipotermia Inducida/instrumentación , Hipotermia Inducida/métodos , Ventilación Liquida , Masculino , Mitocondrias Cardíacas/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Poro de Transición de la Permeabilidad Mitocondrial , Contracción Miocárdica , Isquemia Miocárdica/complicaciones , Isquemia Miocárdica/patología , Isquemia Miocárdica/fisiopatología , Miocardio/metabolismo , Conejos , Daño por Reperfusión/etiología , Daño por Reperfusión/patología , Daño por Reperfusión/fisiopatología , Factores de Tiempo , Disfunción Ventricular Izquierda/etiología , Disfunción Ventricular Izquierda/patología , Disfunción Ventricular Izquierda/fisiopatologíaRESUMEN
The purpose of the present study was to investigate the potential cardioprotective effects of an original approach based on the properties of the X chromosome-linked Inhibitor of Apoptosis (XIAP), the most effective endogenous inhibitor of apoptosis. For this purpose, the C-terminal part of XIAP (BIR3 and RING domains) was fused to the protein transduction domain (PTD) of the HIV1 transactivator of transcription, which confers to fused protein the ability to cross cell membranes. This protein, so-called PTD-BIR3/RING, was administered intravenously in C57BL/6J mice subjected to 30 min coronary artery occlusion and 24 h of reperfusion. Administration of PTD-BIR3/RING at 5 min before and 30 min after the onset of reperfusion reduced infarct size vs control (23+/-2% vs 41+/-4% and 27+/-4% vs 41+/-3%, respectively, p<0.05). Similar reduction in infarct size was observed when PTD-BIR3/RING was administered prior to ischemia (28+/-1% vs 44+/-3%). In addition to inhibition of caspase-3 and -9 activities, PTD-BIR3/RING induced an inhibition of caspase-8 and several other actors of the apoptotic pathways. In conclusion, this study demonstrates that the administration of PTD-BIR3/RING reduces myocardial infarct size even when injected during reperfusion through interruption of caspase activation by pharmacologically mimicking endogenous XIAP.
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Imitación Molecular , Infarto del Miocardio/prevención & control , Proteínas Recombinantes de Fusión/metabolismo , Proteína Inhibidora de la Apoptosis Ligada a X/metabolismo , Animales , Western Blotting , Peso Corporal , Caspasas/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Infarto del Miocardio/enzimología , Infarto del Miocardio/patología , Estructura Terciaria de Proteína , Ratas , Proteína Inhibidora de la Apoptosis Ligada a X/químicaRESUMEN
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.
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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
OBJECTIVE: Cardiac stem cell transplantation as a potential means of regenerating infarcted myocardium is currently receiving a great deal of interest. However, data on these endogenous cardiac precursors are primarily derived from animal studies, and their clinical relevance still remains elusive. METHODS: We prospectively screened 32 endomyocardial biopsies harvested from heart transplant recipients (off rejection episodes) and 18 right appendage biopsies collected during coronary artery bypass surgery, and processed the tissue specimens for the immunohistochemical detection of markers of stemness (c-kit, MDR-1, Isl-1), hematopoietic origin (CD45), mast cells (tryptase), endothelial cells (CD105), and cardiac lineage (Nkx2.5). Confocal microscopy was used for colocalization experiments. Three right appendage biopsies were also cultured for 2 to 3 weeks, at the completion of which c-kit-positive cells were sorted by flow cytometry. RESULTS: In endomyocardial biopsies, a median number of 2.7 (1.8-4) c-kit-positive cells/mm(2) were found, and this number was even significantly smaller in right appendage biopsies (1 [0.5-1.8] c-kit-positive cell/mm(2), P = .01). All of these c-kit-positive cells co-stained for CD45 and were more specifically identified as mast cells by their positive staining for the specific tryptase marker. However, none of the c-kit-positive cells expressed the markers of stemness MDR-1 and Isl-1 or colocalized with CD105. Flow cytometry confirmed the small number of c-kit-positive cells in cultured right atrial appendages. CONCLUSION: These data raise a cautionary note on the therapeutic exploitation of cardiac stem cells in patients with ischemic cardiomyopathy, who may be the elective candidates for regenerative therapy.
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Apéndice Atrial/patología , Biopsia con Aguja/métodos , Miocitos Cardíacos/patología , Células Madre/fisiología , Adulto , Separación Celular/métodos , Células Cultivadas , Puente de Arteria Coronaria , Endocardio/patología , Femenino , Técnica del Anticuerpo Fluorescente , Trasplante de Corazón/patología , Humanos , Masculino , Microscopía Confocal , Persona de Mediana Edad , Miocardio/patología , Miocitos Cardíacos/trasplante , Probabilidad , Sensibilidad y Especificidad , Estadísticas no Paramétricas , Trasplante de Células Madre , Células Madre/ultraestructuraRESUMEN
BACKGROUND: Lumen loss in graft arteriosclerosis is the consequence of the development of a thick neointima and constrictive arterial remodeling. The latter is due to adventitial chronic inflammation and excessive perivascular collagen deposition. We reasoned that blockade of the portal of entry of inflammatory effectors may constitute a strategy to prevent constrictive arterial remodeling. METHODS AND RESULTS: We found that an anti-angiogenic therapy (ABT-510 nonapeptide), devoid of direct immunomodulatory properties, dramatically reduced adventitial angiogenesis by 66% (P<0.0001) in the rat aortic interposition model of graft arteriosclerosis. The associated decreased entry of inflammatory cells (44%; P<0.00001) resulted in drastic reduction of collagen deposition (57%; P<0.0001) thereby preventing subsequent adventitial constrictive remodeling and reduction of lumen surface area (5.26+/-0.74 vs. 8.58+/-2.48 microm2; Control vs. ABT-510-treated rats; P<0.0001). ABT-510 had no effect on the development of the neointima. CONCLUSION: This work supports the idea that targeting angiogenesis may act synergistically with conventional immunosuppressive therapy in preventing graft arteriosclerosis, a crucial feature of chronic graft rejection.
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Inhibidores de la Angiogénesis/uso terapéutico , Aorta/trasplante , Arteriosclerosis/prevención & control , Animales , Aorta/efectos de los fármacos , Aorta/patología , Aorta/ultraestructura , Colágeno/análisis , Citometría de Flujo , Prueba de Cultivo Mixto de Linfocitos , Masculino , Modelos Animales , Ratas , Ratas Endogámicas BN , Ratas Endogámicas Lew , Trasplante de Piel/patología , Trasplante de Piel/fisiología , Trasplante Homólogo , Trasplante IsogénicoRESUMEN
Sirolimus has been associated with high-range proteinuria when used in replacement of calcineurin inhibitors in renal transplant recipients with chronic allograft nephropathy (CAN). Primary FSGS was demonstrated previously in some such patients, but the coexistence of CAN lesions made the interpretation uneasy. However, nephrotic syndrome and FSGS were observed recently in three patients who received sirolimus de novo, without medical history of primary FSGS or CAN. Markers of podocyte differentiation were studied in kidney biopsies of the three patients who received sirolimus de novo and of five patients who switched to sirolimus. All patients developed FSGS lesions of classic type (not otherwise specified), but only switched patients exhibited advanced sclerotic lesions. Immunohistochemistry showed that some podocytes in FSGS lesions had absent or diminished expression of the podocyte-specific epitopes synaptopodin and p57, reflecting dedifferentiation, and had acquired expression of cytokeratin and PAX2, reflecting a immature fetal phenotype. Such a pattern of epitope expression provides evidence for podocyte dysregulation. Moreover, a decrease in vascular endothelial growth factor expression was observed in some glomeruli. In conclusion, sirolimus induces FSGS that is responsible for proteinuria in some transplant patients.
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Glomeruloesclerosis Focal y Segmentaria/inducido químicamente , Inmunosupresores/efectos adversos , Trasplante de Riñón , Complicaciones Posoperatorias/inducido químicamente , Sirolimus/efectos adversos , Adulto , Biopsia , Femenino , Glomeruloesclerosis Focal y Segmentaria/patología , Humanos , Inmunosupresores/administración & dosificación , Masculino , Persona de Mediana Edad , Sirolimus/administración & dosificaciónRESUMEN
OBJECTIVES: The present study evaluated the effect of phosphorylcholine (PC) immunization on the extent of experimental atherosclerosis. BACKGROUND: Immunization against oxidized lipoprotein (oxLDL) or Streptococcus pneumoconiae reduces atherosclerosis. Phosphorylcholine is the main epitope recognized by both antipneumococcus and anti-oxLDL antibodies. Therefore we reasoned that PC-specific antibodies might play an important role in atherogenesis. METHODS: Apolipoprotein E knockout mice were immunized with PC every second week over 4 months. At the end of the study, serum antibodies directed to either PC or oxLDL were measured. Splenic and peritoneal B cells were analyzed by flow cytometry. Aortic root atherosclerotic lesions were quantified by morphometry and phenotyped by immunohistochemistry. Immune and control sera were also tested for their effect on foam cell formation in macrophage culture in the presence of oxLDL. RESULTS: The PC-immunized mice showed 3-fold increase in titers of anti-PC and -oxLDL antibodies compared with control mice (p < 0.01). The PC-immunized mice also showed a significant increase in the number of splenic mature B cells. The extent of atherosclerotic aorta root lesions was reduced by >40% in the PC-immunized mice (p < 0.01). Immunohistochemistry showed reduced expression of major histocompatibility complex class II antigens (p < 0.05) and the presence of B-cell clusters in plaques of PC-immunized mice. Finally, PC-immune serum was able to reduce macrophage-derived foam cell formation in the presence of oxLDL in vitro. CONCLUSIONS: Phosphorylcholine immunization drives a specific humoral immune response that reduces foam cell formation in vitro and is atheroprotective in vivo.
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Anticuerpos/uso terapéutico , Aterosclerosis/inmunología , Lipoproteínas LDL/inmunología , Fosforilcolina/inmunología , Streptococcus pneumoniae/inmunología , Animales , Anticuerpos/sangre , Formación de Anticuerpos/fisiología , Aterosclerosis/fisiopatología , Aterosclerosis/terapia , Linfocitos B/fisiología , Células Cultivadas , Femenino , Hemocianinas/inmunología , Sueros Inmunes/farmacología , Inmunización Pasiva/métodos , Lipoproteínas LDL/metabolismo , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Ratones , Ratones Noqueados , Vacunación/métodosRESUMEN
Although parietal podocytes along the Bowman's capsule have been described by electron microscopy in the normal human kidney, their molecular composition remains unknown. Ten human normal kidneys that were removed for cancer were assessed for the presence and the extent of parietal podocytes along the Bowman's capsule. The expression of podocyte-specific proteins (podocalyxin, glomerular epithelial protein-1, podocin, nephrin, synaptopodin, and alpha-actinin-4), podocyte synthesized proteins (vascular endothelial growth factor and novH), transcription factors (WT1 and PAX2), cyclin-dependent kinase inhibitor p57, and intermediate filaments (cytokeratins and vimentin) was tested. In addition, six normal fetal kidneys were studied to track the ontogeny of parietal podocytes. The podocyte protein labeling detected parietal podocytes in all of the kidneys, was found in 76.6% on average of Bowman's capsule sections, and was prominent at the vascular pole. WT1 and p57 were expressed in some parietal cells, whereas PAX2 was present in all or most of them, so some parietal cells coexpressed WT1 and PAX2. Furthermore, parietal podocytes coexpressed WT1 and podocyte proteins. Cytokeratin-positive cells covered a variable part of the capsule and did not express podocyte proteins. Tuft-capsular podocyte bridges were present in 15.5 +/- 3.7% of the glomerular sections. Parietal podocytes often covered the juxtaglomerular arterioles and were present within the extraglomerular mesangium. Parietal podocytes were present in fetal kidneys. Parietal podocytes that express the same epitopes as visceral podocytes do exist along Bowman's capsule in the normal adult kidney. They are a constitutive cell type of the Bowman's capsule. Therefore, their role in physiology and pathology should be investigated.