RESUMEN
BACKGROUND: In modern biotechnology, there is a need for pausing cell lines by cold storage to adapt large-scale cell cultures to the variable demand for their products. We compared various cell culture media/solutions for cold storage of Vero-B4 kidney cells, a cell line widely used in biotechnology. RESULTS: Cold storage in RPMI 1640 medium, a recommended cell culture medium for Vero-B4 cells, surprisingly, strongly enhanced cold-induced cell injury in these cells in comparison to cold storage in Krebs-Henseleit buffer or other cell culture media (DMEM, L-15 and M199). Manufacturer, batch, medium supplements and the most likely components with concentrations outside the range of the other media/solutions (vitamin B12, inositol, biotin, p-aminobenzoic acid) did not cause this aggravation of cold-induced injury in RPMI 1640. However, a modified Krebs-Henseleit buffer with a low calcium concentration (0.42 mM), a high concentration of inorganic phosphate (5.6 mM), and glucose (11.1 mM; i.e. concentrations as in RPMI 1640) evoked a cell injury and loss of metabolic function corresponding to that observed in RPMI 1640. Deferoxamine improved cell survival and preserved metabolic function in modified Krebs-Henseleit buffer as well as in RPMI 1640. Similar Ca2+ and phosphate concentrations did not increase cold-induced cell injury in the kidney cell line LLC-PK1, porcine aortic endothelial cells or rat hepatocytes. However, more extreme conditions (Ca2+ was nominally absent and phosphate concentration raised to 25 mM as in the organ preservation solution University of Wisconsin solution) also increased cold-induced injury in rat hepatocytes and porcine aortic endothelial cells. CONCLUSION: These data suggest that the combination of low calcium and high phosphate concentrations in the presence of glucose enhances cold-induced, iron-dependent injury drastically in Vero-B4 cells, and that a tendency for this pathomechanism also exists in other cell types.
Asunto(s)
Medios de Cultivo/farmacología , Adenosina/farmacología , Alopurinol/farmacología , Animales , Calcio/química , Calcio/farmacología , Supervivencia Celular/efectos de los fármacos , Chlorocebus aethiops , Deferoxamina/farmacología , Células Endoteliales/citología , Células Endoteliales/efectos de los fármacos , Glutatión/farmacología , Hepatocitos/citología , Hepatocitos/efectos de los fármacos , Insulina/farmacología , Células LLC-PK1 , Soluciones Preservantes de Órganos/farmacología , Fosfatos/química , Fosfatos/farmacología , Rafinosa/farmacología , Ratas , Porcinos , Temperatura , Células VeroRESUMEN
OBJECTIVE: Aim of this study was to evaluate a new histidine-tryptophan-ketoglutarate (HTK)-based preservation solution on chronic isograft injury in comparison to traditional HTK solution. METHODS: Hearts of C57BL/6J (H-2b) mice were stored for 15 h in 0-4 °C cold preservation solution and then transplanted heterotopically into C57BL/6J (H-2b) mice. Three groups were evaluated: HTK, the base solution of a new preservation solution and hearts without cold ischemia (control). Time to restoration of heartbeat was measured (re-beating time). Strength of the heartbeat was palpated daily and scored on a 4-level scale (palpation score). Animals were sacrificed after 60 days of observation (24 h for TGF-ß expression). The transplanted hearts were evaluated histologically for myocardial damage, vasculopathy and interstitial fibrosis. TGF-ß expression was assessed immunohistologically. All investigators were blinded to the groups. ANOVA and LSD post hoc test were used for statistical analysis. RESULTS: The re-beating time was significantly shorter in hearts stored in the new solution (10.3±2.6 min vs. HTK 14.2±4.1 min; p<0.05). The palpation score was significantly higher in hearts stored in the new solution (2.3±0.4 vs. HTK 1.6±0.5; p<0.01). Hearts stored in the new solution showed a lower myocardial injury score (1.8±0.2 vs. HTK 2.2±0.7), less interstitial fibrosis (4.8±1.9% vs. HTK 8.5±3.8%, p<0.05), less vasculopathy (14.7±6.9% vs. 22.0±23.2%; p=0.06) and lower TGF-ß1-expression (6.6±1.4% vs. HTK 12.0±4.6%). CONCLUSION: The new HTK-based solution reduces the chronic isograft injury. This protective effect is likely achieved through several modifications and supplements into the new solution like N-acetyl-L-histidine, glycine, alanine, arginine and sucrose.
Asunto(s)
Isquemia Fría/métodos , Trasplante de Corazón/métodos , Corazón/efectos de los fármacos , Soluciones Preservantes de Órganos , Alanina/química , Animales , Fibrosis Endomiocárdica/prevención & control , Glicina/química , Corazón/fisiología , Histidina/química , Ácidos Cetoglutáricos/química , Masculino , Ratones , Ratones Endogámicos C57BL , Modelos Animales , Soluciones Preservantes de Órganos/química , Soluciones Preservantes de Órganos/farmacología , Factor de Crecimiento Transformador beta/biosíntesis , Factor de Crecimiento Transformador beta1/biosíntesis , Trasplante Heterotópico , Trasplante Isogénico , Triptófano/químicaRESUMEN
BACKGROUND: The mouse abdominal heart transplantation model is a basic and important immunological research model. We developed a technique for placing entire everting sutures instead of half inverting and half everting sutures for anastomosis between donor and recipients' caval veins. The purpose of this study was to evaluate this modified method. METHODS: Each technique was used in 25 mice subjected to isogenic abdominal heart transplantation. Recipient operation time, graft warm ischaemia time, time of caval anastomosis, and re-beating time were recorded. After transplantation, the heartbeat was palpated through the abdominal wall once a day for 100 days. RESULTS: Recipient operation time (40.7 ± 2.5 min vs. 44.3 ± 2.3 min, p < 0.01), cava-caval anastomosis time (8.4 ± 1.3 min vs. 12.1 ± 1.2 min, p < 0.01), and warm ischaemia time were significantly shorter (23.4 ± 1.7 min vs. 27.2 ± 1.6 min, p < 0.01) with the modified technique. Re-beating time was 1.2 ± 0.4 minutes with the modified technique vs. 1.5 ± 0.5 minutes (p = 0.04). There was a tendency for less surgical complications in the modified group, but there were no differences in survival rates. CONCLUSION: The new suturing technique for mouse cardiac transplantation facilitates easier anastomosis of the outflow tract, thereby reducing operation, warm ischaemia, and re-beating times.
Asunto(s)
Trasplante de Corazón/métodos , Microcirugia , Técnicas de Sutura , Anastomosis Quirúrgica/métodos , Animales , Aorta Abdominal/cirugía , Masculino , Ratones , Ratones Endogámicos C57BL , Modelos Animales , Vena Cava Inferior/cirugíaRESUMEN
Despite the introduction of arterial stiffness measurements in the European recommendation, pulse wave velocity (PWV) and augmentation index (AI) are still not used routinely in clinical practice. It would be of advantage if such measurements were done in the sitting position as is done for blood pressure. The aim of this study was to evaluate whether there is a difference in stiffness parameters in sitting vs. supine position. Arterial stiffness was measured in 24 healthy volunteers and 20 patients with cardiovascular disease using three different devices: SphygmoCor (Atcor Medical, Sydney, Australia), Arteriograph (TensioMed, Budapest, Hungary) and Vascular Explorer (Enverdis, Jena, Germany). Three measurements were performed in supine position followed by three measurements in sitting position. Methods were compared using correlation and Bland-Altman analysis. There was a significant correlation between PWV in supine and sitting position (Arteriograph: P<0.0001, r=0.93; Vascular Explorer; P<0.0001, r=0.87). There were significant correlations between AI sitting and AI supine using Arteriograph (P<0.0001, r=0.97), Vascular Explorer (P<0.0001, r=0.98) and SphygmoCor (P<0.0001, r=0.96). When analyzed by Bland-Altman, PWV and AI measurements in supine vs. sitting showed good agreement. There was no significant difference in PWV obtained with the three different devices (Arteriograph 7.5±1.6 m s(-1), Vascular Explorer 7.3±0.9 m s(-1), SphygmoCor 7.0±1.8 m s(-1)). AI was significantly higher using the Arteriograph (17.6±15.0%) than Vascular Explorer and SphygmoCor (10.2±15.1% and 10.3±18.1%, respectively). The close agreement between sitting and supine measurements suggests that both PWV and AI can be reliably measured in the sitting position.
Asunto(s)
Posicionamiento del Paciente , Postura/fisiología , Resistencia Vascular/fisiología , Adulto , Anciano , Arteria Braquial/fisiopatología , Enfermedad Coronaria/fisiopatología , Femenino , Frecuencia Cardíaca/fisiología , Humanos , Enfermedades Renales/fisiopatología , Masculino , Persona de Mediana EdadRESUMEN
AIMS: The aim of this study was to evaluate a new histidine-tryptophan-ketoglutarate (HTK)-based cold preservation solution in comparison with traditional HTK solution in a mouse cardiac transplant model and to assess the impact of chloride ions and of iron chelators. METHODS AND RESULTS: After 24 h cold ischaemia, traditional HTK-preserved hearts survived up to 13 days (4.4 ± 1.7 days; n = 8)). Hearts stored in the new solution without iron chelators (N46) showed significantly prolonged survival up to 2 months (N46: 11.9 ± 8.7 days; P < 0.01; n = 7) and with iron chelators (DesfLK) up to 3 months (N46 DesfLK: 12.7 ± 13.7 days; n = 6). Re-beating time was significantly shorter with the new solution (HTK: 14.5 ± 5.9 min, N46: 9.2 ± 2.7 min, N46 DesfLK: 7.1 ± 3.7 min; P < 0.01). The new solution showed significantly decreased release of creatine kinase (HTK: 25998 ± 8471 U/L, N46: 13829 ± 7679 U/L, N46 DesfLK: 3093 ± 597 U/L; P < 0.01 and n = 7 each) and lactate dehydrogenase (HTK: 5391 ± 1062 U/L, N46: 3428 ± 1890 U/L, P < 0.05; N46 DesfLK: 682 ± 344 U/L, P < 0.01) and decreased histological evidence of injury. A chloride-poor variant of the new solution showed inferior graft survival. CONCLUSION: The new solution markedly attenuates myocardial injury and yields better graft survival than traditional HTK solution. The presence of chloride ions is crucial for heart preservation. Some protective effects are obviously caused by iron chelators.
Asunto(s)
Criopreservación/métodos , Supervivencia de Injerto/fisiología , Corazón , Soluciones Preservantes de Órganos/farmacología , Preservación de Órganos/métodos , Animales , Apoptosis , Cloruros/análisis , Cloruros/fisiología , Isquemia Fría , F2-Isoprostanos/metabolismo , Glucosa/química , Glucosa/farmacología , Histidina/farmacología , Quelantes del Hierro/farmacología , Ácidos Cetoglutáricos , Masculino , Manitol/química , Manitol/farmacología , Ratones , Ratones Endogámicos C57BL , Miocardio/patología , Soluciones Preservantes de Órganos/química , Cloruro de Potasio/química , Cloruro de Potasio/farmacología , Procaína/química , Procaína/farmacología , Triptófano/farmacologíaRESUMEN
BACKGROUND: Cardiovascular disease is one of the major causes of mortality and morbidity in patients with end-stage renal disease (ESRD). It is characterized by multiple left ventricular abnormalities, referred to as 'uraemic cardiomyopathy'. The aim of the study was to investigate uraemic cardiac disease in a mouse model of chronic renal failure induced by subtotal nephrectomy and to evaluate the impact of the tyrosine kinase inhibitor imatinib and its antifibrotic as well as functional properties on the extent of the disease. METHODS: Male BALB/c mice were sham operated (SH) or subtotally nephrectomized and either left untreated (5/6) or treated with imatinib (5/6+I: 10 mg/kg/day p.o.) for up to 24 weeks. Cardiac and arterial structure and function were analysed using echocardiography, histology, extent of lipid peroxidation and myography, respectively. RESULTS: Subtotal nephrectomy resulted in cardiac dysfunction characterized by reduced fractional shortening (SH: 21.6 +/- 4.7%; 5/6: 11.1 +/- 2.4%; 5/6+I: 8.4 +/- 2.7%; P < 0.05) and ejection fraction (SH: 38.8 +/- 4.5%; 5/6: 26.1 +/- 2.8%; 5/6+I: 18.6 +/- 2.6%; P < 0.05) after 24 weeks. This was associated with impaired endothelium-dependent vasodilatation in mesenteric resistance vessels and elevated cardiac malondialdehyde concentrations as a marker of lipid peroxidation. In this model, the continuous application of the tyrosine kinase inhibitor imatinib was associated with less myocardial fibrosis (SH: 2.52 +/- 0.34%; 5/6: 5.50 +/- 0.18%; 5/6+I: 3.52 +/- 0.52%; P < 0.05), but did not preserve myocardial function. CONCLUSIONS: Uraemic cardiac disease in BALB/c results in fibrosis, oxidative damage and endothelial dysfunction. However, the anti-fibrotic activity of imatinib did not ameliorate cardiac dysfunction. Thus, our data suggest that uraemic cardiac disease in this mouse model is driven by oxidative damage and endothelial dysfunction.
Asunto(s)
Enfermedades Cardiovasculares/tratamiento farmacológico , Piperazinas/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Pirimidinas/farmacología , Uremia/tratamiento farmacológico , Animales , Benzamidas , Presión Sanguínea/efectos de los fármacos , Enfermedades Cardiovasculares/etiología , Enfermedades Cardiovasculares/patología , Enfermedades Cardiovasculares/fisiopatología , Modelos Animales de Enfermedad , Endotelio Vascular/fisiopatología , Fibrosis , Mesilato de Imatinib , Fallo Renal Crónico/complicaciones , Fallo Renal Crónico/tratamiento farmacológico , Fallo Renal Crónico/patología , Fallo Renal Crónico/fisiopatología , Masculino , Ratones , Ratones Endogámicos BALB C , Nefrectomía , Estrés Oxidativo , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Uremia/complicaciones , Uremia/patología , Uremia/fisiopatologíaRESUMEN
PURPOSE: The purpose of this study was to analyse the outcome and its influencing factors in patients whose therapy was converted from calcineurin inhibitors (CNI) to sirolimus (SRL) due to chronic allograft nephropathy (CAN). MATERIALS AND METHODS: Therapies of 78 patients (44 men) with CAN from three European transplant centres were converted from CNI therapy to SRL and followed 24 months. Slopes for creatinine clearance before and after conversion were calculated. Influencing factors were analysed by a multivariance analysis. RESULTS: The slope of the creatinine clearance improved significantly (-0.90 vs. -0.34 ml min(-1) month(-1); p < 0.01). In patients whose therapy was converted from cyclosporine A (CyA) to SRL, the slope improved significantly, whereas conversion from Tacrolimus (Tac) to SRL did not affect the slope. The benefit was more pronounced in (1) patients with low or moderate baseline creatinine clearance, (2) patients receiving SRL after conversion without additional mycophenolate mofetil and (3) patients with low or moderate proteinuria. CONCLUSION: Conversion from CyA to SRL but not from Tac to CRL is associated with a reduced loss of renal allograft function in patients with CAN.
Asunto(s)
Inhibidores de la Calcineurina , Ciclosporina/efectos adversos , Inmunosupresores/uso terapéutico , Enfermedades Renales/terapia , Trasplante de Riñón/efectos adversos , Sirolimus/uso terapéutico , Adulto , Estudios de Cohortes , Femenino , Humanos , Enfermedades Renales/diagnóstico , Enfermedades Renales/etiología , Masculino , Persona de Mediana Edad , Estudios Retrospectivos , Factores de Riesgo , Resultado del TratamientoRESUMEN
BACKGROUND: Serum creatinine (SCR) and blood urea nitrogen (BUN) determine the glomerular filtration rate (GFR) improperly in acute renal failure. Serum cystatin C (CYS) has the potential to be a more precise marker for GFR. The aim of this study was to compare the sensitivity of SCR, BUN and CYS with respect to the detection of acute renal failure in mice. METHODS: In an ischaemia reperfusion (I/R) injury model, mice suffered 60-min left kidney ischaemia and right nephrectomy. In a nephrectomy model, mice were nephrectomized to a different extent: from unilateral (3/6Nx) to bilateral nephrectomy (BiNx). Blood samples were collected 2, 12 or 24 h post-op. RESULTS: SCR, BUN and CYS increased significantly in the I/R-model in comparison to sham mice and 3/6Nx mice at 12 and 24 h post-op (SCR P = 0.009; BUN P < 0.001 and CYS P < 0.004). There were no significant differences in all three markers between 3/6Nx and sham-operated mice. In graded nephrectomy, BUN and CYS showed already significantly the loss of kidney in 4/6Nx mice 12 h post-op [BUN (mg/dl): sham 26.4 +/- 3.5, 4/6Nx 52.3 +/- 13.4, P < 0.01; CYS (mg/l): sham 0.08 +/- 0.03, 4/6Nx 0.15 +/- 0.04, P < 0.01], whereas SCR was only significantly increased in 5/6Nx and BiNx mice 24 h post-op [SCR (mg/dl): sham 0.39 +/- 0.05, 4/6Nx 0.52 +/- 0.07, P = 0.13, 5/6Nx 1.00 +/- 0.29, P < 0.01]. In the longitudinal experiment, CYS showed the renal damage significantly earlier and to a larger extent (2 h: SCR 57 +/- 15%, BUN 40 +/- 16%, CYS 295 +/- 143%, P <0.001). CONCLUSIONS: CYS can be used as a reliable and precise marker for renal function in mouse models. CYS is more sensitive than SCR, and it shows renal damage earlier than SCR and BUN.
Asunto(s)
Lesión Renal Aguda/sangre , Lesión Renal Aguda/diagnóstico , Cistatina C/sangre , Daño por Reperfusión/sangre , Lesión Renal Aguda/etiología , Animales , Biomarcadores/sangre , Nitrógeno de la Urea Sanguínea , Creatinina/sangre , Modelos Animales de Enfermedad , Masculino , Ratones , Ratones Endogámicos BALB C , Daño por Reperfusión/complicacionesRESUMEN
BACKGROUND: Ezetimibe has shown efficacy in the therapy of hypercholesterolemia in renal transplant patients. This is the first study investigating the effect of ezetimibe on renal function in kidney transplant recipients. METHODS: Fifty-six patients with statin-resistant hypercholesterolemia (total cholesterol >200 mg/dl) after renal transplantation received additional ezetimibe therapy (10 mg/day) for 12 months. A group receiving statin therapy (n=28) served as controls in this prospective study. RESULTS: Total cholesterol and LDL cholesterol concentrations decreased significantly in the ezetimibe-treated patients but remained stable in the control group (delta total cholesterol: -24+/-49 mg/dl vs 19+/-49 mg/dl, P<0.01; delta LDL: -30+/-39 mg/dl vs -3+/-31 mg/dl, P<0.01). Mean creatinine clearance remained stable in ezetimibe-treated patients but decreased significantly in control group (delta Cockcroft-Gault: 0.9+/-7.3 ml/min vs - 4.8+/-12.8 ml/min, P=0.025; delta Modification of Diet in Renal Disease: -0.4+/-6.2 ml/min/1.73 m(2) vs 4.7+/-8.8 ml/min/1.73 m(2), P=0.033). CONCLUSIONS: The data of our prospective case-control study suggest that ezetimibe appears to ameliorate the decline of renal function after renal transplantation.