RESUMEN
Simultaneous pancreas kidney transplantation (SPKT) is the treatment of choice for patients with type 1 diabetes and end-stage renal disease. Rapamycin and mycophenolate mofetil (MMF) have been used for maintenance immunosuppression with tacrolimus in SPKT; however, long-term outcomes are lacking. From September 2000 through December 2009, 170 SPKT recipients were enrolled in a randomized, prospective trial receiving Rapamycin (n = 84) or MMF (n = 86). All patients received dual induction therapy with thymoglobulin and daclizumab, and low-dose maintenance tacrolimus and corticosteroids. Compared to MMF, rates of freedom from first biopsy-proven acute kidney or pancreas rejection were superior for Rapamycin at year 1 (kidney: 100% vs. 88%; P = 0.001; pancreas: 99% vs. 92%; P = 0.04) and at year 10 (kidney: 88% vs. 71%, P = 0.01; pancreas: 99% vs. 89%, P = 0.01). The higher rates of rejection were associated with withholding MMF (vs. Rapamycin, p = 0.009), generally for gastrointestinal or bone marrow toxicity. There was no significant difference in creatinine, proteinuria, c-peptide, viral infections, lymphoproliferative disorders or posttransplant diabetes. HbA1C and lipid levels were normal in both groups, although higher in the Rapamycin arm. There were no significant differences in patient or allograft survival. In this 10-year SPKT study, Rapamycin in combination with tacrolimus was better tolerated and more effective than MMF. Overall, the patient and allograft survival were equivalent.
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Rechazo de Injerto/tratamiento farmacológico , Inmunosupresores/uso terapéutico , Trasplante de Riñón , Ácido Micofenólico/análogos & derivados , Trasplante de Páncreas , Sirolimus/uso terapéutico , Tacrolimus/uso terapéutico , Adolescente , Adulto , Niño , Preescolar , Femenino , Supervivencia de Injerto/efectos de los fármacos , Humanos , Lactante , Masculino , Persona de Mediana Edad , Ácido Micofenólico/uso terapéutico , Pronóstico , Estudios Prospectivos , Factores de Tiempo , Trasplante Homólogo , Adulto JovenRESUMEN
Sphingomyelin phosphodiesterase acid-like 3b (SMPDL3b) is a lipid raft enzyme that regulates plasma membrane (PM) fluidity. Here we report that SMPDL3b excess, as observed in podocytes in diabetic kidney disease (DKD), impairs insulin receptor isoform B-dependent pro-survival insulin signaling by interfering with insulin receptor isoforms binding to caveolin-1 in the PM. SMPDL3b excess affects the production of active sphingolipids resulting in decreased ceramide-1-phosphate (C1P) content as observed in human podocytes in vitro and in kidney cortexes of diabetic db/db mice in vivo. Podocyte-specific Smpdl3b deficiency in db/db mice is sufficient to restore kidney cortex C1P content and to protect from DKD. Exogenous administration of C1P restores IR signaling in vitro and prevents established DKD progression in vivo. Taken together, we identify SMPDL3b as a modulator of insulin signaling and demonstrate that supplementation with exogenous C1P may represent a lipid therapeutic strategy to treat diabetic complications such as DKD.
Asunto(s)
Antígenos CD/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 3/metabolismo , Nefropatías Diabéticas/patología , Insulina/metabolismo , Receptor de Insulina/metabolismo , Esfingomielina Fosfodiesterasa/metabolismo , Animales , Caveolina 1/metabolismo , Línea Celular , Membrana Celular/metabolismo , Ceramidas/metabolismo , Ceramidas/uso terapéutico , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 3/genética , Nefropatías Diabéticas/tratamiento farmacológico , Modelos Animales de Enfermedad , Femenino , Humanos , Masculino , Ratones , Ratones Noqueados , Podocitos/citología , Podocitos/metabolismo , Isoformas de Proteínas/metabolismo , Transducción de Señal , Resultado del TratamientoRESUMEN
AIMS/HYPOTHESIS: Inhibition of c-jun N-terminal kinase (JNK) favours pancreatic islet function and survival. Since two JNK isoforms are present in the pancreas (JNK1 and JNK2), we addressed their specific roles in experimental islet transplantation. METHODS: C57BL/6J (wild-type [WT]), Jnk1 (also known as Mapk8)(-/-) and Jnk2 (also known as Mapk9)(-/-) mice were used as donor/recipients in a syngeneic islet transplantation model. Islet cell composition, function, viability, production of cytokines and of vascular endothelial growth factor (VEGF) were also studied in vitro. RESULTS: Jnk1 ( -/- ) islets secreted more insulin in response to glucose and were more resistant to cytokine-induced cell death compared with WT and Jnk2 (-/-) islets (p < 0.01). Cytokines reduced VEGF production in WT and Jnk2 (-/-) but not Jnk1 ( -/- ) islets; VEGF blockade restored Jnk1 ( -/- ) islet susceptibility to cytokine-induced cell death. Transplantation of Jnk1 ( -/- ) or WT islets into WT recipients made diabetic had similar outcomes. However, Jnk1 ( -/- ) recipients of WT islets had shorter time to diabetes reversal (17 vs 55 days in WT, p = 0.033), while none of the Jnk2 (-/-) recipients had diabetes reversal (0% vs 71% in WT, p = 0.0003). Co-culture of WT islets with macrophages from each strain revealed a discordant cytokine production. CONCLUSIONS/INTERPRETATION: We have shown a deleterious effect of JNK2 deficiency on islet graft outcome, most likely related to JNK1 activation, suggesting that specific JNK1 blockade may be superior to general JNK inhibition, particularly when administered to transplant recipients.
Asunto(s)
Islotes Pancreáticos/citología , Islotes Pancreáticos/enzimología , Proteína Quinasa 8 Activada por Mitógenos/metabolismo , Animales , Supervivencia Celular/efectos de los fármacos , Técnicas de Cocultivo , Citocinas/farmacología , Diabetes Mellitus Experimental/enzimología , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/patología , Diabetes Mellitus Experimental/cirugía , Supervivencia de Injerto , Islotes Pancreáticos/cirugía , Trasplante de Islotes Pancreáticos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteína Quinasa 8 Activada por Mitógenos/deficiencia , Proteína Quinasa 8 Activada por Mitógenos/genética , Proteína Quinasa 9 Activada por Mitógenos/deficiencia , Proteína Quinasa 9 Activada por Mitógenos/genética , Proteína Quinasa 9 Activada por Mitógenos/metabolismo , FosforilaciónRESUMEN
Loss of podocytes by apoptosis characterizes the early stages of diabetic nephropathy. To examine its mechanism we studied glomeruli and podocytes isolated from db/db mice with early diabetic nephropathy and albuminuria. Phosphorylation of AKT (protein kinase B, a key survival protein) was found to be lower in the glomeruli of 12 week old db/db compared to db/+ mice. In vitro, insulin phosphorylated AKT solely in podocytes from db/+ mice. Serum deprivation and exposure to tumor necrosis factor-alpha significantly compromised cell viability in podocytes from db/db but not from db/+ mice, and this was associated with a significant decrease in AKT phosphorylation. Inhibition of AKT was necessary to achieve the same degree of cell death in db/+ podocytes. Our study shows that podocyte inability to respond to insulin and susceptibility to cell death may partially account for the decreased podocyte number seen in early diabetic nephropathy.
Asunto(s)
Nefropatías Diabéticas/enzimología , Nefropatías Diabéticas/patología , Podocitos/enzimología , Podocitos/patología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Albuminuria/enzimología , Albuminuria/patología , Animales , Apoptosis , Recuento de Células , Regulación hacia Abajo , Insulina/farmacología , Glomérulos Renales/enzimología , Glomérulos Renales/patología , Ratones , Ratones Endogámicos , Fosforilación/efectos de los fármacos , Podocitos/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
INTRODUCTION: Many cytoprotective agents have been reported to improve islet isolation and transplantation outcomes. However, several of these agents improve all cell subsets within an islet preparation; selection of non-beta-cell components (eg, acinar cells) may have a negative effect on beta-cell function and survival. In this study, we examined the effect of prolactin (PRL) supplementation in the culture medium to determine whether it exerted beta-cell-selective cytoprotection on islet viability and function. MATERIALS AND METHODS: Human islets were precultured with or without recombinant human PRL (500 microg/L) for 48 hours. The fractional viability and cellular composition of non-beta-cell and beta-cell-specific components were assessed using FACS and Laser Scanning Cytometry (LSC). Islet potency was assessed in vivo by transplantation into chemically induced diabetic immunodeficient mice. RESULTS: The relative viable beta-cell mass and the relative islet beta-cell content in the PRL group were 28% higher (P = .018) and 19% higher (P = .029) than the control group, respectively. All transplanted mice achieved normoglycemia in both groups, indicating that PRL treatment did not alter islet function. CONCLUSION: PRL treatment improved beta-cell-specific viability and survival of human islets in vitro. The development of novel beta-cell-specific cytoprotective strategies may be of assistance in improving islet transplantation.
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Supervivencia Celular/efectos de los fármacos , Diabetes Mellitus Experimental/cirugía , Células Secretoras de Insulina/citología , Prolactina/farmacología , Animales , Técnicas de Cultivo de Célula , Modelos Animales de Enfermedad , Humanos , Células Secretoras de Insulina/efectos de los fármacos , Células Secretoras de Insulina/trasplante , Trasplante de Islotes Pancreáticos , Ratones , Ratones DesnudosRESUMEN
We found that ROP Os/+ (Os/+) mice had diffuse glomerulosclerosis and glomerular hypertrophy and that their mesangial cells (the vascular smooth muscle cells of the glomerulus) displayed an apparent sclerosing phenotype. Since mesangial cells are the major source of scar tissue in glomerulosclerosis, we postulated that the sclerosis phenotype was carried by mesangial cell progenitors and that this phenotype could be derived from the bone marrow (BM). Therefore, we transplanted BM from Os/+ mice into congenic ROP +/+ mice (+/+ mice), which have normal glomeruli. We found that glomeruli of +/+ recipients of Os/+ marrow contained the Os/+ genotype, were hypertrophied, and contained increased extracellular matrix. Clones of recipient glomerular mesangial cells with the donor genotype were found in all +/+ recipients that developed mesangial sclerosis and glomerular hypertrophy, whereas +/+ recipients of +/+ BM had normal glomeruli. Thus, the sclerotic (Os/+) or normal (+/+) genotype and phenotype were present in, and transmitted by, BM-derived progenitors. These data show that glomerular mesangial cell progenitors are derived from the BM and can deliver a disease phenotype to normal glomeruli. Glomerular lesions may therefore be perpetuated or aggravated, rather than resolved, by newly arriving progenitor cells exhibiting a disease phenotype.
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Trasplante de Médula Ósea , Mesangio Glomerular/citología , Trasplante de Células Madre Hematopoyéticas , Glomérulos Renales/patología , Animales , Femenino , Genotipo , Hematopoyesis , Hipertrofia , Tolerancia Inmunológica , Metaloproteinasa 2 de la Matriz/genética , Ratones , Músculo Liso Vascular/citología , EsclerosisRESUMEN
Systemic lupus erythematosus, which predominantly affects young women, frequently is complicated by renal involvement. The presence of acute glomerulonephritis significantly adds to morbidity and mortality. Based on currently published clinical trials, induction therapy with cyclophosphamide combined with pulse corticosteroids is an efficacious treatment option to preserve renal function, and long-term data are available to support this choice. Mycophenolate mofetil is a promising new agent, and recent data suggest that it is at least as efficacious as cyclophosphamide in the induction and maintenance phase, but with fewer side effects. Cell-depleting agents may be added in patients who fail the traditional regimens, preferentially in the setting of one of the ongoing clinical trials. The number of treatment regimens that the clinician can choose from when confronted with a patient with severe lupus nephritis has increased significantly, and more options are on the horizon. This promises more efficacious and better tolerable therapies, but it also puts an additional obligation on the physician to consider risk-to-benefit ratio, patient preference and adherence, feasibility, and cost; and to engage the patient in an active discussion about the different alternatives.
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Nefritis Lúpica/tratamiento farmacológico , Ciclofosfamida/uso terapéutico , Evaluación Preclínica de Medicamentos , Humanos , Índice de Severidad de la EnfermedadRESUMEN
BACKGROUND: Chronic nephrotoxicity is an important adverse effect of cyclosporine A (CsA) therapy. Tubulo-interstitial lesions and arteriolopathy are common histologic findings. Glomerular lesions are also described, but they are of variable severity. The aim of our study is to determine whether CsA has a direct effect on mesangial cells and whether the cellular response depends on the genetic background. METHODS: We studied mesangial cells isolated from mice susceptible (ROP/Le-+Es1(b)+Es1(a), ROP) and resistant to glomerulosclerosis (B6SJLF1, C57). We previously showed that sclerosis-prone and sclerosis-resistant phenotypes are maintained in vitro. We examined whether CsA exposure directly affected extracellular matrix turnover in mesangial cells and whether the response is determined by the genetic background. Extracellular matrix synthesis and degradation were studied by proline incorporation, ELISA, reverse transcription-polymerase chain reaction, zymography, and reverse zymography. We chose a CsA dose that induced neither cytotoxicity nor apoptosis (1 microg/ml). RESULTS: At the dose of 1 microg/ml total collagen accumulation was increased in ROP but not in C57 cells. Matrix metalloproteinase (MMP)-2 activity and mRNA levels were selectively decreased in ROP cells. CsA exposure did not affect tissue inhibitors of MMP (TIMP)-1 and -2 activity or TGF-beta1 mRNA expression and protein synthesis in either cell line. CONCLUSION: CsA increases total collagen accumulation in mesangial cells from sclerosis-prone mice by decreasing MMP-2 activity, but does not affect cells from sclerosis-resistant mice. Thus, CsA directly affects mesangial cells, but only those with a permissive genetic background for glomerulosclerosis.
Asunto(s)
Ciclosporina/farmacología , Matriz Extracelular/fisiología , Mesangio Glomerular/fisiología , Glomerulonefritis/genética , Metaloproteinasa 2 de la Matriz/metabolismo , Inhibidor Tisular de Metaloproteinasa-1/metabolismo , Inhibidor Tisular de Metaloproteinasa-2/metabolismo , Animales , Apoptosis , Colágeno/genética , Susceptibilidad a Enfermedades , Matriz Extracelular/efectos de los fármacos , Mesangio Glomerular/citología , Mesangio Glomerular/efectos de los fármacos , Inmunidad Innata , Metaloproteinasa 2 de la Matriz/genética , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Especificidad de la Especie , Inhibidor Tisular de Metaloproteinasa-1/genética , Inhibidor Tisular de Metaloproteinasa-2/genética , Transcripción Genética , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
We used a rat model of pancreas cold preservation to assess its effects on islets. Glands were surgically retrieved and stored in University of Wisconsin (UW) solution for 3 hours (Short) or 18 hours (Long) cold ischemia time (CIT). Islet yield was significantly lower in the Long-CIT than the Short-CIT group, as well as islet recovery after overnight culture (P < .01). Islet cell viability after isolation was significantly reduced in the Long-CIT group (P < .05). Reversal of diabetes following transplantation of suboptimal islet grafts occurred earlier in the Short-CIT group than the Long-CIT. All animals in the Short-CIT group and 80% in the Long-CIT group achieved euglycemia. Freshly isolated islets showed a significant increase of JNK and p38 (P < .05) phosphorylation in Long-CIT compared with Short-CIT. Histopathological assessment of the pancreas showed a significantly higher injury score. Proteomic analysis of pancreatic tissue led to identification of 5 proteins consistently differentially expressed between Short-CIT and Long-CIT. Better understanding of the molecular pathways involved in this phenomenon will be of assistance in defining targeted interventions to improve organ use in the clinical arena.
Asunto(s)
Islotes Pancreáticos/citología , Páncreas/citología , Adenosina , Alopurinol , Animales , Supervivencia Celular , Glutatión , Insulina , Isquemia , Islotes Pancreáticos/patología , Masculino , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Soluciones Preservantes de Órganos , Páncreas/irrigación sanguínea , Páncreas/patología , Fosfotransferasas/metabolismo , Rafinosa , Ratas , Ratas Endogámicas Lew , Recolección de Tejidos y Órganos/métodosRESUMEN
AIMS/HYPOTHESIS: Activation of c-jun N-terminal kinase (JNK) has been described in islet isolation and engraftment, making JNK a key target in islet transplantation. The objective of this study was to investigate if JNK inhibition with a cell-permeable TAT peptide inhibitor (L-JNKI) protects functional beta cell mass in human islets and affects AKT and its substrates in islet cells. METHODS: The effect of L-JNKI (10 micromol/l) on islet count, mitochondrial membrane potential, glucose-stimulated insulin release and phosphorylation of both AKT and its substrates, as well as on reversal of diabetes in immunodeficient diabetic Nu/Nu mice was studied. RESULTS: In vitro, L-JNKI reduced the islet loss in culture and protected from cell death caused by acute cytokine exposure. In vivo, treatment of freshly isolated human islets and diabetic Nu/Nu mice recipients of such islets resulted in improved functional beta cell mass. We showed that L-JNKI activates AKT and downregulates glycogen synthase kinase-3 beta (GSK-3B) in human islets exposed to cytokines, while other AKT substrates were unaffected, suggesting that a specific AKT/GSK-3B regulation by L-JNKI may represent one of its mechanisms of cytoprotection. CONCLUSIONS/INTERPRETATION: In conclusion, we have demonstrated that targeting JNK in human pancreatic islets results in improved functional beta cell mass and in the regulation of AKT/GSK3B activity.
Asunto(s)
Glucógeno Sintasa Quinasa 3/metabolismo , Células Secretoras de Insulina/efectos de los fármacos , Proteínas Quinasas JNK Activadas por Mitógenos/antagonistas & inhibidores , Péptidos/farmacología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Animales , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Terapia Combinada , Citocinas/farmacología , Diabetes Mellitus Experimental/tratamiento farmacológico , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/cirugía , Humanos , Células Secretoras de Insulina/citología , Células Secretoras de Insulina/metabolismo , Islotes Pancreáticos/citología , Islotes Pancreáticos/efectos de los fármacos , Islotes Pancreáticos/metabolismo , Trasplante de Islotes Pancreáticos/métodos , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Ratones , Ratones Desnudos , Péptidos/uso terapéutico , Fosforilación/efectos de los fármacos , Trasplante Heterólogo , Resultado del TratamientoRESUMEN
Poor outcomes have been reported in African Americans and Hispanics compared to Caucasians with lupus nephritis. The purpose of this retrospective analysis was to identify independent predictors of outcomes in African Americans and Hispanics with lupus nephritis. In total, 93 African Americans, 100 Hispanics, and 20 Caucasians with a mean age of 28 +/- 13 years and an annual household income of 32.9 +/- 17.3 (in 1000 US dollars) were studied. World Health Organization (WHO) lupus nephritis classes II, III, IV, and V were seen in 9, 13, 52, and 26%, respectively. Important baseline differences were higher mean arterial pressure (MAP) in African Americans compared to Hispanics and Caucasians (107 +/- 19, 102 +/- 15, and 99 +/- 13 mmHg, P < 0.05), and higher serum creatinine (1.66 +/- 1.3, 1.25 +/- 1.0, and 1.31 +/- 1.0 mg/dl, P < 0.025). African Americans had lower hematocrit compared to Hispanics and Caucasians (29 +/- 5, and 31 +/- 6, and 32 +/- 7%, P < 0.05), and lower annual household income (30.8 +/- 14.9, 33.1 +/- 15.9, and 42.2 +/- 29.3 in 1000 US dollars; P < 0.05). Lower prevalence of WHO class IV was seen in Caucasians (30%) compared to Hispanics (57%, P = 0.03) and African Americans (51%, P = 0.09). Development of doubling creatinine or end-stage renal disease was higher in African Americans and Hispanics than in Caucasians (31, 18, and 10%; P < 0.05), as was the development of renal events or death (34, 20, and 10%; P < 0.025). Our results suggest that both biological factors indicating an aggressive disease and low household income are common in African Americans and Hispanics with lupus nephritis, and outcomes in these groups are worse than in Caucasians.
Asunto(s)
Fallo Renal Crónico/mortalidad , Nefritis Lúpica/complicaciones , Nefritis Lúpica/etnología , Nefritis Lúpica/mortalidad , Adolescente , Adulto , Negro o Afroamericano/estadística & datos numéricos , Presión Sanguínea , Estudios de Casos y Controles , Estudios de Cohortes , Creatinina/sangre , Femenino , Hispánicos o Latinos/estadística & datos numéricos , Humanos , Inmunosupresores/uso terapéutico , Renta , Fallo Renal Crónico/etnología , Fallo Renal Crónico/patología , Nefritis Lúpica/clasificación , Nefritis Lúpica/diagnóstico , Nefritis Lúpica/epidemiología , Nefritis Lúpica/patología , Nefritis Lúpica/terapia , Masculino , Prevalencia , Estudios Retrospectivos , Análisis de Supervivencia , Población Blanca/estadística & datos numéricosRESUMEN
Cyclosporin A (CsA) nephropathy is associated with altered expression of apoptosis regulatory genes such as Fas-ligand and Bcl-2 family members in the glomerular, tubulointerstitial, and vascular compartments. Both hepatocyte growth factor (HGF) and insulin-like growth factor (IGF-I) protect against apoptosis, and HGF specifically up-regulates Bcl-xL, a protein that regulates apoptosis. We investigated whether Bcl-xL and Fas/Fas-ligand were regulated by CsA in cultured podocytes and whether CsA-induced apoptosis was prevented by HGF or IGF-I. A murine podocyte cell line was treated with CsA in the presence or absence of HGF or IGF-I. Apoptosis was quantitated by ELISA and by flow cytometry; Bcl-xL, Fas, and Fas-ligand were measured by Western blotting. Inhibitors of MAP kinase/ERK kinase (MEK)-1 and of phosphatidylinositol 3'-kinase (PI3'-K) were used to determine the signaling pathways involved in Bcl-xL regulation. Apoptosis was induced by CsA in a dose- and time-dependent fashion. CsA also decreased Bcl-xL levels. HGF, but not IGF-I, prevented apoptosis and restored Bcl-xL levels. The regulation of Bcl-xL by HGF was mediated by the PI3'-K but not by the MEK-1 pathway. In summary, we showed that CsA induces apoptosis in podocytes. Apoptosis was prevented by pretreatment with HGF but not IGF-I. Decreased apoptosis appeared to be mediated by regulation of Bcl-xL via the PI3'-K pathway. Our data suggest that the effect of CsA on podocytes may contribute to the glomerular damage and that HGF could provide protection.
Asunto(s)
Apoptosis/efectos de los fármacos , Ciclosporina/farmacología , Factor de Crecimiento de Hepatocito/farmacología , Factor I del Crecimiento Similar a la Insulina/farmacología , Glomérulos Renales/efectos de los fármacos , Animales , Células Cultivadas , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Proteína Ligando Fas , Femenino , Flavonoides/farmacología , Glomérulos Renales/citología , Glomérulos Renales/metabolismo , MAP Quinasa Quinasa 1 , Glicoproteínas de Membrana/efectos de los fármacos , Glicoproteínas de Membrana/metabolismo , Ratones , Quinasas de Proteína Quinasa Activadas por Mitógenos/antagonistas & inhibidores , Quinasas de Proteína Quinasa Activadas por Mitógenos/fisiología , Fosfatidilinositol 3-Quinasas/fisiología , Inhibidores de las Quinasa Fosfoinosítidos-3 , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/fisiología , Proteínas Proto-Oncogénicas c-bcl-2/efectos de los fármacos , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Factores de Tiempo , Proteína bcl-X , Receptor fas/efectos de los fármacos , Receptor fas/metabolismoRESUMEN
Hepatocyte growth factor (HGF) is a potent mitogen for tubular cells. Experimental injury to the kidney is associated with HGF release both locally and by distant organs stimulated by circulating 'injurins'. In this study, the serum HGF concentration was measured in patients with acute renal failure (ARF). Normal subjects and chronic renal failure patients either not on dialysis or on regular dialysis treatment served as controls. Human mesangial cells were incubated with sera from ARF patients and controls. The serum HGF concentration was strikingly increased in ARF patients (478 +/- 68 ng/dl) and was normal in chronic renal failure patients not on dialysis (20 +/- 3 ng/dl) and in those on regular dialysis treatment (25 +/- 3 ng/dl). Serum of ARF patients strongly stimulated HGF release from mesangial cells (1,384 +/- 55 ng/ml) in comparison with normal serum (67 +/- 10 ng/ml). These results indicate that in ARF HGF participates in tubular repair both as an endocrine factor, released in the circulation, and as a paracrine substance, diffusing to the tubules from the mesangium.
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Lesión Renal Aguda/sangre , Factor de Crecimiento de Hepatocito/sangre , Fallo Renal Crónico/sangre , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores/sangre , Células Cultivadas , Creatinina/sangre , Medios de Cultivo Condicionados , Femenino , Mesangio Glomerular/citología , Mesangio Glomerular/metabolismo , Factor de Crecimiento de Hepatocito/metabolismo , Humanos , Masculino , Persona de Mediana Edad , Valores de ReferenciaRESUMEN
BACKGROUND: Immunosuppressant therapy is thought to be a major contributor to post-transplant bone disease. Histological data and serum parameters suggest that Cyclosporin A (CsA) treatment causes osteopenia as a result of an altered bone turnover, but the pathogenic mechanisms of this process remain unclear. We investigate if CsA affects cell turnover and extracellular matrix (ECM) synthesis and degradation in MC3T3-E1 osteoblasts, as a surrogate model for in vivo events. METHODS: Cells were exposed to increasing doses of CsA (0, 0.5, 1 and 5 microg/ml). Proliferation was evaluated by bromodeoxyuridine (BrdU) incorporation, viability by Trypan Blue exclusion and apoptosis by ELISA. Type I collagen was measured by ELISA and reverse transcription-polymerase chain reaction (RT-PCR), matrix metalloproteinases (MMP) by zymography and RT-PCR, and tissue inhibitors of MMP (TIMP) by reverse zymography. RESULTS: CsA exposure for 48 h decreased osteoblast number in a dose-dependent manner in the absence of apoptosis or cytotoxicity. CsA at a dose of 5 microg/ml for 72 h caused decreased collagen type I mRNA expression and protein accumulation. While MMP-2 remained unaffected, MMP-9 activity increased. TIMP-1 activity was unaffected, while a dose-dependent increase of TIMP-2 was observed. CONCLUSIONS: These data suggest that CsA alters ECM synthesis and degradation in MC3T3-E1 osteoblasts by decreasing type I collagen production and increasing MMP-9 activity. The combination of increased MMP-9 with unchanged TIMP-1 activity could reduce the osteoid matrix available for mineralization. In addition, decreased proliferation could further reduce the number of cells synthesizing new osteoid matrix and thus contribute to the process of bone loss.
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Ciclosporina/farmacología , Matriz Extracelular/metabolismo , Inmunosupresores/farmacología , Osteoblastos/metabolismo , Fosfatasa Alcalina/antagonistas & inhibidores , Animales , Muerte Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Línea Celular , Colágeno/antagonistas & inhibidores , Colágeno/metabolismo , Relación Dosis-Respuesta a Droga , Metaloproteinasa 9 de la Matriz/genética , Metaloproteinasa 9 de la Matriz/metabolismo , Ratones , Osteoblastos/citología , ARN Mensajero/metabolismo , Inhibidor Tisular de Metaloproteinasa-2/genética , Inhibidor Tisular de Metaloproteinasa-2/metabolismoRESUMEN
BACKGROUND: Nephrotoxicity, accelerated atherosclerosis, and graft vascular disease are common complications of cyclosporine long-term treatment characterized by a wide disruption of organ architecture with increased interstitial areas and accumulation of extracellular matrix (ECM). How cyclosporine induces these changes is not clear, but it is conceivable that they are the sum of changes induced at the cell level. METHODS: We studied the effects of cyclosporine on human endothelial (HEC), epithelial (HK-2), and fibroblast (MRC5) cells. Cell proliferation was evaluated by cell counting, apoptosis and collagen production by enzyme-linked immunosorbent assay, and nitric oxide by measuring the concentration of nitrite/nitrate in the cell supernatant. (alpha1)I and (alpha2)IV collagen, matrix metalloprotease-9 (MMP9), and tissue inhibitors of metalloprotease-1 (TIMP-1) mRNA levels were measured by reverse transcription-polymerase chain reaction. Proteolytic activity was evaluated by zymography. RESULTS: Cyclosporine showed a marked antiproliferative and proapoptotic effect on endothelial and epithelial cells. Fibroblast growth was not affected by cyclosporine. Nitric oxide was up-regulated by cyclosporine in epithelial cells and fibroblasts but not in endothelial cells. (alpha1)I and (alpha2)IV collagen synthesis was increased in cyclosporine-treated endothelial and epithelial cells, respectively. Proteolytic activity was increased in endothelial and epithelial cells. TIMP-1 mRNA was up-regulated by cyclosporine in fibroblasts. CONCLUSIONS: Our results demonstrate that cyclosporine exhibits an antiproliferative effect on endothelial and epithelial cells. This effect is associated with induction of apoptosis probably via nitric oxide up-regulation in epithelial cell cultures. Cyclosporine treatment induces ECM accumulation by increasing collagen synthesis in endothelial and epithelial cells and reducing its degradation by up-regulating TIMP-1 expression in fibroblasts. We conclude that cyclosporine affects cell types differently and that the disruption of organ architecture is the result of multiple effects at the cell level.
Asunto(s)
Ciclosporina/farmacología , Endotelio Vascular/citología , Células Epiteliales/efectos de los fármacos , Inmunosupresores/farmacología , Túbulos Renales/citología , Piel/citología , Actinas/genética , Actinas/metabolismo , Capilares/citología , Capilares/metabolismo , División Celular/efectos de los fármacos , Células Cultivadas , Colágeno/genética , Colágeno/metabolismo , Endotelio Vascular/metabolismo , Células Epiteliales/citología , Células Epiteliales/enzimología , Proteínas de la Matriz Extracelular/metabolismo , Fibroblastos/citología , Fibroblastos/enzimología , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Humanos , Trasplante de Riñón/inmunología , Túbulos Renales/metabolismo , Metaloproteinasa 9 de la Matriz/genética , Metaloproteinasa 9 de la Matriz/metabolismo , Óxido Nítrico/biosíntesis , ARN Mensajero/análisis , Piel/metabolismo , Inhibidor Tisular de Metaloproteinasa-1/genética , Inhibidor Tisular de Metaloproteinasa-1/metabolismoRESUMEN
Renal dysfunction is one of the most common and threatening complications in heart transplant recipients. Even if ciclosporin seems to play a central role in inducing renal damage, other factors may concur or predispose to renal injury. In order to identify factors responsible for renal dysfunction, we retrospectively studied a cohort of 114 cardiac transplant recipients during a follow-up period of at least 3 years. The patients had a normal renal function before and 0.5 months after heart transplantation. Doubling of baseline serum creatinine or attainment of serum creatinine steadily above 176.8 micromol/l (2.0 mg/dl) was used as criterion to define the end-point renal dysfunction. A series of clinical and laboratory variables were obtained from the patients' charts at different time intervals, and their prognostic value for the occurrence of renal dysfunction was calculated by Cox proportional hazards models. 23 out of 114 patients reached the end point after a median time period of 21 months. High serum triglyceride, alanine aminotransferase, alkaline phosphatase, ciclosporin, urea, glucose, and hemoglobin levels were shown to be associated with the development of renal dysfunction. Four variables, i.e., triglyceride, ciclosporin, urea, and alkaline phosphatase, had an independent prognostic value. Our results confirm a role for ciclosporin in inducing renal dysfunction and identify hyperlipidemia and an increased plasma urea level as risk factors for renal dysfunction in heart transplant recipients.