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1.
Nephrol Dial Transplant ; 32(2): 325-332, 2017 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-27333618

RESUMO

Background: Membranous nephropathy (MN) is a common cause of nephrotic syndrome in adults. Previous genome-wide association studies (GWAS) of 300 000 genotyped variants identified MN-associated loci at HLA-DQA1 and PLA2R1. Methods: We used a combined approach of genotype imputation, GWAS, human leucocyte antigen (HLA) imputation and extension to other aetiologies of chronic kidney disease (CKD) to investigate genetic MN risk variants more comprehensively. GWAS using 9 million high-quality imputed genotypes and classical HLA alleles were conducted for 323 MN European-ancestry cases and 345 controls. Additionally, 4960 patients with different CKD aetiologies in the German Chronic Kidney Disease (GCKD) study were genotyped for risk variants at HLA-DQA1 and PLA2R1. Results: In GWAS, lead variants in known loci [rs9272729, HLA-DQA1, odds ratio (OR) = 7.3 per risk allele, P = 5.9 × 10-27 and rs17830558, PLA2R1, OR = 2.2, P = 1.9 × 10-8] were significantly associated with MN. No novel signals emerged in GWAS of X-chromosomal variants or in sex-specific analyses. Classical HLA alleles (DRB1*0301-DQA1*0501-DQB1*0201 haplotype) were associated with MN but provided little additional information beyond rs9272729. Associations were replicated in 137 GCKD patients with MN (HLA-DQA1: P = 6.4 × 10-24; PLA2R1: P = 5.0 × 10-4). MN risk increased steeply for patients with high-risk genotype combinations (OR > 79). While genetic variation in PLA2R1 exclusively associated with MN across 19 CKD aetiologies, the HLA-DQA1 risk allele was also associated with lupus nephritis (P = 2.8 × 10-6), type 1 diabetic nephropathy (P = 6.9 × 10-5) and focal segmental glomerulosclerosis (P = 5.1 × 10-5), but not with immunoglobulin A nephropathy. Conclusions: PLA2R1 and HLA-DQA1 are the predominant risk loci for MN detected by GWAS. While HLA-DQA1 risk variants show an association with other CKD aetiologies, PLA2R1 variants are specific to MN.

2.
Arterioscler Thromb Vasc Biol ; 34(2): 365-76, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24233489

RESUMO

OBJECTIVE: The risk of cardiovascular disease is increased by up to 33 to 50× in chronic inflammatory states and convention doses of statins may not provide the same cardiovascular protection as in noninflamed patients. This study investigated whether the increase in 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCoA-R)-mediated cholesterol synthesis observed under inflammatory stress was resistant to the action of statins and if so, whether this was because of interference with the sterol regulatory element binding protein cleavage-activating protein pathway. APPROACH AND RESULTS: Inflammatory stress was induced by adding cytokines (interleukin-1ß, tumor necrosis factor-α, and interleukin-6) and lipopolysaccharides to vascular smooth muscle cells in vitro and by subcutaneous casein injection in apolipoprotein E/scavenger receptors class A/CD36 triple knockout mice in vivo. Inflammatory stress exacerbated cholesterol ester accumulation and was accompanied in vitro and in vivo by increased HMGCoA-R mRNA and protein expression mediated via activation of the sterol regulatory element binding protein cleavage-activating protein/sterol regulatory element binding protein-2 pathway. Atorvastatin reduced HMGCoA-R enzymatic activity and intracellular cholesterol synthesis in vitro. However, inflammatory stress weakened these suppressive effects. Atorvastatin at concentrations of 16 µmol/L inhibited HMGCoA-R activity by 50% in vascular smooth muscle cells, but the same concentration resulted in only 30% of HMGCoA-R activity in vascular smooth muscle cells in the presence of interleukin-1ß. Knocking down sterol regulatory element binding protein cleavage-activating protein prevented statin resistance induced by interleukin-1ß, and overexpression of sterol regulatory element binding protein cleavage-activating protein induced statin resistance even without inflammatory stress. In vivo, the amount of atorvastatin required to lower serum cholesterol and decrease aortic lipid accumulation rose from 2 to 10 mg/kg per day in the presence of inflammatory stress. CONCLUSIONS: Increased cholesterol synthesis mediated by HMGCoA-R under inflammatory stress may be one of the mechanisms for intracellular lipid accumulation and statin resistance.


Assuntos
Resistência a Medicamentos , Ácidos Heptanoicos/farmacologia , Hidroximetilglutaril-CoA Redutases/metabolismo , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Hiperlipidemias/tratamento farmacológico , Inflamação/enzimologia , Músculo Liso Vascular/efeitos dos fármacos , Pirróis/farmacologia , Estresse Fisiológico , Animais , Apolipoproteínas E/deficiência , Apolipoproteínas E/genética , Atorvastatina , Antígenos CD36/deficiência , Antígenos CD36/genética , Colesterol/sangue , Colesterol na Dieta , Citocinas/metabolismo , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Retroalimentação Fisiológica , Células Hep G2 , Humanos , Hiperlipidemias/sangue , Hiperlipidemias/enzimologia , Hiperlipidemias/genética , Inflamação/sangue , Mediadores da Inflamação/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculo Liso Vascular/enzimologia , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/enzimologia , Interferência de RNA , Receptores Depuradores Classe A/deficiência , Receptores Depuradores Classe A/genética , Fatores de Tempo , Transfecção
3.
N Engl J Med ; 364(7): 616-26, 2011 Feb 17.
Artigo em Inglês | MEDLINE | ID: mdl-21323541

RESUMO

BACKGROUND: Idiopathic membranous nephropathy is a major cause of the nephrotic syndrome in adults, but its etiologic basis is not fully understood. We investigated the genetic basis of biopsy-proven cases of idiopathic membranous nephropathy in a white population. METHODS: We performed independent genomewide association studies of single-nucleotide polymorphisms (SNPs) in patients with idiopathic membranous nephropathy from three populations of white ancestry (75 French, 146 Dutch, and 335 British patients). The patients were compared with racially matched control subjects; population stratification and quality controls were carried out according to standard criteria. Associations were calculated by means of a chi-square basic allele test; the threshold for significance was adjusted for multiple comparisons (with the Bonferroni method). RESULTS: In a joint analysis of data from the 556 patients studied (398 men), we identified significant alleles at two genomic loci associated with idiopathic membranous nephropathy. Chromosome 2q24 contains the gene encoding M-type phospholipase A(2) receptor (PLA(2)R1) (SNP rs4664308, P=8.6×10(-29)), previously shown to be the target of an autoimmune response. Chromosome 6p21 contains the gene encoding HLA complex class II HLA-DQ alpha chain 1 (HLA-DQA1) (SNP rs2187668, P=8.0×10(-93)). The association with HLA-DQA1 was significant in all three populations (P=1.8×10(-9), P=5.6×10(-27), and P=5.2×10(-36) in the French, Dutch, and British groups, respectively). The odds ratio for idiopathic membranous nephropathy with homozygosity for both risk alleles was 78.5 (95% confidence interval, 34.6 to 178.2). CONCLUSIONS: An HLA-DQA1 allele on chromosome 6p21 is most closely associated with idiopathic membranous nephropathy in persons of white ancestry. This allele may facilitate an autoimmune response against targets such as variants of PLA2R1. Our findings suggest a basis for understanding this disease and illuminate how adaptive immunity is regulated by HLA.


Assuntos
Estudo de Associação Genômica Ampla , Glomerulonefrite Membranosa/genética , Antígenos HLA-DQ/genética , Polimorfismo de Nucleotídeo Único , Receptores da Fosfolipase A2/genética , Alelos , Cromossomos Humanos Par 2 , Cromossomos Humanos Par 6 , Europa (Continente) , Feminino , Genótipo , Cadeias alfa de HLA-DQ , Humanos , Masculino , Razão de Chances , População Branca/genética
4.
Nephrol Dial Transplant ; 29(10): 1864-78, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-24895437

RESUMO

BACKGROUND: Patients with chronic kidney disease (CKD) are unlikely to gain the same benefit from conventional doses of statins as do patients with cardiovascular disease alone. This study investigated whether inflammation accompanying CKD causes statin resistance. METHODS: Inflammatory stress was induced by adding cytokines and lipopolysaccharide (LPS) to human mesangial cells (HMCs) in vitro, and in vivo by subcutaneous casein injection in apolipoprotein E, scavenger receptors class A and CD36 triple knockout mice. RESULTS: Inflammatory stress exacerbated cholesterol accumulation and was accompanied in vitro and in vivo by increased HMGCoA reductase (HMGCoA-R) mRNA and protein expression mediated via activation of the sterol regulatory element-binding protein cleavage-activating protein (SCAP)/sterol regulatory element-binding protein 2 pathway. Atorvastatin reduced HMGCoA-R enzymatic activity and intracellular cholesterol synthesis in vitro; however, inflammatory stress weakened these suppressive effects. Atorvastatin at concentrations of 15 µM inhibited HMGCoA-R activity by 50% (IC50) in HMCs, but the same concentration in the presence of interleukin (IL)-1ß resulted in only 30% inhibition of HMGCoA-R activity in HMCs. Knocking down SCAP prevented statin resistance induced by IL-1ß, and overexpression of SCAP-induced statin resistance even without inflammatory stress. In vivo, the amount of atorvastatin required to lower serum cholesterol and decrease kidney lipid accumulation rose from 2 to 10 mg/kg/day in the presence of inflammatory stress. CONCLUSIONS: Inflammatory stress can disrupt HMGCoA-R-mediated cholesterol synthesis resulting in intracellular lipid accumulation and statin resistance.


Assuntos
Retroalimentação Fisiológica/efeitos dos fármacos , Ácidos Heptanoicos/farmacologia , Hidroximetilglutaril-CoA Redutases/metabolismo , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Inflamação/fisiopatologia , Rim/efeitos dos fármacos , Pirróis/farmacologia , Estresse Fisiológico , Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Animais , Apolipoproteínas E/fisiologia , Atorvastatina , Western Blotting , Antígenos CD36/fisiologia , Caseínas/administração & dosagem , Células Cultivadas , Colesterol/sangue , Citocinas/genética , Citocinas/metabolismo , Resistência a Medicamentos , Humanos , Hidroximetilglutaril-CoA Redutases/genética , Rim/enzimologia , Lipopolissacarídeos/administração & dosagem , Masculino , Células Mesangiais/efeitos dos fármacos , Células Mesangiais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteína de Ligação a Elemento Regulador de Esterol 2/genética , Proteína de Ligação a Elemento Regulador de Esterol 2/metabolismo , Resultado do Tratamento , Fator de Necrose Tumoral alfa/farmacologia
5.
Am J Physiol Renal Physiol ; 301(1): F236-43, 2011 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-21511699

RESUMO

Advanced glycation end products (AGEs) is one of the causative factors of diabetic nephropathy, which is associated with lipid accumulation in glomeruli. This study was designed to investigate whether N(ε)-(carboxymethyl) lysine (CML; a member of the AGEs family) increases lipid accumulation by impairing the function of sterol-regulatory element binding protein (SREBP) cleavage-activating protein (SCAP) in human mesangial cells (HMCs). Intracellular cholesterol content was assessed by Oil Red O staining and quantitative assay. The expression of molecules controlling cholesterol homeostasis was examined using real-time quantitative RT-PCR and Western blotting. The activity of Golgi-processing enzymes was determined using enzyme-based methods, and the translocation of SCAP from the endoplasmic reticulum (ER) to the Golgi was detected by confocal microscopy. CML increased cholesterol accumulation in HMCs. Exposure to CML increased expression and abnormal translocation of SCAP from the ER to the Golgi even in the presence of a high concentration of LDL. The increased SCAP translocation carried more SREBP-2 to the Golgi for activation by proteolytic cleavages, enhancing transcription of 3-hydroxy-3-methylclutaryl-CoA reductase and the LDL receptor. CML increased Golgi mannosidase activity, which may enhance glycosylation of SCAP. This prolonged the half-life and enhanced recycling of SCAP between the ER and the Golgi. The effects of CML were blocked by inhibitors of Golgi mannosidases. AGEs (CML) increased lipid synthesis and uptake, thereby causing foam cell formation via increasing transcription and protein glycosylation of SCAP in HMCs. These data imply that inhibitors of Golgi-processing enzymes might have a potential renoprotective role in prevention of mesangial foam cell formation.


Assuntos
Células Espumosas/fisiologia , Mesângio Glomerular/metabolismo , Produtos Finais de Glicação Avançada/farmacologia , Complexo de Golgi/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Western Blotting , Células Cultivadas , Colesterol/metabolismo , Retículo Endoplasmático/metabolismo , Mesângio Glomerular/citologia , Glicosilação , Humanos , Hidroximetilglutaril-CoA Redutases/metabolismo , Lisina/análogos & derivados , Lisina/farmacologia , Microscopia Confocal , Microssomos/metabolismo , Plasmídeos , RNA/biossíntese , RNA/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Receptores de LDL/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , alfa-Manosidase/metabolismo
6.
Am J Physiol Renal Physiol ; 301(4): F713-22, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21795641

RESUMO

Both lipids and inflammation play important roles in the progression of kidney disease. This study was designed to investigate whether inflammation exacerbates lipid accumulation via LDL receptors (LDLr), thereby causing renal injury in C57BL/6J mice, apolipoprotein E (ApoE) knockout (KO) mice, and ApoE/CD36/scavenger receptor A triple KO mice. The mice were given a subcutaneous casein injection to induce inflammatory stress. After 14 wk, terminal blood samples were taken for renal function, lipid profiles, amyloid A (SAA), and IL-6 assays. Lipid accumulation in kidneys was visualized by oil red O staining. Fibrogenic molecule expression in kidneys was examined. There was a significant increase in serum SAA and IL-6 in the all casein-injected mice compared with respective controls. Casein injection reduced serum total cholesterol, LDL cholesterol, and HDL cholesterol and caused lipid accumulation in kidneys from three types of mice. The expression of LDLr and its regulatory proteins sterol-responsive element-binding protein (SREBP) 2 and SREBP cleavage-activating protein (SCAP) were upregulated in inflamed mice compared with controls. Casein injection induced renal fibrosis accompanied by increased expression of fibrogenic molecules in the triple KO mice. These data imply that inflammation exacerbates lipid accumulation in the kidney by diverting lipid from the plasma to the kidney via the SCAP-SREBP2-LDLr pathway and causing renal injury. Low blood cholesterol levels, resulting from inflammation, may be associated with high risk for chronic renal fibrosis.


Assuntos
Inflamação/metabolismo , Nefropatias/metabolismo , Nefropatias/patologia , Metabolismo dos Lipídeos , Animais , Apolipoproteínas E/genética , Antígenos CD36/genética , Caseínas/efeitos adversos , Colesterol/sangue , Colesterol/metabolismo , Doença Crônica , Progressão da Doença , Fibrose , Inflamação/genética , Interleucina-6/sangue , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Nefropatias/genética , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de LDL/metabolismo , Receptores Depuradores Classe A/genética , Proteína Amiloide A Sérica/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 2/metabolismo
7.
Lipids Health Dis ; 10: 110, 2011 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-21718499

RESUMO

BACKGROUND: Chronic systemic inflammation and abnormal free fatty acid metabolism are closely related to ectopic lipid deposition. In this study, we investigate if inflammation tissue-specifically disrupts lipogenesis and lipolysis in nonadipose tissues and adipose tissue, resulting in ectopic lipid deposition in C57BL/6J mice. METHODS: We used casein injection in C57BL/6J mice to induce a chronic systemic inflammatory stress in vivo. Serum was analyzed for free fatty acid and cytokines. Insulin sensitivities were evaluated by glucose and insulin tolerance tests. Liver, muscle, adipose tissues were taken for lipid analysis. Real-time polymerase chain reaction and western blotting were used to examine the gene and protein expression of molecules involved in adipogenesis and lipolysis in tissues. RESULTS: Casein injection elevated serum levels of IL-6 and SAA in mice, which are associated with increased lipid accumulation in liver and muscle, suggesting that chronic systemic inflammation induces ectopic lipid deposition in nonadipose tissues. The inflammatory stress upregulated mRNA and protein expression of sterol regulatory element binding protein 1, fatty acid synthase, and acetyl CoA carboxylase alpha, while inhibited these molecules expression in adipose. Interestingly, in the same experimental setting, inflammation increased triglyceride lipase and hormone-sensitive lipase expression in white adipose tissue. Inflammation also induced insulin resistance and increased serum free fatty acid levels in C57BL/6J mice. CONCLUSIONS: Chronic systemic inflammation increased lipogenesis in nonadipose tissues and lipolysis in white adipose tissue, resulting in ectopic lipid deposition in nonadipose tissues. This disturbed free fatty acid homeostasis and caused insulin resistance in C57BL/6J mice.


Assuntos
Inflamação/induzido quimicamente , Metabolismo dos Lipídeos , Estresse Fisiológico , Acetil-CoA Carboxilase/genética , Acetil-CoA Carboxilase/metabolismo , Tecido Adiposo Branco/enzimologia , Tecido Adiposo Branco/metabolismo , Tecido Adiposo Branco/patologia , Animais , Glicemia , Caseínas , Ácido Graxo Sintase Tipo I/genética , Ácido Graxo Sintase Tipo I/metabolismo , Ácidos Graxos não Esterificados/sangue , Expressão Gênica , Insulina/sangue , Resistência à Insulina , Interleucina-6/sangue , Fígado/enzimologia , Fígado/metabolismo , Fígado/patologia , Camundongos , Camundongos Endogâmicos C57BL , Músculo Esquelético/enzimologia , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Proteína Amiloide A Sérica/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 1/genética , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo
8.
Am J Physiol Heart Circ Physiol ; 298(6): H1646-51, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20348217

RESUMO

Inflammatory stress accelerates the progression of atherosclerosis. Sirolimus, a new immunosuppressive agent, has been shown to have pleiotropic antiatherosclerotic effects. In this study we hypothesized that sirolimus inhibits 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR)-mediated cholesterol synthesis in human vascular smooth muscle cells (VSMCs) under inflammatory stress. Using radioactive assay, we demonstrated that sirolimus inhibited the increase of interleukin-1beta (IL-1beta)-induced cholesterol synthesis in VSMCs. Further studies showed that sirolimus inhibited both the HMGR gene and protein expression in VSMCs treated with or without IL-1beta. These effects were mediated by inhibiting the gene expression of sterol regulatory element-binding protein-2 (SREBP-2) and SREBP-2 cleavage-activating protein (SCAP) as checked by real-time PCR, Western blot analysis, and confocal microscopy for the observation of decreased protein translocation of the SCAP/SREBP-2 complex from the endoplasmic reticulum (ER) to the Golgi. Insulin-induced gene-1 (Insig-1) is a key ER protein controlling the feedback regulation of HMGR at transcriptional and posttranscriptional levels. We demonstrated that sirolimus increased Insig-1 expression which may bind to the SCAP, preventing the exit of SCAP-SREBP complexes from the ER. The increased Insig-1 also accelerated HMGR protein degradation in VSMCs as shown by pulse-chase analysis. In conclusion, sirolimus inhibits cholesterol synthesis induced by inflammatory stress through the downregulation of HMGR expression and the acceleration of HMGR protein degradation. These findings may improve our understanding of the molecular mechanisms of the antiatherosclerosis properties of sirolimus.


Assuntos
Colesterol/metabolismo , Imunossupressores/farmacologia , Inflamação/metabolismo , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Sirolimo/farmacologia , Acil Coenzima A/metabolismo , Aterosclerose/prevenção & controle , Células Cultivadas , Vasos Coronários/efeitos dos fármacos , Vasos Coronários/metabolismo , Vasos Coronários/patologia , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Humanos , Inflamação/patologia , Interleucina-1beta/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Músculo Liso Vascular/patologia , Proteína de Ligação a Elemento Regulador de Esterol 2/metabolismo
9.
Hepatology ; 48(3): 770-81, 2008 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-18752326

RESUMO

UNLABELLED: The prevailing theory in non-alcoholic fatty liver disease (NAFLD) is the "two-hit" hypothesis. The first hit mainly consists of lipid accumulation, and the second is subsequent systemic inflammation. The current study was undertaken to investigate whether inflammatory stress exacerbates lipid accumulation in liver and its underlying mechanisms. We used interleukin-1beta (IL-1beta) and tumor necrosis factor alpha (TNF-alpha) stimulation in human hepatoblastoma cell line (HepG2) cells and primary hepatocytes in vitro, and casein injection in apolipoprotein E knockout mice in vivo to induce inflammatory stress. The effects of inflammatory stress on cholesterol accumulation were examined by histochemical staining and a quantitative intracellular cholesterol assay. The gene and protein expressions of molecules involved in cholesterol trafficking were examined by real-time polymerase chain reaction (PCR) and western blot. Cytokine production in the plasma of apolipoprotein E knockout mice was measured by enzyme-linked immunosorbent assay. Our results showed that inflammatory stress increased cholesterol accumulation in hepatic cells and in the livers of apolipoprotein E knockout mice. Further analysis showed that inflammatory stress increased the expression of low-density lipoprotein (LDL) receptor (LDLr), sterol regulatory element-binding protein (SREBP) cleavage activating protein (SCAP), and SREBP-2. Confocal microscopy showed that IL-1beta increased the translocation of SCAP/SREBP-2 complex from endoplasmic reticulum (ER) to Golgi in HepG2 cells, thereby activating LDLr gene transcription. IL-1beta, TNF-alpha, and systemic inflammation induced by casein injection also inhibited expression of adenosine triphosphate-binding cassette transporter A1 (ABCA1), peroxisome proliferator-activated receptor-alpha (PPAR-alpha), and liver X receptor-alpha (LXRalpha). This inhibitory effect may cause cholesterol efflux reduction. CONCLUSION: Inflammatory stress up-regulates LDLr-mediated cholesterol influx and down-regulates ABCA1-mediated cholesterol efflux in vivo and in vitro. This may exacerbate the progression of NAFLD by disrupting cholesterol trafficking control, especially during the second hit phase of liver damage.


Assuntos
Colesterol/metabolismo , Fígado Gorduroso/metabolismo , Hepatócitos/metabolismo , Inflamação/metabolismo , Metabolismo dos Lipídeos/fisiologia , Transportador 1 de Cassete de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Apolipoproteínas E/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/metabolismo , Modelos Animais de Doenças , Fígado Gorduroso/patologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/patologia , Humanos , Inflamação/induzido quimicamente , Inflamação/patologia , Interleucina-1beta/efeitos adversos , Metabolismo dos Lipídeos/efeitos dos fármacos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Receptores X do Fígado , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores Nucleares Órfãos , PPAR alfa/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores de LDL/metabolismo , Fator de Necrose Tumoral alfa/efeitos adversos
10.
J Surg Res ; 157(2): 216-22, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19482293

RESUMO

BACKGROUND: Histological assessment of intraportally transplanted islets in experimental rodent models is limited by the wide dissemination of islets throughout the liver. We describe a technique of highly selective intraportal islet transplantation, which increases the density of transplanted islets and hence facilitates their histological analysis and validate this model as a means of quantitative histological analysis of islet graft survival. We also compared the number of islets visualized histologically with that of nonabsorbable dextran beads, representing the number of transplanted islets there would have been if there had been no graft loss. MATERIALS AND METHODS: Diabetic Lewis rats or nondiabetic Sprague-Dawley rats were transplanted with 500 or 1000 syngeneic islets or an equivalent number of beads either into the main branch (MB), or selectively into the right branch (RB) of the portal vein. RESULTS: Islet transplantation led to an identical fall in blood glucose levels whichever technique was used. The graft area and number of islets recovered for histological analysis was 3- to 4-fold higher when islets were transplanted using the RB technique. Quantitative histological graft analysis demonstrated that 46% to 61% of intraportally transplanted islets were lost compared with corresponding bead graft sizes. Fewer islets were lost when a greater islet mass was transplanted. CONCLUSIONS: Selective islet transplantation increases the concentration of islets and hence facilitates islet recovery after intraportal transplantation without detrimental effects on transplantation outcome. This technique, when combined with bead transplantation and digital image analysis, provides an accurate method for estimating the number of islets surviving intra-portal transplantation.


Assuntos
Diabetes Mellitus Experimental/cirurgia , Transplante das Ilhotas Pancreáticas/métodos , Veia Porta , Animais , Glicemia/metabolismo , Dextranos , Diabetes Mellitus Experimental/sangue , Modelos Animais de Doenças , Sobrevivência de Enxerto/fisiologia , Injeções Intravenosas/métodos , Transplante das Ilhotas Pancreáticas/patologia , Fígado/patologia , Masculino , Microesferas , Tamanho do Órgão/fisiologia , Ratos , Ratos Endogâmicos Lew , Ratos Sprague-Dawley , Estreptozocina , Resultado do Tratamento
11.
Nephrol Dial Transplant ; 23(6): 1876-85, 2008 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-18281317

RESUMO

BACKGROUND: Monocyte recruitment into the mesangium and foam cell formation are recognized features of glomerular injury. External signals encountered by infiltrating mononuclear cells may determine their behaviour and thereby potentially influence disease outcome. Having previously demonstrated that monocytes are activated by exposure to matrix secreted by mesangial cells, we set out to determine whether matrix activation of monocytes led to expression of a macrophage phenotype. METHODS: THP-1 mononuclear cells were incubated for up to 120 h (5 days) with 500 microg/ml solublized matrix extracted from cultured human mesangial cells or with phorbol myristate ester (PMA-positive control) or albumin (negative control). Expression of peroxisome proliferator activated receptor-gamma (PPAR-gamma) and of scavenger receptors was used as a marker of monocyte to macrophage differentiation. The presence of functional scavenger receptors was examined by assessing cellular uptake of Dil-labelled acetylated (Ac)-LDL by flow cytometry. Matrix-mediated LDL oxidation was assessed using agarose gel electrophoresis to determine mobility shifts. RESULTS: Matrix activation was associated with an increase in the expression of PPAR-gamma, scavenger receptor-B (CD36) and scavenger receptor-A mRNA with a corresponding increase in PPAR-gamma protein. Matrix-activated cells incubated with Ac-LDL demonstrated foam cell formation, whilst incubation with Dil-labelled Ac-LDL led to an increase in mean fluorescence intensity of 373 +/- 34.8% (P < 0.005) as compared to albumin (100%) and PMA (423 +/- 55.8%) (P < 0.005). This could be inhibited by the addition of excess unlabelled ligand, suggesting specific involvement of scavenger receptors. Incubation of LDL with mesangial matrix in the absence of mesangial cells or monocytes led to enhanced electrophoretic mobility of the recovered lipoprotein on agarose gel, an effect that could be inhibited by the addition of anti-oxidants. CONCLUSION: Exposure to mesangial cell matrix induces expression of monocyte characteristics associated with a macrophage phenotype and promotes oxidation of LDL, thereby converting this lipoprotein to a scavenger receptor ligand. These observations may help to explain foam cell formation in the mesangium in the context of glomerular disease.


Assuntos
Células Espumosas/metabolismo , Mesângio Glomerular/fisiologia , Peroxidação de Lipídeos/fisiologia , Monócitos/fisiologia , Receptores Depuradores/metabolismo , Sequência de Bases , Biomarcadores/metabolismo , Western Blotting , Antígenos CD36/genética , Antígenos CD36/metabolismo , Movimento Celular , Células Cultivadas , Citometria de Fluxo , Células Espumosas/citologia , Regulação da Expressão Gênica , Mesângio Glomerular/citologia , Humanos , Líquido Intracelular/metabolismo , Dados de Sequência Molecular , Probabilidade , RNA Mensageiro/análise , Receptores Depuradores/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Estatísticas não Paramétricas
13.
Transplantation ; 84(8): 1029-36, 2007 Oct 27.
Artigo em Inglês | MEDLINE | ID: mdl-17989609

RESUMO

BACKGROUND: Sirolimus is a potent immunosuppressive agent, which is associated with dyslipidemia in clinical transplantation. The present study was undertaken to investigate the potential hepatocyte mechanisms by which sirolimus causes dyslipidemia. METHODS: Using both a quantitative assay of intracellular cholesterol and an [3H]-labeled cholesterol efflux assay, we studied the effect of sirolimus on cholesterol accumulation and cholesterol efflux in HepG2 cells in the absence or presence of inflammatory stress induced by interleukin-1beta. The gene and protein expression of molecules involved in cholesterol homeostasis were examined by real-time reverse-transcription polymerase chain reaction and Western blotting. RESULTS: Sirolimus inhibited low-density lipoprotein (LDL) receptor (LDLr)-mediated cholesterol ester accumulation induced by interleukin-1beta in HepG2 cells. This inhibitory effect was mediated by down-regulation of sterol regulatory element-binding proteins (SREBP) cleavage activating protein (SCAP) and SREBP-2 mRNA expression. Using confocal microscopy, we demonstrated that sirolimus reduced translocation of SCAP-SREBP2 complex from endoplasmic reticulum to Golgi for activation, thereby inhibiting LDLr gene transcription. Reduction of LDLr in the liver may result in a delay of LDL-cholesterol clearance from circulation causing an increase of plasma cholesterol concentration. Furthermore, sirolimus increased cholesterol efflux mediated by adenosine triphosphate-binding cassette transporter A1 gene expression by increasing peroxisome proliferator-activated receptor-alpha and liver X receptor-alpha gene and protein expression. Increased cholesterol efflux from HepG2 cells may increase high-density lipoprotein cholesterol level and also contribute to apolipoprotein B lipoprotein formation by enhancing transfer of high-density lipoprotein cholesterol to apolipoprotein B lipoproteins. CONCLUSIONS: This study demonstrates that the increase of LDL cholesterol by sirolimus is partly due to the reduction of LDLr on hepatocytes.


Assuntos
LDL-Colesterol/metabolismo , Dislipidemias/induzido quimicamente , Hepatócitos/efeitos dos fármacos , Imunossupressores/farmacologia , Receptores de LDL/antagonistas & inibidores , Sirolimo/farmacologia , Linhagem Celular , Dislipidemias/metabolismo , Retículo Endoplasmático/química , Retículo Endoplasmático/metabolismo , Complexo de Golgi/química , Complexo de Golgi/metabolismo , Hepatócitos/metabolismo , Homeostase/efeitos dos fármacos , Humanos , Imunossupressores/efeitos adversos , Peptídeos e Proteínas de Sinalização Intracelular/análise , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/análise , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/metabolismo , Transporte Proteico , RNA Mensageiro/análise , RNA Mensageiro/metabolismo , Receptores de LDL/genética , Receptores de LDL/metabolismo , Sirolimo/efeitos adversos , Proteína de Ligação a Elemento Regulador de Esterol 2/análise , Proteína de Ligação a Elemento Regulador de Esterol 2/antagonistas & inibidores , Proteína de Ligação a Elemento Regulador de Esterol 2/metabolismo
14.
Cell Transplant ; 16(5): 505-16, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-17708340

RESUMO

A large proportion of islets are lost after transplantation partly due to a lack of functional vasculature. Islets revascularize from host tissue but the process takes up to 2 weeks and has been suggested to result in reduced vascular density in engrafted islets. We describe a method for observing and quantifying the revascularization of intraportally transplanted islets that includes number, density, and branching of islet capillaries. Syngeneic islets were transplanted selectively into the two right posterior lobes of the liver of adult Lewis rats. Sections of the livers were dual stained for insulin and Bandeiraea simplicifolia and analyzed for islet morphology, area, and vascular density from day 0 to day 14 posttransplant and compared to native islets. Vascular density was 1431 +/- 75.7 vessels/mm2 in native islets and fell to 325.3 +/- 30.8 vessels/mm2 (p < 0.001) by day 1 posttransplant and subsequently increased until day 14 when it was significantly higher than in native islets (2612.5 +/- 107.8 vessels/mm2, p < 0.001). The percentage of islet area occupied by vascular space was 9.1 +/- 0.9% in native islets. After falling to 2.3 +/- 0.3% (p < 0.001) 1 day posttransplant this rose to supranormal levels (21.5 +/- 0.8%, p < 0.001) by day 14. The index of capillary branching was 0.771 +/- 0.017 in native islets and fell to 0.465 +/- 0.02 (p = 0.001) by day 3 but returned to native values by day 7 posttransplantation (0.726 +/- 0.03). This technique provides a robust method for tracking and quantifying the revascularization of intraportally transplanted islets, which should enable the comparison of different strategies aimed at accelerating islet revascularization.


Assuntos
Transplante das Ilhotas Pancreáticas , Ilhotas Pancreáticas/irrigação sanguínea , Neovascularização Fisiológica , Sistema Porta , Animais , Glicemia/análise , Ilhotas Pancreáticas/citologia , Ratos , Ratos Endogâmicos Lew , Fatores de Tempo
15.
Arterioscler Thromb Vasc Biol ; 26(5): 1150-5, 2006 May.
Artigo em Inglês | MEDLINE | ID: mdl-16543490

RESUMO

OBJECTIVE: Although inflammation is a recognized feature of atherosclerosis, the impact of inflammation on cellular cholesterol homeostasis is unclear. This study focuses on the molecular mechanisms by which inflammatory cytokines disrupt low-density lipoprotein (LDL) receptor regulation. METHODS AND RESULTS: IL-1beta enhanced transformation of vascular smooth muscle cells into foam cells by increasing uptake of unmodified LDL via LDL receptors and by enhancing cholesterol esterification as demonstrated by Oil Red O staining and direct assay of intracellular cholesterol concentrations. In the absence of IL-1beta, a high concentration of LDL decreased LDL receptor promoter activity, mRNA synthesis and protein expression. However, IL-1beta enhanced LDL receptor expression, overriding the suppression usually induced by a high concentration of LDL and inappropriately increasing LDL uptake. Exposure to IL-1beta also caused overexpression of the sterol regulatory element binding protein (SREBP) cleavage-activating protein (SCAP), and enhanced its translocation from the endoplasmic reticulum to the Golgi, where it is known to cleave SREBP, thereby enhancing LDL receptor gene expression. CONCLUSIONS: These observations demonstrate that IL-1beta disrupts cholesterol-mediated LDL receptor feedback regulation, permitting intracellular accumulation of unmodified LDL and causing foam cell formation. The implication of these findings is that inflammatory cytokines may contribute to intracellular LDL accumulation without previous modification of the lipoprotein.


Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Interleucina-1/farmacologia , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Receptores de LDL/genética , Células Cultivadas , Retículo Endoplasmático/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Lipoproteínas LDL/metabolismo , Proteínas de Membrana/metabolismo , Regiões Promotoras Genéticas , Esterol O-Aciltransferase/genética , Esterol O-Aciltransferase/metabolismo , Proteínas de Ligação a Elemento Regulador de Esterol/metabolismo
17.
Transplantation ; 75(12): 1954-9, 2003 Jun 27.
Artigo em Inglês | MEDLINE | ID: mdl-12829893

RESUMO

BACKGROUND: Hypoxia in the portal vein may compromise the survival of intraportally transplanted pancreatic islets. We therefore examined the effect of inspired oxygen on the outcome of islet transplantation. METHODS: Blood glucose concentrations, glucose tolerance, and the size and number of surviving islets were measured in diabetic rats housed for 48 hr under hyperoxic (100% O(2)), hypoxic (11% O(2)), or normoxic (21%O(2)) conditions after intraportal transplantation of 350, 500, 700, or 1,000 syngeneic islets. RESULTS: In normoxic diabetic rats, the smallest graft size to consistently restore normoglycemia was 1,000 islets. A graft size of 700 islets was effective in only three of nine animals, whereas 500 islets were ineffective in all eight animals undergoing transplantation. In contrast, in hyperoxically housed rats, graft sizes of 700 or 500 islets restored normoglycemia in eight of nine or five of eight animals, respectively. In those animals that became normoglycemic, the glucose tolerance of the hyperoxically treated rats receiving 700 islets was almost identical to that of normoxically housed animals receiving 1,000 islets. The average size of the islets 6 weeks after transplantation was the same in livers of hyperoxic and control rats. However, the total islet area and number of islets engrafted in hyperoxic rats was significantly increased when compared with livers from normoxic animals receiving the same graft size, so the area in hyperoxic rats receiving 700 islets was not significantly different from normoxic recipients of 1,000 islets. CONCLUSIONS: Hyperoxia posttransplantation increases the number of islets that survive the engraftment process and allows normalization of plasma glucose levels with a smaller number of transplanted islets.


Assuntos
Diabetes Mellitus Experimental/cirurgia , Sobrevivência de Enxerto/fisiologia , Hiperóxia , Transplante das Ilhotas Pancreáticas/fisiologia , Animais , Glicemia/metabolismo , Diabetes Mellitus Experimental/sangue , Teste de Tolerância a Glucose , Transplante das Ilhotas Pancreáticas/métodos , Transplante das Ilhotas Pancreáticas/patologia , Sistema Porta , Ratos , Fatores de Tempo , Transplante Isogênico
18.
Cell Transplant ; 13(7-8): 801-8, 2004.
Artigo em Inglês | MEDLINE | ID: mdl-15690982

RESUMO

Because hypoxia may compromise the survival of intraportally transplanted pancreatic islets, we have measured portal blood flow and both portal and hepatic oxygenation in normal and diabetic rats breathing graded inspired oxygen concentrations. Portal blood flow and hepatic tissue oxygenation were measured using a transonic flowmeter and near infrared spectroscopy while gas analysis was carried out on portal venous blood samples. The effects of breathing 13%, 21%, 50%, or 100% oxygen were compared in animals with steptozotocin-induced diabetes and in controls. In diabetic rats breathing 21% oxygen, portal blood flow was significantly lower than in controls (7.2+/-0.7 vs. 9.1+/-0.8 ml/min, p < 0.05). In both groups, breathing 100% oxygen significantly increased portal flow (to 8.4+/-1.0 and 12.2+/-0.7 ml/min, respectively). This effect was not secondary to hepatic arterial vasoconstriction because it was not prevented by hepatic artery ligation. In controls, breathing 100% oxygen increased portal pO2 from 5.0+/-0.9 to 14.4+/-1.4 kPa (p < 0.05) and portal venous oxygen saturation (PSaO2) from 53.9+/-12.1% to 92.9+/-1.4% (p < 0.05), a value not significantly different from peripheral (arterial) saturation. Similarly, in diabetic animals pO2 rose from 5.6+/-0.3 to 11.7+/-0.4 kPa (p < 0.01) and SO2 from 55.5+/-5.2% to 88.5+/-0.6% (p < 0.05). Hepatic oxyhemoglobin rose and deoxyhemoglobin fell reciprocally as a function of the inspired oxygen concentration. Improved hepatic oxygenation observed in animals breathing oxygen-enriched gas mixtures results from an increase in splanchnic blood flow coupled with a marked increase in portal oxygen saturation. This effective arterialization of portal blood may have important consequences for the success of intraportal transplantation of pancreatic islets.


Assuntos
Diabetes Mellitus Experimental/fisiopatologia , Hiperóxia , Circulação Hepática/efeitos dos fármacos , Fígado/irrigação sanguínea , Oxigênio/farmacologia , Veia Porta/fisiologia , Animais , Gasometria , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Hemoglobinas/análise , Artéria Hepática/fisiologia , Hiperóxia/induzido quimicamente , Hipóxia/fisiopatologia , Hipóxia/prevenção & controle , Ligadura , Fígado/fisiologia , Circulação Hepática/fisiologia , Masculino , Oxigênio/metabolismo , Consumo de Oxigênio/efeitos dos fármacos , Consumo de Oxigênio/fisiologia , Oxiemoglobinas/análise , Ratos , Ratos Endogâmicos Lew
20.
J Endocrinol ; 219(3): 195-204, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24029730

RESUMO

Inflammatory stress is closely related to metabolic disease and insulin resistance. The precise cellular mechanism linking obesity and diabetes is largely unknown, but about 14-20% of obese individuals develop diabetes. In this study, we investigated whether chronic inflammation exacerbated glucose metabolism disorder by impairing ß cell function in high-fat diet (HFD)-fed C57BL/6J mice. We used s.c. casein injection to induce chronic inflammation in HFD-fed C57BL/6J mice; 14 weeks on a HFD resulted in weight gain, hyperlipidemia, and low insulin sensitivity in these mice which nevertheless had normal blood glucose and serum inflammatory cytokines levels. Casein injection in the background of HFD elevated serum tumor necrosis factor α (TNFα) and serum amyloid A levels and increased TNFα and MCP1 expression in the adipose tissue, liver, and muscle of HFD-fed mice. Chronic inflammation induced by casein injection further decreased insulin sensitivity and insulin signaling, resulting in insulin deficiency and hyperglycemia in these mice. Islet mass and insulin content were markedly increased in HFD mice. However, in contrast with HFD-fed alone, chronic inflammation in HFD-fed mice decreased both islet mass and insulin content, reduced the genetic expression of insulin synthesis and secretion, and increased ß cell apoptosis. We conclude that chronic inflammation exacerbated glucose metabolism disorders by impairing ß cell function in HFD-fed C57BL/6J mice, suggesting that this mechanism may operate in obese individuals with chronic inflammation, making them prone to hyperglycemia.


Assuntos
Dieta Hiperlipídica/efeitos adversos , Modelos Animais de Doenças , Transtornos do Metabolismo de Glucose/imunologia , Resistência à Insulina , Células Secretoras de Insulina/imunologia , Obesidade/complicações , Tecido Adiposo Branco/imunologia , Tecido Adiposo Branco/metabolismo , Animais , Apoptose , Caseínas/administração & dosagem , Quimiocina CCL2/metabolismo , Transtornos do Metabolismo de Glucose/complicações , Transtornos do Metabolismo de Glucose/patologia , Transtornos do Metabolismo de Glucose/fisiopatologia , Hiperlipidemias/complicações , Hiperlipidemias/etiologia , Injeções Subcutâneas , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/patologia , Fígado/imunologia , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Músculo Esquelético/imunologia , Músculo Esquelético/metabolismo , Obesidade/etiologia , Proteína Amiloide A Sérica/análise , Fator de Necrose Tumoral alfa/sangue , Fator de Necrose Tumoral alfa/metabolismo
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