RESUMO
Hemolytic uremic syndrome (HUS) is a thrombotic microangiopathy with manifestations of hemolytic anemia, thrombocytopenia, and renal impairment. Genetic studies have shown that mutations in complement regulatory proteins predispose to non-Shiga toxin-associated HUS (non-Stx-HUS). We undertook genetic analysis on membrane cofactor protein (MCP), complement factor H (CFH), and factor I (IF) in 156 patients with non-Stx-HUS. Fourteen, 11, and 5 new mutational events were found in MCP, CFH, and IF, respectively. Mutation frequencies were 12.8%, 30.1%, and 4.5% for MCP, CFH, and IF, respectively. MCP mutations resulted in either reduced protein expression or impaired C3b binding capability. MCP-mutated patients had a better prognosis than CFH-mutated and nonmutated patients. In MCP-mutated patients, plasma treatment did not impact the outcome significantly: remission was achieved in around 90% of both plasma-treated and plasma-untreated acute episodes. Kidney transplantation outcome was favorable in patients with MCP mutations, whereas the outcome was poor in patients with CFH and IF mutations due to disease recurrence. This study documents that the presentation, the response to therapy, and the outcome of the disease are influenced by the genotype. Hopefully this will translate into improved management and therapy of patients and will provide the way to design tailored treatments.
Assuntos
Fator H do Complemento/genética , Fator I do Complemento/genética , Síndrome Hemolítico-Urêmica/genética , Proteína Cofatora de Membrana/genética , Mutação , Transfusão de Componentes Sanguíneos , Complemento C3b/genética , Complemento C3b/metabolismo , Fator H do Complemento/biossíntese , Fator I do Complemento/biossíntese , Feminino , Frequência do Gene/genética , Genótipo , Síndrome Hemolítico-Urêmica/metabolismo , Síndrome Hemolítico-Urêmica/terapia , Humanos , Transplante de Rim , Masculino , Proteína Cofatora de Membrana/biossíntese , Plasma , Ligação Proteica/genética , Biossíntese de Proteínas/genética , Recidiva , Toxina Shiga , Resultado do TratamentoRESUMO
Endothelial injury is the central factor in the events leading to thrombotic microangiopathy (TMA); however, the mechanisms involved are not fully understood. Here we investigate the role of neutrophils (PMNs) and of complement activation in inducing microvascular damage and loss of thromboresistance in TMA associated with ADAMTS-13 deficiency. PMNs isolated during the acute phase of the disease released excessive amounts of reactive-oxygen species (ROS), N-derived oxidants and proteinases and induced damage and thromboresistance loss in human microvascular endothelial cell line (HMEC-1) ex vivo. Endothelial cytotoxicity and thromboresistance loss was also induced by TMA serum. Complement-derived products were responsible for the above effects: in fact, TMA serum caused C3 and Membrane Attack Complex (MAC) deposition on HMEC-1 and its cytotoxic effect was abolished by complement inhibition. TMA serum caused surface expression of P-selectin on HMEC-1 which may promote PMN adhesion and resulted in increased PMN cytotoxicity, indicating that complement may have a role in PMN activation. In addition, TMA serum stimulated control PMNs to release ROS and proteinases, and to cause endothelial cell cytotoxicity. All of the above effects were abrogated by complement inactivation. These data document for the first time that complement-initiated PMN activation and endothelial injury may have a crucial role in microvascular thrombosis of TMA associated with ADAMTS-13 deficiency.
Assuntos
Proteínas ADAM/deficiência , Ativação do Complemento/fisiologia , Síndrome Hemolítico-Urêmica/etiologia , Púrpura Trombocitopênica Trombótica/etiologia , Proteína ADAMTS13 , Linhagem Celular , Endotélio Vascular/patologia , Humanos , Ativação de Neutrófilo/imunologia , Oxidantes/sangue , Peptídeo Hidrolases/sangue , Espécies Reativas de Oxigênio/sangueRESUMO
BACKGROUND: During hemodialysis, vascular reactivity is impaired, which can be corrected by lowering dialysate temperature. It has also been shown that nitric oxide (NO) is related to intradialytic hypotension. As NO synthesis may be temperature-dependent, this study addressed the influence of dialysate temperature on the NO synthetic capacity of plasma. METHODS: NO synthetic capacity was studied during hemodialysis with a dialysate temperature of 37.5 degrees C (dialysis-37.5 degrees C) and programmed extracorporeal blood cooling (cool dialysis; Blood Temperature Monitor; Fresenius C) in 12 stable patients. NO synthetic capacity was assessed ex vivo by [3H]L-citrulline formation from [3H]L-arginine in cultured endothelial cells after incubation with plasma samples obtained during the respective sessions. RESULTS: Core temperature decreased (-0.32 +/- 0.10 degrees C) and energy transfer rate was significantly lower (-27.5 +/- 2.8 W; p < 0.05) during cool dialysis compared to dialysis-37.5 degrees C (0.19 +/- 0.06 degrees C and -0.8 +/- 1.2 W respectively; p < 0.05). Systolic blood pressure decreased during dialysis-37.5 degrees C (-19 +/- 4 mm Hg; p < 0.05), but not during cool dialysis (-6 +/- 5 mm Hg). NO synthetic capacity increased during dialysis-37.5 degrees C (55.5 +/- 9.3 to 73.5 +/- 10.2 pmol/10(5) cells; p < 0.05), in contrast to cool dialysis (67.3 +/- 11.1 to 66.2 +/- 10.8 pmol/10(5) cells). CONCLUSION: The stimulatory effect of uremic plasma on endothelial NO synthesis was augmented during dialysis-37.5 degrees C but not during cool dialysis.
Assuntos
Óxido Nítrico Sintase/metabolismo , Óxido Nítrico/biossíntese , Diálise Renal/efeitos adversos , Temperatura , Idoso , Arginina/análise , Arginina/metabolismo , Endotélio Vascular/enzimologia , Transferência de Energia , Feminino , Hemodinâmica , Humanos , Nefropatias/enzimologia , Nefropatias/metabolismo , Nefropatias/terapia , Masculino , Pessoa de Meia-Idade , Óxido Nítrico Sintase Tipo IIIRESUMO
Less nitric oxide (NO)-dependent vasodilation and excess formation of reactive oxygen species could explain poor placenta perfusion in preeclampsia, but the pathways involved are unknown. We tested the hypothesis that reduced NO activity and increased oxidative stress in preeclamptic placenta is related to a low bioavailability of l-arginine. Placental endothelial NO synthase (ecNOS) expression (by immunoperoxidase) and activity (by diaphorase and [(3)H]L-citrulline formation) were comparable in normotensive pregnancy and in preeclampsia, whereas nitrotyrosine staining, a marker of peroxynitrite, was stronger in preeclamptic villi, confirming previously reported data. Oxidative tissue damage was documented in preeclamptic villi by strong 4-hydroxynonenal-lysine staining (by immunoperoxidase), which closely colocalized with nitrotyrosine. Concentration of the NO precursor l-arginine (by HPLC) in umbilical blood and in villous tissue was lower in preeclampsia than in normotensive pregnancy. This was not caused by a defective l-arginine transport, because gene expression of the CAT-1, 4F2hc, and LAT-1 cationic amino acid transporters (by real-time reverse-transcription polymerase chain reaction [RT-PCR]) was normal. Instead, gene expression (by real-time RT-PCR) and protein tissue content (by immunoperoxidase and Western blot) of arginase II-the enzyme that degrades arginine to ornithine-were higher in preeclamptic villi than in normotensive pregnancy. These results provide a biochemical explanation for defective NO activity and increased oxidative stress in preeclamptic placenta. In normal placenta, adequate concentration of l-arginine orients ecNOS toward NO. In preeclampsia, a lower than normal l-arginine concentration caused by arginase II overexpression redirects ecNOS toward peroxynitrite.
Assuntos
Arginina/metabolismo , Óxido Nítrico Sintase/metabolismo , Ácido Peroxinitroso/biossíntese , Pré-Eclâmpsia/enzimologia , Adulto , Sistemas de Transporte de Aminoácidos Básicos/metabolismo , Arginase/metabolismo , Feminino , Humanos , Peroxidação de Lipídeos , Troca Materno-Fetal , NADPH Desidrogenase/análise , Óxido Nítrico Sintase/análise , Óxido Nítrico Sintase Tipo III , Estresse Oxidativo , Placenta/enzimologia , Placenta/metabolismo , Pré-Eclâmpsia/metabolismo , Pré-Eclâmpsia/patologia , Gravidez , Espécies Reativas de Nitrogênio/metabolismoRESUMO
BACKGROUND: Mutations in factor H (HF1) have been reported in a consistent number of diarrhoea-negative, non-Shiga toxin-associated cases of haemolytic uraemic syndrome (D-HUS). However, most patients with D-HUS have no HF1 mutations, despite decreased serum concentrations of C3. Our aim, therefore, was to assess whether genetic abnormalities in other complement regulatory proteins are involved. METHODS: We screened genes that encode the complement regulatory proteins-ie, factor H related 5, complement receptor 1, and membrane cofactor protein (MCP)-by PCR-single-strand conformation polymorphism (PCR-SSCP) and by direct sequencing, in 25 consecutive patients with D-HUS, an abnormal complement profile, and no HF1 mutation, from our International Registry of Recurrent and Familial HUS/TTP (HUS/thrombotic thrombocytopenic purpura). FINDINGS: We identified a heterozygous mutation in MCP, a surface-bound complement regulator, in two patients with a familial history of HUS. The mutation causes a change in three aminoacids at position 233-35 and insertion of a premature stop-codon, which results in loss of the transmembrane domain of the protein and severely reduced cell-surface expression of MCP. INTERPRETATION: Results of previous studies on HF1 indicate an association between HF1 deficiency and D-HUS. Our findings of an MCP mutation in two related patients suggest that impaired regulation of complement activation might be a factor in the pathogenesis of genetic forms of HUS. MCP could be a second putative candidate gene for D-HUS. The protein is highly expressed in the kidney and plays a major part in regulation of glomerular C3 activation. We propose, therefore, that reduced expression of MCP in response to complement-activating stimuli could prevent restriction of complement deposition on glomerular endothelial cells, leading to microvascular cell damage and tissue injury.