RESUMO
Genetic alterations in the complement system have been linked to a variety of diseases, including atypical hemolytic uremic syndrome (aHUS), C3 glomerulopathy (C3G), and age-related macular degeneration (AMD). We performed sequence analysis of the complement genes complement factor H (CFH), complement factor I (CFI), and complement C3 (C3) in 866 aHUS/C3G and 697 AMD patients. In total, we identified 505 low-frequency alleles, representing 121 unique variants, of which 51 are novel. CFH contained the largest number of unique low-frequency variants (n = 64; 53%), followed by C3 (n = 32; 26%) and CFI (n = 25; 21%). A substantial number of variants were found in both patients groups (n = 48; 40%), while 41 (34%) variants were found only in aHUS/C3G and 32 (26%) variants were AMD specific. Genotype-phenotype correlations between the disease groups identified a higher frequency of protein altering alleles in short consensus repeat 20 (SCR20) of factor H (FH), and in the serine protease domain of factor I (FI) in aHUS/C3G patients. In AMD, a higher frequency of protein-altering alleles was observed in SCR3, SCR5, and SCR7 of FH, the SRCR domain of FI, and in the MG3 domain of C3. In conclusion, we observed a substantial overlap of variants between aHUS/C3G and AMD; however, there is a distinct clustering of variants within specific domains.
Assuntos
Síndrome Hemolítico-Urêmica Atípica/genética , Complemento C3/genética , Fator I do Complemento/genética , Genótipo , Glomerulonefrite Membranosa/genética , Degeneração Macular/genética , Fenótipo , Síndrome Hemolítico-Urêmica Atípica/metabolismo , Síndrome Hemolítico-Urêmica Atípica/fisiopatologia , Estudos de Coortes , Complemento C3/metabolismo , Fator H do Complemento/genética , Fator H do Complemento/metabolismo , Fator I do Complemento/metabolismo , Predisposição Genética para Doença , Glomerulonefrite Membranosa/metabolismo , Glomerulonefrite Membranosa/fisiopatologia , Humanos , Degeneração Macular/metabolismo , Degeneração Macular/fisiopatologiaRESUMO
Atypical haemolytic uraemic syndrome (aHUS) is associated with (genetic) alterations in alternative complement pathway. Nevertheless, comprehensive evidence that the complement system in aHUS patients is more prone to activation is still lacking. Therefore, we performed a thorough analysis of complement activation in acute phase and in remission of this disease. Complement activation patterns of the aHUS patients in acute phase and in remission were compared to those of healthy controls. Background levels of complement activation products C3b/c, C3bBbP and terminal complement complex (TCC) were measured using enzyme-linked immunosorbent assay (ELISA) in ethylenediamine tetraacetic acid (EDTA) plasma. In vitro-triggered complement activation in serum samples was studied using zymosan-coating and pathway-specific assay. Furthermore, efficiencies of the C3b/c, C3bBbP and TCC generation in fluid phase during spontaneous activation were analysed. Patients with acute aHUS showed elevated levels of C3b/c (P < 0·01), C3bBbP (P < 0·0001) and TCC (P < 0·0001) in EDTA plasma, while values of patients in remission were normal, compared to those of healthy controls. Using data from a single aHUS patient with complement factor B mutation we illustrated normalization of complement activation during aHUS recovery. Serum samples from patients in remission showed normal in vitro patterns of complement activation and demonstrated normal kinetics of complement activation in the fluid phase. Our data indicate that while aHUS patients have clearly activated complement in acute phase of the disease, this is not the case in remission of aHUS. This knowledge provides important insight into complement regulation in aHUS and may have an impact on monitoring of these patients, particularly when using complement inhibition therapy.
Assuntos
Síndrome Hemolítico-Urêmica Atípica/imunologia , Ativação do Complemento , Complemento C3b/metabolismo , Complexo de Ataque à Membrana do Sistema Complemento/metabolismo , Doença Aguda , Adolescente , Adulto , Síndrome Hemolítico-Urêmica Atípica/sangue , Síndrome Hemolítico-Urêmica Atípica/patologia , Síndrome Hemolítico-Urêmica Atípica/terapia , Estudos de Casos e Controles , Criança , Pré-Escolar , Ativação do Complemento/efeitos dos fármacos , Fator B do Complemento/metabolismo , Fator H do Complemento/metabolismo , Via Alternativa do Complemento/efeitos dos fármacos , Feminino , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Plasma Rico em Plaquetas , Isoformas de Proteínas/sangue , Indução de Remissão , Diálise Renal , Zimosan/farmacologiaRESUMO
Promising renal replacement therapies include the development of a bioartificial kidney using functional human kidney cell models. In this study, human conditionally immortalized proximal tubular epithelial cell (ciPTEC) lines originating from kidney tissue (ciPTEC-T1 and ciPTEC-T2) were compared to ciPTEC previously isolated from urine (ciPTEC-U). Subclones of all ciPTEC isolates formed tight cell layers on Transwell inserts as determined by transepithelial resistance, inulin diffusion, E-cadherin expression and immunocytochemisty. Extracellular matrix genes collagen I and -IV α1 were highly present in both kidney tissue derived matured cell lines (p<0.001) compared to matured ciPTEC-U, whereas matured ciPTEC-U showed a more pronounced fibronectin I and laminin 5 gene expression (p<0.01 and p<0.05, respectively). Expression of the influx carrier Organic Cation Transporter 2 (OCT-2), and the efflux pumps P-glycoprotein (P-gp), Multidrug Resistance Protein 4 (MRP4) and Breast Cancer Resistance Protein (BCRP) were confirmed in the three cell lines using real-time PCR and Western blotting. The activities of OCT-2 and P-gp were sensitive to specific inhibition in all models (p<0.001). The highest activity of MRP4 and BCRP was demonstrated in ciPTEC-U (p<0.05). Finally, active albumin reabsorption was highest in ciPTEC-T2 (p<0.001), while Na(+)-dependent phosphate reabsorption was most abundant in ciPTEC-U (p<0.01). In conclusion, ciPTEC established from human urine or kidney tissue display comparable functional PTEC specific transporters and physiological characteristics, providing ideal human tools for bioartificial kidney development.
Assuntos
Órgãos Bioartificiais , Túbulos Renais Proximais/citologia , Rins Artificiais , Urina/citologia , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/antagonistas & inibidores , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/biossíntese , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/biossíntese , Transportadores de Cassetes de Ligação de ATP/metabolismo , Caderinas/biossíntese , Moléculas de Adesão Celular/biossíntese , Técnicas de Cultura de Células , Linhagem Celular , Colágeno Tipo I/biossíntese , Colágeno Tipo I/metabolismo , Fibronectinas/biossíntese , Humanos , Inulina/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/biossíntese , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/metabolismo , Fator 2 de Transcrição de Octâmero/antagonistas & inibidores , Fator 2 de Transcrição de Octâmero/biossíntese , Fator 2 de Transcrição de Octâmero/metabolismo , Engenharia Tecidual , Migração Transendotelial e Transepitelial/fisiologia , CalininaRESUMO
During chronic kidney disease (CKD), drug metabolism is affected leading to changes in drug disposition. Furthermore, there is a progressive accumulation of uremic retention solutes due to impaired renal clearance. Here, we investigated whether uremic toxins can influence the metabolic functionality of human conditionally immortalized renal proximal tubule epithelial cells (ciPTEC) with the focus on UDP-glucuronosyltransferases (UGTs) and mitochondrial activity. Our results showed that ciPTEC express a wide variety of metabolic enzymes, including UGTs. These enzymes were functionally active as demonstrated by the glucuronidation of 7-hydroxycoumarin (7-OHC; K(m) of 12±2µM and a V(max) of 76±3pmol/min/mg) and p-cresol (K(m) of 33±13µM and a V(max) of 266±25pmol/min/mg). Furthermore, a wide variety of uremic toxins, including indole-3-acetic acid, indoxyl sulfate, phenylacetic acid and kynurenic acid, reduced 7-OHC glucuronidation with more than 30% as compared with controls (p<0.05), whereas UGT1A and UGT2B protein expressions remained unaltered. In addition, our results showed that several uremic toxins inhibited mitochondrial succinate dehydrogenase (i.e. complex II) activity with more than 20% as compared with controls (p<0.05). Moreover, indole-3-acetic acid decreased the reserve capacity of the electron transport system with 18% (p<0.03). In conclusion, this study shows that multiple uremic toxins inhibit UGT activity and mitochondrial activity in ciPTEC, thereby affecting the metabolic capacity of the kidney during CKD. This may have a significant impact on drug and uremic retention solute disposition in CKD patients.
Assuntos
Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/metabolismo , Rim/metabolismo , Mitocôndrias/metabolismo , Uremia/metabolismo , Linhagem Celular , Cresóis/metabolismo , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/enzimologia , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/genética , Transporte de Elétrons , Glucuronosiltransferase/genética , Glucuronosiltransferase/metabolismo , Humanos , Rim/enzimologia , Mitocôndrias/enzimologia , Mitocôndrias/genética , Preparações Farmacêuticas/metabolismo , Succinato Desidrogenase/genética , Succinato Desidrogenase/metabolismo , Umbeliferonas/metabolismo , Uremia/enzimologia , Uremia/genéticaAssuntos
Anticorpos Monoclonais Humanizados/farmacologia , Síndrome Hemolítico-Urêmica Atípica/tratamento farmacológico , Proteínas do Sistema Complemento/análise , Hemoglobinúria Paroxística/tratamento farmacológico , Complicações Hematológicas na Gravidez/tratamento farmacológico , Anticorpos Monoclonais Humanizados/sangue , Anticorpos Monoclonais Humanizados/uso terapêutico , Proteínas do Sistema Complemento/metabolismo , Feminino , Sangue Fetal/química , Humanos , Lactente , Recém-Nascido , Troca Materno-Fetal , Circulação Placentária , GravidezRESUMO
Complement convertases are enzymatic complexes that play a central role in sustaining and amplification of the complement cascade. Impairment of complement function leads directly or indirectly to pathological conditions, including higher infection rate, kidney diseases, autoimmune- or neurodegenerative diseases and ischaemia-reperfusion injury. An assay for direct measurement of activity of the convertases in patient sera is not available. Existing assays testing convertase function are based on purified complement components and, thus, convertase formation occurs under non-physiological conditions. We designed a new assay, in which C5 blocking compounds enabled separation of the complement cascade into two phases: the first ending at the stage of C5 convertases and the second ending with membrane attack complex formation. The use of rabbit erythrocytes or antibody-sensitized sheep erythrocytes as the platforms for convertase formation enabled easy readout based on measurement of haemolysis. Thus, properties of patient sera could be studied directly regarding convertase activity and membrane attack complex formation. Another advantage of this assay was the possibility to screen for host factors such as C3 nephritic factor and other anti-complement autoantibodies, or gain-of-function mutations, which prolong the half-life of complement convertases. Herein, we present proof of concept, detailed description and validation of this novel assay.
Assuntos
Convertases de Complemento C3-C5/análise , Eritrócitos/enzimologia , Imunoensaio/métodos , Animais , Autoanticorpos/imunologia , Fator Nefrítico do Complemento 3/imunologia , Convertases de Complemento C3-C5/imunologia , Via Alternativa do Complemento/imunologia , Proteínas do Sistema Complemento/imunologia , Eritrócitos/imunologia , Cobaias , Meia-Vida , Humanos , Coelhos , OvinosRESUMO
Hyperhomocysteinaemia has been identified as a risk factor for cerebrovascular, peripheral vascular and coronary heart disease. Elevated levels of plasma homocysteine can result from genetic or nutrient-related disturbances in the trans-sulphuration or re-methylation pathways for homocysteine metabolism. 5, 10-Methylenetetrahydrofolate reductase (MTHFR) catalyzes the reduction of 5, 10-methylenetetrahydrofolate to 5-methyltetrahydrofolate, the predominant circulatory form of folate and carbon donor for the re-methylation of homocysteine to methionine. Reduced MTHFR activity with a thermolabile enzyme has been reported in patients with coronary and peripheral artery disease. We have identified a common mutation in MTHFR which alters a highly-conserved amino acid; the substitution occurs at a frequency of approximately 38% of unselected chromosomes. The mutation in the heterozygous or homozygous state correlates with reduced enzyme activity and increased thermolability in lymphocyte extracts; in vitro expression of a mutagenized cDNA containing the mutation confirms its effect on thermolability of MTHFR. Finally, individuals homozygous for the mutation have significantly elevated plasma homocysteine levels. This mutation in MTHFR may represent an important genetic risk factor in vascular disease.
Assuntos
Mutação , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/deficiência , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/genética , Doenças Vasculares/genética , Adulto , Sequência de Bases , DNA Complementar , Estabilidade Enzimática , Escherichia coli/metabolismo , Feminino , Homocisteína/metabolismo , Humanos , Rim/metabolismo , Fígado/metabolismo , Linfócitos/metabolismo , Masculino , Metilenotetra-Hidrofolato Redutase (NADPH2) , Pessoa de Meia-Idade , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Quebeque , Fatores de Risco , Temperatura , Doenças Vasculares/epidemiologiaRESUMO
Primary hypomagnesaemia is composed of a heterogeneous group of disorders characterized by renal or intestinal Mg(2+) wasting, often associated with disturbances in Ca(2+) excretion. We identified a putative dominant-negative mutation in the gene encoding the Na(+), K(+)-ATPase gamma-subunit (FXYD2), leading to defective routing of the protein in a family with dominant renal hypomagnesaemia.
Assuntos
Túbulos Renais Distais/metabolismo , Deficiência de Magnésio/genética , Magnésio/metabolismo , ATPase Trocadora de Sódio-Potássio/deficiência , Processamento Alternativo , Sequência de Aminoácidos , Animais , Células COS , Chlorocebus aethiops , Cromossomos Humanos Par 11/genética , DNA Complementar/genética , Genes Dominantes , Vetores Genéticos , Humanos , Deficiência de Magnésio/sangue , Mamíferos/metabolismo , Camundongos , Dados de Sequência Molecular , Nucleopoliedrovírus/genética , Subunidades Proteicas , Transporte Proteico , Ratos , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência , ATPase Trocadora de Sódio-Potássio/química , ATPase Trocadora de Sódio-Potássio/genética , ATPase Trocadora de Sódio-Potássio/metabolismo , Especificidade da Espécie , Spodoptera/citologia , Spodoptera/metabolismo , TransfecçãoRESUMO
Introduction: Since 2016, kidney transplantation in patients with atypical hemolytic uremic syndrome (aHUS) in the Netherlands is performed without eculizumab prophylaxis. Eculizumab is given in case of posttransplant aHUS recurrence. Eculizumab therapy is monitored in the CUREiHUS study. Methods: All participating kidney transplant patients who received eculizumab therapy for a suspected posttransplant aHUS recurrence were evaluated. Overall recurrence rate was monitored prospectively at Radboud University Medical Center. Results: In the period from January 2016 until October 2020, we included 15 (12 females, 3 males; median age 42 years, range 24-66 years) patients with suspected aHUS recurrence after kidney transplantation in this study. The time interval to recurrence showed a bimodal distribution. Seven patients presented early after transplantation (median 3 months, range 0.3-8.8 months), with typical aHUS features: rapid loss of estimated glomerular filtration rate (eGFR) and laboratory signs of thrombotic microangiopathy (TMA). Eight patients presented late (median 46 months, range 18-69 months) after transplantation. Of these, only 3 patients had systemic TMA, whereas 5 patients presented with slowly deteriorating eGFR without systemic TMA. Treatment with eculizumab resulted in improvement or stabilization of eGFR in 14 patients. Eculizumab discontinuation was tried in 7 patients; however, it was successful only in 3. At the end of the follow-up (median 29 months, range 3-54 months after start of eculizumab), 6 patients had eGFR <30 ml/min per 1.73 m2. Graft loss had occurred in 3 of them. Overall, aHUS recurrence rate without eculizumab prophylaxis was 23%. Conclusions: Rescue treatment of posttransplant aHUS recurrence is effective; however, some patients suffer from irreversible loss of kidney function, likely caused by delayed diagnosis and treatment and/or too aggressive discontinuation of eculizumab. Physicians should be aware that recurrence of aHUS can present without evidence of systemic TMA.
RESUMO
Mitochondrial complex I is the largest multi-protein enzyme complex of the oxidative phosphorylation system. Seven subunits of this complex are encoded by the mitochondrial and the remainder by the nuclear genome. We review the natural disease course and signs and symptoms of 130 patients (four new cases and 126 from literature) with mutations in nuclear genes encoding structural complex I proteins or those involved in its assembly. Complex I deficiency caused by a nuclear gene defect is usually a non-dysmorphic syndrome, characterized by severe multi-system organ involvement and a poor prognosis. Age at presentation may vary, but is generally within the first year of life. The most prevalent symptoms include hypotonia, nystagmus, respiratory abnormalities, pyramidal signs, dystonia, psychomotor retardation or regression, failure to thrive, and feeding problems. Characteristic symptoms include brainstem involvement, optic atrophy and Leigh syndrome on MRI, either or not in combination with internal organ involvement and lactic acidemia. Virtually all children ultimately develop Leigh syndrome or leukoencephalopathy. Twenty-five percent of the patients died before the age of six months, more than half before the age of two and 75 % before the age of ten years. Some patients showed recovery of certain skills or are still alive in their thirties . No clinical, biochemical, or genetic parameters indicating longer survival were found. No clear genotype-phenotype correlations were observed, however defects in some genes seem to be associated with a better or poorer prognosis, cardiomyopathy, Leigh syndrome or brainstem lesions.
Assuntos
Núcleo Celular/genética , Doenças Mitocondriais/genética , Mutação , Complexo I de Transporte de Elétrons/deficiência , Complexo I de Transporte de Elétrons/genética , Estudos de Associação Genética , Humanos , Mitocôndrias/genéticaRESUMO
A 34-year-old man of North African descent was referred to the emergency department because of malignant hypertension (220/113 mmHg), acute visual disturbances and acute kidney failure (serum creatinine 14.0 mg/dL). Blood analysis was compatible with thrombotic microangiopathy (TMA). Kidney biopsy confirmed this diagnosis with histological changes including intimal edema, arteriolar thrombi, and severe tubulointerstitial damage. Fundoscopy showed hypertensive retinopathy stage IV. Subsequent biochemical screening revealed normal complement testing and a marked elevation in homocysteine concentration (161 µmol/L; normal value 7-15 µmol/L). Other secondary causes of TMA were excluded. Further genetic testing for cobalamin C (cblC) deficiency showed no pathogenic mutations in the MMACHC gene. However, a homozygous c.665C>T polymorphism (NM_005957.4) in the methylenetetrahydrofolate reductase (MTHFR) gene was found explaining the severe hyperhomocysteinemia due to reduced activity of MTHFR. Additional genetic testing for alternative complement pathway proteins showed mutations in the genes encoding factor H and factor B, both categorized as possibly pathogenic using mutation prediction software. This is the first described case of TMA in a patient with severe hyperhomocysteinemia caused by a genetic defect other than cblC. We postulate that endothelial damage due to hyperhomocysteinemia and hypertension could have triggered the TMA episode in this patient with two possible predisposing pathogenic mutations in the alternative complement pathway. Furthermore, our case demonstrates the need for complete full diagnostic testing in patients with TMA.
Assuntos
Hiper-Homocisteinemia , Microangiopatias Trombóticas , Injúria Renal Aguda/diagnóstico , Injúria Renal Aguda/etiologia , Injúria Renal Aguda/patologia , Adulto , Humanos , Hipertensão/diagnóstico , Hipertensão/etiologia , Rim/patologia , Masculino , Metilenotetra-Hidrofolato Redutase (NADPH2)/genética , Oxirredutases/genética , Complexo Vitamínico B/uso terapêuticoRESUMO
The oxidative phosphorylation (OXPHOS) system, comprising five enzyme complexes, is located in the inner membrane of mitochondria and is the final biochemical pathway in oxidative ATP production. Defects in this energy-generating system can cause a wide range of clinical symptoms; these diseases are often progressive and multisystemic. Numerous genes have been implicated in OXPHOS deficiencies and many mutations have been described. However, in a substantial number of patients with decreased enzyme activities of two or more OXPHOS complexes, no mutations in the mitochondrial DNA or in nuclear genes known to be involved in these disorders have been found. In this study, four nuclear candidate genes--NIPSNAP1, GBAS, CHCHD1 and METT11D1--were screened for mutations in 22 patients with a combined enzymatic deficiency of primarily the OXPHOS complexes I, III and IV to determine whether a mutation in one of these genes could explain the mitochondrial disorder. For each variant not yet reported as a polymorphism, 100 control samples were screened for the presence of the variant. This way we identified 14 new polymorphisms and 2 presumably non-pathogenic mutations. No mutations were found that could explain the mitochondrial disorder in the patients investigated in this study. Therefore, the genetic defect in these patients must be located in other nuclear genes involved in mtDNA maintenance, transcription or translation, in import, processing or degradation of nuclear encoded mitochondrial proteins, or in assembly of the OXPHOS system.
Assuntos
Metabolismo Energético/genética , Proteínas de Membrana/genética , Erros Inatos do Metabolismo/genética , Metiltransferases/genética , Doenças Mitocondriais/genética , Mutação , Proteínas Nucleares/genética , Fosfoproteínas/genética , Polimorfismo Genético , Proteínas/genética , Estudos de Casos e Controles , Células Cultivadas , Análise Mutacional de DNA , Fibroblastos/enzimologia , Estudos de Associação Genética , Predisposição Genética para Doença , Testes Genéticos/métodos , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas de Membrana/metabolismo , Erros Inatos do Metabolismo/enzimologia , Doenças Mitocondriais/enzimologia , Dados de Sequência Molecular , Músculo Esquelético/enzimologia , Proteínas Nucleares/metabolismo , Fosforilação Oxidativa , Fenótipo , Fosfoproteínas/metabolismo , Proteínas/metabolismoRESUMO
Shiga-toxin (Stx)-producing Escherichia coli hemolytic-uremic syndrome (STEC-HUS) is one of the most common causes of acute kidney injury in children. Stx-mediated endothelial injury initiates the cascade leading to thrombotic microangiopathy (TMA), still the exact pathogenesis remains elusive. Interestingly, there is wide variability in clinical presentation and outcome. One explanation for this could be the enhancement of TMA through other factors. We hypothesize that heme, as released during extensive hemolysis, contributes to the etiology of TMA. Plasma levels of heme and its scavenger hemopexin and degrading enzyme heme-oxygenase-1 (HO-1) were measured in 48 STEC-HUS patients. Subsequently, the effect of these disease-specific heme concentrations, in combination with Stx, was assessed on primary human glomerular microvascular endothelial cells (HGMVECs). Significantly elevated plasma heme levels up to 21.2 µM were found in STEC-HUS patients compared to controls and were inversely correlated with low or depleted plasma hemopexin levels (R2 -0.74). Plasma levels of HO-1 are significantly elevated compared to controls. Interestingly, especially patients with high heme levels (n = 12, heme levels above 75 quartile range) had high plasma HO-1 levels with median of 332.5 (86-720) ng/ml (p = 0.008). Furthermore, heme is internalized leading to a significant increase in reactive oxygen species production and stimulated both nuclear translocation of NF-κB and increased levels of its target gene (tissue factor). In conclusion, we are the first to show elevated heme levels in patients with STEC-HUS. These increased heme levels mediate endothelial injury by promoting oxidative stress and a pro-inflammatory and pro-thrombotic state. Hence, heme may be a contributing and driving factor in the pathogenesis of STEC-HUS and could potentially amplify the cascade leading to TMA.
Assuntos
Suscetibilidade a Doenças , Heme/metabolismo , Síndrome Hemolítico-Urêmica/etiologia , Síndrome Hemolítico-Urêmica/metabolismo , Escherichia coli Shiga Toxigênica/fisiologia , Apoptose , Biomarcadores , Criança , Pré-Escolar , Células Endoteliais/metabolismo , Feminino , Heme Oxigenase-1/metabolismo , Síndrome Hemolítico-Urêmica/diagnóstico , Síndrome Hemolítico-Urêmica/terapia , Humanos , Lactente , Masculino , Oxirredução , Fenótipo , Transporte Proteico , Espécies Reativas de Oxigênio/metabolismo , Estresse Fisiológico , Tromboplastina/metabolismoRESUMO
This review deals with podocyte proteins that play a significant role in the structure and function of the glomerular filter. Genetic linkage studies has identified several genes involved in the development of nephrotic syndrome and contributed to the understanding of the pathophysiology of glomerular proteinuria and/or focal segmental glomerulosclerosis. Here, we describe already well-characterized genetic diseases due to mutations in nephrin, podocin, CD2AP, alpha-actinin-4, WT1, and laminin beta2 chain, as well as more recently identified genetic abnormalities in TRPC6, phospholipase C epsilon, and the proteins encoded by the mitochondrial genome. In addition, the role of the proteins which have shown to be important for the structure and functions by gene knockout studies in mice, are also discussed. Furthermore, some rare syndromes with glomerular involvement, in which molecular defects have been recently identified, are briefly described. In summary, this review updates the current knowledge of genetic causes of congenital and childhood nephrotic syndrome and provides new insights into mechanisms of glomerular dysfunction.
Assuntos
Marcadores Genéticos/genética , Glomerulosclerose Segmentar e Focal/genética , Glomérulos Renais/fisiopatologia , Podócitos , Actinina/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas do Citoesqueleto/genética , Genoma , Glomerulosclerose Segmentar e Focal/fisiopatologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Laminina/genética , Proteínas de Membrana/genética , Mutação , Fosfoinositídeo Fosfolipase C/genética , Proteinúria/genética , Canais de Cátion TRPC/genética , Canal de Cátion TRPC6 , Proteínas WT1/genéticaRESUMO
PURPOSE: To identify the biochemical and molecular genetic defect in a 16-year-old patient presenting with apical hypertrophic cardiomyopathy and neuropathy suspected for a mitochondrial disorder. METHODS: Measurement of the mitochondrial energy-generating system (MEGS) capacity in muscle and enzyme analysis in muscle and fibroblasts were performed. Relevant parts of the mitochondrial DNA were analysed by sequencing. Transmitochondrial cybrids were obtained by fusion of 143B206 TK(-) rho zero cells with patient-derived enucleated fibroblasts. Immunoblotting techniques were applied to study the complex V assembly. RESULTS: A homoplasmic nonsense mutation m.8529G-->A (p.Trp55X) was found in the mitochondrial ATP8 gene in the patient's fibroblasts and muscle tissue. Reduced complex V activity was measured in the patient's fibroblasts and muscle tissue, and was confirmed in cybrid clones containing patient-derived mitochondrial DNA. Immunoblotting after blue native polyacrylamide gel electrophoresis showed a lack of holocomplex V and increased amounts of mitochondrial ATP synthase subcomplexes. An in-gel activity assay of ATP hydrolysis showed activity of free F(1)-ATPase in the patient's muscle tissue and in the cybrid clones. CONCLUSION: We describe the first pathogenic mutation in the mitochondrial ATP8 gene, resulting in an improper assembly and reduced activity of the complex V holoenzyme.
Assuntos
Cardiomiopatia Hipertrófica/enzimologia , Cardiomiopatia Hipertrófica/genética , Códon sem Sentido , Genes Mitocondriais , ATPases Mitocondriais Próton-Translocadoras/deficiência , ATPases Mitocondriais Próton-Translocadoras/genética , Doenças do Sistema Nervoso/enzimologia , Doenças do Sistema Nervoso/genética , Adolescente , Sequência de Aminoácidos , Sequência de Bases , Primers do DNA/genética , Humanos , Células Híbridas , Masculino , Doenças Mitocondriais/enzimologia , Doenças Mitocondriais/genética , ATPases Mitocondriais Próton-Translocadoras/química , Dados de Sequência Molecular , Homologia de Sequência de AminoácidosRESUMO
C3 glomerulopathy is a rare renal disease that has been distinguished as a renal disease for about 10 years. It is caused by an excessive activation of the alternative complement pathway in the circulation, which leads to deposition of complement factor C3 in glomeruli. It is diagnosed based on clinical presentation, histological patterns in a kidney biopsy and tests of the complement pathways. It can closely resemble immune complexmediated glomerulonephritis and postinfectious glomerulonephritis. Renal failure develops in up to half of all patients within 10 years after presentation. A curative treatment is not available. Treatment relies on renoprotective measures, occasional immunosuppressive medication and experimental novel complement inhibitors. Because the disease is rare, its care and cure are concentrated in centers of expertise. Here we provide an overview of the state-ofthe-art diagnosis and treatment of C3 glomerulopathy in a center of expertise in the Netherlands.
Assuntos
Ativação do Complemento/fisiologia , Complemento C3/metabolismo , Glomerulonefrite/diagnóstico , Glomerulonefrite/tratamento farmacológico , Glomérulos Renais/metabolismo , Via Alternativa do Complemento/imunologia , Via Alternativa do Complemento/fisiologia , Glomerulonefrite/imunologia , Humanos , Rim/patologia , Glomérulos Renais/patologiaRESUMO
We report a 5-year-old child carrying polymerase gamma (POLG1) mutations, but strikingly normal oxidative phosphorylation analysis in muscle, fibroblasts and liver. Mutations in POLG1 have so far been described in children with severe combined oxidative phosphorylation (OXPHOS) deficiencies and with the classical Alpers-Huttenlocher syndrome. The patient presented with a delayed psychomotor development and ataxia during the first two years of life. From the third year of life he developed epilepsy and regression in development, together with symptoms of visual impairment and sensorineuronal deafness. Cerebrospinal fluid showed elevated lactic acid and protein concentrations. An elder brother had died due to combined OXPHOS deficiencies. Despite the clinical similarity with the elder brother, except for liver involvement, the OXPHOS system analysis in a frozen muscle biopsy was normal. For this reason a fresh muscle biopsy was performed, which has the advantage of the possibility of measuring the substrate oxidation rates and ATP production, part of the mitochondrial energy-generating system (MEGS). During the same session, biopsies of liver and fibroblasts were taken. These three tissues showed normal measurements of the MEGS capacity. Based on the phenotype of Alpers-Huttenlocher syndrome in the elder brother, we decided to screen the POLG1 gene. Mutation analysis showed compound heterozygosity with two known mutations, A467T and G848S. The normal MEGS capacity in this patient expands the already existing complexity and heterogeneity of the childhood POLG1 patients and, on the basis of the high frequency of POLG1 mutations in childhood, warrants a liberal strategy with respect to mutation analysis.
Assuntos
DNA Polimerase Dirigida por DNA/genética , Esclerose Cerebral Difusa de Schilder/diagnóstico , Fibroblastos/enzimologia , Fígado/enzimologia , Músculo Esquelético/enzimologia , Mutação , Fosforilação Oxidativa , Biomarcadores/sangue , Biomarcadores/urina , Biópsia , Pré-Escolar , Análise Mutacional de DNA , DNA Polimerase gama , Esclerose Cerebral Difusa de Schilder/complicações , Esclerose Cerebral Difusa de Schilder/enzimologia , Esclerose Cerebral Difusa de Schilder/genética , Progressão da Doença , Predisposição Genética para Doença , Heterozigoto , Humanos , Masculino , Linhagem , FenótipoRESUMO
We determined the molecular basis of cystathionine beta-synthase (CBS) deficiency in a partially pyridoxine-responsive homocystinuria patient. Direct sequencing of the entire CBS cDNA revealed the presence of a homozygous G1330A transition. This mutation causes an amino acid change from aspartic acid to asparagine (D444N) in the regulatory domain of the protein and abolishes a TaqI restriction site at DNA level. Despite the homozygous mutation, CBS activities in extracts of cultured fibroblasts of this patient were not in the homozygous but in the heterozygous range. Furthermore, we observed no stimulation of CBS activity by S-adenosylmethionine, contrary to a threefold stimulation in control fibroblast extract. The mutation was introduced in an E. coli expression system and CBS activities were measured after addition of different S-adenosylmethionine concentrations (0-200 microM). Again, we observed a defective stimulation of CBS activity by S-adenosylmethionine in the mutated construct, whereas the normal construct showed a threefold stimulation in activity. These data suggest that this D444N mutation interferes in S-adenosylmethionine regulation of CBS. Furthermore, it indicates the importance of S-adenosylmethionine regulation of the transsulfuration pathway in homocysteine homeostasis in humans.
Assuntos
Cistationina beta-Sintase/deficiência , Cistationina beta-Sintase/genética , Regulação Enzimológica da Expressão Gênica , Homocistinúria/genética , Mutação Puntual , Piridoxina/uso terapêutico , S-Adenosilmetionina/farmacologia , Adulto , Sequência de Aminoácidos , Asparagina , Ácido Aspártico , Sequência de Bases , Cistationina beta-Sintase/metabolismo , DNA/sangue , Primers do DNA , Feminino , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Heterozigoto , Homocistinúria/enzimologia , Homozigoto , Humanos , Masculino , Dados de Sequência Molecular , Linhagem , Reação em Cadeia da Polimerase , Valores de ReferênciaRESUMO
Defective control of the alternative route of the complement system is an important cause of the non-diarrhoea haemolytic uraemic syndrome (HUS). On the endothelial surface, mutations in HF1, MCP and IF predispose to development ofHUS. Uncontrolled complement activation on the surface of endothelial cells will damage these cells extensively. Plasmapheresis can be an effective, although temporary, treatment for mutations in HF1 and IF. HUS frequently recurs after renal transplantation in patients with HF1 or IF mutations but not in patients with a mutation of MCP. These genetic defects can be detected by routine diagnostics.
Assuntos
Via Alternativa do Complemento/genética , Proteínas do Sistema Complemento/genética , Síndrome Hemolítico-Urêmica/genética , Mutação , Predisposição Genética para Doença , HumanosRESUMO
Septic acute kidney injury (AKI) associates with poor survival rates and often requires renal replacement therapy. Glucocorticoids may pose renal protective effects in sepsis via stimulation of mitochondrial function. Therefore, we studied the mitochondrial effects of dexamethasone in an experimental inflammatory proximal tubule epithelial cell model. Treatment of human proximal tubule epithelial cells with lipopolysaccharide (LPS) closely resembles pathophysiological processes during endotoxaemia, and led to increased cytokine excretion rates and cellular reactive oxygen species levels, combined with a reduced mitochondrial membrane potential and respiratory capacity. These effects were attenuated by dexamethasone. Dexamethasone specifically increased the expression and activity of mitochondrial complex V (CV), which could not be explained by an increase in mitochondrial mass. Finally, we demonstrated that dexamethasone acidified the intracellular milieu and consequently reversed LPS-induced alkalisation, leading to restoration of the mitochondrial function. This acidification also provides an explanation for the increase in CV expression, which is expected to compensate for the inhibitory effect of the acidified environment on this complex. Besides the mechanistic insights into the beneficial effects of dexamethasone during renal cellular inflammation, our work also supports a key role for mitochondria in this process and, hence, provides novel therapeutic avenues for the treatment of AKI.