RESUMO
Susceptibility and protection against human autoimmune diseases, including type I diabetes, multiple sclerosis, and Goodpasture disease, is associated with particular human leukocyte antigen (HLA) alleles. However, the mechanisms underpinning such HLA-mediated effects on self-tolerance remain unclear. Here we investigate the molecular mechanism of Goodpasture disease, an HLA-linked autoimmune renal disorder characterized by an immunodominant CD4+ T-cell self-epitope derived from the α3 chain of type IV collagen (α3135-145). While HLA-DR15 confers a markedly increased disease risk, the protective HLA-DR1 allele is dominantly protective in trans with HLA-DR15 (ref. 2). We show that autoreactive α3135-145-specific T cells expand in patients with Goodpasture disease and, in α3135-145-immunized HLA-DR15 transgenic mice, α3135-145-specific T cells infiltrate the kidney and mice develop Goodpasture disease. HLA-DR15 and HLA-DR1 exhibit distinct peptide repertoires and binding preferences and present the α3135-145 epitope in different binding registers. HLA-DR15-α3135-145 tetramer+ T cells in HLA-DR15 transgenic mice exhibit a conventional T-cell phenotype (Tconv) that secretes pro-inflammatory cytokines. In contrast, HLA-DR1-α3135-145 tetramer+ T cells in HLA-DR1 and HLA-DR15/DR1 transgenic mice are predominantly CD4+Foxp3+ regulatory T cells (Treg cells) expressing tolerogenic cytokines. HLA-DR1-induced Treg cells confer resistance to disease in HLA-DR15/DR1 transgenic mice. HLA-DR15+ and HLA-DR1+ healthy human donors display altered α3135-145-specific T-cell antigen receptor usage, HLA-DR15-α3135-145 tetramer+ Foxp3- Tconv and HLA-DR1-α3135-145 tetramer+ Foxp3+CD25hiCD127lo Treg dominant phenotypes. Moreover, patients with Goodpasture disease display a clonally expanded α3135-145-specific CD4+ T-cell repertoire. Accordingly, we provide a mechanistic basis for the dominantly protective effect of HLA in autoimmune disease, whereby HLA polymorphism shapes the relative abundance of self-epitope specific Treg cells that leads to protection or causation of autoimmunity.
Assuntos
Doença Antimembrana Basal Glomerular/imunologia , Autoimunidade/imunologia , Linfócitos T Reguladores/imunologia , Animais , Doença Antimembrana Basal Glomerular/patologia , Sequência de Bases , Linfócitos T CD4-Positivos/imunologia , Colágeno Tipo IV/química , Colágeno Tipo IV/imunologia , Citocinas/imunologia , Feminino , Fatores de Transcrição Forkhead/metabolismo , Subtipos Sorológicos de HLA-DR/imunologia , Antígeno HLA-DR1/imunologia , Humanos , Epitopos Imunodominantes , Masculino , Camundongos , Camundongos Transgênicos , Modelos MolecularesRESUMO
BACKGROUND: Abnormalities in serum potassium are a well known complication of chronic kidney disease (CKD), but little is known about their impact on inpatient outcomes. AIMS: To better understand the role of dyskalaemia in hospital in-patients, we assessed the epidemiology of potassium disorders among CKD patients, and the association between admission potassium and inpatient mortality or intensive care unit (ICU) requirement. METHODS: This retrospective hospital-based cohort study (n = 11 156) included patients with an estimated glomerular filtration rate (eGFR) <60 mL/min/1.73 m2 admitted to Austin Health between 2014 and 2018 and who had an admission potassium value. Dialysis patients or those with a renal transplant were excluded. Multivariate logistic analysis was conducted to identify factors associated with hyperkalaemia (≥5.5 mmol/L) and hypokalaemia (<3.5 mmol/L). Odds ratios for inpatient mortality and ICU admission between potassium categories were obtained by multivariate regression with adjustments for demographics, renal function and comorbidities. RESULTS: Hyperkalaemia and hypokalaemia were present in 6.86% and 2.94% of hospital admissions respectively. In multivariate regression male sex, lower eGFR, diabetes and cardiac failure were associated with higher odds of hyperkalaemia. Thiazide diuretics, loop diuretics, infectious disease and endocrine pathology were associated with higher odds of hypokalaemia. A U-shaped association was noted between potassium and inpatient mortality. Potassium <4.0 mmol/L and ≥5.0 mmol/L was associated with increased mortality. Only patients with potassium ≥5.5 mmol/L had increased ICU admission risk. CONCLUSION: Derangements in potassium frequently occur in CKD inpatients and are independently associated with higher mortality and ICU requirement. Further studies are required to determine whether interventions to maintain normokalaemia improve outcomes in this population.
Assuntos
Hiperpotassemia , Insuficiência Renal Crônica , Estudos de Coortes , Humanos , Hiperpotassemia/epidemiologia , Masculino , Potássio , Prevalência , Insuficiência Renal Crônica/diagnóstico , Insuficiência Renal Crônica/epidemiologia , Estudos RetrospectivosRESUMO
BACKGROUND: Driving is a complex task requiring multiple cognitive domains and the musculoskeletal system. Cognitive dysfunction is associated with driving impairment. Dialysis patients are known to have a high prevalence of cognitive impairment and other comorbidities, and may be at risk of driving impairment. No Australian guidelines address driving safety in dialysis patients. AIMS: To estimate the proportion of dialysis patients who were driving and those at risk of driving impairment, and to investigate the agreement between objective and subjective markers of risk. METHODS: This single-centre study involved dialysis patients voluntarily completing two questionnaires relating to risk of driving impairment; the first questionnaire focussed on objective markers, and the second questionnaire focussed on subjective markers. Risk of driving impairment was established using pre-determined criteria, and the agreement between objective and subjective markers was estimated using Cohen kappa. RESULTS: A total of 44.8% (99/221) of patients participated; 76.8% (76/99) of participants were driving, and 76.3% (58/76) of drivers were at risk of driving impairment. Factors associated with at-risk driving included post dialysis dizziness, leg weakness or numbness, falling asleep while driving and hypoglycaemia. Sixteen patients reported collisions since commencing dialysis. The questionnaires displayed slight agreement (Cohen kappa = 0.20) between objective and subjective markers. CONCLUSIONS: Dialysis patients are at risk of driving impairment based on self-reported questionnaire responses. Discrepancies between patients' perceptions and objective markers were apparent. Further research into appropriate risk assessments, as well as development of guidelines to aid in determining driving safety in dialysis patients, is needed.
Assuntos
Condução de Veículo , Disfunção Cognitiva , Falência Renal Crônica , Acidentes de Trânsito , Humanos , Falência Renal Crônica/epidemiologia , Falência Renal Crônica/terapia , Diálise Renal , Inquéritos e QuestionáriosRESUMO
Graft-derived cell-free DNA (donor-derived cell-free DNA) is an emerging marker of kidney allograft injury. Studies examining the clinical validity of this biomarker have previously used the graft fraction, or proportion of total cell-free DNA that is graft-derived. The present study evaluated the diagnostic validity of absolute measurements of graft-derived cell-free DNA, as well as calculated graft fraction, for the diagnosis of graft dysfunction. Plasma graft-derived cell-free DNA, total cell-free DNA, and graft fraction were correlated with biopsy diagnosis as well as individual Banff scores. Sixty-one samples were included in the analysis. For the diagnosis of antibody mediated rejection, the receiver-operator characteristic area under the curves of graft-derived cell-free DNA and graft fraction were 0.91 (95% CI 0.82-0.98) and 0.89 (95% CI 0.79-0.98), respectively. Both measures did not diagnose borderline or type 1A cellular mediated rejection. Graft fraction was associated with a broader range of Banff lesions, including lesions associated with cellular mediated rejection, while graft-derived cell-free DNA appeared more specific for antibody mediated rejection. Limitations of this study include a small sample size and lack of a validation cohort. The capacity for absolute quantification, and lower barriers to implementation of this methodology recommend it for further study.
Assuntos
Ácidos Nucleicos Livres/sangue , Rejeição de Enxerto/diagnóstico , Rejeição de Enxerto/genética , Transplante de Rim , Adulto , Estudos Transversais , Feminino , Humanos , Imunossupressores/administração & dosagem , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Transplante HomólogoRESUMO
Activation of the heterotrimeric energy-sensing kinase AMP-activated protein kinase (AMPK) has been reported to improve experimental diabetic kidney disease. We examined the effect of type 1 diabetes in wild-type (WT) mice and mice lacking the ß1 subunit of AMPK (AMPK ß1-/- mice), which have reduced AMPK activity in kidneys and other organs. Diabetes was induced using streptozotocin (STZ) and the animals followed up for 4 weeks. Hyperglycaemia was more severe in diabetic AMPK ß1-/- mice, despite the absence of any difference in serum levels of insulin, adiponectin and leptin. There was no change in AMPK activity in the kidneys of diabetic WT mice by AMPK activity assay, or phosphorylation of either the αT172 activation site on the α catalytic subunit of AMPK or the AMPK-specific phosphosite S79 on acetyl CoA carboxylase 1 (ACC1). Phosphorylation of the inhibitory αS485 site on the α subunit of AMPK was significantly increased in the WT diabetic mice compared to non-diabetic controls. Despite increased plasma glucose levels in the diabetic AMPK ß1-/- mice, there were fewer myofibroblasts in the kidneys compared to diabetic WT mice, as evidenced by reduced α-smooth muscle actin (α-SMA) protein by Western blot, mRNA by qRT-PCR and fewer α-SMA-positive cells by immunohistochemical staining. Albuminuria was also reduced in the AMPK ß1-/- mice. In contrast to previous studies, therefore, myofibroblasts were reduced in the kidneys of AMPK ß1-/- diabetic mice compared to diabetic WT mice, despite increased circulating glucose, suggesting that AMPK can worsen renal fibrosis in type 1 diabetes.
Assuntos
Proteínas Quinases Ativadas por AMP/fisiologia , Diabetes Mellitus Experimental/patologia , Diabetes Mellitus Tipo 1/patologia , Rim/patologia , Miofibroblastos/fisiologia , Proteínas Quinases Ativadas por AMP/deficiência , Proteínas Quinases Ativadas por AMP/genética , Albuminúria/metabolismo , Albuminúria/patologia , Animais , Glicemia/metabolismo , Creatinina/metabolismo , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 1/enzimologia , Nefropatias Diabéticas/metabolismo , Nefropatias Diabéticas/patologia , Rim/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , FosforilaçãoRESUMO
BACKGROUND: Expression of genes regulating fatty acid metabolism is reduced in tubular epithelial cells from kidneys with tubulointerstitial fibrosis (TIF), thus decreasing the energy produced by fatty acid oxidation (FAO). Acetyl-CoA carboxylase (ACC), a target for the energy-sensing AMP-activating protein kinase (AMPK), is the major controller of the rate of FAO within cells. Metformin has a well described antifibrotic effect, and increases phosphorylation of ACC by AMPK, thereby increasing FAO. METHODS: We evaluated phosphorylation of ACC in cell and mouse nephropathy models, as well as the effects of metformin administration in mice with and without mutations that reduce ACC phosphorylation. RESULTS: Reduced phosphorylation of ACC on the AMPK site Ser79 occurred in both tubular epithelial cells treated with folate to mimic cellular injury and in wild-type (WT) mice after induction of the folic acid nephropathy model. When this effect was exaggerated in mice with knock-in (KI) Ser to Ala mutations of the phosphorylation sites in ACC, lipid accumulation and fibrosis increased significantly compared with WT. The effect of ACC phosphorylation on fibrosis was confirmed in the unilateral ureteric obstruction model, which showed significantly increased lipid accumulation and fibrosis in the KI mice. Metformin use was associated with significantly reduced fibrosis and lipid accumulation in WT mice. In contrast, in the KI mice, the drug was associated with worsened fibrosis. CONCLUSIONS: These data indicate that reduced phosphorylation of ACC after renal injury contributes to the development of TIF, and that phosphorylation of ACC is required for metformin's antifibrotic action in the kidney.
Assuntos
Proteínas Quinases Ativadas por AMP/efeitos dos fármacos , Acetil-CoA Carboxilase/metabolismo , Nefropatias/patologia , Metformina/farmacologia , Oxirredução/efeitos dos fármacos , Proteínas Quinases Ativadas por AMP/metabolismo , Análise de Variância , Animais , Biópsia por Agulha , Células Cultivadas , Modelos Animais de Doenças , Humanos , Imuno-Histoquímica , Resistência à Insulina/fisiologia , Nefropatias/metabolismo , Túbulos Renais/citologia , Túbulos Renais/metabolismo , Metabolismo dos Lipídeos/efeitos dos fármacos , Metformina/metabolismo , Camundongos , Camundongos Knockout , Análise Multivariada , Fosforilação , Distribuição Aleatória , Reação em Cadeia da Polimerase em Tempo RealRESUMO
The high-energy requirement of the kidney and the importance of energy metabolism in renal physiology has been appreciated for decades, but only recently has there emerged a strong link between impaired renal energy metabolism and chronic kidney disease (CKD). The mechanisms underlying the association between changes in energy metabolism and progression of CKD, however, remain poorly understood. A new study from Qiu and colleagues reported in the Journal of Pathology has advanced this understanding by showing that, after renal injury, the energy sensor AMPK inhibits epithelial-mesenchymal transition and inflammation, processes important in the pathogenesis of CKD. Furthermore, this study identifies an interaction between AMPK and CK2ß as an important mechanism in the anti-fibrotic effect. CK2ß has previously been shown to interact with STK11 (also known as LKB1) to regulate cellular polarity. These findings are consistent with the known roles of the LKB1-AMPK pathway in sustaining cellular energy homeostasis and epithelial cell polarity, and add to growing evidence linking the suppression of energy metabolism to CKD. They emphasize the importance of energy metabolism in general and the LKB1-AMPK axis in particular as key investigational and therapeutic targets in the battle against CKD.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Caseína Quinase II/metabolismo , Transdiferenciação Celular , Células Epiteliais/enzimologia , Transição Epitelial-Mesenquimal , Túbulos Renais Proximais/enzimologia , Nefrite Intersticial/prevenção & controle , Animais , HumanosRESUMO
BACKGROUND/AIMS: Intravascular volume expansion due to sodium retention is involved in the pathogenesis of obesity-related hypertension. Institution of high fat diet (HFD) feeding leads to an initial state of positive sodium balance due to enhanced tubular reabsorption of sodium, but which tubular sodium transporters are responsible for this remains undefined. METHODS: C57/Bl6 mice were fed control or HFD for 3 weeks. Blood pressures were recorded by tail cuff method. Sodium transporter expression and phosphorylation were determined by Western blotting. In vivo activity of NCC was determined using natriuretic responses to hydrochlorothiazide. Expression of NCC mRNA was determined using qPCR. RESULTS: At 3 weeks HFD mice had significant weight gains compared to control mice, but blood pressures were not yet elevated. There were no changes in expression or phosphorylation of the bumetanide-sensitive cotransporter, NKCC2, or in expression of subunits of the amiloride-sensitive ion channel, ENaC. However, there were significant increases in mRNA and protein expression of the thiazide-sensitive co-transporter, NCC, in kidneys from HFD mice. Consistent with this, HFD mice had increased in vivo activity of NCC. CONCLUSIONS: Increased expression of NCC promotes the sodium loading response to institution of HFD feeding before onset of hypertension.
Assuntos
Gorduras na Dieta/efeitos adversos , Hidroclorotiazida/farmacologia , Obesidade/metabolismo , Receptores de Droga/biossíntese , Simportadores de Cloreto de Sódio/biossíntese , Cloreto de Sódio na Dieta/efeitos adversos , Sódio/metabolismo , Animais , Gorduras na Dieta/administração & dosagem , Camundongos , Camundongos Endogâmicos C57BL , Obesidade/induzido quimicamente , Obesidade/patologia , Cloreto de Sódio na Dieta/administração & dosagemRESUMO
AIM: In the Australian state of Victoria, the Renal Health Clinical Network (RHCN) of the Department of Health Victoria established a Renal Key Performance Indicator (KPI) Working Group in 2011. The group developed four KPIs related to chronic kidney disease and dialysis. A transplant working group of the RHCN developed two additional KPIs. The aim was to develop clinical indicators to measure performance of renal services to drive service improvement. METHODS: A data collection and benchmarking programme was established, with data provided monthly to the Department using a purpose-designed website portal. The KPI Working Group is responsible for analysing data each quarter and ensuring indicators remain accurate and relevant. Each indicator has clear definitions and targets, and assess (i) patient education, (ii) timely creation of vascular access for haemodialysis, (iii) proportion of patients dialysing at home, (iv) incidence of dialysis-related peritonitis, (v) incidence of pre-emptive renal transplantation, and (vi) timely listing of patients for deceased donor transplantation. RESULTS: Most KPIs have demonstrated improved performance over time with limited gains notably in two: the proportion of patients dialysing at home (KPI 3) and timely listing patients for transplantation (KPI 6). CONCLUSION: KPI implementation has been established in Victoria for 2 years, providing performance data without additional funding. The six Victorian KPIs are measurable, relevant and modifiable, and implementation relies on enthusiasm and goodwill of physicians and nurses involved in collecting data. The KPIs require further evaluation, but adoption of a similar programme by other jurisdictions could lead to improved national outcomes.
Assuntos
Transplante de Rim/normas , Nefrologia/normas , Padrões de Prática Médica/normas , Avaliação de Processos em Cuidados de Saúde/normas , Melhoria de Qualidade/normas , Indicadores de Qualidade em Assistência à Saúde/normas , Diálise Renal/normas , Insuficiência Renal Crônica/terapia , Benchmarking/normas , Acessibilidade aos Serviços de Saúde/normas , Disparidades em Assistência à Saúde/normas , Humanos , Transplante de Rim/efeitos adversos , Transplante de Rim/tendências , Educação de Pacientes como Assunto/normas , Padrões de Prática Médica/tendências , Avaliação de Processos em Cuidados de Saúde/tendências , Avaliação de Programas e Projetos de Saúde , Melhoria de Qualidade/tendências , Indicadores de Qualidade em Assistência à Saúde/tendências , Diálise Renal/efeitos adversos , Diálise Renal/tendências , Insuficiência Renal Crônica/diagnóstico , Resultado do Tratamento , Vitória , Listas de EsperaRESUMO
The co-transporter activity of Na(+)-K(+)-2Cl(-) 1 (NKCC1) is dependent on phosphorylation. In this study we show the energy-sensing kinase AMPK inhibits NKCC1 activity. Three separate AMPK activators (AICAR, Phenformin and A-769662) inhibited NKCC1 flux in a variety of nucleated cells. Treatment with A-769662 resulted in a reduction of NKCC1(T212/T217) phosphorylation, and this was reversed by treatment with the non-selective AMPK inhibitor Compound C. AMPK dependence was confirmed by treatment of AMPK null mouse embryonic fibroblasts, where A-769662 had no effect on NKCC1 mediated transport. AMPK was found to directly phosphorylate a recombinant human-NKCC1 N-terminal fragment (1-293) with the phosphorylated site identified as S77. Mutation of Serine 77 to Alanine partially prevented the inhibitory effect of A-769662 on NKCC1 activity. In conclusion, AMPK can act to reduce NKCC1-mediated transport. While the exact mechanism is still unclear there is evidence for both a direct effect on phosphorylation of S77 and reduced phosphorylation of T212/217.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Aminoimidazol Carboxamida/análogos & derivados , Fenformin/farmacologia , Pironas/farmacologia , Ribonucleotídeos/farmacologia , Membro 2 da Família 12 de Carreador de Soluto/metabolismo , Tiofenos/farmacologia , Proteínas Quinases Ativadas por AMP/genética , Alanina/metabolismo , Aminoimidazol Carboxamida/farmacologia , Animais , Compostos de Bifenilo , Linhagem Celular , Cães , Regulação da Expressão Gênica/efeitos dos fármacos , Células HEK293 , Humanos , Células Madin Darby de Rim Canino , Camundongos , Fosforilação , Mutação Puntual , Transporte Proteico/efeitos dos fármacos , Serina/metabolismo , Membro 2 da Família 12 de Carreador de Soluto/genéticaRESUMO
Enhanced tubular reabsorption of salt is important in the pathogenesis of obesity-related hypertension, but the mechanisms remain poorly defined. To identify changes in the regulation of salt transporters in the kidney, C57BL/6 mice were fed a 40% fat diet [high-fat diet (HFD)] or a 12% fat diet (control diet) for 14 wk. Compared with control diet-fed mice, HFD-fed mice had significantly greater elevations in weight, blood pressure, and serum insulin and leptin levels. When we examined Na(+) transporter expression, Na(+)-K(+)-2Cl(-) cotransporter (NKCC2) was unchanged in whole kidney and reduced in the cortex, Na(+)-Cl(-) cotransporter (NCC) and α-epithelial Na(+) channel (ENaC) and γ-ENaC were unchanged, and ß-ENaC was reduced. Phosphorylation of NCC was unaltered. Activating phosphorylation of NKCC2 at S126 was increased 2.5-fold. Activation of STE-20/SPS1-related proline-alanine-rich protein kinase (SPAK)/oxidative stress responsive 1 kinase (OSR1) was increased in kidneys from HFD-fed mice, and enhanced phosphorylation of NKCC2 at T96/T101 was evident in the cortex. Increased activity of NKCC2 in vivo was confirmed with diuretic experiments. HFD-fed mice had reduced activating phosphorylation of AMP-activated protein kinase (AMPK) in the renal cortex. In vitro, activation of AMPK led to a reduction in phospho-SPAK/phospho-OSR1 in AMPK(+/+) murine embryonic fibroblasts (MEFs), but no effect was seen in AMPK(-/-) MEFs, indicating an AMPK-mediated effect. Activation of the with no lysine kinase/SPAK/OSR1 pathway with low-NaCl solution invoked a greater elevation in phospho-SPAK/phospho-OSR1 in AMPK(-/-) MEFs than in AMPK(+/+) MEFs, consistent with a negative regulatory effect of AMPK on SPAK/OSR1 phosphorylation. In conclusion, this study identifies increased phosphorylation of NKCC2 on S126 as a hitherto-unrecognized mediator of enhanced Na(+) reabsorption in obesity and identifies a new role for AMPK in regulating the activity of SPAK/OSR1.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Obesidade/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Cloreto de Sódio na Dieta/metabolismo , Membro 1 da Família 12 de Carreador de Soluto/metabolismo , Animais , Canais Epiteliais de Sódio/metabolismo , Rim/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , FosforilaçãoRESUMO
AMG 416 (velcalcetide), a novel peptide agonist of the calcium-sensing receptor, lowers plasma parathyroid hormone in preclinical uremic animal models and in normal healthy individuals. Here, we studied its efficacy in hemodialysis patients suffering from secondary hyperparathyroidism. Major inclusion criteria were hemodialysis for at least 3 months, serum parathyroid hormone over 300 pg/ml, a corrected serum calcium of 9.0 mg/dl or more, and stable doses of vitamin D analogs for at least 3 weeks prior to screening. Twenty-eight patients were enrolled in one of five cohorts (5, 10, 20, 40, 60 mg). Cohorts 1-3 (four patients each) were treated in a two-period crossover design, while cohorts 4 and 5 (eight patients each) were randomized 1:1 to AMG 416 or placebo. Patients were admitted to a clinical research unit following hemodialysis and studied for 3 days prior to discharge for hemodialysis. Single intravenous doses of AMG 416 from 5 to 60 mg were well tolerated, and plasma levels increased in a dose-related manner. AMG 416 treatment was associated with significant, dose-dependent reductions in serum parathyroid hormone and fibroblast growth factor 23. Compared with placebo, all dose groups of 10 mg or more were associated with attenuation in the rise in serum phosphate during the interdialytic period. Dose-dependent reductions in serum calcium were observed and were well tolerated. Thus, AMG 416 represents a novel therapeutic approach for the treatment of secondary hyperparathyroidism in hemodialysis patients.
Assuntos
Hiperparatireoidismo Secundário/tratamento farmacológico , Peptídeos/uso terapêutico , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Injeções Intravenosas , Falência Renal Crônica/complicações , Falência Renal Crônica/terapia , Masculino , Pessoa de Meia-Idade , Peptídeos/administração & dosagem , Diálise RenalRESUMO
BACKGROUND: We describe a novel approach that harnesses the ubiquity of copy number deletion polymorphisms in human genomes to definitively detect and quantify chimeric DNA in clinical samples. Unlike other molecular approaches to chimerism analysis, the copy number deletion (CND) method targets genomic loci (>50 base pairs in length) that are wholly absent from wild-type (i.e., self) background DNA sequences in a sex-independent manner. METHODS: Bespoke quantitative PCR (qPCR) CND assays were developed and validated using a series of DNA standards and chimeric plasma DNA samples collected from 2 allogeneic kidney transplant recipients and 12 pregnant women. Assay performance and informativeness were assessed using appropriate statistical methods. RESULTS: The CND qPCR assays showed high sensitivity, precision, and reliability for linear quantification of DNA chimerism down to 16 genomic equivalents (i.e., 106 pg). Fetal fraction (%) in 12 singleton male pregnancies was calculated using the CND qPCR approach, which showed closer agreement with single-nucleotide polymorphism-based massively parallel sequencing than the SRY (sex determining region Y) (Y chromosome) qPCR assay. The latter consistently underestimated the fetal fraction relative to the other methods. We also were able to measure biological changes in plasma nonself DNA concentrations in 2 renal transplant recipients. CONCLUSIONS: The CND qPCR technique is suitable for measurement of chimerism for monitoring of rejection in allogeneic organ transplantation and quantification of the cell-free fetal DNA fraction in maternal plasma samples used for noninvasive prenatal genetic testing.
Assuntos
Quimera/genética , Variações do Número de Cópias de DNA , Humanos , Limite de Detecção , Reação em Cadeia da Polimerase/métodos , Reprodutibilidade dos TestesRESUMO
Calcific coronary artery stenosis is a complex disease associated with adverse outcomes and suboptimal percutaneous treatment. Calcium plaque modification has emerged as a key strategy to tackle the issues that accompany calcific stenosis - namely reduced device deliverability, unpredictable lesion characteristics, and difficult dilatation. Atherectomy has traditionally been the treatment modality of choice for heavily calcified coronary stenoses. Contemporary technologies have emerged to aid with planning, preparation, and treatment of calcified coronary stenosis in an attempt to improve procedural success and long-term outcomes. In this State Of The Art Review, we synthesize the body of data surrounding the diagnosis, imaging, and treatment of calcific coronary disease, with a focus on i) intravascular imaging, ii) calcific lesion preparation, iii) treatment modalities including atherectomy, and iv) updated treatment algorithms for the management of calcified coronary stenosis.
RESUMO
Acute kidney injury (AKI) disrupts energy metabolism. Targeting metabolism through AMP-activated protein kinase (AMPK) may alleviate AKI. ATX-304, a pan-AMPK activator, was evaluated in C57Bl/6 mice and tubular epithelial cell (TEC) cultures. Mice received ATX-304 (1â¯mg/g) or control chow for 7 days before cisplatin-induced AKI (CI-AKI). Primary cultures of tubular epithelial cells (TECs) were pre-treated with ATX-304 (20⯵M, 4â¯h) prior to exposure to cisplatin (20⯵M, 23â¯h). ATX-304 increased acetyl-CoA carboxylase phosphorylation, indicating AMPK activation. It protected against CI-AKI measured by serum creatinine (control 0.05 + 0.03â¯mM vs ATX-304 0.02 + 0.01â¯mM, P = 0.03), western blot for neutrophil gelatinase-associated lipocalin (NGAL) (control 3.3 + 1.8-fold vs ATX-304 1.2 + 0.55-fold, P = 0.002), and histological injury (control 3.5 + 0.59 vs ATX-304 2.7 + 0.74, P = 0.03). In TECs, pre-treatment with ATX-304 protected against cisplatin-mediated injury, as measured by lactate dehydrogenase release, MTS cell viability, and cleaved caspase 3 expression. ATX-304 protection against cisplatin was lost in AMPK-null murine embryonic fibroblasts. Metabolomic analysis in TECs revealed that ATX-304 (20⯵M, 4â¯h) altered 66/126 metabolites, including fatty acids, tricarboxylic acid cycle metabolites, and amino acids. Metabolic studies of live cells using the XFe96 Seahorse analyzer revealed that ATX-304 increased the basal TEC oxygen consumption rate by 38%, whereas maximal respiration was unchanged. Thus, ATX-304 protects against cisplatin-mediated kidney injury via AMPK-dependent metabolic reprogramming, revealing a promising therapeutic strategy for AKI.
Assuntos
Proteínas Quinases Ativadas por AMP , Injúria Renal Aguda , Cisplatino , Camundongos Endogâmicos C57BL , Animais , Injúria Renal Aguda/induzido quimicamente , Injúria Renal Aguda/prevenção & controle , Injúria Renal Aguda/metabolismo , Injúria Renal Aguda/patologia , Proteínas Quinases Ativadas por AMP/metabolismo , Camundongos , Masculino , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Cultivadas , Substâncias Protetoras/farmacologia , Fosforilação , Compostos de Bifenilo , Pironas , TiofenosRESUMO
Salt reabsorption is the major energy-requiring process in the kidney, and AMP-activated protein kinase (AMPK) is an important regulator of cellular metabolism. Mice with targeted deletion of the ß1-subunit of AMPK (AMPK-ß1(-/-) mice) had significantly increased urinary Na(+) excretion on a normal salt diet. This was associated with reduced expression of the ß-subunit of the epithelial Na(+) channel (ENaC) and increased subapical tubular expression of kidney-specific Na(+)-K(+)-2Cl(-) cotransporter 2 (NKCC2) in the medullary thick ascending limb of Henle. AMPK-ß1(-/-) mice fed a salt-deficient diet were able to conserve Na(+), but renin secretion increased 180% compared with control mice. Cyclooxygenase-2 mRNA also increased in the kidney cortex, indicating greater signaling through the macula densa tubular salt-sensing pathway. To determine whether the increase in renin secretion was due to a change in regulation of fatty acid metabolism by AMPK, mice with a mutation of the inhibitory AMPK phosphosite in acetyl-CoA carboxylase 1 [ACC1-knockin (KI)(S79A) mice] were examined. ACC1-KI(S79A) mice on a normal salt diet had no increase in salt loss or renin secretion, and expression of NKCC2, Na(+)-Cl(-) cotransporter, and ENaC-ß were similar to those in control mice. When mice were placed on a salt-deficient diet, however, renin secretion and cortical expression of cyclooxygenase-2 mRNA increased significantly in ACC1-KI(S79A) mice compared with control mice. In summary, our data suggest that renin synthesis and secretion are regulated by AMPK and coupled to metabolism by phosphorylation of ACC1.
Assuntos
Proteínas Quinases Ativadas por AMP/genética , Acetil-CoA Carboxilase/metabolismo , Renina/sangue , Proteínas Quinases Ativadas por AMP/deficiência , Acetil-CoA Carboxilase/genética , Animais , Canais Epiteliais de Sódio/biossíntese , Camundongos , Fosforilação , Renina/biossíntese , Sódio/urina , Cloreto de Sódio na Dieta/administração & dosagemRESUMO
Kidney transplant recipients undergo lifelong monitoring of allograft function and evaluation for transplant complications. The current monitoring paradigm utilizes blood, urine, and tissue markers that are insensitive, nonspecific, or invasive to obtain. As a result, problems are detected late, after significant damage has accrued, and often beyond the time at which complete resolution is possible. Indeed, most kidney transplants eventually fail, usually because of chronic rejection and other undetected injury. There is a clear need for a transplant-specific biomarker that enables a proactive approach to monitoring via early detection of reversible pathology. A biomarker that supports timely and personalized treatment would assist in achieving the ultimate goal of improving allograft survival and limiting therapeutic toxicity to the recipient. Donor-derived cell-free DNA (ddcfDNA) has been proposed as one such transplant biomarker. Although the test is presently utilized most in the United States, it is conceivable that its use will become more widespread. This review covers aspects of ddcfDNA that support informed use of the test by general nephrologists, including the basic biology of ddcfDNA, methodological nuances of testing, and general recommendations for use in the kidney transplant population. Clinical contexts are used to illustrate evidence-supported interpretation of ddcfDNA results and subsequent management. Finally, knowledge gaps and areas for further study are discussed.
Assuntos
Ácidos Nucleicos Livres , Transplante de Rim , Humanos , Transplante de Rim/efeitos adversos , Rejeição de Enxerto/diagnóstico , Rejeição de Enxerto/prevenção & controle , Rejeição de Enxerto/etiologia , Doadores de Tecidos , BiomarcadoresRESUMO
Resolving-type macrophages prevent chronic inflammation by clearing apoptotic cells through efferocytosis. These macrophages are thought to rely mainly on oxidative phosphorylation, but emerging evidence suggests a possible link between efferocytosis and glycolysis. To gain further insight into this issue, we investigated molecular-cellular mechanisms involved in efferocytosis-induced macrophage glycolysis and its consequences. We found that efferocytosis promotes a transient increase in macrophage glycolysis that is dependent on rapid activation of the enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 2 (PFKFB2), which distinguishes this process from glycolysis in pro-inflammatory macrophages. Mice transplanted with activation-defective PFKFB2 bone marrow and then subjected to dexamethasone-induced thymocyte apoptosis exhibit impaired thymic efferocytosis, increased thymic necrosis, and lower expression of the efferocytosis receptors MerTK and LRP1 on thymic macrophages compared with wild-type control mice. In vitro mechanistic studies revealed that glycolysis stimulated by the uptake of a first apoptotic cell promotes continual efferocytosis through lactate-mediated upregulation of MerTK and LRP1. Thus, efferocytosis-induced macrophage glycolysis represents a unique metabolic process that sustains continual efferocytosis in a lactate-dependent manner. The differentiation of this process from inflammatory macrophage glycolysis raises the possibility that it could be therapeutically enhanced to promote efferocytosis and resolution in chronic inflammatory diseases.
Assuntos
Ácido Láctico , Fagocitose , Animais , Camundongos , c-Mer Tirosina Quinase/metabolismo , Inflamação/metabolismo , Ácido Láctico/metabolismo , Macrófagos/metabolismo , Fagocitose/fisiologiaRESUMO
The anti-fibrotic effect of metformin has been widely demonstrated. Fibrosis in the kidney after injury is associated with reduced expression of genes involved in both fatty acid and glycolytic energy metabolism. We have previously reported that the anti-fibrotic effect of metformin requires phosphoregulation of fatty acid oxidation by AMP-activated protein kinase (AMPK). To determine whether metformin also acts via regulation of glycolysis, we mutated regulatory phosphosites in the PFKFB2 isoform of 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase (PFKFB2), a key regulator of glycolysis in the kidney. Mice with inactivating knockin (KI) mutations of the phosphorylation sites in PFKFB2 (PFKFB2 KI mice), which reduces the ability to increase the rate of glycolysis following stimulation, were used. Metformin was administered via drinking water to mice with a unilateral ureteric obstruction (UUO) model of renal fibrosis. In the PFKFB2 KI mice treated with metformin, there was decreased fibrosis and macrophage infiltration following UUO as assessed by Western blot for fibronectin and RT-PCR for α-smooth muscle actin, collagen 3, and F4.80, and confirmed by histology. Expression of the inducible PFKFB3 isoform was increased with metformin in UUO in both WT and PFKFB2 KI mice. There was no significant difference between WT and PFKFB2 KI mice treated with metformin in the degree of fibrosis following UUO in any of the Western blot or RT-PCR parameters that were measured. These data show that inhibition of the regulation of glycolysis by PFKFB2 does not diminish the anti-fibrotic effect of metformin in a model of renal fibrosis.
Assuntos
Nefropatias , Metformina , Obstrução Ureteral , Animais , Camundongos , Modelos Animais de Doenças , Fibrose , Rim/patologia , Nefropatias/tratamento farmacológico , Nefropatias/genética , Nefropatias/complicações , Metformina/farmacologia , Metformina/metabolismo , Mutação , Fosforilação , Obstrução Ureteral/complicaçõesRESUMO
To better understand the role of the urea-to-creatinine ratio in chronic kidney disease patients, we assessed the epidemiology of the urea-to-creatinine ratio among hospitalised chronic kidney disease patients, and the association between the urea-to-creatinine ratio and inpatient clinical outcomes. This retrospective cohort study (n = 11,156) included patients with at least two eGFR values < 60 mL/min/1.73m2 measured greater than 90-days apart and admitted to a tertiary hospital between 2014 and 2019. Dialysis and renal transplant patients were excluded. Adjusted odds ratios for factors associated with an elevated urea-to-creatinine ratio were calculated. Multivariate regression was conducted to identify the relationship between elevated UCR and inpatient mortality, intensive care admission, hospital readmission and hospital length-of-stay. Urea-to-creatinine ratio > 100 was present in 27.67% of hospital admissions. Age ≥ 65 years, female gender, gastrointestinal tract bleeding, heart failure, acute kidney injury and lower serum albumin were associated with elevated urea-to-creatinine ratio. Higher urea-to-creatinine ratio level was associated with greater rates of inpatient mortality, hospital readmission within 30-days and longer hospital length-of-stay. Despite this, there was no statistically significant association between higher urea-to-creatinine ratio and intensive care unit admission. Elevated urea-to-creatinine ratio is associated with poor clinical outcomes in chronic kidney disease inpatients. This warrants further investigation to understand the pathophysiological basis for this relationship and to identify effective interventions.