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ADN Mitocondrial , Enfermedad Pulmonar Obstructiva Crónica , Humanos , ADN Mitocondrial/genética , ADN Mitocondrial/orina , Enfermedad Pulmonar Obstructiva Crónica/orina , Enfermedad Pulmonar Obstructiva Crónica/genética , Enfermedad Pulmonar Obstructiva Crónica/diagnóstico , Masculino , Femenino , Persona de Mediana Edad , Anciano , Estudios de Cohortes , Biomarcadores/orina , EspirometríaRESUMEN
Urinary DNA is widely studied as a non-invasive marker for monitoring of kidneys after transplantation or the progression of urinary tract tumors. The quantity of urinary DNA especially of mitochondrial origin has been reported to mirror kidney damage in various renal diseases and their models. Processing of samples might affect urinary DNA concentrations but the details are not clear. Samples of urine were collected from fifteen healthy volunteers. DNA was extracted from the whole urine, but also from the supernatant after centrifugation at 1600 g and 16000 g. In addition, we have analyzed the DNA in the microparticles in the pellet after the last spin. DNA was measured using fluorometry and real time PCR targeting nuclear and mitochondrial sequences. Addition of deoxyribonuclease to aliquots of samples enabled the characterization of DNA protection. Centrifugation at 1600 g decreased the concentration of extracted DNA by 66% at least in samples with higher DNA in whole urine. Interestingly, the additional spin at 16000 g did not result in a significant decrease in DNA concentration in the supernatant despite detectable microparticle-associated DNA. Deoxyribonuclease decreases total and nuclear DNA by 26% and 31% in whole urine. The majority of urinary mitochondrial DNA seems to be protected against deoxyribonuclease. Our results indicate high variability in urinary DNA even in healthy probands. Extracellular urinary DNA is partially bound to cell debris or microparticles, but a considerable part is still in the supernatant and is protected against cleavage. Further research should identify the nature of the protection, especially for mitochondrial DNA. Better understanding of the biology of urinary DNA should help its clinical interpretation.
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Líquidos Corporales , ADN Mitocondrial , Humanos , ADN Mitocondrial/genética , ADN Mitocondrial/orina , Mitocondrias , Centrifugación , DesoxirribonucleasasRESUMEN
Sodium-glucose co-transporter 2 (SGLT2) inhibitors improve cardiovascular and renal outcomes in type 2 diabetes mellitus (T2DM) patients. However, the mechanisms by which SGLT2 inhibitors improve the clinical outcomes remain elusive. We evaluated whether empagliflozin, an SGLT2 inhibitor, ameliorates mitochondrial dysfunction and inflammatory milieu of the kidneys in T2DM patients. We prospectively measured copy numbers of urinary and serum mitochondrial DNA (mtDNA) nicotinamide adenine dinucleotide dehydrogenase subunit-1 (mtND-1) and cytochrome-c oxidase 3 (mtCOX-3) and urinary interleukin-1ß (IL-1ß) in healthy volunteers (n = 22), in SGLT2 inhibitor-naïve T2DM patients (n = 21) at baseline, and in T2DM patients after 3 months of treatment with empagliflozin (10 mg, n = 17 or 25 mg, n = 4). Both urinary mtDNA copy numbers and IL-1ß levels were higher in the T2DM group than in healthy volunteers. Baseline copy numbers of serum mtCOX-3 in the T2DM group were lower than those in healthy volunteers. Empagliflozin induced marked reduction in both urinary and serum mtND-1 and mtCOX-3 copy numbers, as well as in urinary IL-1ß. Empagliflozin could attenuate mitochondrial damage and inhibit inflammatory response in T2DM patients. This would explain the beneficial effects of SGLT2 inhibitors on cardiovascular and renal outcomes.
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Diabetes Mellitus Tipo 2 , Inhibidores del Cotransportador de Sodio-Glucosa 2 , Humanos , Inhibidores del Cotransportador de Sodio-Glucosa 2/farmacología , Inhibidores del Cotransportador de Sodio-Glucosa 2/uso terapéutico , Diabetes Mellitus Tipo 2/tratamiento farmacológico , ADN Mitocondrial/orina , Interleucina-1beta , Variaciones en el Número de Copia de ADN , Compuestos de Bencidrilo/farmacología , Compuestos de Bencidrilo/uso terapéutico , Mitocondrias , Hipoglucemiantes/farmacologíaRESUMEN
Mitochondrial injury plays an important role in the occurrence and development of kidney diseases. However, the existing assays to determine mitochondrial function restrict our ability to understand the relationship between mitochondrial dysfunction and kidney damage. These limitations may be overcome by recent findings on urinary mitochondrial DNA (UmtDNA). Elevated UmtDNA level may serve as a surrogate biomarker of mitochondrial dysfunction, kidney damage, and progression and prognosis of kidney diseases. Herein, we review the recent research progress on UmtDNA in kidney diseases diagnosis and highlight the research areas that should be expanded in future as well as discuss the future perspectives.
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ADN Mitocondrial/orina , Enfermedades Renales/diagnóstico , Enfermedades Renales/patología , Mitocondrias/metabolismo , Animales , Humanos , Enfermedades Renales/orinaRESUMEN
Liquid biopsy recently became a very promising diagnostic method that has several advantages over conventional invasive methods. Liquid biopsy may serve as a source of several important biomarkers including cell-free nucleic acids (cf-NAs). Cf-DNA is widely used in prenatal testing in order to characterize fetal genetic disorders. Analysis of cf-DNA may provide information about the mutation profile of tumor cells, while cell-free non-coding RNAs are promising biomarker candidates in the diagnosis and prognosis of cancer. Many of these markers have the potential to help clinicians in therapy selection and in the follow-up of patients. Thus, cf-NA-based diagnostics represent a new path in personalized medicine. Although several reviews are available in the field, most of them focus on a limited number of cf-NA types. In this review, we give an overview about all known cf-NAs including cf-DNA, cf-mtDNA and cell-free non-coding RNA (miRNA, lncRNA, circRNA, piRNA, YRNA, and vtRNA) by discussing their biogenesis, biological function and potential as biomarker candidates in liquid biopsy. We also outline possible future directions in the field.
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Ácidos Nucleicos Libres de Células/genética , Exosomas/genética , Feto/metabolismo , Biopsia Líquida/métodos , Medicina de Precisión/métodos , Biomarcadores de Tumor/sangre , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Biomarcadores de Tumor/orina , Ácidos Nucleicos Libres de Células/sangre , Ácidos Nucleicos Libres de Células/metabolismo , Ácidos Nucleicos Libres de Células/orina , ADN Mitocondrial/sangre , ADN Mitocondrial/genética , ADN Mitocondrial/metabolismo , ADN Mitocondrial/orina , Exosomas/metabolismo , Femenino , Feto/patología , Humanos , MicroARNs/sangre , MicroARNs/genética , MicroARNs/metabolismo , Mutación , Embarazo , Pronóstico , ARN Largo no Codificante/sangre , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , ARN Largo no Codificante/orinaRESUMEN
OBJECTIVE: The aim of this study was to evaluate if urinary sediment cells offered a robust alternative to muscle biopsy for the diagnosis of single mtDNA deletions. METHODS: Eleven adult patients with progressive external ophthalmoplegia and a known single mtDNA deletion were investigated. Urinary sediment cells were used to isolate DNA, which was then subjected to long-range polymerase chain reaction. Where available, the patient`s muscle DNA was studied in parallel. Breakpoint and thus deletion size were identified using both Sanger sequencing and next generation sequencing. The level of heteroplasmy was determined using quantitative polymerase chain reaction. RESULTS: We identified the deletion in urine in 9 of 11 cases giving a sensitivity of 80%. Breakpoints and deletion size were readily detectable in DNA extracted from urine. Mean heteroplasmy level in urine was 38% ± 26 (range 8 - 84%), and 57% ± 28 (range 12 - 94%) in muscle. While the heteroplasmy level in urinary sediment cells differed from that in muscle, we did find a statistically significant correlation between these two levels (R = 0.714, P = 0.031(Pearson correlation)). INTERPRETATION: Our findings suggest that urine can be used to screen patients suspected clinically of having a single mtDNA deletion. Based on our data, the use of urine could considerably reduce the need for muscle biopsy in this patient group.
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ADN Mitocondrial/genética , ADN Mitocondrial/orina , Miopatías Mitocondriales/diagnóstico , Miopatías Mitocondriales/orina , Eliminación de Secuencia/genética , Urinálisis/normas , Adolescente , Adulto , Femenino , Humanos , Masculino , Persona de Mediana Edad , Oftalmoplejía Externa Progresiva Crónica/diagnóstico , Oftalmoplejía Externa Progresiva Crónica/orina , Reacción en Cadena de la Polimerasa , Sensibilidad y Especificidad , Análisis de Secuencia de ADNRESUMEN
BACKGROUNDMitochondrial dysfunction, a proposed mechanism of chronic obstructive pulmonary disease (COPD) pathogenesis, is associated with the leakage of mitochondrial DNA (mtDNA), which may be detected extracellularly in various bodily fluids. Despite evidence for the increased prevalence of chronic kidney disease in COPD subjects and for mitochondrial dysfunction in the kidneys of murine COPD models, whether urine mtDNA (u-mtDNA) associates with measures of disease severity in COPD is unknown.METHODSCell-free u-mtDNA, defined as copy number of mitochondrially encoded NADH dehydrogenase-1 (MTND1) gene, was measured by quantitative PCR and normalized to urine creatinine in cell-free urine samples from participants in the Subpopulations and Intermediate Outcome Measures in COPD Study (SPIROMICS) cohort. Urine albumin/creatinine ratios (UACR) were measured in the same samples. Associations between u-mtDNA, UACR, and clinical disease parameters - including FEV1 % predicted, clinical measures of exercise tolerance, respiratory symptom burden, and chest CT measures of lung structure - were examined.RESULTSU-mtDNA and UACR levels were measured in never smokers (n = 64), smokers without airflow obstruction (n = 109), participants with mild/moderate COPD (n = 142), and participants with severe COPD (n = 168). U-mtDNA was associated with increased respiratory symptom burden, especially among smokers without COPD. Significant sex differences in u-mtDNA levels were observed, with females having higher u-mtDNA levels across all study subgroups. U-mtDNA associated with worse spirometry and CT emphysema in males only and with worse respiratory symptoms in females only. Similar associations were not found with UACR.CONCLUSIONU-mtDNA levels may help to identify distinct clinical phenotypes and underlying pathobiological differences in males versus females with COPD.TRIAL REGISTRATIONThis study has been registered at ClinicalTrials.gov ( NCT01969344).FUNDINGUS NIH, National Heart, Lung and Blood Institute, supplemented by contributions made through the Foundation for the NIH and the COPD Foundation from AstraZeneca/MedImmune, Bayer, Bellerophon Therapeutics, Boehringer-Ingelheim Pharmaceuticals Inc., Chiesi Farmaceutici S.p.A., Forest Research Institute Inc., GlaxoSmithKline, Grifols Therapeutics Inc., Ikaria Inc., Novartis Pharmaceuticals Corporation, Nycomed GmbH, ProterixBio, Regeneron Pharmaceuticals Inc., Sanofi, Sunovion, Takeda Pharmaceutical Company, and Theravance Biopharma and Mylan.
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ADN Mitocondrial/orina , Enfermedad Pulmonar Obstructiva Crónica/orina , Anciano , Biomarcadores/orina , Estudios de Cohortes , Femenino , Humanos , Masculino , Persona de Mediana Edad , Enfermedad Pulmonar Obstructiva Crónica/fisiopatologíaRESUMEN
BACKGROUND: The value of urinary mitochondrial DNA (mtDNA) for assessing kidney injury of anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) was investigated. METHODS: Thirty-nine kidney biopsy-proved myeloperoxidase (MPO)-ANCA associated AAV patients were enrolled and analyzed. RESULTS: The average urinary mtDNA of patients was significantly higher than that of normal controls (3372.74 ± 1859.72 vs. 474.90 ± 123.59 copy/nmol creatinine, p < 0.001). The patients who needed dialysis at disease onset had the highest levels of urinary mtDNA (5072.23 ± 1302.87 copy/nmol creatinine). Urinary mtDNA positively correlated with urinary neutrophil gelatinase-associated lipocalin (R = 0.661, P < 0.001) and negatively correlated with estimated glomerular filtration rate (R = -0.515, P = 0.001). The urinary mtDNA level of crescentic class (4703.08 ± 1744.31 copy/nmol creatinine) was higher than that of mixed class (3258.14 ± 1158.99 copy/nmol creatinine) and focal class (2268.15 ± 1897.63 copy/nmol creatinine). Univariate correlation analysis showed urinary mtDNA positively correlated with interstitial neutrophils (R = 0.471, P = 0.048) and glomerular neutrophils (R = 0.673, P = 0.002) in kidney biopsy. Among 13 patients who needed hemodialysis at disease onset, 10 patients who got renal recovery had higher urinary mtDNA than 3 patients who remained dialysis dependent (5455.20 ± 1174.64 vs. 3795.67 ± 893.34 copy/nmol creatinine, p = 0.047). CONCLUSIONS: Urinary mtDNA increases in AAV with kidney injury, and its levels correlate with the severity of kidney injury and neutrophils infiltration in pathology.
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Lesión Renal Aguda/orina , Vasculitis Asociada a Anticuerpos Citoplasmáticos Antineutrófilos/orina , ADN Mitocondrial/orina , Lesión Renal Aguda/diagnóstico , Lesión Renal Aguda/metabolismo , Vasculitis Asociada a Anticuerpos Citoplasmáticos Antineutrófilos/diagnóstico , Vasculitis Asociada a Anticuerpos Citoplasmáticos Antineutrófilos/metabolismo , Biomarcadores/metabolismo , Biomarcadores/orina , ADN Mitocondrial/metabolismo , Femenino , Tasa de Filtración Glomerular , Humanos , Lipocalina 2/metabolismo , Lipocalina 2/orina , Masculino , Persona de Mediana Edad , Peroxidasa/metabolismo , Peroxidasa/orinaRESUMEN
BACKGROUND: Ischaemia-reperfusion (IR) injury is an important determinant of delayed graft function (DGF) affecting allograft function. Mitochondrial DNA (mtDNA) is released upon cell death and platelet activation into the extracellular environment and has been suggested to be a biomarker in several diseases. Whether extracellular mtDNA accumulates in plasma and/or urine upon renal IR and predisposes DGF is unknown. METHODS: C57BL/6J wild-type mice were subjected to renal IR. In addition, an observational case-control study was set up enrolling 43 patients who underwent kidney transplantation. One day post-IR in mice and a few days following renal transplantation in human, blood and urine were collected. Patients were stratified into DGF and non-DGF groups. RESULTS: mtDNA-encoded genes accumulate in urine and plasma in both mice subjected to renal IR injury and in humans following renal transplantation. In human renal transplant recipients, cold ischaemia time and renal function correlate with urinary mtDNA levels. Urinary mtDNA levels but not urinary nuclear DNA levels were significantly higher in the DGF group compared with the non-DGF group. Multiple receiver operating characteristic curves revealed significant diagnostic performance for mtDNA-encoded genes cytochrome c oxidase III (COXIII); nicotinamide adenine dinucleotide hydrogen subunit 1 (NADH-deh); mitochondrially encoded, mitochondrially encoded nicotinamide adenine dinucleotide dehydrogenase 2 (MT-ND2) with an area under the curve of, respectively, 0.71 [P = 0.03; 95% confidence interval (CI) 0.54-0.89], 0.75 (P = 0.01; 95% CI 0.58-0.91) and 0.74 (P = 0.02; 95% CI 0.58-0.89). CONCLUSIONS: These data suggest that renal ischaemia time determines the level of mtDNA accumulation in urine, which associates with renal allograft function and the diagnosis of DGF following renal transplantation.
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Biomarcadores/orina , ADN Mitocondrial/orina , Funcionamiento Retardado del Injerto/diagnóstico , Trasplante de Riñón/efectos adversos , Daño por Reperfusión/complicaciones , Animales , Estudios de Casos y Controles , Funcionamiento Retardado del Injerto/etiología , Funcionamiento Retardado del Injerto/orina , Femenino , Supervivencia de Injerto , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Curva ROC , Receptores de Trasplantes , Trasplante HomólogoRESUMEN
Mitochondrial injury plays important roles in the pathogenesis of various kidney diseases. However, mitochondrial injury in IgA nephropathy (IgAN) remains largely unexplored. Here, we examined the associations among mitochondrial injury, IgAN, and treatment outcomes. We prospectively enrolled patients with IgAN and age-/sex-matched healthy volunteers (HVs) as controls (n = 31 each). Urinary copy numbers of the mitochondrial DNA (mtDNA) genes cytochrome-c oxidase-3 (COX3) and nicotinamide adenine dinucleotide dehydrogenase subunit-1 (ND1) were measured. Urinary mtDNA levels were elevated in the IgAN group compared with that in HVs (p < 0.001). Urinary ND1 levels were significantly higher in the low proteinuria group than in the high proteinuria group (p = 0.027). Changes in urinary levels of ND1 and COX3 were positively correlated with changes in proteinuria (p = 0.038 and 0.024, respectively) and inversely correlated with changes in the estimated glomerular filtration rate (p = 0.033 and 0.017, respectively) after medical treatment. Mitochondrial injury played important roles in IgAN pathogenesis and may be involved in early-stage glomerular inflammation, prior to pathological changes and increased proteinuria. The correlation between changes in urinary mtDNA and proteinuria suggest that these factors may be promising biomarkers for treatment outcomes in IgAN.
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Variaciones en el Número de Copia de ADN , ADN Mitocondrial , Glomerulonefritis por IGA , Mitocondrias , Adulto , ADN Mitocondrial/genética , ADN Mitocondrial/orina , Femenino , Glomerulonefritis por IGA/genética , Glomerulonefritis por IGA/orina , Humanos , Masculino , Persona de Mediana Edad , Mitocondrias/genética , Mitocondrias/metabolismo , Proteínas Mitocondriales/genética , Estudios ProspectivosRESUMEN
INTRODUCTION: Cell-free deoxyribonucleic acid DNA (cf-DNA) in urine is promising due to the advantage of urine as an easily obtained and non-invasive sample source over tissue and blood. In clinical practice, it is important to identify non-invasive biomarkers of chronic kidney disease (CKD) in monitoring and surveillance of disease progression. Information is limited, however, regarding the relationship between urine and plasma cf-DNA and the renal outcome in CKD patients. METHODS: One hundred and thirty-one CKD patients were enrolled between January 2016 and September 2018. Baseline urine and plasma cell-free mitochondrial DNA (cf-mtDNA) and cell-free nuclear DNA (cf-nDNA) were isolated using quantitative real-time PCR. Estimated glomerular filtration rate (eGFR) measurement was performed at baseline and 6-month follow-up. Favorable renal outcome was defined as eGFR at 6 months minus baseline eGFR> = 0. Receiver operator characteristics (ROC) curve analysis was performed to assess different samples of cf-DNA to predict favorable renal outcomes at 6 months. A multivariate linear regression model was used to evaluate independent associations between possible predictors and different samples of cf-DNA. RESULTS: Patients with an advanced stage of CKD has significantly low plasma cf-nDNA and high plasma neutrophil gelatinase-associated lipocalin (NGAL) levels. Low urine cf-mtDNA, cf-nDNA levels and low plasma NGAL were significantly correlated with favorable renal outcomes at 6 months. The urine albumin-creatinine ratio (ACR) or urine protein-creatinine ratio (PCR) level is a robust predictor of cf-mtDNA and cf-nDNA in CKD patients. Baseline urine levels of cf-mtDNA and cf-nDNA could predict renal outcomes at 6 months. CONCLUSIONS: Urinary cf-mtDNA and cf-nDNA may provide novel prognostic biomarkers for renal outcome in CKD patients. The levels of plasma cf-nDNA and plasma NGAL are significantly correlated with the severity of CKD.
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Ácidos Nucleicos Libres de Células/orina , ADN Mitocondrial/orina , Insuficiencia Renal Crónica/fisiopatología , Adulto , Anciano , Albuminuria/orina , Área Bajo la Curva , Biomarcadores/sangre , Biomarcadores/orina , Ácidos Nucleicos Libres de Células/sangre , Creatinina/orina , ADN Mitocondrial/sangre , Progresión de la Enfermedad , Femenino , Tasa de Filtración Glomerular , Humanos , Lipocalina 2/sangre , Masculino , Persona de Mediana Edad , Valor Predictivo de las Pruebas , Pronóstico , Curva ROCRESUMEN
OBJECTIVE: Obesity is an independent risk factor for chronic kidney disease. Recently, urinary mitochondrial DNA (mtDNA) has been used as a surrogate marker of mitochondrial damage in various kidney diseases. However, there are no data regarding its use in patients with obesity or the change in urinary mtDNA copy number after surgery. DESIGN: We prospectively recruited age- and sex-matched healthy volunteers and patients with obesity (n = 22 in each group: nine men and 13 women). The copy number of urinary and serum mtDNA nicotinamide adenine dinucleotide dehydrogenase subunit-1 (mtND-1) and cytochrome-c oxidase 3 (mtCOX-3) was measured using quantitative PCR. We measured urinary mtDNA and body weight and carried out laboratory tests, 6 months after surgery. RESULTS: Urinary mtND-1 copy number was significantly higher in the obese group than in healthy volunteers. However, urinary mtCOX-3 and serum ND-1 copy numbers in the obese group did not differ from that in the healthy volunteers. When patients with obesity were divided into two groups, according to their baseline mtND-1 copy number, bariatric surgery reduced the mtND-1 copy number (P = 0.006) in the high baseline mtDNA copy-number group. The change in urinary mtND-1 copy number was correlated with a change in urinary albumin (r = 0.478, P = 0.025). CONCLUSIONS: Obesity is associated with elevated urinary mtND-1 copy number. Bariatric surgery reduces the elevated urinary mtND-1 copy number in patients with obesity. This suggests that bariatric surgery could attenuate mitochondrial damage in the kidney cells of patients with obesity.
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Cirugía Bariátrica , ADN Mitocondrial/orina , Dosificación de Gen , Obesidad/genética , Adulto , Complejo IV de Transporte de Electrones/sangre , Complejo IV de Transporte de Electrones/orina , Femenino , Tasa de Filtración Glomerular , Humanos , Masculino , NADH Deshidrogenasa/sangre , NADH Deshidrogenasa/orina , Estudios ProspectivosRESUMEN
BACKGROUND: Mitochondrial dysfunction and chronic sterile inflammation are common features of type 2 diabetes. Therefore, we aimed to investigate whether mitochondrial DNA (mtDNA) could be a biomarker implicated in the progression of type 2 diabetes and diabetic nephropathy and explore the underlying mechanism. MATERIAL AND METHODS: We developed a method for relative quantification of mtDNA content in clinical practice. qRT-PCR was used to measure the mtDNA content both in vivo in CD-1 mice with diabetes induction by streptozotocin and in vitro in murine endothelial cells and conditionally immortalized mouse podocytes. By pumping mtDNA into the mouse circulation, the effect of mtDNA on the kidney was assessed in mice. In patients with type 2 diabetes (n = 42; 24 males; mean age 57.9 ± 12.00 years), plasma mtDNA was evaluated. RESULTS: Plasma mtDNA content was significantly decreased in patients with type 2 diabetes, particularly those with significant proteinuria. In vitro, high glucose treatment suppressed intracellular mtDNA content and facilitated the extracellular release of mtDNA, so excessive circulatory mtDNA induced by high glucose might be filtered through the kidney and then into urine. Indeed, urinary mtDNA content was significantly increased in both diabetic patients and mice. Moreover, by pumping excess mtDNA into circulation in mice, filtered mtDNA could trigger inflammation and induce kidney injury. CONCLUSION: Excessive mtDNA filtered through the kidney under diabetic conditions may be involved in chronic renal inflammation. Reduced plasma mtDNA content and increased urinary mtDNA/creatinine ratio might play a potential role as an early biomarker of diabetic nephropathy.
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Biomarcadores/orina , ADN Mitocondrial/genética , ADN Mitocondrial/orina , Diabetes Mellitus Experimental/complicaciones , Diabetes Mellitus Tipo 2/complicaciones , Nefropatías Diabéticas/diagnóstico , Inflamación/diagnóstico , Animales , Estudios de Casos y Controles , Nefropatías Diabéticas/etiología , Nefropatías Diabéticas/orina , Femenino , Estudios de Seguimiento , Humanos , Inflamación/etiología , Inflamación/orina , Masculino , Ratones , Persona de Mediana Edad , PronósticoRESUMEN
BACKGROUND: The mitochondrial DNA m.3243A>G mutation is one the most prevalent mutation causing mitochondrial disease in adult patients. Several cohort studies have used heteroplasmy levels in urinary epithelial cells (UEC) to correlate the genotype of the patients to the clinical severity. However, the interpretation of these data is hampered by a lack of knowledge on the intra-patient variability of the heteroplasmy levels. The goal of this study was to determine the day-to-day variation of the heteroplasmy levels in UEC. METHODS: Fifteen carriers of the m.3243A>G mutation collected five urine samples in a 14-day window. Heteroplasmy levels of the m.3243A>G mutation were determined in these samples. Data from the national cohort study, including Newcastle Mitochondrial Disease Adult Scale scores and clinical diagnosis, were used. RESULTS: In the samples of six patients, heteroplasmy levels were within a 5% margin. In the samples collected from five patients, the margin was >20%. CONCLUSION: Heteroplasmy levels of UEC in carriers of the m.3243A>G mutation have a significant day-to-day variation. The interpretation of a correlation between heteroplasmy levels in urine and disease severity is therefore not reliable. Therefore, heteroplasmy levels in UEC should not be used as a prognostic biomarker in these patients.
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Variación Biológica Individual , ADN Mitocondrial/genética , Enfermedades Mitocondriales/genética , Polimorfismo de Nucleótido Simple , Urotelio/metabolismo , Adulto , Anciano , Biomarcadores/orina , ADN Mitocondrial/orina , Heterocigoto , Humanos , Masculino , Persona de Mediana Edad , Enfermedades Mitocondriales/orinaRESUMEN
Cell free DNA (cfDNA) in plasma has been described as a potential diagnostic indicator for a variety of clinical conditions, including neonatal hypoxia. Neonatal hypoxia or perinatal asphyxia is a severe medical condition caused by a temporary interruption in oxygen availability during birth. Previously, we have reported temporal changes of cfDNA detected in blood in a newborn piglet model of perinatal asphyxia. However, cfDNA can also be found in other body liquids, opening for a less invasive diagnostic prospective. The objective of this study was to test and establish a reliable method for the isolation and quantification of cfDNA from urine and to explore changes in the quantities of cfDNA using a newborn piglet model of asphyxia. Animals were exposed to hypoxia-reoxygenation (n = 6), hypoxia-reoxygenation + hypothermia (n = 6) or were part of the sham-operated control group (n = 6) and urine samples (n = 18) were collected at 570 minutes post-intervention. Two alternative applications of cfDNA measurement were tested, an indirect method comprising a centrifugation step together with DNA extraction with magnetic beads versus a direct assessment based on two centrifugation steps. CfDNA concentrations were determined by a fluorescent assay using PicoGreen and by qRT-PCR. Genomic (gDNA) and mitochondrial DNA (mtDNA) cfDNA were determined in parallel, taking into account potential differences in the rates of damages caused by oxidative stress. In contrast to previous publications, our results indicate that the direct method is insufficient. Application of the indirect method obtained with the fluorescence assay revealed mean cfDNA levels (SD) of 1.23 (1.76) ng/ml for the hypoxia samples, 4.47 (6.15) ng/ml for the samples exposed to hypoxia + hypothermia and 2.75 (3.62) ng/ml for the control animals. The mean cfDNA levels in piglets exposed to hypoxia + hypothermia revealed significantly higher cfDNA amounts compared to mean cfDNA levels in the samples purely exposed to hypoxia (p < 0.05); however, no significant difference could be determined when compared to the control group (p = 0.09). Application of the indirect method by qRT-PCR revealed mean cfDNA levels of mtDNA and gDNA at the detection limit of the technique and thus no reliable statistics could be performed between the observed cfDNA levels in the investigated groups. The methodology for detection and monitoring of cfDNA in urine has to be further optimized before it can be applied in a clinical setting in the future.
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Asfixia Neonatal/diagnóstico , Ácidos Nucleicos Libres de Células/aislamiento & purificación , Hipoxia/complicaciones , Animales , Animales Recién Nacidos , Asfixia Neonatal/etiología , Asfixia Neonatal/terapia , Asfixia Neonatal/orina , Biomarcadores/sangre , Biomarcadores/orina , Ácidos Nucleicos Libres de Células/sangre , Ácidos Nucleicos Libres de Células/orina , ADN Mitocondrial/orina , Modelos Animales de Enfermedad , Estudios de Factibilidad , Femenino , Voluntarios Sanos , Humanos , Hipotermia Inducida/efectos adversos , Límite de Detección , Oxígeno/administración & dosificación , Proyectos Piloto , PorcinosRESUMEN
BACKGROUND: Urinary mitochondrial DNA (mtDNA) fragment level has been proposed as a biomarker of chronic kidney disease (CKD). In this study, we determine the relation between urinary mtDNA level and rate of renal function deterioration in non-diabetic CKD. METHODS: We recruited 102 non-diabetic CKD patients (43 with kidney biopsy that showed non-specific nephrosclerosis). Urinary mtDNA level was measured and compared to baseline clinical and pathological parameters. The patients were followed 48.3 ± 31.8 months for renal events (need of dialysis or over 30% reduction in estimated glomerular filtration rate [eGFR]). RESULTS: The median urinary mtDNA level was 1519.42 (inter-quartile range 511.81-3073.03) million copy/mmol creatinine. There were significant correlations between urinary mtDNA level and baseline eGFR (r = 0.429, p < 0.001), proteinuria (r = 0.368, p < 0.001), severity of glomerulosclerosis (r = - 0.537, p < 0.001), and tubulointerstitial fibrosis (r = - 0.374, p = 0.014). The overall rate of eGFR decline was - 2.18 ± 5.94 ml/min/1.73m2 per year. There was no significant correlation between the rate of eGFR decline and urinary mtDNA level. By univariate analysis, urinary mtDNA level predicts dialysis-free survival, but the result became insignificant after adjusting for clinical and histological confounding factors. CONCLUSION: Urinary mtDNA levels have no significant association with the rate of renal function decline in non-diabetic CKD, although the levels correlate with baseline renal function, proteinuria, and the severity of histological damage. Urinary mtDNA level may be a surrogate marker of permanent renal damage in non-diabetic CKD.
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ADN Mitocondrial/orina , Tasa de Filtración Glomerular , Nefroesclerosis/patología , Insuficiencia Renal Crónica/patología , Insuficiencia Renal Crónica/orina , Adulto , Anciano , Biomarcadores/orina , Femenino , Fibrosis , Humanos , Glomérulos Renales/patología , Túbulos Renales/patología , Masculino , Persona de Mediana Edad , Proteinuria/orina , Insuficiencia Renal Crónica/fisiopatologíaRESUMEN
Mitochondrial disease associated with the pathogenic m.3243A>G variant is a common, clinically heterogeneous, neurogenetic disorder. Using multiple linear regression and linear mixed modelling, we evaluated which commonly assayed tissue (blood N = 231, urine N = 235, skeletal muscle N = 77) represents the m.3243A>G mutation load and mitochondrial DNA (mtDNA) copy number most strongly associated with disease burden and progression. m.3243A>G levels are correlated in blood, muscle and urine (R2 = 0.61-0.73). Blood heteroplasmy declines by ~2.3%/year; we have extended previously published methodology to adjust for age. In urine, males have higher mtDNA copy number and ~20% higher m.3243A>G mutation load; we present formulas to adjust for this. Blood is the most highly correlated mutation measure for disease burden and progression in m.3243A>G-harbouring individuals; increasing age and heteroplasmy contribute (R2 = 0.27, P < 0.001). In muscle, heteroplasmy, age and mtDNA copy number explain a higher proportion of variability in disease burden (R2 = 0.40, P < 0.001), although activity level and disease severity are likely to affect copy number. Whilst our data indicate that age-corrected blood m.3243A>G heteroplasmy is the most convenient and reliable measure for routine clinical assessment, additional factors such as mtDNA copy number may also influence disease severity.
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ADN Mitocondrial/análisis , Genes Mitocondriales , Enfermedades Mitocondriales/genética , Mutación , Adulto , Factores de Edad , Anciano , Variaciones en el Número de Copia de ADN , Análisis Mutacional de ADN , ADN Mitocondrial/sangre , ADN Mitocondrial/orina , Progresión de la Enfermedad , Femenino , Humanos , Modelos Lineales , Masculino , Persona de Mediana Edad , Músculo Esquelético/metabolismo , Análisis de Regresión , Factores SexualesRESUMEN
BACKGROUND: Mitochondrial dysfunction plays an important role in the pathogenesis and progression of chronic kidney disease (CKD). We study the relation between urinary mitochondrial DNA (mtDNA) levels and renal dysfunction in non-diabetic CKD. METHODS: We recruited 32 CKD patients (20 had hypertensive nephrosclerosis, 12 had IgA nephropathy). Urinary supernatant mtDNA level was measured and compared to baseline clinical and pathological parameters. The patients were followed 57.8⯱â¯30.5â¯months for renal function decline. RESULTS: The average urinary supernatant mtDNA level was 222.0⯱â¯210.3â¯copy/µL. There was a modest but significant correlation between urinary mtDNA level and proteinuria (Spearman's râ¯=â¯0.387, pâ¯=â¯0.035), but not any other baseline clinical or pathological parameter. Urinary mtDNA level had a significant inverse correlation with the slope of GFR decline (râ¯=â¯-0.402, pâ¯=â¯0.023). Urinary mtDNA level is a predictor of renal survival even after adjusting for baseline proteinuria with multivariate Cox analysis. In this model, every increase in urinary mtDNA by 100â¯copy/µL confers a 25.0% increase in risk of doubling of serum creatinine or need of dialysis (95%CI, 0.7% to 55.1%). CONCLUSION: Mitochondrial DNA is readily detectable in the urinary supernatant of non-diabetic CKD, and its level correlates with the rate of renal function decline and predicts the risk of doubling of serum creatinine or need of dialysis. Further studies are needed to determine the value of urinary supernatant mtDNA level as a prognostic indicator of non-diabetic CKD.
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ADN Mitocondrial/orina , Insuficiencia Renal Crónica/orina , Adulto , Anciano , Femenino , Humanos , Masculino , Persona de Mediana Edad , Adulto JovenRESUMEN
BACKGROUND: Recent animal studies have shown that mitochondrial dysfunction initiates and accelerates renal injury in sepsis, but its role in sepsis remains unknown. Mitochondrial stress or dying cells can lead to fragmentation of the mitochondrial genome, which is considered a surrogate marker of mitochondrial dysfunction. Therefore, we evaluated the efficiency of urinary mitochondrial DNA (UmtDNA) as a marker of renal dysfunction during sepsis-induced acute kidney injury (AKI). METHODS: We isolated DNA from plasma and urine of patients. mtDNA levels were quantified by quantitative PCR. Sepsis patients were divided into no AKI, mild AKI, and severe AKI groups according to RIFLE criteria. Additionally, cecal ligation and puncture (CLP) was established in rats to evaluate the association between UmtDNA and mitochondrial function. RESULTS: A total of 52 (49.5%) developed AKI among enrolled sepsis patients. Increased systemic mtDNA did not correlate with systemic inflammation or acute renal dysfunction in sepsis patients, while AKI did not have an additional effect on circulating mtDNA levels. In contrast, UmtDNA was significantly enriched in severe AKI patients compared with that in the mild AKI or no AKI group, positively correlated with plasma creatinine, urinary neutrophil gelatinase-associated lipocalin, and kidney injury molecule-1, and inversely with the estimated glomerular filtration rate. Additionally, UmtDNA increased in rats following CLP-induced sepsis. UmtDNA was predictive of AKI development and correlated with plasma creatinine and blood urea nitrogen in the rat sepsis model. Finally, the UmtDNA level was inversely correlated with the cortical mtDNA copy number and relative expression of mitochondrial gene in the kidney. CONCLUSION: An elevated UmtDNA level correlates with mitochondrial dysfunction and renal injury in sepsis patients, indicating renal mitochondrial injury induced by sepsis. Therefore, UmtDNA may be regarded as a valuable biomarker for the occurrence of AKI and the development of mitochondria-targeted therapies following sepsis-induced AKI.
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Lesión Renal Aguda/orina , ADN Mitocondrial/orina , Riñón/metabolismo , Riñón/patología , Sepsis/complicaciones , Sepsis/orina , Adenosina Trifosfato/metabolismo , Adulto , Animales , Femenino , Tasa de Filtración Glomerular/fisiología , Humanos , Masculino , Persona de Mediana Edad , Embarazo , ARN Mensajero/genética , Ratas , Ratas Sprague-DawleyRESUMEN
Background: Mitochondrial dysfunction plays an important role in the pathogenesis and progression of diabetic nephropathy (DN). We study the relation between urinary and intra-renal mitochondrial deoxyribonucleic acid (mtDNA) levels and renal dysfunction in DN. Methods: We recruited 92 patients with biopsy-proven DN. Urinary sediment, urinary supernatant and intra-renal mtDNA levels were measured and compared with baseline renal biopsy, kidney scarring and renal function decline in the subsequent 24 months. Results: mtDNA could be detected in all urine supernatant, urine sediment and renal biopsy specimens. There was a modest but statistically significant inverse correlation between urinary supernatant and intra-renal mtDNA levels (r = -0.453, P = 0.012). Urinary supernatant mtDNA level had modest but statistically significant correlations, inversely with estimated glomerular filtration rate (r = -0.214, P = 0.04), and positively with interstitial fibrosis (r = 0.300, P = 0.005). Intra-renal mtDNA had significant inverse correlation with interstitial fibrosis (r = -0.537, P = 0.003). However, there was no significant relation between renal function decline and urinary supernatant, urinary sediment or intra-renal mtDNA levels. Conclusions: mtDNA is readily detectable in urinary supernatant and kidney tissue, and their levels correlate with renal function and scarring in DN. Further studies are needed to determine the accuracy of urinary supernatant mtDNA level as a prognostic indicator of DN, as well as its role in other kidney diseases.