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1.
Int J Mol Sci ; 22(15)2021 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-34360944

RESUMO

Endothelial and epithelial barrier function is crucial for the maintenance of physiological processes. The barrier paracellular permeability depends on the composition and spatial distribution of the cell-to-cell tight junctions (TJ). Here, we provide an experimental workflow that yields several layers of physiological data in the setting of a single endothelial cell monolayer. Human umbilical vein endothelial cells were grown on Transwell filters. Transendothelial electrical resistance (TER) and 10 kDa FITC dextran flux were measured using Alanyl-Glutamine (AlaGln) as a paracellular barrier modulator. Single monolayers were immunolabelled for Zonula Occludens-1 (ZO-1) and Claudin-5 (CLDN5) and used for automated immunofluorescence imaging. Finally, the same monolayers were used for single molecule localization microscopy (SMLM) of ZO-1 and CLDN5 at the nanoscale for spatial clustering analysis. The TER increased and the paracellular dextran flux decreased after the application of AlaGln and these functional changes of the monolayer were mediated by an increase in the ZO-1 and CLDN5 abundance in the cell-cell interface. At the nanoscale level, the functional and protein abundance data were accompanied by non-random increased clustering of CLDN5. Our experimental workflow provides multiple data from a single monolayer and has wide applicability in the setting of paracellular studies in endothelia and epithelia.


Assuntos
Permeabilidade Capilar , Junções Íntimas/metabolismo , Claudina-5/metabolismo , Dextranos/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Proteína da Zônula de Oclusão-1/metabolismo
2.
Medicine (Baltimore) ; 100(25): e26403, 2021 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-34160425

RESUMO

INTRODUCTION: Sepsis and septic shock are the most severe forms of infection affecting predominantly elderly people, preterm and term neonates, and young infants. Even in high-income countries sepsis causes about 8% of admissions to pediatric intensive care units (PICUs). Early diagnosis, rapid anti-infective treatment, and prompt hemodynamic stabilization are crucial for patient survival. In this context, it is essential to identify the causative pathogen as soon as possible to optimize antimicrobial treatment. To date, culture-based diagnostic procedures (e.g., blood cultures) represent the standard of care. However, they have 2 major problems: on the one hand, in the case of very small sample volumes (and thus usually in children), they are not sufficiently sensitive. On the other hand, with a time-to-result of 2 to 5 days, blood cultures need a relatively long time for the anti-infective therapy to be calculated. To overcome these problems, culture-independent molecular diagnostic procedures such as unbiased sequence analysis of circulating cell-free DNA (cfDNA) from plasma samples of septic patients by next-generation sequencing (NGS) have been tested successfully in adult septic patients. However, these results still need to be transferred to the pediatric setting. METHODS: The Next GeneSiPS-Trial is a prospective, observational, non-interventional, multicenter study used to assess the diagnostic performance of an NGS-based approach for the identification of causative pathogens in (preterm and term) neonates (d1-d28, n = 50), infants (d29 to <1 yr, n = 50), and toddlers (1 yr to <5 yr, n = 50) with suspected or proven severe sepsis or septic shock (according to the pediatric sepsis definition) by the use of the quantitative sepsis indicating quantifier (SIQ) score in comparison to standard of care (culture-based) microbiological diagnostics. Potential changes in anti-infective treatment regimens based on these NGS results will be estimated retrospectively by a panel of 3 independent clinical specialists. DISCUSSION: Neonates, infants, and young children are significantly affected by sepsis. Fast and more sensitive diagnostic approaches are urgently needed. This prospective, observational, non-interventional, multicenter study seeks to evaluate an NGS-based approach in critically ill children suffering from sepsis. TRIAL REGISTRATION: DRKS-ID: DRKS00015705 (registered October 24, 2018). https://www.drks.de/drks_web/navigate.do?navigationId=trial.HTML&TRIAL_ID=DRKS00015705.


Assuntos
Bacteriemia/diagnóstico , Bactérias/isolamento & purificação , Sequenciamento de Nucleotídeos em Larga Escala , Técnicas de Diagnóstico Molecular/métodos , Choque Séptico/diagnóstico , Antibacterianos/uso terapêutico , Bacteriemia/tratamento farmacológico , Bacteriemia/microbiologia , Bactérias/genética , Hemocultura , Pré-Escolar , DNA Bacteriano/isolamento & purificação , Diagnóstico Precoce , Feminino , Humanos , Lactente , Recém-Nascido , Unidades de Terapia Intensiva Pediátrica , Masculino , Estudos Multicêntricos como Assunto , Estudos Observacionais como Assunto , Estudos Prospectivos , Estudos Retrospectivos , Índice de Gravidade de Doença , Choque Séptico/sangue , Choque Séptico/tratamento farmacológico , Choque Séptico/microbiologia
3.
Pediatr Nephrol ; 36(8): 2473-2484, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-33759004

RESUMO

BACKGROUND: Non-HLA antibodies against endothelial targets have been implicated in the pathogenesis of antibody-mediated rejection (ABMR), but data in pediatric patients are scarce. METHODS: We retrospectively analyzed a carefully phenotyped single-center (University Children's Hospital Heidelberg, Germany) cohort of 62 pediatric kidney transplant recipients (mean age at transplantation, 8.6 ± 5.0 years) at increased risk of graft function deterioration. Patients had received their transplant between January 1, 1999, and January 31, 2010. We examined at time of late index biopsies (more than 1-year post-transplant, occurring after January 2004) the association of antibodies against the angiotensin II type 1 receptor (AT1R), the endothelin type A receptor (ETAR), the MHC class I chain-like gene A (MICA), and vimentin in conjunction with overall and complement-binding donor-specific HLA antibodies (HLA-DSA) with graft histology and function. RESULTS: We observed a high prevalence (62.9%) of non-HLA antibody positivity. Seventy-two percent of HLA-DSA positive patients showed additional positivity for at least one non-HLA antibody. Antibodies against AT1R, ETAR, and MICA were associated with the histological phenotype of ABMR. The cumulative load of HLA-DSA and non-HLA antibodies in circulation was related to the degree of microinflammation in peritubular capillaries. Non-HLA antibody positivity was an independent non-invasive risk factor for graft function deterioration (adjusted hazard ratio 6.38, 95% CI, 2.11-19.3). CONCLUSIONS: Our data indicate that the combined detection of antibodies to HLA and non-HLA targets may allow a more comprehensive assessment of the patients' immune responses against the kidney allograft and facilitates immunological risk stratification.

4.
Pediatr Pulmonol ; 55(11): 3057-3066, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32833345

RESUMO

BACKGROUND: Pulmonary alveolar proteinosis (PAP) is a heterogeneous condition with more than 100 different underlying disorders that need to be differentiated to target therapeutic options, which are generally limited. METHODS: The clinical course of two brothers with pathogenic variants in the methionyl-tRNA synthetase (MARS)1 gene was compared to previously published patients. Functional studies in patient-derived fibroblasts were performed and therapeutic options evaluated. RESULTS: The younger brother was diagnosed with PAP at the age of 1 year. Exome sequencing revealed the homozygous MARS1 variant p.(Arg598Cys), leading to interstitial lung and liver disease (ILLD). At 2 years of age, following surgery hypoglycemia was detected, the pulmonary condition deteriorated, and the patient developed multiorgan failure. Six therapeutic whole lung lavages (WLL) were necessary to improve respiratory insufficiency. Methionine supplementation was started and a high protein diet ensured, leading to complete respiratory recovery. The older brother, homozygous for the same MARS1 variant, had a long-known distinct eating preference of methionine-rich food and showed a less severe clinical phenotype. Decreased aminoacylation activity confirmed the pathogenicity of p.(Arg598Cys) in vitro. In agreement with our review of currently published ILLD patients, the presence of hepatopathy, developmental delay, muscular hypotonia, and anemia support the multisystemic character of the disease. CONCLUSIONS: Catabolic events can provoke a severe deterioration of the pulmonary situation in ILLD with a need for repetitive WLL. Although the precise role of oral methionine supplementation and high protein intake are unknown, we observed an apparent treatment benefit, which needs to be evaluated systematically in controlled trials.


Assuntos
Lavagem Broncoalveolar , Proteínas na Dieta/administração & dosagem , Metionina tRNA Ligase/genética , Metionina/administração & dosagem , Proteinose Alveolar Pulmonar/terapia , Insuficiência Respiratória/terapia , Criança , Pré-Escolar , Humanos , Hepatopatias/genética , Hepatopatias/terapia , Doenças Pulmonares Intersticiais/genética , Doenças Pulmonares Intersticiais/terapia , Masculino , Proteinose Alveolar Pulmonar/genética , Insuficiência Respiratória/genética
5.
Front Cell Dev Biol ; 8: 583, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32754590

RESUMO

Despite widespread drug exposure, for example during gestation or in prematurely born children, organ-specific developmental toxicity of most drugs is poorly understood. Developmental and functional abnormalities are a major cause of kidney diseases during childhood; however, the potential causal relationship to exposure with nephrotoxic drugs during nephrogenesis is widely unknown. To identify developmental nephrotoxic drugs in a large scale, we established and performed an automated high-content screen to score for phenotypic renal alterations in the Tg(wt1b:EGFP) zebrafish line. During early nephrogenesis, embryos were exposed to a compound library of approved drugs. After treatment, embryos were aligned within microtiter plates using 3D-printed orientation tools enabling the robust acquisition of consistent dorsal views of pronephric kidneys by automated microscopy. To qualitatively and quantitatively score and visualize phenotypes, we developed software tools for the semi-automated analysis, processing and visualization of this large image-based dataset. Using this scoring scheme, we were able to categorize compounds based on their potential developmental nephrotoxic effects. About 10% of tested drugs induced pronephric phenotypes including glomerular and tubular malformations, or overall changes in kidney morphology. Major chemical compound groups identified to cause glomerular and tubular alterations included dihydropyridine derivatives, HMG CoA reductase inhibitors, fibrates, imidazole, benzimidazole and triazole derivatives, corticosteroids, glucocorticoids, acetic acid derivatives and propionic acid derivatives. In conclusion, the presented study demonstrates the large-scale screening of kidney-specific toxicity of approved drugs in a live vertebrate embryo. The associated technology and tool-sets can be easily adapted for other organ systems providing a unique platform for in vivo large-scale assessment of organ-specific developmental toxicity or other biomedical applications. Ultimately, the presented data and associated visualization and browsing tools provide a resource for potentially nephrotoxic drugs and for further investigations.

6.
Cells ; 9(5)2020 05 20.
Artigo em Inglês | MEDLINE | ID: mdl-32443839

RESUMO

Automated high-throughput workflows allow for chemical toxicity testing and drug discovery in zebrafish disease models. Due to its conserved structural and functional properties, the zebrafish pronephros offers a unique model to study renal development and disease at larger scale. Ideally, scoring of pronephric phenotypes includes morphological and functional assessments within the same larva. However, to efficiently upscale such assays, refinement of existing methods is required. Here, we describe the development of a multiparametric in vivo screening pipeline for parallel assessment of pronephric morphology, kidney function and heart rate within the same larva on a single imaging platform. To this end, we developed a novel 3D-printed orientation tool enabling multiple consistent orientations of larvae in agarose-filled microplates. Dorsal pronephros imaging was followed by assessing renal clearance and heart rates upon fluorescein isothiocyanate (FITC)-inulin microinjection using automated time-lapse imaging of laterally positioned larvae. The pipeline was benchmarked using a set of drugs known to induce developmental nephrotoxicity in humans and zebrafish. Drug-induced reductions in renal clearance and heart rate alterations were detected even in larvae exhibiting minor pronephric phenotypes. In conclusion, the developed workflow enables rapid and semi-automated in vivo assessment of multiple morphological and functional parameters.


Assuntos
Bioensaio/métodos , Testes de Função Cardíaca , Frequência Cardíaca/fisiologia , Rim/fisiologia , Pronefro/anatomia & histologia , Peixe-Zebra/anatomia & histologia , Peixe-Zebra/fisiologia , Animais , Embrião não Mamífero/fisiologia , Fluoresceína-5-Isotiocianato/metabolismo , Larva/fisiologia , Pronefro/embriologia , Peixe-Zebra/embriologia
8.
Biochem Biophys Res Commun ; 527(2): 432-439, 2020 06 25.
Artigo em Inglês | MEDLINE | ID: mdl-32334837

RESUMO

In zebrafish, cilia movement within the Kupffer's vesicle (KV) generates a fluid flow responsible for accumulating nodal signals exclusively in the left lateral plate mesoderm, thereby initiating left-right patterning (LRP). Defects in LRP cause devastating congenital disorders including congenital heart malformations due to organ mis-positioning. We identified the miR-103/107 family to be involved in regulating LRP. Depletion of miR-103/107 in zebrafish embryos resulted in malpositioned and malformed visceral organs and hearts due to disturbed LRP gene expression, indicating early defects in LRP. Additionally, loss of miR-103/107 affected KV morphogenesis and cilia formation without disturbing endoderm development. Human fibroblasts depleted of miR-103a/107 often failed to extend cilia or developed shorter cilia, indicating functional conservation between species. We identified arl6, araf and foxH1 as direct targets of miR-103/107 providing a mechanistic link to cilia development and nodal signal titration. We describe a new microRNA family controlling KV development and hence influencing establishment of internal organ asymmetry.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Peixe-Zebra/genética , Animais , Padronização Corporal , Linhagem Celular , Cílios/genética , Embrião não Mamífero/anormalidades , Embrião não Mamífero/metabolismo , Coração/embriologia , Humanos , Mesoderma/embriologia , Mesoderma/metabolismo , Peixe-Zebra/embriologia
9.
Eur Arch Otorhinolaryngol ; 277(4): 1139-1147, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32020311

RESUMO

PURPOSE: The aim of this study was to determine whether there were differences in decannulation rates and time to decannulation in children depending on the indication for tracheostomy, age, and maturity at birth. STUDY DESIGN: Retrospective chart review and prospective interview by questionnaire. METHODS: The medical records of 106 pediatric patients (age 0-18 years) tracheostomized between January 1 1999 and January 1 2019 were reviewed. Patients were divided into three different groups depending on the indication for tracheostomy: unsafe airway (37.7%), long-term respiratory dependence (50.9%), or bronchopulmonary toilet for aspirations (11.3%). RESULTS: 40 patients were successfully decannulated. The time-dependent decannulation rate after 2 and 5 years was 28.3% and 40.5% for patients with an unsafe airway, 42.4% and 66.8% for patients with long-term respiratory dependence, and 41.7% and 70.8% for patients needing bronchopulmonary toilet, respectively. After 2 and 5 years, patients aged 0-12 months at the time of tracheostomy were decannulated in 13.1% and 50.2% of cases, 1-5-year-olds in 35.3% and 48.2% of cases, 6-10-year-olds in 70% and 70% of cases, and 11-18-year-olds in 66.6% and 66.6% of cases, respectively. However, in a multivariate analysis, prematurity was found to be the only significant unfavorable variable (p = 0.013). Maturely born patients had an odds ratio of 3.87 (95% CI 1.32-11.33) for successful decannulation. This effect was present only in the first 5 years of life. CONCLUSION: Factors indicating problems with decannulation are an unsafe airway, a young age at the time of tracheostomy, and prematurity at birth.


Assuntos
Remoção de Dispositivo , Traqueostomia , Adolescente , Criança , Pré-Escolar , Humanos , Lactente , Recém-Nascido , Razão de Chances , Estudos Prospectivos , Estudos Retrospectivos
10.
Int J Mol Sci ; 21(4)2020 Feb 11.
Artigo em Inglês | MEDLINE | ID: mdl-32054020

RESUMO

Acute kidney injury (AKI) in critically ill children and adults is associated with significant short- and long-term morbidity and mortality. As serum creatinine- and urine output-based definitions of AKI have relevant limitations, there is a persistent need for better diagnostics of AKI. Nuclear magnetic resonance (NMR) spectroscopy allows for analysis of metabolic profiles without extensive sample manipulations. In the study reported here, we examined the diagnostic accuracy of NMR urine metabolite patterns for the diagnosis of neonatal and pediatric AKI according to the Kidney Disease: Improving Global Outcomes (KDIGO) definition. A cohort of 65 neonatal and pediatric patients (0-18 years) with established AKI of heterogeneous etiology was compared to both a group of apparently healthy children (n = 53) and a group of critically ill children without AKI (n = 31). Multivariate analysis identified a panel of four metabolites that allowed diagnosis of AKI with an area under the receiver operating characteristics curve (AUC-ROC) of 0.95 (95% confidence interval 0.86-1.00). Especially urinary citrate levels were significantly reduced whereas leucine and valine levels were elevated. Metabolomic differentiation of AKI causes appeared promising but these results need to be validated in larger studies. In conclusion, this study shows that NMR spectroscopy yields high diagnostic accuracy for AKI in pediatric patients.


Assuntos
Injúria Renal Aguda/urina , Injúria Renal Aguda/diagnóstico , Adolescente , Biomarcadores/urina , Criança , Pré-Escolar , Ácido Cítrico/urina , Feminino , Humanos , Lactente , Recém-Nascido , Leucina/urina , Espectroscopia de Ressonância Magnética , Masculino , Metabolômica , Projetos Piloto , Urinálise , Valina/urina
11.
Genet Med ; 22(3): 511-523, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31680123

RESUMO

PURPOSE: Fetal akinesia has multiple clinical subtypes with over 160 gene associations, but the genetic etiology is not yet completely understood. METHODS: In this study, 51 patients from 47 unrelated families were analyzed using next-generation sequencing (NGS) techniques aiming to decipher the genomic landscape of fetal akinesia (FA). RESULTS: We have identified likely pathogenic gene variants in 37 cases and report 41 novel variants. Additionally, we report putative pathogenic variants in eight cases including nine novel variants. Our work identified 14 novel disease-gene associations for fetal akinesia: ADSSL1, ASAH1, ASPM, ATP2B3, EARS2, FBLN1, PRG4, PRICKLE1, ROR2, SETBP1, SCN5A, SCN8A, and ZEB2. Furthermore, a sibling pair harbored a homozygous copy-number variant in TNNT1, an ultrarare congenital myopathy gene that has been linked to arthrogryposis via Gene Ontology analysis. CONCLUSION: Our analysis indicates that genetic defects leading to primary skeletal muscle diseases might have been underdiagnosed, especially pathogenic variants in RYR1. We discuss three novel putative fetal akinesia genes: GCN1, IQSEC3 and RYR3. Of those, IQSEC3, and RYR3 had been proposed as neuromuscular disease-associated genes recently, and our findings endorse them as FA candidate genes. By combining NGS with deep clinical phenotyping, we achieved a 73% success rate of solved cases.


Assuntos
Doenças Fetais/genética , Fatores de Troca do Nucleotídeo Guanina/genética , Proteínas de Ligação a RNA/genética , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Transativadores/genética , Adolescente , Adulto , Artrogripose/genética , Artrogripose/patologia , Criança , Pré-Escolar , Variações do Número de Cópias de DNA/genética , Feminino , Doenças Fetais/patologia , Predisposição Genética para Doença , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Lactente , Recém-Nascido , Masculino , Doenças Musculares/genética , Doenças Musculares/patologia , Adulto Jovem
12.
Int J Mol Sci ; 20(6)2019 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-30875791

RESUMO

The zebrafish is being increasingly used in biomedical research and drug discovery to conduct large-scale compound screening. However, there is a lack of accessible methodologies to enable automated imaging and scoring of tissue-specific phenotypes at enhanced resolution. Here, we present the development of an automated imaging pipeline to identify chemical modifiers of glomerular cyst formation in a zebrafish model for human cystic kidney disease. Morpholino-mediated knockdown of intraflagellar transport protein Ift172 in Tg(wt1b:EGFP) embryos was used to induce large glomerular cysts representing a robustly scorable phenotypic readout. Compound-treated embryos were consistently aligned within the cavities of agarose-filled microplates. By interfacing feature detection algorithms with automated microscopy, a smart imaging workflow for detection, centring and zooming in on regions of interests was established, which enabled the automated capturing of standardised higher resolution datasets of pronephric areas. High-content screening datasets were processed and analysed using custom-developed heuristic algorithms implemented in common open-source image analysis software. The workflow enables highly efficient profiling of entire compound libraries and scoring of kidney-specific morphological phenotypes in thousands of zebrafish embryos. The demonstrated toolset covers all the aspects of a complex whole organism screening assay and can be adapted to other organs, specimens or applications.


Assuntos
Proteínas de Transporte/genética , Processamento de Imagem Assistida por Computador/métodos , Rim/diagnóstico por imagem , Doenças Renais Policísticas/diagnóstico por imagem , Bibliotecas de Moléculas Pequenas/administração & dosagem , Proteínas Adaptadoras de Transdução de Sinal , Algoritmos , Animais , Proteínas do Citoesqueleto , Modelos Animais de Doenças , Embrião não Mamífero/metabolismo , Técnicas de Silenciamento de Genes , Humanos , Rim/metabolismo , Especificidade de Órgãos , Fenótipo , Doenças Renais Policísticas/metabolismo , Bibliotecas de Moléculas Pequenas/metabolismo , Software , Fluxo de Trabalho , Peixe-Zebra
13.
Pediatr Res ; 85(5): 678-686, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30745571

RESUMO

BACKGROUND: Serum creatinine (SCr)- or urine output-based definitions of acute kidney injury (AKI) have important limitations in neonates. This study evaluates the diagnostic value of urinary biomarkers in very low-birth-weight (VLBW) infants receiving indomethacin for closure of a patent ductus arteriosus (PDA). METHODS: Prospective cohort study in 14 indomethacin-treated VLBW infants and 18 VLBW infants without indomethacin as controls. Urinary biomarkers were measured before, during, and after indomethacin administration. RESULTS: Indomethacin therapy was associated with significantly higher SCr concentrations at 36, 84, and 120 h compared to controls. At 36 h, three indomethacin-treated patients met the criteria for neonatal modified Kidney Disease: Improving Global Outcomes (KDIGO) AKI. The product of urinary tissue inhibitor of metalloproteinase-2 and insulin-like growth factor-binding protein 7 ([TIMP-2]•[IGFBP7]) was significantly elevated in the AKI subgroup at 12 h (P < 0.05), hence 24 h earlier than the increase in SCr. Urinary neutrophil gelatinase-associated lipocalin (NGAL) and calprotectin were significantly increased in the indomethacin group at 12 h (P < 0.05), irrespective of fulfillment of the AKI criteria. Urinary kidney injury molecule-1 (KIM-1) was not significantly altered. CONCLUSION: While urinary [TIMP-2]•[IGFBP7] proves valuable for the early diagnosis of neonatal modified KDIGO-defined AKI, elevated urinary NGAL and calprotectin concentrations in indomethacin-treated VLBW infants not fulfilling the AKI criteria may indicate subclinical kidney injury.


Assuntos
Injúria Renal Aguda/urina , Fármacos Cardiovasculares/efeitos adversos , Permeabilidade do Canal Arterial/tratamento farmacológico , Indometacina/efeitos adversos , Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina/urina , Inibidor Tecidual de Metaloproteinase-2/urina , Injúria Renal Aguda/induzido quimicamente , Biomarcadores/sangue , Biomarcadores/urina , Estudos de Casos e Controles , Creatinina/sangue , Creatinina/urina , Estudos Transversais , Permeabilidade do Canal Arterial/complicações , Permeabilidade do Canal Arterial/urina , Feminino , Humanos , Recém-Nascido de Baixo Peso , Recém-Nascido , Recém-Nascido de muito Baixo Peso , Complexo Antígeno L1 Leucocitário/urina , Lipocalina-2/urina , Masculino , Estudos Prospectivos , Resultado do Tratamento
14.
Front Pediatr ; 6: 183, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30003073

RESUMO

Genetic disorders account for a wide range of renal diseases emerging during childhood and adolescence. Due to the utilization of modern biochemical and biomedical techniques, the number of identified disease-associated genes is increasing rapidly. Modeling of congenital human disease in animals is key to our understanding of the biological mechanism underlying pathological processes and thus developing novel potential treatment options. The zebrafish (Danio rerio) has been established as a versatile small vertebrate organism that is widely used for studying human inherited diseases. Genetic accessibility in combination with elegant experimental methods in zebrafish permit modeling of human genetic diseases and dissecting the perturbation of underlying cellular networks and physiological processes. Beyond its utility for genetic analysis and pathophysiological and mechanistic studies, zebrafish embryos, and larvae are amenable for phenotypic screening approaches employing high-content and high-throughput experiments using automated microscopy. This includes large-scale chemical screening experiments using genetic models for searching for disease-modulating compounds. Phenotype-based approaches of drug discovery have been successfully performed in diverse zebrafish-based screening applications with various phenotypic readouts. As a result, these can lead to the identification of candidate substances that are further examined in preclinical and clinical trials. In this review, we discuss zebrafish models for inherited kidney disease as well as requirements and considerations for the technical realization of drug screening experiments in zebrafish.

15.
Am J Physiol Renal Physiol ; 315(4): F861-F869, 2018 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-29513070

RESUMO

Endothelial injury with consecutive microangiopathy and endothelial dysfunction plays a central role in the pathogenesis of the postenteropathic hemolytic uremic syndrome (D + HUS). To identify new treatment strategies, we examined the regenerative potential of endothelial progenitor cells (EPCs) in an in vitro model of Shiga toxin (Stx) 2a-induced glomerular endothelial injury present in D + HUS and the mechanisms of EPC-triggered endothelial regeneration. We simulated the proinflammatory milieu present in D + HUS by priming human renal glomerular endothelial cells (HRGECs) with tumor necrosis factor-α before stimulation with Stx2a. This measure led to a time- and concentration-dependent decrease of HRGEC viability of human renal glomerular endothelial cells as detected by a colorimetric assay. Coincubation with EPCs (104-105 cells/ml) under dynamic flow conditions led to a significant improvement of cell viability in comparison to untreated monolayers (0.45 ± 0.06 vs. 0.16 ± 0.04, P = 0.003). A comparable regenerative effect of EPCs was observed in a coculture model using cell culture inserts (0.41 ± 0.05 vs. 0.16 ± 0.04, P = 0.003) associated with increased concentrations of vascular endothelial growth factor, insulin-like growth factor I, fibroblast growth factor-2, and hepatocyte growth factor in the supernatant. Treatment of Stx2a-injured monolayers with a combination of these growth factors imitated this effect. EPCs did not show distinct sings of migration and angiogenic tube formation in functional assays. These data demonstrate that EPCs significantly improve endothelial viability after Stx2a-induced injury in vitro and that this effect is associated with the release of growth factors by EPCs.


Assuntos
Células Progenitoras Endoteliais/efeitos dos fármacos , Endotélio Vascular/efeitos dos fármacos , Regeneração/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Células Progenitoras Endoteliais/metabolismo , Endotélio Vascular/metabolismo , Humanos , Glomérulos Renais/efeitos dos fármacos , Glomérulos Renais/metabolismo , Neovascularização Fisiológica/efeitos dos fármacos , Toxina Shiga II/farmacologia , Células-Tronco/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/efeitos dos fármacos
16.
Nephrol Dial Transplant ; 33(6): 1065-1072, 2018 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-29444269

RESUMO

Background: We analysed in a carefully phenotyped cohort of paediatric patients the association of serum angiotensin II type 1 receptor antibodies (AT1R-Ab) with specific histological lesions and with graft function and survival in conjunction with overall and complement-binding donor-specific human leucocyte antigen donor-specific antibodies (HLA-DSA). Methods: Sera of 62 patients at the time of renal graft biopsy for clinical indication >1 year post-transplant were assessed for AT1R-Ab by enzyme-linked immunosorbent assay (ELISA) and for DSA and C1q-fixing DSA by single-antigen bead technology. Results: Serum AT1R-Ab concentration was significantly higher in antibody-mediated rejection (ABMR) than in T-cell-mediated rejection or control. By receiver operating characteristic (ROC) curve analysis, the optimal AT1R-Ab cut-off value discriminating between patients with features of ABMR and those without was 9.5 U/mL. A total of 6 of 28 patients (21.4%) with ABMR were only positive for AT1R-Ab. Patients with AT1R-Ab and HLA-DSA double positivity had a significantly higher vascular micro-inflammation score than DSA-negative patients. The 5-year graft survival was only 59% in the AT1R-Ab-positive group compared with 87% in the AT1R-Ab-negative group. Patients with AT1R-Ab and HLA-DSA double positivity tended to have a more rapid decline of estimated glomerular filtration rate (eGFR) than patients who were only positive for AT1R-Ab or HLA-DSA. In a multivariate Cox regression model of non-invasive factors, C1q-positive HLA-DSA, eGFR and AT1R-Ab positivity were significantly associated with accelerated graft function decline. Conclusions: Serum AT1R-Ab positivity in the context of an indication biopsy >1 year post-transplant is associated with the histopathology of ABMR and is an independent non-invasive risk factor for adverse graft outcome.


Assuntos
Anticorpos/efeitos adversos , Rejeição de Enxerto/mortalidade , Sobrevivência de Enxerto , Antígenos HLA/imunologia , Transplante de Rim/efeitos adversos , Receptor Tipo 1 de Angiotensina/imunologia , Transplantados/estatística & dados numéricos , Adolescente , Adulto , Anticorpos/imunologia , Criança , Feminino , Taxa de Filtração Glomerular , Rejeição de Enxerto/etiologia , Rejeição de Enxerto/patologia , Humanos , Masculino , Receptor Tipo 1 de Angiotensina/sangue , Estudos Retrospectivos , Medição de Risco , Fatores de Risco , Taxa de Sobrevida , Doadores de Tecidos
17.
Eur J Pediatr ; 176(6): 745-755, 2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-28409285

RESUMO

Early identification of patients with acute kidney injury (AKI) being at high risk for adverse outcome can influence medical treatment. This study compares urinary calprotectin, kidney injury molecule-1 (KIM-1), and neutrophil gelatinase-associated lipocalin (NGAL) for their performance in predicting mortality and need for renal replacement therapy (RRT) in pediatric AKI patients. Urinary biomarker concentrations were assessed prospectively in 141 subjects aged 0-18 years including 55 patients with established AKI according to pediatric Risk, Injury, Failure, Loss, and End-stage kidney disease (pRIFLE) criteria, 27 patients without AKI, and 59 healthy children. Within the AKI group, receiver operating characteristic (ROC) curve analysis revealed moderate to poor performance of calprotectin and KIM-1 in the prediction of 30-day mortality (calprotectin area under the curve (AUC) 0.55; KIM-1 AUC 0.55) and 3-month mortality (calprotectin AUC 0.61; KIM-1 AUC 0.60) and fair performance in the prediction of RRT requirement (calprotectin AUC 0.72; KIM-1 AUC 0.71). Urinary NGAL showed good performance in predicting 30-day (AUC 0.79) and 3-month (AUC 0.81) mortality and moderate performance in predicting RRT (AUC 0.61). CONCLUSIONS: Whereas urinary calprotectin and KIM-1 can be useful for the prediction of RRT, urinary NGAL has a good diagnostic performance in predicting mortality in pediatric patients with AKI of heterogeneous etiology. What is known: • There is increasing evidence that urinary biomarkers like neutrophil gelatinase-associated lipocalin (NGAL) and kidney injury molecule-1 (KIM-1) are valuable for the prediction of adverse outcome in adult acute kidney injury (AKI), whereas data on pediatric AKI is scarce. What is new: • Urinary calprotectin and KIM-1 do not predict mortality in our heterogeneous pediatric AKI cohort, but they show moderate performance in the prediction of dialysis. • Urinary NGAL is a good predictor of mortality performing better than pRIFLE stage, eGFR, or creatinine, but it shows moderate performance in the prediction of dialysis.


Assuntos
Injúria Renal Aguda/diagnóstico , Receptor Celular 1 do Vírus da Hepatite A/metabolismo , Complexo Antígeno L1 Leucocitário/urina , Lipocalina-2/urina , Injúria Renal Aguda/mortalidade , Injúria Renal Aguda/terapia , Injúria Renal Aguda/urina , Adolescente , Biomarcadores/urina , Criança , Pré-Escolar , Estudos Transversais , Técnicas de Apoio para a Decisão , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Prognóstico , Estudos Prospectivos , Curva ROC , Terapia de Substituição Renal , Sensibilidade e Especificidade , Índice de Gravidade de Doença
18.
Pediatr Nephrol ; 31(12): 2353-2363, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27286687

RESUMO

BACKGROUND: Urinary calprotectin and neutrophil gelatinase-associated lipocalin (NGAL) have recently been identified as promising biomarkers for the differentiation of prerenal and intrinsic acute kidney injury (AKI) in adults. In the study reported here we examined the diagnostic accuracy of calprotectin, NGAL, and kidney injury molecule 1 (KIM-1) in pediatric patients. METHODS: Urinary calprotectin, NGAL, and KIM-1 concentrations were assessed in a study population of 139 pediatric subjects including 39 patients with intrinsic AKI, 14 with prerenal AKI, and 86 non-AKI subjects. RESULTS: Median urinary calprotectin and NGAL concentrations were higher in patients with intrinsic AKI than in those with prerenal AKI (calprotectin by 22-fold, NGAL by 9-fold). Receiver operating characteristic (ROC) curve analyses for the differentiation of intrinsic and prerenal AKI resulted in an area under the curve (AUC) of 0.90 [95 % confidence interval (CI) 0.81-0.98] for calprotectin and 0.73 (95 % CI 0.58-0.87) for NGAL. Median urinary KIM-1 concentrations were not significantly different between patients with prerenal AKI and those with intrinsic disease (P = 0.98; AUC 0.50, 95 % CI, 0.35-0.65). The AUC for the fractional excretion of sodium (FENa) and proteinuria was 0.78 (95 % CI 0.63-0.92) and 0.77 (CI 0.65-0.90), respectively. CONCLUSIONS: Urinary calprotectin outperforms NGAL, KIM-1, FENa, and proteinuria as a biomarker for the differentiation of prerenal and intrinsic AKI in pediatric patients with a high diagnostic accuracy.


Assuntos
Injúria Renal Aguda/diagnóstico , Injúria Renal Aguda/urina , Biomarcadores/urina , Estudos de Coortes , Estudos Transversais , Diagnóstico Diferencial , Feminino , Receptor Celular 1 do Vírus da Hepatite A/análise , Humanos , Lactente , Recém-Nascido , Complexo Antígeno L1 Leucocitário/urina , Lipocalina-2/urina , Masculino , Proteinúria/etiologia , Proteinúria/urina , Reprodutibilidade dos Testes , Sódio/urina
19.
Pediatr Nephrol ; 31(7): 1157-66, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-26928311

RESUMO

BACKGROUND: We investigated the prognostic value of overall and complement-binding donor-specific HLA antibodies (DSA) in pediatric patients undergoing clinically indicated graft biopsies and their association with graft outcome and specific histological lesions. METHODS: Sera of 62 patients at time of indication biopsy ≥1 year posttransplant were assessed for DSA and C1q-fixing DSA by single-antigen bead (SAB) technology. RESULTS: Twenty-six patients (42 %) were DSA-positive at time of indication biopsy and nine (15 %) were C1q-positive. At 4 years postbiopsy, patients with C1q-positivity had a low graft survival (11 %) compared to DSA-positive, C1q-negative patients (82 %, p = 0.001) and to DSA-negative patients (88 %, p < 0.001). The majority (89 %) of C1q-positive patients were diagnosed with active chronic antibody-mediated rejection (ABMR). C1q DSA-positivity [adjusted hazard ratio (HR) 6.35], presence of transplant glomerulopathy (HR 9.54), and estimated glomerular filtration rate (eGFR) at the time of indication biopsy (HR 0.91) were risk factors for subsequent graft loss. CONCLUSIONS: The presence of C1q-positive DSA in the context of an indication biopsy identifies a subgroup of pediatric renal transplant recipients with a markedly increased risk of subsequent graft loss. Because a fraction of DSA-positive patients escape rejection or graft dysfunction, the C1q assay increases the specificity of a positive DSA result regarding unfavorable transplant outcome.


Assuntos
Anticorpos/imunologia , Ativação do Complemento/imunologia , Complemento C1q/imunologia , Rejeição de Enxerto/imunologia , Antígenos HLA/imunologia , Transplante de Rim , Adolescente , Criança , Estudos de Coortes , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Masculino , Prognóstico , Estudos Retrospectivos , Transplantados
20.
PLoS One ; 10(11): e0143628, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26606754

RESUMO

BACKGROUND: The G1 cell cycle inhibitors tissue inhibitor of metalloproteinase-2 (TIMP-2) and insulin-like growth factor-binding protein 7 (IGFBP7) have been identified as promising biomarkers for the prediction of adverse outcomes including renal replacement therapy (RRT) and mortality in critically ill adult patients who develop acute kidney injury (AKI). However, the prognostic value of urinary TIMP-2 and IGFBP7 in neonatal and pediatric AKI for adverse outcome has not been investigated yet. METHODS: The product of the urinary concentration of TIMP-2 and IGFBP7 ([TIMP-2]•[IGFBP7]) was assessed by a commercially available immunoassay (NephroCheck™) in a prospective cohort study in 133 subjects aged 0-18 years including 46 patients with established AKI according to pRIFLE criteria, 27 patients without AKI (non-AKI group I) and 60 apparently healthy neonates and children (non-AKI group II). AKI etiologies were: dehydration/hypovolemia (n = 7), hemodynamic instability (n = 7), perinatal asphyxia (n = 9), septic shock (n = 7), typical hemolytic-uremic syndrome (HUS; n = 5), interstitial nephritis (n = 5), vasculitis (n = 4), nephrotoxic injury (n = 1) and renal vein thrombosis (n = 1). RESULTS: When AKI patients were classified into pRIFLE criteria, 6/46 (13%) patients fulfilled the criteria for the category "Risk", 13/46 (28%) for "Injury", 26/46 (57%) for "Failure" and 1/46 (2%) for "Loss". Patients in the "Failure" stage had a median 3.7-fold higher urinary [TIMP-2]•[IGFBP7] compared to non-AKI subjects (P<0.001). When analyzed for AKI etiology, highest [TIMP-2]•[IGFBP7] values were found in patients with septic shock (P<0.001 vs. non-AKI I+II). Receiver operating characteristic (ROC) curve analyses in the AKI group revealed good performance of [TIMP-2]•[IGFBP7] in predicting 30-day (area under the curve (AUC) 0.79; 95% CI, 0.61-0.97) and 3-month mortality (AUC 0.84; 95% CI, 0.67-0.99) and moderate performance in predicting RRT (AUC 0.67; 95% CI, 0.50-0.84). CONCLUSIONS: This study shows that urinary [TIMP-2]•[IGFBP7] has a good diagnostic performance in predicting adverse outcomes in neonatal and pediatric AKI of heterogeneous etiology.


Assuntos
Injúria Renal Aguda/diagnóstico , Injúria Renal Aguda/urina , Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina/urina , Inibidor Tecidual de Metaloproteinase-2/urina , Injúria Renal Aguda/etiologia , Injúria Renal Aguda/mortalidade , Adolescente , Fatores Etários , Biomarcadores , Estudos de Casos e Controles , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Recém-Nascido , Estimativa de Kaplan-Meier , Masculino , Avaliação de Resultados da Assistência ao Paciente , Prognóstico , Estudos Prospectivos , Curva ROC
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