Zebrafish as a Model for Drug Screening in Genetic Kidney Diseases

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

Urinary NMR Profiling in Pediatric Acute Kidney Injury—A Pilot Study

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

Urinary Tissue Inhibitor of Metalloproteinase-2 (TIMP-2) • Insulin-Like Growth Factor-Binding Protein 7 (IGFBP7) Predicts Adverse Outcome in Pediatric Acute Kidney Injury.

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.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).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).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

A Smart Imaging Workflow for Organ-Specific Screening in a Cystic Kidney Zebrafish Disease Model

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

Urinary [TIMP-2]•[IGFBP7] in non-AKI and AKI subjects stratified for age.

<p>Boxplots of urinary [TIMP-2]•[IGFBP7] for different age ranges for the non-AKI group I (white boxes), non-AKI group II (light grey boxes) and the AKI group (dark grey boxes). Selected age groups were composed as follows: 0–28 days (non-AKI group I, n = 4; non-AKI group II, n = 18; AKI group, n = 14), 29 days—2 years (non-AKI group I, n = 9; non-AKI group II, n = 4; AKI group, n = 10), 2–5 years (non-AKI group I, n = 7; non-AKI group II, n = 13; AKI group, n = 9), 6–11 years (non-AKI group I, n = 4; non-AKI group II, n = 17; AKI group, n = 4), 12–18 years (non-AKI group I, n = 3; non-AKI group II, n = 8; AKI group, n = 9). The lower and upper edges of the box represent the first and third quartile, respectively, while the horizontal line within the box indicates the median. The vertical length of the box represents the interquartile range (IQR). The most extreme sample values (within a distance of 1.5 x IQR) are the endpoints of the whiskers. Outliers (1.5–3.0 x IQR outside the box) are shown as dots, extremes (> 3.0 x IQR) as triangles. Unit for [TIMP-2]•[IGFBP7] is (ng/mL)²/1,000. Abbreviations: d, days; yrs, years. *P<0.05 vs. AKI of same age group, **P<0.05 vs. age group 0–28 days and 29 days– 2 years of non-AKI group II by Kruskal-Wallis test and Dunn’s multiple comparison test.</p

Characteristics of the study population.

<p>Numeric data are presented as median and interquartile range due to non-normal distribution. Statistical tests used for the individual parameters are presented in the statistics section. Unit for [TIMP-2]•[IGFBP7] is (ng/mL)²/1,000. Abbreviations: AKI, acute kidney injury; SCr, serum creatinine; eCCl, estimated creatinine clearance; CrP, C-reactive protein; RRT, renal replacement therapy; ICU, intensive care unit.</p><p>Characteristics of the study population.</p

Urinary [TIMP-2]•[IGFBP7] in established AKI according to pRIFLE classification.

<p>Boxplots of urinary [TIMP-2]•[IGFBP7] for the AKI cohort (n = 46) stratified for the different pRIFLE stages. Groups were composed as follows: non-AKI I+II (n = 87), “Risk” (n = 6), “Injury” (n = 13), “Failure” (n = 26), “Loss” (n = 1). For explanation of boxplots and unit for [TIMP-2]•[IGFBP7] see legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143628#pone.0143628.g001" target="_blank">Fig 1</a>. P<0.001 for “Failure” vs. non-AKI I+II by Kruskal-Wallis test and Dunn’s multiple comparison test.</p

Urinary [TIMP-2]•[IGFBP7] in established AKI of heterogeneous etiology.

<p>Boxplots of urinary [TIMP-2]•[IGFBP7] for the different AKI etiologies. Cause of AKI is listed under the boxplot, number of patients of each group in brackets. For explanation of boxplots and unit for [TIMP-2]•[IGFBP7] see legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143628#pone.0143628.g001" target="_blank">Fig 1</a>. Abbreviations: HUS, hemolytic uremic syndrome. *P<0.001 by Kruskal-Wallis test and Dunn’s multiple comparison test.</p