Corticosteroids alter kidney development and increase glomerular filtration rate in larval zebrafish (Danio rerio).

Pharmaceutical drugs and other chemicals can impact organogenesis, either during pregnancy or by postnatal exposure of very preterm infants. Corticosteroids are administered to pregnant women at risk of preterm delivery in order to reduce neonatal morbidity and mortality. In addition, high-dose corticosteroid exposure of very preterm infants regularly serves to maintain blood pressure and to prevent and treat bronchopulmonary dysplasia, a form of chronic lung disease in prematurely born infants. Despite clinical benefits, there is increasing evidence of corticosteroid-mediated short- and long-term detrimental developmental effects, especially in the kidney. Here, we performed a detailed morphological and functional analysis of corticosteroid-mediated effects on pronephros development in larval zebrafish. 24 hours post fertilization (hpf) transgenic Tg(wt1b: EGFP) zebrafish larvae were exposed to a set of natural and synthetic corticosteroids (hydrocortisone, dexamethasone, 6α-methylprednisolone, betamethasone, prednisolone, fludrocortisone, 11-deoxycorticosterone) with varying glucocorticoid and mineralocorticoid potency for 24 hours at different concentrations. A semi-automated, multiparametric in vivo workflow enabled simultaneous assessment of kidney morphology, renal FITC-inulin clearance, and heart rate within the same larva. All corticosteroids exerted significant morphological and functional effects on pronephros development, including a significant hypertrophy of the pronephric glomeruli as well as dose-dependent increases in FITC-inulin clearance as a marker of glomerular filtration rate. In conclusion, the present study demonstrates a significant impact of corticosteroid exposure on kidney development and function in larval zebrafish. Hence, these studies underline that corticosteroid exposure of the fetus and the preterm neonate should be carefully considered due to potential short- and long-term harm to the kidney.

An Experimental Workflow for Studying Barrier Integrity, Permeability, and Tight Junction Composition and Localization in a Single Endothelial Cell Monolayer: Proof of Concept.

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.

In vivo High-Content Screening in Zebrafish for Developmental Nephrotoxicity of Approved Drugs.

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.

A Multiparametric Assay Platform for Simultaneous In Vivo Assessment of Pronephric Morphology, Renal Function and Heart Rate in Larval Zebrafish.

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.

miR-103/107 regulates left-right asymmetry in zebrafish by modulating Kupffer's vesicle development and ciliogenesis.

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.

miR-128 regulates non-myocyte hyperplasia, deposition of extracellular matrix and Islet1 expression during newt cardiac regeneration.

Cardiovascular disease is a global scourge to society, with novel therapeutic approaches required in order to alleviate the suffering caused by sustained cardiac damage. MicroRNAs (miRNAs) are being touted as one such approach in the fight against heart disease, acting as possible post-transcriptional molecular triggers responsible for invoking cardiac regeneration. To further ones understanding of miRNAs and cardiac regeneration, it is prudent to learn from organisms that can intrinsically regenerate their hearts following injury. Using the red-spotted newt, an adult chordate capable of cardiac regeneration, we decided to delve deeper into the role miRNAs play during this process. RNA isolated from regenerating newt heart samples, was used in a microarray screen, to identify significantly expressed candidate miRNAs during newt cardiac regeneration. We performed quantitative qPCR analysis on several conserved miRNAs and found one in particular, miR-128, to be significantly elevated when cardiac hyperplasia is at its peak following injury. In-situ hybridisation techniques revealed a localised expression pattern for miR-128 in the cardiomyocytes and non-cardiomyocytes in close proximity to the regeneration zone and in vivo knockdown studies revealed a regulatory role for miR-128 in proliferating non-cardiomyocyte populations and extracellular matrix deposition. Finally, 3'UTR reporter assays revealed Islet1 as a biological target for miR-128, which was confirmed further through in vivo Islet1 transcriptional and translational expression analysis in regenerating newt hearts. From these studies we conclude that miR-128 regulates both cardiac hyperplasia and Islet1 expression during newt heart regeneration and that this information could be translated into future mammalian cardiac studies.