Zebrafish (Danio rerio) larva as an in vivo vertebrate model to study renal function.

There is an increasing interest in using zebrafish (Danio rerio) larva as a vertebrate screening model to study drug disposition. As the pronephric kidney of zebrafish larvae shares high similarity with the anatomy of nephrons in higher vertebrates including humans, we explored in this study whether 3- to 4-day-old zebrafish larvae have a fully functional pronephron. Intravenous injection of fluorescent polyethylene glycol and dextran derivatives of different molecular weight revealed a cutoff of 4.4-7.6 nm in hydrodynamic diameter for passive glomerular filtration, which is in agreement with corresponding values in rodents and humans. Distal tubular reabsorption of a FITC-folate conjugate, covalently modified with PEG2000, via folate receptor 1 was shown. Transport experiments of fluorescent substrates were assessed in the presence and absence of specific inhibitors in the blood systems. Thereby, functional expression in the proximal tubule of organic anion transporter oat (slc22) multidrug resistance-associated protein mrp1 (abcc1), mrp2 (abcc2), mrp4 (abcc4), and zebrafish larva p-glycoprotein analog abcb4 was shown. In addition, nonrenal clearance of fluorescent substrates and plasma protein binding characteristics were assessed in vivo. The results of transporter experiments were confirmed by extrapolation to ex vivo experiments in killifish (Fundulus heteroclitus) proximal kidney tubules. We conclude that the zebrafish larva has a fully functional pronephron at 96 h postfertilization and is therefore an attractive translational vertebrate screening model to bridge the gap between cell culture-based test systems and pharmacokinetic experiments in higher vertebrates.NEW & NOTEWORTHY The study of renal function remains a challenge. In vitro cell-based assays are approved to study, e.g., ABC/SLC-mediated drug transport but do not cover other renal functions such as glomerular filtration. Here, in vivo studies combined with in vitro assays are needed, which are time consuming and expensive. In view of these limitations, our proof-of-concept study demonstrates that the zebrafish larva is a translational in vivo test model that allows for mechanistic investigations to study renal function.

Zebrafish Larvae as an in vivo Model for Antimicrobial Activity Tests against Intracellular Salmonella.

INTRODUCTION: Blood infections from multi-drug-resistant Salmonella pose a major health burden. This is especially true because Salmonella can survive and replicate intracellularly, and the development of new treatment strategies is dependent on expensive and time-consuming in vivo trials. The aim of this study was to develop a Salmonella-infection model that makes it possible to directly observe Salmonella infections of macrophages in vivo and to use this model to test the effect of antimicrobials against intra- and extracellular Salmonella in order to close the gap between in vitro and rodent-infection models. METHODS: We established suitable Salmonella-infection conditions using genetically engineered zebrafish and Salmonella-expressing fluorescent proteins (green fluorescent protein (GFP) and/or mCherry). RESULTS: We detected Salmonella inside and outside zebrafish larvae macrophages. Administration of the cell-impermeable antibiotic tobramycin removed Salmonella residing outside macrophages but did not affect Salmonella in macrophages, whereas ceftriaxone successfully cleared both types of Salmonella. Salmonella inside and outside macrophages experienced substantial DNA damage after administration of fluoroquinolones consistent with the excellent cell penetration of these antibiotics. CONCLUSIONS: The zebrafish-larvae model enables testing of antimicrobials for efficacy against extra- and intracellular Salmonella in a complex in vivo environment. This model thus might serve for antimicrobial lead optimization prior to using rodent models.

Incorporation of phosphatidylserine improves efficiency of lipid based gene delivery systems.

The essential homeostatic process of dead cell clearance (efferocytosis) is used by viruses in an act of apoptotic mimicry. Among others, virions leverage phosphatidylserine (PS) as an essential "eat me" signal in viral envelopes to increase their infectivity. In a virus-inspired biomimetic approach, we demonstrate that PS can be incorporated into non-viral lipid nanoparticle (LNP) pDNA/mRNA constructs to enhance cellular transfection. The inclusion of the bioactive PS leads to an increased ability of LNPs to deliver nucleic acids in vitro to cultured HuH-7 hepatocellular carcinoma cells resulting in a 6-fold enhanced expression of a transgene. Optimal PS concentrations are in the range of 2.5 to 5% of total lipids. PS-decorated mRNA-LNPs show a 5.2-fold enhancement of in vivo transfection efficiency as compared to mRNA-LNPs devoid of PS. Effects were less pronounced for PS-decorated pDNA-LNPs (3.2-fold increase). Incorporation of small, defined amounts of PS into gene delivery vectors opens new avenues for efficient gene therapy and can be easily extended to other therapeutic systems.