Aluminum and ABC transporter activity.

Aluminum is the third most common element on Earth´s crust and despite its wide use in our workaday life it has been associated with several health risks after overexposure. In the present study the impact of aluminum salts upon ABC transporter activity was studied in the P-GP-expressing human blood-brain barrier cell line hCMEC/D3, in MDCKII cells overexpressing BCRP and MRP2, respectively, and in freshly isolated, functionally intact kidney tubules from Atlantic killifish (Fundulus heteroclitus), which express the analog ABC transporters, P-gp, Bcrp and Mrp2. In contrast to previous findings with heavy metals salts (cadmium(II) chloride or mercury(II) chloride), which have a strong inhibitory effect on ABC transporter activity, or zinc(II) chloride and sodium arsenite, which have a stimulatory effect upon ABC transport function, the results indicate no modulatory effect of aluminum salts on the efflux activity of the human ABC transporters P-GP, BCRP and MRP2 nor on the analog transporters P-gp, Bcrp and Mrp2.

Zebrafish (Danio rerio) larvae as a predictive model to study gentamicin-induced structural alterations of the kidney.

Nephrotoxicity is an important drug safety aspect to be assessed during drug discovery and development. To study renal toxicity, in vitro cell-based assays are often used. Unfortunately, translating the results of such cell assays to vertebrates including human remains challenging. Therefore, we aim to evaluate whether zebrafish larvae (ZFL) could serve as a vertebrate screening model to detect gentamicin-induced changes of kidney glomeruli and proximal tubules. To validate the model, we compared the results of ZFL with those obtained from kidney biopsies of gentamicin-treated mice. We used transgenic zebrafish lines expressing enhanced green fluorescent proteins in the glomerulus to visualize glomerular damage. Synchrotron radiation-based computed tomography (SRµCT) is a label-free approach providing three-dimensional representations of renal structures with micrometre resolution. Clinically used gentamicin concentrations induce nephrotoxicity and affect glomerular and proximal tubular morphology. Findings were confirmed in mice and ZFL. There was a strong correlation between fluorescent signals in ZFL, SRµCT- derived descriptors of glomerular and proximal tubular morphology and the histological analysis of mouse kidney biopsies. A combination of SRµCT and confocal microscopy provides unprecedented insights into anatomical structures of the zebrafish kidney. Based on our findings, we suggest to use ZFL as a predictive vertebrate screening model to study drug-induced nephrotoxicity and to bridge the gap between cell culture-based test systems and experiments in mammals.

Nephrotoxicity of iopamidol is associated with mitochondrial impairment in human cell and teleost models.

Iopamidol is a nonionic, low-osmolar iodinated contrast agent used for angiography. Its clinical use is associated with renal dysfunction. Patients suffering from preexisting kidney disease have an increased risk of renal failure upon iopamidol administration. Studies in animals confirmed renal toxicity, but the involved mechanisms remain unclear. Therefore, the aim of the present study was to use human embryonic kidney cells (HEK293T) as a general cell model of mitochondrial damage, as well as, zebrafish larvae, and isolated proximal tubules of killifish to investigate factors promoting renal tubular toxicity of iopamidol with a focus on mitochondrial damage. Results from in vitro HEK293T cell-based assays indicate that iopamidol affects mitochondrial function Treatment with iopamidol induces ATP depletion, reduces the mitochondrial membrane potential, and elevates mitochondrial superoxide and reactive oxygen species accumulation. Similar results were obtained with gentamicin sulfate and cadmium chloride, two well-known model compounds associated with renal tubular toxicity. Confocal microscopy confirms changes in mitochondrial morphology, such as mitochondrial fission. Importantly, these results were confirmed in proximal renal tubular epithelial cells using ex vivo and in vivo teleost models. In conclusion, this study provides evidence for iopamidol-induced mitochondrial damage in proximal renal epithelial cells. Teleost models allow studying proximal tubular toxicity with translational relevance for humans.

Sodium arsenite but not aluminum chloride stimulates ABC transporter activity in renal proximal tubules of killifish (Fundulus heteroclitus).

ABC export proteins including Multidrug resistance-related protein 2 (Mrp2) serve as detoxification mechanism in renal proximal tubules due to active transport of xenobiotics and metabolic waste products into primary urine. The environmental pollutants aluminum and arsenic interfere with a multitude of regulatory mechanisms in the body and here their impact on ABC transporter function was studied. NaAsO2 but not AlCl3 rapidly stimulated Mrp2-mediated Texas Red (TR) transport in isolated renal proximal tubules from killifish, a well-established laboratory model for the determination of efflux transporter activity by utilizing fluorescent substrates for the ABC transporters of interest and confocal microscopy followed by image analysis. This observed stimulation remained unaffected by the translation inhibitor cycloheximide (CHX), but it was abrogated by antagonists and inhibitors of the endothelin receptor type B (ETB)/nitric oxide synthase (NOS)/protein kinase C (PKC) signaling pathway. NaAsO2-triggered effects were abolished as a consequence of PKCα inhibition through Gö6976 and PKCα inhibitor peptide C2-4. Phosphatidylinositol 3-kinase (PI3K) inhibitor LY 294,002 as well as the mammalian target of rapamycin (mTOR) inhibitor rapamycin suppressed NaAsO2-triggered stimulation of luminal TR transport. In addition, the stimulatory effect of NaAsO2 was abolished by GSK650394, an inhibitor of serum- and glucocorticoid-inducible kinase 1 (SGK1), which is an important downstream target. Environmentally relevant concentrations of NaAsO2 further stimulated transport function of P-glycoprotein (P-gp), Multidrug resistance-related protein 4 (Mrp4) and Breast cancer resistance protein (Bcrp) while AlCl3 was ineffective. To our knowledge, this is the first report engaging in the impact of NaAsO2 on efflux transporter signaling and it may contribute to the understanding of defense mechanisms versus this worrying pollutant.

In vivo acoustic manipulation of microparticles in zebrafish embryos.

In vivo micromanipulation using ultrasound is an exciting technology with promises for cancer research, brain research, vasculature biology, diseases, and treatment development. In the present work, we demonstrate in vivo manipulation of gas-filled microparticles using zebrafish embryos as a vertebrate model system. Micromanipulation methods often are conducted in vitro, and they do not fully reflect the complex environment associated in vivo. Four piezoelectric actuators were positioned orthogonally to each other around an off-centered fluidic channel that allowed for two-dimensional manipulation of intravenously injected microbubbles. Selective manipulation of microbubbles inside a blood vessel with micrometer precision was achieved without interfering with circulating blood cells. Last, we studied the viability of zebrafish embryos subjected to the acoustic field. This successful high-precision, in vivo acoustic manipulation of intravenously injected microbubbles offers potentially promising therapeutic options.

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.

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.

Virus-Derived Peptides for Hepatic Enzyme Delivery.

Recently, a lipopeptide derived from the hepatitis B virus (HBV) large surface protein has been developed as an HBV entry inhibitor. This lipopeptide, called MyrcludexB (MyrB), selectively binds to the sodium taurocholate cotransporting polypeptide (NTCP) on the basolateral membrane of hepatocytes. Here, the feasibility of coupling therapeutic enzymes to MyrB was investigated for the development of enzyme delivery strategies. Hepatotropic targeting shall enable enzyme prodrug therapies and detoxification procedures. Here, horseradish peroxidase (HRP) was conjugated to MyrB via maleimide chemistry, and coupling was validated by SDS-PAGE and reversed-phase HPLC. The specificity of the target recognition of HRP-MyrB could be shown in an NTCP-overexpressing liver parenchymal cell line, as demonstrated by competitive inhibition with an excess of free MyrB and displayed a strong linear dependency on the applied HRP-MyrB concentration. In vivo studies in zebrafish embryos revealed a dominating interaction of HRP-MyrB with scavenger endothelial cells vs xenografted NTCP expressing mammalian cells. In mice, radiolabeled 125I-HRP-MyrBy, as well as the non-NTCP targeted control HRP-peptide-construct (125I-HRP-alaMyrBy) demonstrated a strong liver accumulation confirming the nonspecific interaction with scavenger cells. Still, MyrB conjugation to HRP resulted in an increased and NTCP-mediated hepatotropism, as revealed by competitive inhibition. In conclusion, the model enzyme HRP was successfully conjugated to MyrB to achieve NTCP-specific targeting in vitro with the potential for ex vivo diagnostic applications. In vivo, target specificity was reduced by non-NTCP-mediated interactions. Nonetheless, tissue distribution experiments in zebrafish embryos provide mechanistic insight into underlying scavenging processes indicating partial involvement of stabilin receptors.