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Defects in the glomerular filtration barrier (GFB) play a major role in the onset of human renal diseases. Highly ramified glomerular cells named podocytes are a critical component of the GFB. Injury to podocytes results in abnormal excretion of plasma proteins, which can lead to chronic kidney disease. The conserved paired nephron of larval zebrafish is an excellent model for assessing glomerular function and injury. The efficacy of two known podocyte toxins was tested to refine models of acute podocyte injury in larval zebrafish. The validated compound was then used to test a novel assay of the dynamics of abnormal protein excretion. Injected adriamycin was found to be unsuitable for induction of glomerular injury due to off-target cardiovascular toxicity. In contrast, puromycin treatment resulted in a loss of discriminative filtration, measured by excretion of 70 kDa dextran, and podocyte effacement confirmed by electron microscopy. The dynamics of dextran excretion during puromycin injury modeled the onset of glomerular damage within 24 hours postinjection. These data validate puromycin for induction of acute podocyte injury in zebrafish larvae and describe a semihigh-throughput assay for quantifying the dynamics of abnormal protein excretion.

The ability to kill individual or groups of cells in vivo is important for studying cellular processes and their physiological function. Cell-specific genetically encoded photosensitizing proteins, such as KillerRed, permit spatiotemporal optogenetic ablation with low-power laser light. We report dramatically improved resolution and speed of cell targeting in the zebrafish kidney through the use of a selective plane illumination microscope (SPIM). Furthermore, through the novel incorporation of a Bessel beam into the SPIM imaging arm, we were able to improve on targeting speed and precision. The low diffraction of the Bessel beam coupled with the ability to tightly focus it through a high NA lens allowed precise, rapid targeting of subsets of cells at anatomical depth in live, developing zebrafish kidneys. We demonstrate that these specific targeting strategies significantly increase the speed of optoablation as well as fish survival.

Zebrafish provide an excellent model in which to assess the role of the renin-angiotensin system in renal development, injury, and repair. In contrast to mammals, zebrafish kidney organogenesis terminates with the mesonephros. Despite this, the basic functional structure of the nephron is conserved across vertebrates. The relevance of teleosts for studies relating to the regulation of the renin-angiotensin system was established by assessing the phenotype and functional regulation of renin-expressing cells in zebrafish. Transgenic fluorescent reporters for renin ( ren ), smooth muscle actin ( acta2 ), and platelet-derived growth factor receptor-beta ( pdgfrb ) were studied to determine the phenotype and secretory ultrastructure of perivascular renin-expressing cells. Whole kidney ren transcription responded to altered salinity, pharmacological renin-angiotensin system inhibition, and renal injury. Mesonephric ren -expressing cells occupied niches at the preglomerular arteries and afferent arterioles, forming intermittent epithelioid-like multicellular clusters exhibiting a granular secretory ultrastructure. In contrast, renin cells of the efferent arterioles were thin bodied and lacked secretory granules. Renin cells expressed the perivascular cell markers acta2 and pdgfrb Transcriptional responses of ren to physiological challenge support the presence of a functional renin-angiotensin system and are consistent with the production of active renin. The reparative capability of the zebrafish kidney was harnessed to demonstrate that ren transcription is a marker for renal injury and repair. Our studies demonstrate substantive conservation of renin regulation across vertebrates, and ultrastructural studies of renin cells reveal at least two distinct morphologies of mesonephric perivascular ren -expressing cells., (Copyright © 2017 the American Physiological Society.)

The copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction has proven to be a pivotal advance in chemical ligation strategies with applications ranging from polymer fabrication to bioconjugation. However, application in vivo has been limited by the inherent toxicity of the copper catalyst. Herein, we report the application of heterogeneous copper catalysts in azide-alkyne cycloaddition processes in biological systems ranging from cells to zebrafish, with reactions spanning from fluorophore activation to the first reported in situ generation of a triazole-containing anticancer agent from two benign components, opening up many new avenues of exploration for CuAAC chemistry., (© 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Although renin is a critical regulatory enzyme of the cardiovascular system, its roles in organogenesis and the establishment of cardiovascular homeostasis remain unclear. Mammalian renin-expressing cells are widespread in embryonic kidneys but are highly restricted, specialized endocrine cells in adults. With a functional pronephros, embryonic zebrafish are ideal for delineating the developmental functions of renin-expressing cells and the mechanisms governing renin transcription. Larval zebrafish renin expression originates in the mural cells of the juxtaglomerular anterior mesenteric artery and subsequently at extrarenal sites. The role of renin was determined by assessing responses to renin-angiotensin system blockade, salinity variation, and renal perfusion ablation. Renin expression did not respond to renal flow ablation but was modulated by inhibition of angiotensin-converting enzyme and altered salinity. Our data in larval fish are consistent with conservation of renin's physiological functions. Using transgenic renin reporter fish, with mindbomb and cloche mutants, we show that Notch signaling and the endothelium are essential for developmental renin expression. After inhibition of angiogenesis, renin-expressing cells precede angiogenic sprouts. Arising from separate lineages, but relying on mutual interplay with endothelial cells, renin-expressing cells are among the earliest mural cells observed in larval fish, performing both endocrine and paracrine functions., (Copyright © 2015 the American Physiological Society.)

Zebrafish, a well-established vertebrate model, offer unique advantages for assessing renal function and physiology. Assays determining renal glomerular function based on cardiovascular erythrocyte flow and reduction of injected FITC-inulin were developed, each validated using the nephrotoxin gentamicin. Bland–Atlman analysis showed a strong association between measurements of the rate of inulin excretion and that of fluorescent reduction from the arterial vasculature. Reduced renal clearance of inulin, resulting from gentamicin or NaCl loading, was concurrent with reduced erythrocyte velocity, and yolk sac and pericardium oedema. These techniques, assessing pronephric function, highlight the potential for in vivo physiological study in this genetically tractable model.

The bioavailability of trace elements in fishmeal diets is influenced by their chemical forms and dietary anti-nutritional factors. In formulated fish feed, supplemented organically bound minerals may be more bioavailable than inorganic minerals. A 10-week feeding trial was undertaken with rainbow trout (Oncorhynchus mykiss) to determine whether the inclusion of organically bound selenium (Se) and zinc (Zn) improved uptake and assimilation of these elements compared to commonly used inorganic forms. The three diets tested included a control diet, no added Zn or Se; an organic Se-yeast and Zn-proteinate supplemented diet; and an inorganic sodium selenite and Zn-sulphate supplemented diet. The endpoints tested were apparent digestibility, whole body levels, tissue distribution and Se- and Zn-dependent enzyme activities. Digestibility of residual Se in the basal diet was 54.2 +/- 1.0% and supplemented Se-yeast was significantly more digestible than selenite (p < 0.05). Digestibility of residual Zn was 21.9 +/- 2.0% and no significant difference was found between the treatments (p = 0.89). Whole body Se was raised by both Se sources and to a greatest extent by Se-yeast (p < 0.001). Zn-sulphate, and to a lesser extent Zn-proteinate, both raised whole body Zn (p < 0.05). Dietary Zn in the basal diet was found to be above requirements, yet Zn-sulphate had a significantly greater retention than Zn-proteinate in those tissues that responded to Zn supplementation. Se-yeast significantly raised Se in all tissues to a greater extent than selenite, except in the pyloric caeca and liver where the greatest increases were by selenite. Only Se-yeast elevated Se-dependent thioredoxin reductase activity (p < 0.05) and neither forms of Se affected glutathione peroxidise activity (p = 0.059). Alkaline phosphatase and carboxypeptidase B were not affected by Zn supplementation (p = 0.51 and p = 0.88 respectively). In all aspects, Se-yeast was found to be a highly bioavailable form of Se in comparison to selenite. Because of its superior bioavailability, organically bound Se would be a preferred Se source for supplementation of fishmeal trout diets than selenite.

Intersexuality has been found in both males and females of the marine/estuarine amphipod, Echinogammarus marinus, at polluted and reference sites in East Scotland. Polluted sites had significantly more intersex specimens than reference sites, however the cause of intersexuality is unclear. Discriminant analysis of morphometric data showed that normal male specimens from the most polluted site resembled pooled intersex males, suggesting that subtle endocrine disruption (ED) maybe occurring in these otherwise apparently normal males. The main discriminating character was gnathopod size, recognised to be under androgenic gland control. The association of distinctive morphometry with intersexuality may provide a new approach to biomarkers of ED in crustaceans. The opportunities for other novel biomarkers, for example biochemical or behavioural markers, may also be explored through study of intersex animals.