Comparative Studies of Renin-Null Zebrafish and Mice Provide New Functional Insights.

BACKGROUND: The renin-angiotensin system is highly conserved across vertebrates, including zebrafish, which possess orthologous genes coding for renin-angiotensin system proteins, and specialized mural cells of the kidney arterioles, capable of synthesising and secreting renin. METHODS: We generated zebrafish with CRISPR-Cas9-targeted knockout of renin (ren-/-) to investigate renin function in a low blood pressure environment. We used single-cell (10×) RNA sequencing analysis to compare the transcriptome profiles of renin lineage cells from mesonephric kidneys of ren-/- with ren+/+ zebrafish and with the metanephric kidneys of Ren1c-/- and Ren1c+/+ mice. RESULTS: The ren-/- larvae exhibited delays in larval growth, glomerular fusion and appearance of a swim bladder, but were viable and withstood low salinity during early larval stages. Optogenetic ablation of renin-expressing cells, located at the anterior mesenteric artery of 3-day-old larvae, caused a loss of tone, due to diminished contractility. The ren-/- mesonephric kidney exhibited vacuolated cells in the proximal tubule, which were also observed in Ren1c-/- mouse kidney. Fluorescent reporters for renin and smooth muscle actin (Tg(ren:LifeAct-RFP; acta2:EGFP)), revealed a dramatic recruitment of renin lineage cells along the renal vasculature of adult ren-/- fish, suggesting a continued requirement for renin, in the absence of detectable angiotensin metabolites, as seen in the Ren1YFP Ren1c-/- mouse. Both phenotypes were rescued by alleles lacking the potential for glycosylation at exon 2, suggesting that glycosylation is not essential for normal physiological function. CONCLUSIONS: Phenotypic similarities and transcriptional variations between mouse and zebrafish renin knockouts suggests evolution of renin cell function with terrestrial survival.

Dietary Oxidative Distress: A Review of Nutritional Challenges as Models for Poultry, Swine and Fish.

The redox system is essential for maintaining cellular homeostasis. When redox homeostasis is disrupted through an increase of reactive oxygen species or a decrease of antioxidants, oxidative distress occurs resulting in multiple tissue and systemic responses and damage. Poultry, swine and fish, raised in commercial conditions, are exposed to different stressors that can affect their productivity. Some dietary stressors can generate oxidative distress and alter the health status and subsequent productive performance of commercial farm animals. For several years, researchers used different dietary stressors to describe the multiple and detrimental effects of oxidative distress in animals. Some of these dietary challenge models, including oxidized fats and oils, exposure to excess heavy metals, soybean meal, protein or amino acids, and feeding diets contaminated with mycotoxins are discussed in this review. A better understanding of the oxidative distress mechanisms associated with dietary stressors allows for improved understanding and evaluation of feed additives as mitigators of oxidative distress.

Investigating the RAS can be a fishy business: interdisciplinary opportunities using Zebrafish.

The renin-angiotensin system (RAS) is highly conserved, and components of the RAS are present in all vertebrates to some degree. Although the RAS has been studied since the discovery of renin, its biological role continues to broaden with the identification and characterization of new peptides. The evolutionarily distant zebrafish is a remarkable model for studying the kidney due to its genetic tractability and accessibility for in vivo imaging. The zebrafish pronephros is an especially useful kidney model due to its structural simplicity yet complex functionality, including capacity for glomerular and tubular filtration. Both the pronephros and mesonephros contain renin-expressing perivascular cells, which respond to RAS inhibition, making the zebrafish an excellent model for studying the RAS. This review summarizes the physiological and genetic tools currently available for studying the zebrafish kidney with regards to functionality of the RAS, using novel imaging techniques such as SPIM microscopy coupled with targeted single cell ablation and synthesis of vasoactive RAS peptides.

Pericytes in the renal vasculature: roles in health and disease.

In the dense circulatory system of the kidney, as in all vascularized tissues, pericytes enwrap capillaries and microvessels to regulate angiogenesis, stabilize microvascular networks and control blood flow by vasoconstriction. Specialized renal pericytes known as mesangial cells provide physical support to glomerular capillaries, whereas a subset of juxtaglomerular arteriolar pericytes control the local blood pressure in the glomerulus via contraction and influence systemic blood pressure by secreting renin. Similar to pericytes from many other organs, cultured human renal pericytes give rise to mesenchymal stem/stromal cells, suggesting a role of perivascular cells in renal homeostasis and regeneration. On the other hand, pericytes directly contribute to renal fibrosis, and mesangial cells may have an essential role in the development of glomerulosclerosis and other nephropathies. From their early emergence in the renal embryonic rudiment to their distribution in diverse perivascular niches in the adult organ, we review the anatomy and function of pericytes in the healthy and diseased kidney. Many aspects of the ontogeny, specification and functional specialization of renal pericytes remain elusive. The development of powerful models in the easily accessible and genetically tractable zebrafish will help to uncover the multiple facets of these cells.

The Efficacy of Puromycin and Adriamycin for Induction of Glomerular Failure in Larval Zebrafish Validated by an Assay of Glomerular Permeability Dynamics.

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.

Zebrafish mesonephric renin cells are functionally conserved and comprise two distinct morphological populations.

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.

Copper Catalysis in Living Systems and In†Situ Drug Synthesis.

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.

Renin expression in developing zebrafish is associated with angiogenesis and requires the Notch pathway and endothelium.

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.

Techniques for the in vivo assessment of cardio-renal function in zebrafish (Danio rerio) larvae.

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. Bland­Atlman 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.

Endocrine disruption in a marine amphipod? Field observations of intersexuality and de-masculinisation.

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.