Insights from the rat genome sequence

The availability of the rat genome sequence, and detailed three-way comparison of the rat, mouse and human genomes, is revealing a great deal about mammalian genome evolution. Together with recent developments in cloning technologies, this heralds an important phase in rat research

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

Abnormal neonatal sodium handling in skin precedes hypertension in the SAME rat

We discovered high Na(+) and water content in the skin of newborn Sprague-Dawley rats, which reduced ~ 2.5-fold by 7 days of age, indicating rapid changes in extracellular volume (ECV). Equivalent changes in ECV post birth were also observed in C57Bl/6 J mice, with a fourfold reduction over 7 days, to approximately adult levels. This established the generality of increased ECV at birth. We investigated early sodium and water handling in neonates from a second rat strain, Fischer, and an Hsd11b2-knockout rat modelling the syndrome of apparent mineralocorticoid excess (SAME). Despite Hsd11b2(-/-) animals exhibiting lower skin Na(+) and water levels than controls at birth, they retained ~ 30% higher Na(+) content in their pelts at the expense of K(+) thereafter. Hsd11b2(-/-) neonates exhibited incipient hypokalaemia from 15 days of age and became increasingly polydipsic and polyuric from weaning. As with adults, they excreted a high proportion of ingested Na(+) through the kidney, (56.15 ± 8.21% versus control 34.15 ± 8.23%; n = 4; P < 0.0001), suggesting that changes in nephron electrolyte transporters identified in adults, by RNA-seq analysis, occur by 4 weeks of age. Our data reveal that Na(+) imbalance in the Hsd11b2(-/-) neonate leads to excess Na(+) storage in skin and incipient hypokalaemia, which, together with increased, glucocorticoid-induced Na(+) uptake in the kidney, then contribute to progressive, volume contracted, salt-sensitive hypertension. Skin Na(+) plays an important role in the development of SAME but, equally, may play a key physiological role at birth, supporting post-natal growth, as an innate barrier to infection or as a rudimentary kidney

Renal disease pathophysiology and treatment:contributions from the rat

The rat has classically been the species of choice for pharmacological studies and disease modeling, providing a source of high-quality physiological data on cardiovascular and renal pathophysiology over many decades. Recent developments in genome engineering now allow us to capitalize on the wealth of knowledge acquired over the last century. Here, we review rat models of hypertension, diabetic nephropathy, and acute and chronic kidney disease. These models have made important contributions to our understanding of renal diseases and have revealed key genes, such as Ace and P2rx7, involved in renal pathogenic processes. By targeting these genes of interest, researchers are gaining a better understanding of the etiology of renal pathologies, with the promised potential of slowing disease progression or even reversing the damage caused. Some, but not all, of these target genes have proved to be of clinical relevance. However, it is now possible to generate more sophisticated and appropriate disease models in the rat, which can recapitulate key aspects of human renal pathology. These advances will ultimately be used to identify new treatments and therapeutic targets of much greater clinical relevance

Development of NASA's Sample Cartridge Assembly: Summary of GEDS Design, Development Testing, and Thermal Analyses

NASA's Sample Cartridge Assembly (SCA) project is responsible for designing and validating a payload that contains materials research samples in a sealed environment. The SCA will be heated in the European Space Agency's (ESA) Low Gradient Furnace (LGF) that is housed inside the Material Science Research Rack (MSRR) located on the International Space Station (ISS). The first Principle Investigator (PI) to utilize the SCA will focus on Gravitational Effects on Distortion in Sintering (GEDS) research. This paper will give a summary of the design and development test effort for the GEDS SCA and will discuss the role of thermal analysis in developing test profiles to meet the science and engineering requirements. Lessons learned will be reviewed and salient design features that may differ for each PI will be discussed

scRNA Transcription Profile of Adult Zebrafish Podocytes Using a Novel Reporter Strain

Background/Aims: The role of podocytes is well conserved across species from drosophila to teleosts, and mammals. Identifying the molecular markers that actively maintain the integrity of the podocyte will enable a greater understanding of the changes that lead to damage. Methods: We generated transgenic zebrafish, expressing fluorescent reporters driven by the podocin promoter, for the visualization and isolation of podocytes. We have conducted single cell RNA sequencing (scRNA-seq) on isolated podocytes from a zebrafish reporter line. Results: We demonstrated that the LifeAct-TagRFP-T fluorescent reporter faithfully replicated podocin expression in vivo. We were also able to show spontaneous GCaMP6s fluorescence using light sheet (single plane illumination) microscopy. We identified many podocyte transcripts, encoding proteins related to calcium-binding and actin filament assembly, in common with those expressed in human and mouse mature podocytes. Conclusion: We describe the establishment of novel transgenic zebrafish and their use to identify and isolate podocyte cells for the preparation of a scRNA-seq library from normal podocytes. The scRNA-seq data identifies distinct populations of cells and potential gene switching between clusters. These data provide a foundation for future comparative studies and for exploiting the zebrafish as a model for kidney development, disease, injury and repair

Transcription controls growth, cell kinetics and cholesterol supply to sustain ACTH responses

Chronic ACTH exposure is associated with adrenal hypertrophy and steroidogenesis. The underlying molecular processes in mice have been analysed by microarray, histological and immunohistochemical techniques. Synacthen infused for 2 weeks markedly increased adrenal mass and plasma corticosterone levels. Microarray analysis found greater than 2-fold changes in expression of 928 genes (P < 0.001; 397 up, 531 down). These clustered in pathways involved in signalling, sterol/lipid metabolism, cell proliferation/hypertrophy and apoptosis. Signalling genes included some implicated in adrenal adenomas but also upregulated genes associated with cyclic AMP and downregulated genes associated with aldosterone synthesis. Sterol metabolism genes were those promoting cholesterol supply (Scarb1, Sqle, Apoa1) and disposal (Cyp27a1, Cyp7b1). Oil red O staining showed lipid depletion consistent with reduced expression of genes involved in lipid synthesis. Genes involved in steroidogenesis (Star, Cyp11a1, Cyp11b1) were modestly affected (P < 0.05; <1.3-fold). Increased Ki67, Ccna2, Ccnb2 and Tk1 expression complemented immunohistochemical evidence of a 3-fold change in cell proliferation. Growth arrest genes, Cdkn1a and Cdkn1c, which are known to be active in hypertrophied cells, were increased >4-fold and cross-sectional area of fasciculata cells was 2-fold greater. In contrast, genes associated with apoptosis (eg Casp12, Clu,) were downregulated and apoptotic cells (Tunel staining) were fewer (P < 0.001) and more widely distributed throughout the cortex. In summary, long-term steroidogenesis with ACTH excess is sustained by genes controlling cholesterol supply and adrenal mass. ACTH effects on adrenal morphology and genes controlling cell hypertrophy, proliferation and apoptosis suggest the involvement of different cell types and separate molecular pathways

The Segond fracture occurs at the site of lowest sub-entheseal trabecular bone volume fraction on the tibial plateau.

In a series of human cadaveric experiments, Dr. Paul Segond first described the avulsion injury occurring at the anterolateral tibial plateau that later took his name. The fracture is thought to arise as a consequence of excessive tibia internal rotation which often also elicits damage to other connective tissue of the knee. The exact mechanism behind the avulsion is, however, unclear. A number of ligamentous structures have been proposed in separate studies to insert into the Segond fragment. Suggestions include the iliotibial band (ITB), biceps femoris and the controversial 'anterolateral ligament' (ALL). Despite increasing knowledge of tibial plateau bony microarchitecture in both healthy and disease states, no studies have yet, to our knowledge, considered the role of tibial sub-entheseal bone structure in pathogenesis of the Segond fracture. The goal of this study was thus to elucidate the differences in trabecular properties at regions across the tibial plateau in order to provide an explanation for the susceptibility of the anterolateral region to avulsion injury. Twenty human tibial plateaus from cadaveric donors were dissected and imaged using a Nikon-XTH225-μCT scanner with <80 μm isotropic voxel size. Scans were reconstructed using MicroView 3D Image Viewer and Analysis Tool. Subsequent virtual biopsy at ten anatomically defined regions of interest (ROI) generated estimates of bone volume fraction ('bone volume divided by total volume' (BV/TV)). The overall mean BV/TV value across all 20 tibiae and all 10 ROIs was 0.271. Univariate repeated-measurements ANOVA demonstrated that BV/TV values differed between ROIs. BV/TV values at the Segond site (Sα, Sβ or Sγ) were lower than all other ROIs at 0.195, 0.192 and 0.193, respectively. This suggests that, notwithstanding inter- and intra-specimen variation, the Segond site tends to have a lower trabecular bone volume fraction than entheseal sites elsewhere on the tibia. Since BV/TV correlates with tensile and torsional strength, the lower BV/TV at the Segond site could equate to a region of local weakness in certain individuals which predisposes them to an avulsion injury following the application of force from excessive internal rotation. The low BV/TV recorded at the Segond site also challenges the idea that the fracture occurs due to pull from a discrete 'anterolateral ligament', as the tension exerted focally would be expected to elicit a hypertrophic response in line with Frost's Mechanostat hypothesis. Our data would instead agree with the aforementioned reports of the fibrous band at the Segond site being part of a broader insertion of an 'anterolateral complex'