4D in vivo imaging of glomerular barrier function in a zebrafish podocyte injury model

AimZebrafish larvae with their simplified pronephros are an ideal model to study glomerular physiology. Although several groups use zebrafish larvae to assess glomerular barrier function, temporary or slight changes are still difficult to measure. The aim of this study was to investigate the potential of in vivo two‐photon microscopy (2‐PM) for long‐term imaging of glomerular barrier function in zebrafish larvae.MethodsAs a proof of principle, we adapted the nitroreductase/metronidazole model of targeted podocyte ablation for 2‐PM. Combination with a strain, which expresses eGFP‐vitamin D‐binding protein in the blood plasma, led to a strain that allowed induction of podocyte injury with parallel assessment of glomerular barrier function. We used four‐dimensional (4D) 2‐PM to assess eGFP fluorescence over 26 h in the vasculature and in tubules of multiple zebrafish larvae (5 days post‐fertilization) simultaneously.ResultsBy 4D 2‐PM, we observed that, under physiological conditions, eGFP fluorescence was retained in the vasculature and rarely detected in proximal tubule cells. Application of metronidazole induced podocyte injury and cell death as shown by TUNEL staining. Induction of podocyte injury resulted in a dramatic decrease of eGFP fluorescence in the vasculature over time (about 50% and 90% after 2 and 12 h respectively). Loss of vascular eGFP fluorescence was paralleled by an endocytosis‐mediated accumulation of eGFP fluorescence in proximal tubule cells, indicating proteinuria.ConclusionWe established a microscopy‐based method to monitor the dynamics of glomerular barrier function during induction of podocyte injury in multiple zebrafish larvae simultaneously over 26 h.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/136708/1/apha12754.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/136708/2/apha12754_am.pd

Evaluation of endogenous miRNA reference genes across different zebrafish strains, developmental stages and kidney disease models

The majority of kidney diseases arise from the loss of podocytes and from morphological changes of their highly complex foot process architecture, which inevitably leads to a reduced kidney filtration and total loss of kidney function. It could have been shown that microRNAs (miRs) play a pivotal role in the pathogenesis of podocyte-associated kidney diseases. Due to their fully functioning pronephric kidney, larval zebrafish have become a popular vertebrate model, to study kidney diseases in vivo. Unfortunately, there is no consensus about a proper normalization strategy of RT-qPCR-based miRNA expression data in zebrafish. In this study we analyzed 9 preselected candidates dre-miR-92a-3p, dre-miR-206-3p, dre-miR-99-1, dre-miR-92b-3p, dre-miR-363-3p, dre-let-7e, dre-miR-454a, dre-miR-30c-5p, dre-miR-126a-5p for their capability as endogenous reference genes in zebrafish experiments. Expression levels of potential candidates were measured in 3 different zebrafish strains, different developmental stages, and in different kidney disease models by RT-qPCR. Expression values were analyzed with NormFinder, BestKeeper, GeNorm, and DeltaCt and were tested for inter-group differences. All candidates show an abundant expression throughout all samples and relatively high stability. The most stable candidate without significant inter-group differences was dre-miR-92b-3p making it a suitable endogenous reference gene for RT-qPCR-based miR expression zebrafish studies

Lidar observations in relation to the atmospheric winds aloft

Laser radar observations of clear tropopause compared to other data on atmospheric winds alof

Efeitos do Exercício Físico e da Terapia Estrogênica Sobre a Reatividade Vascular de Aorta de Ratas Espontaneamente Hipertensas Ovariectomizadas: Papel do Sistema Renina-angiotensina

As doenças cardiovasculares representam as principais causas de morbidade e mortalidade no mundo, inclusive em mulheres no período pós-menopausa. Com a diminuição na produção dos estrógenos, observa-se o aparecimento e a elevação de vários fatores que podem aumentar o risco de desenvolvimento dessas doenças. Dentre esses fatores, o aumento da atividade ou up regulation do Sistema Renina-Angiotensina (SRA) tem se destacado como um importante mediador na fisiopatologia de várias doenças, tal qual, a hipertensão. Apesar de vários estudos experimentais relatarem efeitos benéficos da reposição hormonal com estrogênio, especificamente 17β-estradiol (E2), sobre a redução do risco cardiovascular, os resultados de estudos clínicos são inconclusivos. Desta forma, modificações no estilo de vida se fazem necessárias, como a incorporação da prática regular de exercícios físicos. Muitos estudos têm demonstrado que o exercício físico pode influenciar positivamente sobre os principais fatores de risco cardiovascular, inclusive em mulheres na pós-menopausa. O objetivo do presente estudo foi analisar os efeitos do treinamento físico crônico de natação e da terapia estrogênica sobre a reatividade vascular de aorta de ratas espontaneamente hipertensas (SHR) ovariectomizadas com foco na modulação efetuada pelo SRA. Os experimentos foram conduzidos com ratas SHR Sham e ovariectomizadas, as quais foram divididas aleatoriamente em cinco grupos: SHAM (S), ovariectomizadas (OVX), ovariectomizadas+TE2 (OE2), ovariectomizadas+natação (ON) e ovariectomizadas TE2+N (OE2+N). A reposição com E2 foi realizada por meio de injeções s.c. contendo 5 µg de 17β-estradiol três vezes por semana. O protocolo de treinamento de natação foi realizado por sessenta minutos diários, de forma continua, cinco vezes por semana. Tanto a terapia quanto o treinamento tiveram duração de oito semanas. Quarenta e oito horas após a última sessão de treinamento e/ou tratamento, as ratas tiveram a Pressão Arterial Sistólica (PAS) aferida e após sacrificadas, o sangue foi coletado para dosagem de angiotensina II (Ang II). Para o estudo funcional de reatividade de aorta, avaliou-se a resposta vasoconstritora à angiotensina II e vasodilatadora à Ang-(1-7), ainda efetuando bloqueios farmacológicos para elucidar o mecanismo de ação. A avaliação da expressão das proteínas do SRA em aorta foi realizada por Western Blotting. Os resultados encontrados demonstram que, o grupo ON e OE2+N apresentaram aumento nos níveis plasmáticos de Ang II, que não foi acompanhado por aumento na PAS. A ovariectomia causou um aumento da resposta vasoconstritora a Ang II e diminuição da vasodilatação de Ang-(1-7), que foi prevenida pelo exercício físico ou pela sua associação com a TE2. Além disso, o abrandamento da resposta vasoconstritora a Ang II, assim como o aumento da vasodilatação a Ang-(1-7) parece ter ocorrido por um mecanismo envolvendo o receptor AT2 e Mas, que tiveram sua expressão aumentada no grupo ON e OE2+N. No grupo ON a eficiência funcional destes receptores foi auxiliada pelo aumento na capacidade anti-oxidante, efetuada pela superóxido dismutase. Pôde-se concluir que tanto o treinamento físico quanto a reposição com E2 exercem efeitos cardioprotetores, e a prática regular do exercício físico pode ser uma excelente alternativa à terapia estrogênica em mulheres na pós-menopausa, haja vista que a associação da TE2 ao exercício não promoveu efeitos somatórios

Prolonged podocyte depletion in larval zebrafish resembles mammalian focal and segmental glomerulosclerosis

Focal and segmental glomerulosclerosis (FSGS) is a histological pattern frequently found in patients with nephrotic syndrome that often progress to end-stage kidney disease. The initial step in development of this histologically defined entity is injury and ultimately depletion of podocytes, highly arborized interdigitating cells on the glomerular capillaries with important function for the glomerular filtration barrier. Since there are still no causal therapeutic options, animal models are needed to develop new treatment strategies. Here, we present an FSGS-like model in zebrafish larvae, an eligible vertebrate model for kidney research. In a transgenic zebrafish strain, podocytes were depleted, and the glomerular response was investigated by histological and morphometrical analysis combined with immunofluorescence staining and ultrastructural analysis by transmission electron microscopy. By intravenous injection of fluorescent high-molecular weight dextran, we confirmed leakage of the size selective filtration barrier. Additionally, we observed severe podocyte foot process effacement of remaining podocytes, activation of proximal tubule-like parietal epithelial cells identified by ultrastructural cytomorphology, and expression of proximal tubule markers. These activated cells deposited extracellular matrix on the glomerular tuft which are all hallmarks of FSGS. Our findings indicate that glomerular response to podocyte depletion in larval zebrafish resembles human FSGS in several important characteristics. Therefore, this model will help to investigate the disease development and the effects of potential drugs in a living organism

On the rate of black hole binary mergers in galactic nuclei due to dynamical hardening

We assess the contribution of dynamical hardening by direct three-body scattering interactions to the rate of stellar-mass black hole binary (BHB) mergers in galactic nuclei. We derive an analytic model for the single-binary encounter rate in a nucleus with spherical and disk components hosting a super-massive black hole (SMBH). We determine the total number of encounters $N_{\rm GW}$ needed to harden a BHB to the point that inspiral due to gravitational wave emission occurs before the next three-body scattering event. This is done independently for both the spherical and disk components. Using a Monte Carlo approach, we refine our calculations for $N_{\rm GW}$ to include gravitational wave emission between scattering events. For astrophysically plausible models we find that typically $N_{\rm GW} \lesssim$ 10. We find two separate regimes for the efficient dynamical hardening of BHBs: (1) spherical star clusters with high central densities, low velocity dispersions and no significant Keplerian component; and (2) migration traps in disks around SMBHs lacking any significant spherical stellar component in the vicinity of the migration trap, which is expected due to effective orbital inclination reduction of any spherical population by the disk. We also find a weak correlation between the ratio of the second-order velocity moment to velocity dispersion in galactic nuclei and the rate of BHB mergers, where this ratio is a proxy for the ratio between the rotation- and dispersion-supported components. Because disks enforce planar interactions that are efficient in hardening BHBs, particularly in migration traps, they have high merger rates that can contribute significantly to the rate of BHB mergers detected by the advanced Laser Interferometer Gravitational-Wave Observatory.Comment: 13 pages, 9 figures, accepted for publication in MNRA

Successfully meeting analytical expectations for the fast 0/1-h algorithm for NSTEMI by internal control procedures for cardiac troponin T

Cardiolog

Boundary Terms and Junction Conditions for Generalized Scalar-Tensor Theories

We compute the boundary terms and junction conditions for Horndeski's panoptic class of scalar-tensor theories, and write the bulk and boundary equations of motion in explicitly second order form. We consider a number of special subclasses, including galileon theories, and present the corresponding formulae. Our analysis opens up of the possibility of studying tunnelling between vacua in generalized scalar-tensor theories, and braneworld dynamics. The latter follows because our results are independent of spacetime dimension.Comment: 13 pages, Equation corrected. Thanks to Tsutomu Kobayashi for informing us of the typ

BDNF: mRNA expression in urine cells of patients with chronic kidney disease and its role in kidney function

Podocyte loss and changes to the complex morphology are major causes of chronic kidney disease (CKD). As the incidence is continuously increasing over the last decades without sufficient treatment, it is important to find predicting biomarkers. Therefore, we measured urinary mRNA levels of podocyte genes NPHS1, NPHS2, PODXL and BDNF, KIM-1, CTSL by qRT-PCR of 120 CKD patients. We showed a strong correlation between BDNF and the kidney injury marker KIM-1, which were also correlated with NPHS1, suggesting podocytes as a contributing source. In human biopsies, BDNF was localized in the cell body and major processes of podocytes. In glomeruli of diabetic nephropathy patients, we found a strong BDNF signal in the remaining podocytes. An inhibition of the BDNF receptor TrkB resulted in enhanced podocyte dedifferentiation. The knockdown of the orthologue resulted in pericardial oedema formation and lowered viability of zebrafish larvae. We found an enlarged Bowman's space, dilated glomerular capillaries, podocyte loss and an impaired glomerular filtration. We demonstrated that BDNF is essential for glomerular development, morphology and function and the expression of BDNF and KIM-1 is highly correlated in urine cells of CKD patients. Therefore, BDNF mRNA in urine cells could serve as a potential CKD biomarker

The Imperfect Fluid behind Kinetic Gravity Braiding

We present a standard hydrodynamical description for non-canonical scalar field theories with kinetic gravity braiding. In particular, this picture applies to the simplest galileons and k-essence. The fluid variables not only have a clear physical meaning but also drastically simplify the analysis of the system. The fluid carries charges corresponding to shifts in field space. This shift-charge current contains a spatial part responsible for diffusion of the charges. Moreover, in the incompressible limit, the equation of motion becomes the standard diffusion equation. The fluid is indeed imperfect because the energy flows neither along the field gradient nor along the shift current. The fluid has zero vorticity and is not dissipative: there is no entropy production, the energy-momentum is exactly conserved, the temperature vanishes and there is no shear viscosity. Still, in an expansion around a perfect fluid one can identify terms which correct the pressure in the manner of bulk viscosity. We close by formulating the non-trivial conditions for the thermodynamic equilibrium of this imperfect fluid.Comment: 23 pages plus appendices. New version includes extended discussion on diffusion and dynamics in alternative frames, as well as additional references. v3 reflects version accepted for publication in JHEP: minor comments added regarding suitability to numerical approache