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

Rho-kinase inhibition blunts renal vasoconstriction induced by distinct signaling pathways in vivo

In addition to intracellular calcium, which activates myosin light chain (MLC) kinase, MLC phosphorylation and hence contraction is importantly regulated by MLC phosphatase (MLCP). Recent evidence suggests that distinct signaling cascades of vasoactive hormones interact with the Rho/Rho kinase (ROK) pathway, affecting the activity of MLCP. The present study measured the impact of ROK inhibition on vascular F-actin distribution and on vasoconstriction induced by activation/inhibition of distinct signaling pathways in vivo in the microcirculation of the split hydronephrotic rat kidney. Local application of the ROK inhibitors Y-27632 or HA-1077 induced marked dilation of pre- and postglomerular vessels. Activation of phospholipase C with the endothelin ET B agonist IRL 1620, inhibition of soluble guanylyl cyclase with 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ), or inhibition of adenylyl cyclase with the adenosine A1 agonist N6-cyclopentyladenosine (CPA) reduced glomerular blood flow (GBF) by about 50% through vasoconstriction at different vascular levels. ROK inhibition with Y-27632 or HA-1077, but not protein kinase C inhibition with Ro 31-8220, blunted ET B-induced vasoconstriction. Furthermore, the reduction of GBF and of vascular diameters in response to ODQ or CPA were abolished by pretreatment with Y-27632. ROK inhibitors prevented constriction of preglomerular vessels and of efferent arterioles with equal effectiveness. Confocal microscopy demonstrated that Y-27632 did not change F-actin content and distribution in renal vessels. The results suggest that ROK inhibition might be considered as a potent treatment of renal vasoconstriction, because it interferes with constriction induced by distinct signaling pathways in renal vessels without affecting F-actin structure

Studies of vertical wind profiles at Cape Kennedy, Florida Final report

Vertical wind profiles spectral analysis and numerical wind forecasts at Cape Kenned

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

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

Classical Stability of the Galileon

We consider the classical equations of motion for a single Galileon field with generic parameters in the presence of non-relativistic sources. We introduce the concept of absolute stability of a theory: if one can show that a field at a single point---like infinity for instance---in spacetime is stable, then stability of the field over the rest of spacetime is guaranteed for any positive energy source configuration. The Dvali-Gabadadze-Porrati (DGP) model is stable in this manner, and previous studies of spherically symmetric solutions suggest that certain classes of the single field Galileon (of which the DGP model is a subclass) may have this property as well. We find, however, that when general solutions are considered this is not the case. In fact, when considering generic solutions there are no choices of free parameters in the Galileon theory that will lead to absolute stability except the DGP choice. Our analysis indicates that the DGP model is an exceptional choice among the large class of possible single field Galileon theories. This implies that if general solutions (non-spherically symmetric) exist they may be unstable. Given astrophysical motivation for the Galileon, further investigation into these unstable solutions may prove fruitful.Comment: 23 pages, 3 figure

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

Supersymmetric sound in fluids

We consider the hydrodynamics of supersymmetric fluids. Supersymmetry is broken spontaneously and the low energy spectrum includes a fermionic massless mode, the $\mathit{phonino}$. We use two complementary approaches to describe the system: First, we construct a generating functional from which we derive the equations of motion of the fluid and of the phonino propagating through the fluid. We write the form of the leading corrections in the derivative expansion, and show that the so called diffusion terms in the supercurrent are in fact not dissipative. Second, we use an effective field theory approach which utilizes a non-linear realization of supersymmetry to analyze the interactions between phoninos and phonons, and demonstrate the conservation of entropy in ideal fluids. We comment on possible phenomenological consequences for gravitino physics in the early universe.Comment: Modified introduction and discussion of diffusion terms in the supercurren

Low Serum Testosterone Is Associated with Increased Mortality in Men with Stage 3 or Greater Nephropathy

Background: Chronic kidney disease (CKD) and low serum total testosterone (TT) concentrations are independent predictors of mortality risk in the general population, but their combined potential for improved mortality risk stratification is unknown. Methods: We used data of 1,822 men from the population-based Study of Health in Pomerania followed- up for 9.9 years (median). The direct effects of kidney dysfunction (estimated glomerular filtration rate 2), albuminuria (urinary albumin-creatinine ratio ≧2.5 mg/mmol) and their combination (CKD) on all-cause and cardiovascular mortality were analyzed using multivariable Cox regression models. Serum TT concentrations below the age-specific 10th percentile (by decades) were considered low and were used for further risk stratification. Results: Kidney dysfunction (hazard ratio, HR, 1.40; 95% confidence interval, CI, 1.02–1.92), albuminuria (HR, 1.38; 95% CI, 1.06–1.79), and CKD (HR, 1.42; 95% CI, 1.09–1.84) were associated with increased all-cause mortality risk, while only kidney dysfunction (HR, 2.01; 95% CI, 1.21–3.34) was associated with increased cardiovascular mortality risk after multivariable adjustment. Men with kidney dysfunction and low TT concentrations were identified as high-risk individuals showing a more than 2-fold increased all-cause mortality risk (HR, 2.52; 95% CI, 1.08–5.85). Added to multivariable models, nonsignificant interaction terms suggest that kidney dysfunction and low TT are primarily additive rather than synergistic mortality risk factors. Conclusion: In the case of early loss of kidney function, measured TT concentrations might help to detect high-risk individuals for potential therapeutic interventions and to improve mortality risk assessment and outcome