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

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

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

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

The quantum mechanics of perfect fluids

We consider the canonical quantization of an ordinary fluid. The resulting long-distance effective field theory is derivatively coupled, and therefore strongly coupled in the UV. The system however exhibits a number of peculiarities, associated with the vortex degrees of freedom. On the one hand, these have formally a vanishing strong-coupling energy scale, thus suggesting that the effective theory's regime of validity is vanishingly narrow. On the other hand, we prove an analog of Coleman's theorem, whereby the semiclassical vacuum has no quantum counterpart, thus suggesting that the vortex premature strong-coupling phenomenon stems from a bad identification of the ground state and of the perturbative degrees of freedom. Finally, vortices break the usual connection between short distances and high energies, thus potentially impairing the unitarity of the effective theory.Comment: 35 page

Derrick's theorem beyond a potential

Scalar field theories with derivative interactions are known to possess solitonic excitations, but such solitons are generally unsatisfactory because the effective theory fails precisely where nonlinearities responsible for the solitons are important. A new class of theories possessing (internal) galilean invariance can in principle bypass this difficulty. Here, we show that these galileon theories do not possess stable solitonic solutions. As a by-product, we show that no stable solitons exist for a different class of derivatively coupled theories, describing for instance the infrared dynamics of superfluids, fluids, solids and some k-essence models.Comment: 4 page

Galilean symmetry in the effective theory of inflation: new shapes of non-Gaussianity

We study the consequences of imposing an approximate Galilean symmetry on the Effective Theory of Inflation, the theory of small perturbations around the inflationary background. This approach allows us to study the effect of operators with two derivatives on each field, which can be the leading interactions due to non-renormalization properties of the Galilean Lagrangian. In this case cubic non-Gaussianities are given by three independent operators, containing up to six derivatives, two with a shape close to equilateral and one peaking on flattened isosceles triangles. The four-point function is larger than in models with small speed of sound and potentially observable with the Planck satellite.Comment: 23 pages, 6 figures. v2: minor changes to match JCAP published versio

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