Different laboratories recently reported incongruous results describing the quantification of albumin filtration using two-photon microscopy. We investigated the factors that influence the glomerular sieving coefficient for albumin (GSC A ) in an effort to explain these discordant reports and to develop standard operating procedures for determining GSC A . Multiple factors influenced GSC A , including the kidney depth of image acquisition (10-20 mm was appropriate), the selection of fluorophore (probes emitting longer wavelengths were superior), the selection of plasma regions for fluorescence measurements, the size and molecular dispersion characteristics of dextran polymers if used, dietary status, and the genetic strain of rat. Fasting reduced the GSC A in Simonsen Munich Wistar rats from 0.03560.005 to 0.01660.004 (P,0.01). Frömter Munich Wistar rats had a much lower GSC A in both the fed and the fasted states. Finally, we documented extensive albumin transcytosis with vesicular and tubular delivery to and fusion with the basolateral membrane in S1 proximal tubule cells. In summary, these results help explain the previously conflicting microscopy and micropuncture data describing albumin filtration and highlight the dynamic nature of glomerular albumin permeability. Over the past several years, the roles the glomerular filtration barrier and the proximal tubule play in the development of albuminuria have been debated. 1,2 The present textbook model, in which albumin filtration across a normal glomerulus is thought to be minimal, has recently been challenged. A wide array of genetic, molecular, biochemical, and imaging studies are consistent with proximal tubule cells (PTCs) having a role in reclaiming filtered proteins, including albumin, and thus minimizing proteinuria. These studies include the following: knockout mice lacking Na+-H+ exchanger isoform 3 or chloride channel-5; 3 megalin-cubilin complex defects; 4 Rab 38-mediated tubular proteinuria and albuminuria; 5 statin-mediated inhibition of guanosine triphosphatase prenylation with reduced proximal tubule (PT) endocytosis; 4,6-8 mice lacking FcRn, the IgG and albumin receptor; 9,10 and selective PTC injury using D-serine 11 or the selective expression of diphtheria toxin receptor on PTC. 12 In a preliminary communication, Menzel and colleagues showed that PT reabsorption and transcytosis of podocyte produced and secreted albumin labeled with V5 and hemagglutinin tags. 13 Finally, recently published data 14 using advanced scanning electron microscope imaging techniques indicate that the podocyte slit diaphragm has pores that are much larger than previously determined and are of the size required for albumin filtration.The development of in vivo two-photon microscopy has enabled the direct visualization and quantitation of fluorescent compounds as they filter through the glomerulus and are endocytosed by
Somitogenesis, the formation of the body's primary segmental structure common to all vertebrate development, requires coordination between biological mechanisms at several scales. Explaining how these mechanisms interact across scales and how events are coordinated in space and time is necessary for a complete understanding of somitogenesis and its evolutionary flexibility. So far, mechanisms of somitogenesis have been studied independently. To test the consistency, integrability and combined explanatory power of current prevailing hypotheses, we built an integrated clock-and-wavefront model including submodels of the intracellular segmentation clock, intercellular segmentation-clock coupling via Delta/Notch signaling, an FGF8 determination front, delayed differentiation, clock-wavefront readout, and differential-cell-cell-adhesion-driven cell sorting. We identify inconsistencies between existing submodels and gaps in the current understanding of somitogenesis mechanisms, and propose novel submodels and extensions of existing submodels where necessary. For reasonable initial conditions, 2D simulations of our model robustly generate spatially and temporally regular somites, realistic dynamic morphologies and spontaneous emergence of anterior-traveling stripes of Lfng. We show that these traveling stripes are pseudo-waves rather than true propagating waves. Our model is flexible enough to generate interspecies-like variation in somite size in response to changes in the PSM growth rate and segmentation-clock period, and in the number and width of Lfng stripes in response to changes in the PSM growth rate, segmentation-clock period and PSM length.
This study was designed to develop a zebrafish experimental model to examine defects in retinoic acid signaling caused by embryonic ethanol. Retinoic acid deficiency may be a causative factor leading to a spectrum of birth defects classified as fetal alcohol spectrum disorder (FASD). Experimental support for this hypothesis using Xenopus showed that effects of treatment with ethanol could be partially rescued by adding retinoids during ethanol treatment. Previous studies show that treating zebrafish embryos during gastrulation and somitogenesis stages with a pathophysiological concentration of ethanol (100 mM) produces effects that are characteristic features of FASD. We found that treating zebrafish embryos with retinoic acid at a low concentration (10−9 M) and 100 mM ethanol during gastrulation and somitogenesis stages significantly rescued a spectrum of defects produced by treating embryos with 100 mM ethanol alone. The rescue phenotype that we observed was quantitatively more similar to embryos treated with 10−9 M retinoic acid alone (retinoic acid toxicity) than to untreated or 100 mM ethanol treated embryos. Retinoic acid rescues defects caused by 100 mM ethanol treatment during gastrulation and somitogenesis stages that include early gastrulation cell movements (anterior-posterior axis), craniofacial cartilage formation and ear development. Morphological evidence also suggests that other characteristic features of FASD (e. g., neural axis patterning) are rescued by retinoic acid supplement.
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