scholarly journals “Zebrafishing” for Novel Genes Relevant to the Glomerular Filtration Barrier

2013 ◽  
Vol 2013 ◽  
pp. 1-12 ◽  
Author(s):  
Nils Hanke ◽  
Lynne Staggs ◽  
Patricia Schroder ◽  
Jennifer Litteral ◽  
Susanne Fleig ◽  
...  
Keyword(s):  
Glomerular Filtration ◽  
Barrier Function ◽  
Association Studies ◽  
Genome Wide Association Studies ◽  
Glomerular Filtration Barrier ◽  
Novel Genes ◽  
Zebrafish Model ◽  
Filtration Barrier

Data for genes relevant to glomerular filtration barrier function or proteinuria is continually increasing in an era of microarrays, genome-wide association studies, and quantitative trait locus analysis. Researchers are limited by published literature searches to select the most relevant genes to investigate. High-throughput cell cultures and otherin vitrosystems ultimately need to demonstrate proof in anin vivomodel. Generating mammalian models for the genes of interest is costly and time intensive, and yields only a small number of test subjects. These models also have many pitfalls such as possible embryonic mortality and failure to generate phenotypes or generate nonkidney specific phenotypes. Here we describe anin vivozebrafish model as a simple vertebrate screening system to identify genes relevant to glomerular filtration barrier function. Using our technology, we are able to screen entirely novel genes in 4–6 weeks in hundreds of live test subjects at a fraction of the cost of a mammalian model. Our system produces consistent and reliable evidence for gene relevance in glomerular kidney disease; the results then provide merit for further analysis in mammalian models.

Reduced diffusion of charge-modified, conformationally intact anionic Ficoll relative to neutral Ficoll across the rat glomerular filtration barrier in vivo

2011 ◽  
Vol 301 (4) ◽  
pp. F708-F712 ◽  
Author(s):  
Josefin Axelsson ◽  
Kristinn Sverrisson ◽  
Anna Rippe ◽  
William Fissell ◽  
Bengt Rippe
Keyword(s):  
Glomerular Filtration ◽  
High Performance ◽  
Size Exclusion ◽  
Glomerular Filtration Barrier ◽  
Glomerular Permeability ◽  
Charge Effects ◽  
Filtration Barrier ◽  
A Charge

The glomerular filtration barrier (GFB) is commonly conceived as a negatively charged sieve to proteins. Recent studies, however, indicate that glomerular charge effects are small for anionic, carboxymethylated (CM) dextran vs. neutral dextran. Furthermore, two studies assessing the glomerular sieving coefficients (θ) for negative CM-Ficoll vs. native Ficoll have demonstrated an increased glomerular permeability for CM-Ficoll (Asgeirsson D, Venturoli D, Rippe B, Rippe C. Am J Physiol Renal Physiol 291: F1083–F1089, 2006; Guimarães M, Nikolovski J, Pratt L, Greive K, Comper W. Am Physiol Renal Physiol 285: F1118–F1124, 2003.). The CM-Ficoll used, however, showed a larger Stokes-Einstein radius ( ae) than neutral Ficoll, and it was proposed that the introduction of negative charges in the Ficoll molecule had made it more flexible and permeable. Recently, a negative FITC-labeled CM-Ficoll (CMI-Ficoll) was produced with a conformation identical to that of neutral FITC-Ficoll. Using these probes, we determined their θ:s in anesthetized Wistar rats (259 ± 2.5 g). After blood access had been achieved, the left ureter was cannulated for urine sampling. Either polysaccharide was infused (iv) together with a filtration marker, and urine and plasma were collected. Assessment of θ FITC-Ficoll was achieved by high-performance size-exclusion chromatography (HPSEC). CMI-Ficoll and native Ficoll had identical elugrams on the HPSEC. Diffusion of anionic Ficoll was significantly reduced compared with that of neutral Ficoll across the GFB for molecules of ae ∼20–35 Å, while there were no charge effects for Ficoll of ae = 35–80 Å. The data are consistent with a charge effect present in “small pores,” but not in “large pores,” of the GFB and mimicked those obtained for anionic membranes in vitro for the same probes.

Abstract 516: Knockdown of the Hypertension Associated Gene NOSTRIN Alters Glomerular Barrier Function in Zebrafish (Danio rerio)

Hypertension ◽  
2012 ◽  
Vol 60 (suppl_1) ◽  
Author(s):  
Torsten Kirsch ◽  
Jessica Kaufeld ◽  
Ron Korstanje ◽  
Dirk Hentschel ◽  
Hermann Haller ◽  
...  
Keyword(s):  
Glomerular Filtration ◽  
Barrier Function ◽  
Morphological Changes ◽  
Glomerular Filtration Barrier ◽  
Cell Dysfunction ◽  
Larval Zebrafish ◽  
Filtration Barrier ◽  
Important Regulator ◽  
Glomerular Barrier ◽  
Prime Source

The bioavailability of nitric oxide (NO) has been associated with the development and progression of vascular and renal disease. NOSTRIN (for eNOS Traffic Inducer) has primarily been recognized as one important regulator of eNOS, the prime source of NO in the cardiovascular system, with a possible role in the pathogenesis of pre-eclampsia and the development of increased intrahepatic resistance in liver disease. Here, we identified NOSTRIN in the center of a QTL-overlap region in rat and human trait loci that are associated with hypertension. Glomerular NOSTRIN expression is detectable in podocytes in human and rat glomeruli and podocytic NOSTRIN expression is diminished in hypertensive kidney disease. We show that knockdown of NOSTRIN alters the glomerular filtration barrier function in larval zebrafish, inducing proteinuria and leading to ultrastructural morphological changes on the endothelial as well as epithelial side and the GBM of the glomerular capillary loop. We also demonstrate that NOSTRIN interacts with proteins associated with the podocyte slit membrane. We conclude that NOSTRIN expression is an important factor for the integrity of the glomerular filtration barrier. Disease related alteration of NOSTRIN expression may not only affect the vascular endothelium and therefore contribute to endothelial cell dysfunction but may also contribute to the development of podocyte disease and proteinuria.

scholarly journals Asthma-associated genetic variants induce IL33 differential expression through an enhancer-blocking regulatory region

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Ivy Aneas ◽  
Donna C. Decker ◽  
Chanie L. Howard ◽  
Débora R. Sobreira ◽  
Noboru J. Sakabe ◽  
...  
Keyword(s):  
Association Studies ◽  
Regulatory Region ◽  
Airway Epithelial Cells ◽  
Genome Wide Association Studies ◽  
Airway Epithelial ◽  
Allele Specific ◽  
Enhancer Blocking ◽  
Underlying Mechanisms

AbstractGenome-wide association studies (GWAS) have implicated the IL33 locus in asthma, but the underlying mechanisms remain unclear. Here, we identify a 5 kb region within the GWAS-defined segment that acts as an enhancer-blocking element in vivo and in vitro. Chromatin conformation capture showed that this 5 kb region loops to the IL33 promoter, potentially regulating its expression. We show that the asthma-associated single nucleotide polymorphism (SNP) rs1888909, located within the 5 kb region, is associated with IL33 gene expression in human airway epithelial cells and IL-33 protein expression in human plasma, potentially through differential binding of OCT-1 (POU2F1) to the asthma-risk allele. Our data demonstrate that asthma-associated variants at the IL33 locus mediate allele-specific regulatory activity and IL33 expression, providing a mechanism through which a regulatory SNP contributes to genetic risk of asthma.

P0053A METHOD FOR THE ISOLATION OF FLUORESCENT GLOMERULI FROM ZEBRAFISH LARVAE

2020 ◽  
Vol 35 (Supplement_3) ◽  
Author(s):  
Anna Iervolino ◽  
Tim Lange ◽  
Florian Siegerist ◽  
Maximilian Schindler ◽  
Giovambattista Capasso ◽  
...  
Keyword(s):  
Glomerular Filtration ◽  
Fluorescent Protein ◽  
Transgenic Zebrafish ◽  
Renal Tubules ◽  
Glomerular Filtration Barrier ◽  
Podocyte Injury ◽  
Zebrafish Larvae ◽  
Filtration Barrier ◽  
Glomerular Damage

Abstract Background and Aims The zebrafish is a powerful animal model to study the glomerular morphology and the function of the permselectivity of the glomerular filtration barrier. Since zebrafish larvae develop quickly and can be bred to transparency, in vivo observation of these animals is possible. At 48 hours post fertilization (dpf), zebrafish develop a single filtering glomerulus which is attached to a pair of renal tubules. Like in mammals, the glomerular filtration barrier consists of a fenestrated endothelium, the glomerular basement membrane (GBM) and interdigitating podocyte foot processes bridged by a molecularly conserved slit diaphragm. By the use of genetically modified zebrafish strains with fluorescently labeled podocytes, it is possible to study alterations of the glomerulus during the development of renal disease directly in vivo and in vitro. As an injury model we used the nitroreductase/metronidazole (NTR/MTZ) zebrafish line to induce podocyte apoptosis and detachment from the GBM. Moreover, treatment of these larvae with MTZ induces glomerular injury that mimics focal segmental glomerulosclerosis (FSGS). The aim of our study was to establish a glomeruli isolation method which allows us to identify deregulation of miRNAs and mRNAs in the injured glomeruli by sequencing. Method The transgenic zebrafish strain Cherry (Tg(nphs2:Eco.nfsB-mCherry); mitfaw2/w2; mpv17a9/a9) which expresses the prokaryotic enzyme nitroreductase (NTR) fused to mCherry, a red fluorescent protein, under the control of the podocyte-specific podocin (nphs2) promoter in a transparent zebrafish strain, was used. The NTR/MTZ is a model of cell ablation to mimic podocyte injury. The prodrug MTZ (80 µM) is converted into a cytotoxin by NTR leading to a dose-dependent apoptosis exclusively in NTR-expressing podocytes. To induce podocyte injury, we treated Cherry larvae at 4 days post fertilization with MTZ (80 µM) freshly dissolved in 0.1% DMSO-E3 medium for 48 hours. Control larvae were treated with 0.1% DMSO-E3 medium. The treatment was stopped by a MTZ washout at 6 dpf. In order to perform the miRNA and mRNA sequencing on glomeruli isolated from MTZ-treated and control larvae we tried to establish a method to obtain total RNA samples of good quality. For this purpose, three different approaches were tested and validated: 1) Sieving method, 2) Fluorescence-Activated Cell Sorting method (FACS), and 3) manual isolation of glomeruli by using a micropipette. Results Zebrafish larvae developed a glomerular damage similar to FSGS after MTZ-treatment. MTZ-treated larvae showed severe pericardial edema, a reduction of the nephrin and podocin expression, proteinuria and an increased mortality rate at 8 dpf. After many tests we showed that glomeruli isolation using the sieving method and FACS were not efficient due to contaminations with other organs (sieving) and a loss of a large amount of cells per sample (FACS), respectively. Samples of the required quality for sequencing resulted only from the manual glomeruli isolation. Conclusion Here we describe methods to isolate fluorescent glomeruli from transgenic zebrafish larvae. For our studies, we used the NTZ/MTR kidney disease model in order to identify mRNAs and miRNAs regulated in response to glomerular damage. This technique will further allow to screen for healing drugs in high-throughput experiments.

scholarly journals Podocyte endocytosis in the regulation of the glomerular filtration barrier

2015 ◽  
Vol 309 (5) ◽  
pp. F398-F405 ◽  
Author(s):  
Kazunori Inoue ◽  
Shuta Ishibe
Keyword(s):  
Glomerular Filtration ◽  
Critical Role ◽  
Vital Role ◽  
Glomerular Filtration Barrier ◽  
Filtration Barrier ◽  
Endocytic Machinery ◽  
Health And Disease ◽  
Models Of Disease ◽  
Genetic Mouse Models

Severe defects in the glomerular filtration barrier result in nephrotic syndrome, which is characterized by massive proteinuria. The podocyte, a specialized epithelial cell with interdigitating foot processes separated by a slit diaphragm, plays a vital role in regulating the passage of proteins from the capillary lumen to Bowman's space. Recent findings suggest a critical role for endocytosis in podocyte biology as highlighted by genetic mouse models of disease and human genetic mutations that result in the loss of the integrity of the glomerular filtration barrier. In vitro podocyte studies have also unraveled a plethora of constituents that are differentially internalized to maintain homeostasis. These observations provide a framework and impetus for understanding the precise regulation of podocyte endocytic machinery in both health and disease.

scholarly journals Glomerular endothelial cell fenestrations: an integral component of the glomerular filtration barrier

2009 ◽  
Vol 296 (5) ◽  
pp. F947-F956 ◽  
Author(s):  
Simon C. Satchell ◽  
Filip Braet
Keyword(s):  
Endothelial Cell ◽  
Glomerular Filtration ◽  
In Vitro Models ◽  
Glomerular Injury ◽  
Glomerular Endothelial Cell ◽  
Glomerular Filtration Barrier ◽  
Therapeutic Implications ◽  
Filtration Barrier ◽  
Glomerular Development

Glomerular endothelial cell (GEnC) fenestrations are analogous to podocyte filtration slits, but their important contribution to the glomerular filtration barrier has not received corresponding attention. GEnC fenestrations are transcytoplasmic holes, specialized for their unique role as a prerequisite for filtration across the glomerular capillary wall. Glomerular filtration rate is dependent on the fractional area of the fenestrations and, through the glycocalyx they contain, GEnC fenestrations are important in restriction of protein passage. Hence, dysregulation of GEnC fenestrations may be associated with both renal failure and proteinuria, and the pathophysiological importance of GEnC fenestrations is well characterized in conditions such as preeclampsia. Recent evidence suggests a wider significance in repair of glomerular injury and in common, yet serious, conditions, including diabetic nephropathy. Study of endothelial cell fenestrations is challenging because of limited availability of suitable in vitro models and by the requirement for electron microscopy to image these sub-100-nm structures. However, extensive evidence, from glomerular development in rodents to in vitro studies in human GEnC, points to vascular endothelial growth factor (VEGF) as a key inducer of fenestrations. In systemic endothelial fenestrations, the intracellular pathways through which VEGF acts to induce fenestrations include a key role for the fenestral diaphragm protein plasmalemmal vesicle-associated protein-1 (PV-1). The role of PV-1 in GEnC is less clear, not least because of controversy over existence of GEnC fenestral diaphragms. In this article, the structure-function relationships of GEnC fenestrations will be evaluated in depth, their role in health and disease explored, and the outlook for future study and therapeutic implications of these peculiar structures will be approached.

scholarly journals Humanized TREM2 mice reveal microglia-intrinsic and -extrinsic effects of R47H polymorphism

2018 ◽  
Vol 215 (3) ◽  
pp. 745-760 ◽  
Author(s):  
Wilbur M. Song ◽  
Satoru Joshita ◽  
Yingyue Zhou ◽  
Tyler K. Ulland ◽  
Susan Gilfillan ◽  
...  
Keyword(s):  
Late Onset ◽  
Association Studies ◽  
Amyloid Β ◽  
Genome Wide Association Studies ◽  
Genome Wide ◽  
Soluble Trem2 ◽  
The Common ◽  
Late Onset Dementia

Alzheimer’s disease (AD) is a neurodegenerative disease that causes late-onset dementia. The R47H variant of the microglial receptor TREM2 triples AD risk in genome-wide association studies. In mouse AD models, TREM2-deficient microglia fail to proliferate and cluster around the amyloid-β plaques characteristic of AD. In vitro, the common variant (CV) of TREM2 binds anionic lipids, whereas R47H mutation impairs binding. However, in vivo, the identity of TREM2 ligands and effect of the R47H variant remain unknown. We generated transgenic mice expressing human CV or R47H TREM2 and lacking endogenous TREM2 in the 5XFAD AD model. Only the CV transgene restored amyloid-β–induced microgliosis and microglial activation, indicating that R47H impairs TREM2 function in vivo. Remarkably, soluble TREM2 was found on neurons and plaques in CV- but not R47H-expressing 5XFAD brains, although in vitro CV and R47H were shed similarly via Adam17 proteolytic activity. These results demonstrate that TREM2 interacts with neurons and plaques duing amyloid-β accumulation and R47H impairs this interaction.

scholarly journals PICALM modulates autophagy activity and tau accumulation

2014 ◽  
Vol 5 (1) ◽  
Author(s):  
Kevin Moreau ◽  
Angeleen Fleming ◽  
Sara Imarisio ◽  
Ana Lopez Ramirez ◽  
Jacob L. Mercer ◽  
...  
Keyword(s):  
Genetic Risk ◽  
Association Studies ◽  
Genome Wide Association Studies ◽  
Transgenic Models ◽  
Autophagosome Formation ◽  
Genome Wide ◽  
Autophagy Pathway ◽  
Autophagy Activity

Abstract Genome-wide association studies have identified several loci associated with Alzheimer’s disease (AD), including proteins involved in endocytic trafficking such as PICALM/CALM (phosphatidylinositol binding clathrin assembly protein). It is unclear how these loci may contribute to AD pathology. Here we show that CALM modulates autophagy and alters clearance of tau, a protein which is a known autophagy substrate and which is causatively linked to AD, both in vitro and in vivo. Furthermore, altered CALM expression exacerbates tau-mediated toxicity in zebrafish transgenic models. CALM influences autophagy by regulating the endocytosis of SNAREs, such as VAMP2, VAMP3 and VAMP8, which have diverse effects on different stages of the autophagy pathway, from autophagosome formation to autophagosome degradation. This study suggests that the AD genetic risk factor CALM modulates autophagy, and this may affect disease in a number of ways including modulation of tau turnover.

scholarly journals PD33-01 GLOMERULUS ON-A-CHIP AS A MODEL TO STUDY THE GLOMERULAR FILTRATION BARRIER IN VITRO

2018 ◽  
Vol 199 (4S) ◽  
Author(s):  
Stefano Da Sacco ◽  
Paul Vulto ◽  
Jos Joore ◽  
Roger De Filippo ◽  
Laura Perin
Keyword(s):  
Glomerular Filtration ◽  
Glomerular Filtration Barrier ◽  
Filtration Barrier

scholarly journals Asthma-associated variants induce IL33 differential expression through a novel regulatory region

2020 ◽  
Author(s):  
Ivy Aneas ◽  
Donna C. Decker ◽  
Chanie L. Howard ◽  
Débora R. Sobreira ◽  
Noboru J. Sakabe ◽  
...  
Keyword(s):  
Association Studies ◽  
Regulatory Element ◽  
Regulatory Region ◽  
Airway Epithelial Cells ◽  
Genome Wide Association Studies ◽  
Airway Epithelial ◽  
Allele Specific ◽  
Underlying Mechanisms

ABSTRACTGenome-wide association studies (GWAS) have implicated the IL33 locus in asthma, but the underlying mechanisms remain unclear. Here, we identify a 5 kb region within the GWAS-defined segment that acts as a strong regulatory element in vivo and in vitro. Chromatin conformation capture showed that this 5 kb region loops to the IL33 promoter, potentially regulating its expression. Supporting this notion, we show that genotype at an asthma-associated SNP, rs1888909, located within the 5 kb region, is associated with IL33 gene expression in human airway epithelial cells and IL-33 protein expression in human plasma, potentially through differential binding of OCT-1 (POU2F1) to the asthma-risk allele. Our data demonstrate that asthma-associated variants at the IL33 locus mediate allele-specific regulatory activity and IL33 expression, providing a novel mechanism through which a regulatory SNP contributes to genetic risk of asthma.

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