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Nephron 2018von Hippel-Lindau (VHL) disease is characterized by the development of benign and malignant tumours in many organ systems, including renal cysts and clear cell renal...
BACKGROUND
von Hippel-Lindau (VHL) disease is characterized by the development of benign and malignant tumours in many organ systems, including renal cysts and clear cell renal cell carcinoma. It is not completely understood what underlies the development of renal pathology, and the use of murine Vhl models has been challenging due to limitations in disease conservation. We previously described a zebrafish model bearing inactivating mutations in the orthologue of the human VHL gene.
METHODS
We used histopathological and functional assays to investigate the pronephric and glomerular developmental defects in vhl mutant zebrafish, supported by human cell culture assays.
RESULTS
Here, we report that vhl is required to maintain pronephric tubule and glomerulus integrity in zebrafish embryos. vhl mutant glomeruli are enlarged, cxcr4a+ capillary loops are dilated and the Bowman space is widened. While we did not observe pronephric cysts, the cells of the proximal convoluted and anterior proximal straight tubule are enlarged, periodic acid schiff (PAS) and Oil Red O positive, and display a clear cytoplasm after hematoxylin and eosine staining. Ultrastructural analysis showed the vhl-/- tubule to accumulate large numbers of vesicles of variable size and electron density. Microinjection of the endocytic fluorescent marker AM1-43 in zebrafish embryos revealed an accumulation of endocytic vesicles in the vhl mutant pronephric tubule, which we can recapitulate in human cells lacking VHL.
CONCLUSIONS
Our data indicates that vhl is required to maintain pronephric tubule and glomerulus integrity during zebrafish development, and suggests a role for VHL in endocytic vesicle trafficking.
Topics: Animals; Embryonic Development; Kidney Glomerulus; Kidney Tubules, Proximal; Larva; Mutation; Receptors, Vascular Endothelial Growth Factor; Tumor Suppressor Proteins; Zebrafish; Zebrafish Proteins
PubMed: 29342457
DOI: 10.1159/000484096 -
Fish & Shellfish Immunology Mar 2018The rapid emergence of drug resistance, unfavourable immunosuppression and mounting evidence to suggest the deleterious accumulation of drug breakdown residues within...
The rapid emergence of drug resistance, unfavourable immunosuppression and mounting evidence to suggest the deleterious accumulation of drug breakdown residues within animal tissues has driven a strong desire to move away from these current methods of disease control. Some natural products such as β-glucan, which are extracted from, for example, plants and fungi, are able to modulate the immune system and increase protection against diseases. However, these products are heterogeneous and their effects can be variable thus limiting their applicability and reliability. Carbohydrates were modified via chemical sulphation and these semi-synthetic, sulphated carbohydrates analysed for their immunological activity utilising carp pronephric cells and a carp leucocyte cell line (CLC). A sulphated β(1,4)-glucan, methyl hydroxyethyl cellulose sulphate (MHCS), demonstrated a stimulatory effect on fish immune cells. MHCS induced a range of bioactive effects in carp leucocyte cells whilst not affecting cell viability when cells were exposed for 24 h at concentrations of 1-150 μgml. MHCS stimulated the innate immune system where a significant increase in respiratory burst activity was observed at concentrations 25-250 μgml in comparison to control (sterile water), cellulose ether, MacroGard and zymosan. Also, under in mock bacterial and viral infection conditions i.e. Lipopolysaccharide (LPS) and polyinosinic:polycytidylic acid (Poly(I:C)), MHCS enhanced the immune responses of pronephric cells by stimulating the respiratory burst activity at concentrations 50 and 150 μgml. MHCS also enhanced the expression of cytokines including interleukin 1 beta (IL1β), tumor necrosis factor alpha 1 and 2 (TNFα 1,2), interferons alpha 2 (IFN α2) and inducible nitric oxide synthase (iNOS) in carp pronephric cells. It is proposed that this new semi-synthetic carbohydrate is a potential candidate for the development of a new generation of immunostimulants and adjuvants for use in vaccination strategies in aquaculture.
Topics: Animals; Carbohydrate Metabolism; Carbohydrates; Carps; Cell Line; Immunity, Innate; Leukocytes; Lipopolysaccharides; Methylcellulose; Poly I-C; Sulfates
PubMed: 29289653
DOI: 10.1016/j.fsi.2017.12.047 -
Biochemical and Biophysical Research... Jan 2018Eph/ephrin molecules are widely expressed during embryonic development, and function in a variety of developmental processes. Here we studied the roles of the Eph...
Eph/ephrin molecules are widely expressed during embryonic development, and function in a variety of developmental processes. Here we studied the roles of the Eph receptor EphA7 and its soluble form in Xenopus pronephros development. EphA7 is specifically expressed in pronephric tubules at tadpole stages and knockdown of EphA7 by a translation blocking morpholino led to defects in tubule cell differentiation and morphogenesis. A soluble form of EphA7 (sEphA7) was also identified. Interestingly, the membrane level of claudin6 (CLDN6), a tetraspan transmembrane tight junction protein, was dramatically reduced in the translation blocking morpholino injected embryos, but not when a splicing morpholino was used, which blocks only the full length EphA7. In cultured cells, EphA7 binds and phosphorylates CLDN6, and reduces its distribution at the cell surface. Our work suggests a role of EphA7 in the regulation of cell adhesion during pronephros development, whereas sEphA7 works as an antagonist.
Topics: Animals; Cell Membrane; Claudins; Gene Expression Regulation, Developmental; Gene Knockdown Techniques; Oligodeoxyribonucleotides, Antisense; Pronephros; Receptor, EphA7; Solubility; Xenopus Proteins; Xenopus laevis
PubMed: 29223398
DOI: 10.1016/j.bbrc.2017.12.027 -
Molecular Metabolism Feb 2018Adult human kidneys produce erythropoietin (EPO), which regulates red blood cell formation; however, whether EPO also functions directly on kidney development and...
OBJECTIVE
Adult human kidneys produce erythropoietin (EPO), which regulates red blood cell formation; however, whether EPO also functions directly on kidney development and controls diabetic kidney disease remains unknown. Here we analyzed the role of EPO in kidney development and under hyperglycemic conditions in zebrafish and in humans.
METHODS
Diabetic patients and respective controls were enrolled in two cohorts. Serum EPO level and urine protein change upon human EPO administration were then analyzed. Transient knockdown and permanent knockout of EPO and EPOR in renal TG(WT1B:EGFP) zebrafish were established using the morpholino technology and CRISPR/Cas9 technology. Zebrafish embryos were phenotypically analyzed using fluorescence microscopy, and functional assays were carried out with the help of TexasRed labeled 70 kDa Dextran. Apoptosis was determined using the TUNEL assay and Annexin V staining, and caspase inhibitor zVADfmk was used for rescue experiments.
RESULTS
In type 2 diabetic patients, serum EPO level decreased with the duration of diabetes, which was linked to reduced kidney function. Human recombinant EPO supplementation ameliorated proteinuria in diabetic nephropathy patients. In zebrafish, loss-of-function studies for EPO and EPOR, showed morphological and functional alterations within the pronephros, adversely affecting pronephric structure, leading to slit diaphragm dysfunction by increasing apoptosis within the pronephros. Induction of hyperglycemia in zebrafish embryos induced pronephros alterations which were further worsened upon silencing of EPO expression.
CONCLUSIONS
EPO was identified as a direct renal protective factor, promoting renal embryonic development and protecting kidneys from hyperglycemia induced nephropathy.
Topics: Aged; Animals; Apoptosis; Diabetes Mellitus, Type 2; Diabetic Nephropathies; Erythropoietin; Female; Humans; Kidney; Male; Middle Aged; Proteinuria; Recombinant Proteins; Zebrafish
PubMed: 29203238
DOI: 10.1016/j.molmet.2017.11.006 -
American Journal of Human Genetics Nov 2017Renal agenesis and hypodysplasia (RHD) are major causes of pediatric chronic kidney disease and are highly genetically heterogeneous. We conducted whole-exome sequencing...
Renal agenesis and hypodysplasia (RHD) are major causes of pediatric chronic kidney disease and are highly genetically heterogeneous. We conducted whole-exome sequencing in 202 case subjects with RHD and identified diagnostic mutations in genes known to be associated with RHD in 7/202 case subjects. In an additional affected individual with RHD and a congenital heart defect, we found a homozygous loss-of-function (LOF) variant in SLIT3, recapitulating phenotypes reported with Slit3 inactivation in the mouse. To identify genes associated with RHD, we performed an exome-wide association study with 195 unresolved case subjects and 6,905 control subjects. The top signal resided in GREB1L, a gene implicated previously in Hoxb1 and Shha signaling in zebrafish. The significance of the association, which was p = 2.0 × 10 for novel LOF, increased to p = 4.1 × 10 for LOF and deleterious missense variants combined, and augmented further after accounting for segregation and de novo inheritance of rare variants (joint p = 2.3 × 10). Finally, CRISPR/Cas9 disruption or knockdown of greb1l in zebrafish caused specific pronephric defects, which were rescued by wild-type human GREB1L mRNA, but not mRNA containing alleles identified in case subjects. Together, our study provides insight into the genetic landscape of kidney malformations in humans, presents multiple candidates, and identifies SLIT3 and GREB1L as genes implicated in the pathogenesis of RHD.
Topics: Alleles; Animals; Case-Control Studies; Clustered Regularly Interspaced Short Palindromic Repeats; Congenital Abnormalities; Exome; Female; Genetic Heterogeneity; Genome-Wide Association Study; Genotype; Heredity; Homozygote; Humans; Kidney; Kidney Diseases; Male; Membrane Proteins; Mice; Mutation; Neoplasm Proteins; Phenotype; RNA, Long Noncoding; Urinary Tract; Urogenital Abnormalities; Zebrafish
PubMed: 29100090
DOI: 10.1016/j.ajhg.2017.09.018 -
Endocrinology Dec 2017Although the zebrafish interrenal tissue has been used as a model for steroidogenesis and genesis of the adrenal gland, its specification and morphogenesis remains...
Although the zebrafish interrenal tissue has been used as a model for steroidogenesis and genesis of the adrenal gland, its specification and morphogenesis remains largely unclear. In the present study, we explored how the Wilms tumor 1 (WT1)-expressing cells are segregated from the SF-1-expressing steroidogenic cells in the zebrafish model. The interrenal tissue precursors expressing ff1b, the equivalent of mammalian SF-1, were derived from wt1-expressing pronephric primordia in the zebrafish embryo. Through histochemistry and in situ hybridization, we demonstrated that the size of functionally differentiated interrenal tissue was substantially increased on global inhibition of the Notch signaling pathway and was accompanied by a disrupted segregation between the wt1- and ff1b-expressing cells. As the Notch pathway was conditionally activated during interrenal specification, differentiation, but not ff1b expression, of interrenal tissue was drastically compromised. In embryos deficient for Notch ligands jagged 1b and 2b, transgenic reporter activity of wt1b promoter was detected within the steroidogenic interrenal tissue. In conclusion, our results indicate that Jagged-Notch signaling is required (1) for segregation between wt1-expressing cells and differentiated steroidogenic tissue; and (2) to modulate the extent of functional differentiation in the steroidogenic interrenal tissue.
Topics: Animals; Animals, Genetically Modified; Embryo, Nonmammalian; Gene Expression Regulation, Developmental; Head Kidney; In Situ Hybridization; Interrenal Gland; Jagged-1 Protein; Jagged-2 Protein; Receptors, Notch; Signal Transduction; Steroidogenic Factor 1; Steroids; WT1 Proteins; Zebrafish; Zebrafish Proteins
PubMed: 29029162
DOI: 10.1210/en.2017-00548 -
Archives of Toxicology Jan 2018Prediction and management of drug-induced renal injury (DIRI) rely on the knowledge of the mechanisms of drug insult and on the availability of appropriate animal models...
Prediction and management of drug-induced renal injury (DIRI) rely on the knowledge of the mechanisms of drug insult and on the availability of appropriate animal models to explore it. Zebrafish (Danio rerio) offers unique advantages for assessing DIRI because the larval pronephric kidney has a high homology with its human counterpart and it is fully mature at 3.5 days post-fertilization. Herein, we aimed to evaluate the usefulness of zebrafish larvae as a model of renal tubular toxicity through a comprehensive analysis of the renal alterations induced by the lethal concentrations for 10% of the larvae for gentamicin, paracetamol and tenofovir. We evaluated drug metabolic profile by mass spectrometry, renal function with the inulin clearance assay, the 3D morphology of the proximal convoluted tubule by two-photon microscopy and the ultrastructure of proximal convoluted tubule mitochondria by transmission electron microscopy. Paracetamol was metabolized by conjugation and oxidation with further detoxification with glutathione. Renal clearance was reduced with gentamicin and paracetamol. Proximal tubules were enlarged with paracetamol and tenofovir. All drugs induced mitochondrial alterations including dysmorphic shapes ("donuts", "pancakes" and "rods"), mitochondrial swelling, cristae disruption and/or loss of matrix granules. These results are in agreement with the tubular effects of gentamicin, paracetamol and tenofovir in man and demonstrate that zebrafish larvae might be a good model to assess functional and structural damage associated with DIRI.
Topics: Acetaminophen; Acute Kidney Injury; Animals; Animals, Genetically Modified; Gentamicins; Inactivation, Metabolic; Kidney Function Tests; Kidney Tubules, Proximal; Larva; Mitochondria; Prodrugs; Tenofovir; Toxicity Tests; Zebrafish
PubMed: 28932931
DOI: 10.1007/s00204-017-2063-1 -
Glycobiology Oct 2017Sialic acid acetylesterase (SIAE) removes acetyl moieties from the carbon 9 and 4 hydroxyl groups of sialic acid and recently a debate has been opened on its association...
Sialic acid acetylesterase (SIAE) removes acetyl moieties from the carbon 9 and 4 hydroxyl groups of sialic acid and recently a debate has been opened on its association to autoimmunity. Trying to get new insights on this intriguing enzyme we have studied siae in zebrafish (Danio rerio). In this teleost siae encodes for a polypeptide with a high degree of sequence identity to human and mouse counterparts. Zebrafish Siae behavior upon transient expression in COS7 cells is comparable to human enzyme concerning pH optimum of enzyme activity, subcellular localization and glycosylation. In addition, and as already observed in case of human SIAE, the glycosylated form of the enzyme from zebrafish is released into the culture media. During embryogenesis, in situ hybridization experiments demonstrate that siae transcript is always detectable during development, with a more specific expression in the central nervous system, in pronephric ducts and liver in the more advanced stages of the embryo development. In adult fish an increasing amount of siae mRNA is detectable in heart, eye, muscle, liver, brain, kidney and ovary. These results provide novel information about Siae and point out zebrafish as animal model to better understand the biological role(s) of this rather puzzling enzyme in vertebrates, regarding immune system function and the development of central nervous system.
Topics: Acetylesterase; Animals; COS Cells; Chlorocebus aethiops; Gene Expression Regulation, Developmental; Genome; Humans; Kidney; Liver; Nervous System; RNA, Messenger; Sequence Homology, Nucleic Acid; Zebrafish; Zebrafish Proteins
PubMed: 28922741
DOI: 10.1093/glycob/cwx068 -
Scientific Reports Aug 2017Autosomal dominant polycystic kidney disease (ADPKD) is a common kidney disease caused by mutations in PKD1 or PKD2. Metformin reduces cyst growth in mouse models of...
Autosomal dominant polycystic kidney disease (ADPKD) is a common kidney disease caused by mutations in PKD1 or PKD2. Metformin reduces cyst growth in mouse models of PKD1. However, metformin has not been studied in animal models of PKD2, and the cellular mechanism underlying its effectiveness is not entirely clear. This study investigated the effects of metformin on cyst formation in a zebrafish model of polycystin-2 deficiency resulting from morpholino knockdown of pkd2. We added metformin (2.5 to 20 mM) to the embryo media between 4 and 48 hours post fertilisation and observed pronephric cyst formation by using the wt1b promoter-driven GFP signal in Tg(wt1b:GFP) pkd2 morphants. Metformin inhibited pronephric cyst formation by 42-61% compared with the untreated controls. Metformin also reduced the number of proliferating cells in the pronephric ducts, the degree of dorsal body curvature, and the infiltration of leukocytes surrounding the pronephros. Moreover, metformin treatment increased the phosphorylation of adenosine monophosphate-activated protein kinase (AMPK) and enhanced autophagy in the pronephros. Our data suggest that metformin reduces cyst formation through activation of the AMPK pathway and modulation of defective cellular events such as proliferation and autophagy. These results also imply that metformin could have therapeutic potential for ADPKD treatment.
Topics: AMP-Activated Protein Kinases; Animals; Autophagy; Cell Proliferation; Cloaca; Cysts; Disease Models, Animal; Kidney Tubules; Leukocytes; Metformin; Polycystic Kidney, Autosomal Dominant; Spinal Curvatures; TRPP Cation Channels; Zebrafish
PubMed: 28769124
DOI: 10.1038/s41598-017-07300-x -
Genetics Sep 2017Renal agenesis (RA) is one of the more extreme examples of congenital anomalies of the kidney and urinary tract (CAKUT). Bilateral renal agenesis is almost invariably...
Renal agenesis (RA) is one of the more extreme examples of congenital anomalies of the kidney and urinary tract (CAKUT). Bilateral renal agenesis is almost invariably fatal at birth, and unilateral renal agenesis can lead to future health issues including end-stage renal disease. Genetic investigations have identified several gene variants that cause RA, including , , and However, whereas compound null mutations of genes encoding α and γ retinoic acid receptors (RARs) cause RA in mice, to date there have been no reports of variants in RAR genes causing RA in humans. In this study, we carried out whole exome sequence analysis of two families showing inheritance of an RA phenotype, and in both identified a single candidate gene, Analysis of a zebrafish loss-of-function mutant revealed defects in the pronephric kidney just prior to death, and F0 CRISPR/Cas9 mutagenesis of in the mouse revealed kidney agenesis phenotypes, implicating in this disorder. GREB1L resides in a chromatin complex with RAR members, and our data implicate GREB1L as a coactivator for RARs. This study is the first to associate a component of the RAR pathway with renal agenesis in humans.
Topics: Animals; Congenital Abnormalities; Exome; Female; Humans; Kidney; Kidney Diseases; Loss of Function Mutation; Male; Membrane Proteins; Mice; Neoplasm Proteins; Pedigree; Proteins; Receptors, Retinoic Acid; Zebrafish; Zebrafish Proteins
PubMed: 28739660
DOI: 10.1534/genetics.117.1125