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Arhiv Za Higijenu Rada I Toksikologiju Sep 2015Pesticides used in agriculture can have hazardous effects on aquatic organisms, and amphibians are even more threatened than other aquatic vertebrates. Maneb is widely...
Pesticides used in agriculture can have hazardous effects on aquatic organisms, and amphibians are even more threatened than other aquatic vertebrates. Maneb is widely used to control fungal diseases on crops, fruits, and vegetables. The aim of this study was to investigate the acute toxic effects of maneb on the common (Bufo bufo) and green toad (Pseudepidalea viridis) tadpoles. Tadpoles at the development stage 21 were exposed to maneb (0-5 mg L(-1)) for 120 h. Maneb LC50 values at hour 120 were 1.966 mg L(-1) for B. bufo and 0.332 mg L-1 for P. viridis. To the best of our knowledge, these are the first published LC50 findings for the two species. Visceral oedema and tail deformations were observed in both species. We also observed liver necrosis, pronephric tubule deformations, somite deteriorations, and visceral oedema at maneb concentrations≥0.1 mg L(-1) for B. bufo and ≥0.05 mg L(-1) for P. viridis. Our results show that B. bufo tadpoles have a much higher resilience to maneb than P. viridis tadpoles. This resilience seems to be related to the larger size of the B. bufo tadpoles and their ability to metamorphose faster in adverse conditions. Future research should look into the mechanisms of toxic action of maneb in anurans.
Topics: Animals; Anura; Larva; Maneb; Pesticides; Turkey; Water Pollutants, Chemical
PubMed: 26444339
DOI: 10.1515/aiht-2015-66-2642 -
Kidney & Blood Pressure Research 2016Vitamin C is an antioxidant and acts as a cofactor for several key enzymatic catalytic reactions in animals. Amphibians produce vitamin C in their kidneys, as opposed to...
BACKGROUNDS/AIMS
Vitamin C is an antioxidant and acts as a cofactor for several key enzymatic catalytic reactions in animals. Amphibians produce vitamin C in their kidneys, as opposed to mammals that produce vitamin C in their liver. Gulo serves as a crucial enzyme for vitamin C synthesis in mammals, but the characteristics and localization of its homologous genes during kidney development in Xenopus laevis, an amphibian, remains unknown.
METHODS
We aligned amino acid sequences of Gulo across different species by using bioinformatics methods and detected patterns of expression for Gulo during kidney development by using RT-PCR and in situ hybridization.
RESULTS
We identified a new site on the X. laevis genome, LOC495407. Sequence alignment analysis indicated this fragment is highly conserved and homologous to gulo genes in mammals. RT-PCR and in situ hybridization results reveal that X. laevis gulo is maternally expressed during the early stages of embryonic development, particularly, in the tubules of the pronephros from the middle tail-bud stage and onward in embryos.
CONCLUSION
Gulo is a novel specific marker for pronephros tubules in X. laevis, and may be used as a potential marker for kidney development studies and disease diagnosis in mammals.
Topics: Animals; Biomarkers; Female; Kidney Tubules; L-Gulonolactone Oxidase; Mammals; Pronephros; Sequence Alignment; Xenopus laevis
PubMed: 27832650
DOI: 10.1159/000450561 -
Cell Death & Disease Nov 2016Acute lung injury (ALI) and its more severe form, acute respiratory distress syndrome (ARDS), are life-threatening diseases that are associated with high mortality rates...
Acute lung injury (ALI) and its more severe form, acute respiratory distress syndrome (ARDS), are life-threatening diseases that are associated with high mortality rates due to treatment limitations. Neutrophils play key roles in the pathogenesis of ALI/ARDS by promoting the inflammation and injury of the alveolar microenvironment. To date, in vivo functional approaches have been limited by the inaccessibility to the alveolar sacs, which are located at the anatomical terminal of the respiratory duct in mammals. We are the first to characterize the swim bladder of the zebrafish larva, which is similar to the mammalian lung, as a real-time in vivo model for examining pulmonary neutrophil infiltration during ALI. We observed that the delivery of exogenous materials, including lipopolysaccharide (LPS), Poly IC and silica nanoparticles, by microinjection triggered significant time- and dose-dependent neutrophil recruitment into the swim bladder. Neutrophils infiltrated the LPS-injected swim bladder through the blood capillaries around the pneumatic duct or a site near the pronephric duct. An increase in the post-LPS inflammatory cytokine mRNA levels coincided with the in vivo neutrophil aggregation in the swim bladder. Microscopic examinations of the LPS-injected swim bladders further revealed in situ injuries, including epithelial distortion, endoplasmic reticulum swelling and mitochondrial injuries. Inhibitor screening assays with this model showed a reduction in neutrophil migration into the LPS-injected swim bladder in response to Shp2 inhibition. Moreover, the pharmacological suppression and targeted disruption of Shp2 in myeloid cells alleviated pulmonary inflammation in the LPS-induced ALI mouse model. Additionally, we used this model to assess pneumonia-induced neutrophil recruitment by microinjecting bronchoalveolar lavage fluid from patients into swim bladders; this injection enhanced neutrophil aggregation relative to the control. In conclusion, our findings highlight the swim bladder as a promising and powerful model for mechanistic and drug screening studies of alveolar injuries.
Topics: Acute Lung Injury; Air; Air Sacs; Animals; Cell Movement; Cytokines; Disease Models, Animal; Inflammation; Lipopolysaccharides; Mice; Neutrophil Infiltration; Neutrophils; Protein Tyrosine Phosphatase, Non-Receptor Type 11; Zebrafish
PubMed: 27831560
DOI: 10.1038/cddis.2016.365 -
Journal of Cell Death 2014KillerRed (KR) is a recently discovered fluorescent protein that, when activated with green light, releases reactive oxygen species (ROS) into the cytoplasm, triggering...
KillerRed (KR) is a recently discovered fluorescent protein that, when activated with green light, releases reactive oxygen species (ROS) into the cytoplasm, triggering apoptosis in a KR-expressing cell. This property allows for the use of KR as a means of killing cells in an organism with great temporal and spatial specificity, while minimizing the nonspecific effects that can result from mechanical or chemical exposure damage techniques. Such optogenetic control of cell death, and the resulting ability to induce the targeted death of specific tissues, is invaluable for regeneration/repair studies-particularly in Xenopus laevis, where apoptosis plays a key role in regeneration and repair. We here describe a method by which membrane-bound KR, introduced to Xenopus embryos by mRNA microinjection, can be activated with green light to induce apoptosis in specific organs and tissues, with a focus on the developing eye and pronephric kidney.
PubMed: 25374461
DOI: 10.4137/JCD.S18368 -
PLoS Genetics Apr 2015Lowe syndrome and Dent-2 disease are caused by mutation of the inositol 5-phosphatase OCRL1. Despite our increased understanding of the cellular functions of OCRL1, the...
Lowe syndrome and Dent-2 disease are caused by mutation of the inositol 5-phosphatase OCRL1. Despite our increased understanding of the cellular functions of OCRL1, the underlying basis for the renal tubulopathy seen in both human disorders, of which a hallmark is low molecular weight proteinuria, is currently unknown. Here, we show that deficiency in OCRL1 causes a defect in endocytosis in the zebrafish pronephric tubule, a model for the mammalian renal tubule. This coincides with a reduction in levels of the scavenger receptor megalin and its accumulation in endocytic compartments, consistent with reduced recycling within the endocytic pathway. We also observe reduced numbers of early endocytic compartments and enlarged vacuolar endosomes in the sub-apical region of pronephric cells. Cell polarity within the pronephric tubule is unaffected in mutant embryos. The OCRL1-deficient embryos exhibit a mild ciliogenesis defect, but this cannot account for the observed impairment of endocytosis. Catalytic activity of OCRL1 is required for renal tubular endocytosis and the endocytic defect can be rescued by suppression of PIP5K. These results indicate for the first time that OCRL1 is required for endocytic trafficking in vivo, and strongly support the hypothesis that endocytic defects are responsible for the renal tubulopathy in Lowe syndrome and Dent-2 disease. Moreover, our results reveal PIP5K as a potential therapeutic target for Lowe syndrome and Dent-2 disease.
Topics: Animals; Cell Polarity; Endocytosis; Endosomes; Gene Deletion; Low Density Lipoprotein Receptor-Related Protein-2; Oculocerebrorenal Syndrome; Phosphoric Monoester Hydrolases; Phosphotransferases (Alcohol Group Acceptor); Pronephros; Zebrafish; Zebrafish Proteins
PubMed: 25838181
DOI: 10.1371/journal.pgen.1005058 -
Investigative Ophthalmology & Visual... Jan 2017Joubert syndrome (JBTS) is an autosomal recessive ciliopathy with considerable phenotypic variability. In addition to central nervous system abnormalities, a subset of...
PURPOSE
Joubert syndrome (JBTS) is an autosomal recessive ciliopathy with considerable phenotypic variability. In addition to central nervous system abnormalities, a subset of JBTS patients exhibit retinal dystrophy and/or kidney disease. Mutations in the AHI1 gene are causative for approximately 10% of all JBTS cases. The purpose of this study was to generate ahi1 mutant alleles in zebrafish and to characterize the retinal phenotypes.
METHODS
Zebrafish ahi1 mutants were generated using transcription activator-like effector nucleases (TALENs). Expression analysis was performed by whole-mount in situ hybridization. Anatomic and molecular characterization of photoreceptors was investigated by histology, electron microscopy, and immunohistochemistry. The optokinetic response (OKR) behavior assay was used to assess visual function. Kidney cilia were evaluated by whole-mount immunostaining.
RESULTS
The ahi1lri46 mutation in zebrafish resulted in shorter cone outer segments but did not affect visual behavior at 5 days after fertilization (dpf). No defects in rod morphology or rhodopsin localization were observed at 5 dpf. By 5 months of age, cone degeneration and rhodopsin mislocalization in rod photoreceptors was observed. The connecting cilium formed normally and Cc2d2a and Cep290 localized properly. Distal pronephric duct cilia were absent in mutant fish; however, only 9% of ahi1 mutants had kidney cysts by 5 dpf, suggesting that the pronephros remained largely functional.
CONCLUSIONS
The results indicate that Ahi1 is required for photoreceptor disc morphogenesis and outer segment maintenance in zebrafish.
Topics: Animals; Carrier Proteins; Cell Survival; Cilia; Ciliopathies; DNA Mutational Analysis; Genotype; Immunohistochemistry; In Situ Hybridization; Microscopy, Electron, Transmission; Morphogenesis; Mutation; Proto-Oncogene Proteins; Retinal Photoreceptor Cell Outer Segment; Zebrafish; Zebrafish Proteins
PubMed: 28118669
DOI: 10.1167/iovs.16-20326 -
Developmental Dynamics : An Official... Apr 2016Notch signaling in pronephros development has been shown to regulate establishment of glomus and proximal tubule, but how Notch signal works on competency of pronephric...
Proper Notch activity is necessary for the establishment of proximal cells and differentiation of intermediate, distal, and connecting tubule in Xenopus pronephros development.
BACKGROUND
Notch signaling in pronephros development has been shown to regulate establishment of glomus and proximal tubule, but how Notch signal works on competency of pronephric anlagen during the generation of pronephric components remains to be understood.
RESULTS
We investigated how components of pronephros (glomus, proximal tubule, intermediate tubule, distal tubule, and connecting tubule) were generated in Xenopus embryos by timed overactivation and suppression of Notch signaling. Notch activation resulted in expansion of the glomus and disruption of the proximal tubule formation. Inhibition of Notch signaling reduced expression of wt1 and XSMP-30. In addition, when Notch signaling was overactivated at stage 20 on, intermediate, distal, and connecting tubule markers, gremlin and clcnkb, were decreased while Notch down-regulation increased gremlin and clcnkb. Similar changes were observed with segmental markers, cldn19, cldn14, and rhcg on activation or inhibition of Notch. Although Notch did not affect the expression of pan-pronephric progenitor marker, pax2, its activation inhibited lumen formation in the pronephros.
CONCLUSIONS
Notch signal is essential for glomus and proximal tubule development and inhibition of Notch is critical for the differentiation of the intermediate, distal, and connecting tubule.
Topics: Animals; Antigens, Differentiation; Cell Differentiation; Embryo, Nonmammalian; Gene Expression Regulation, Developmental; Kidney Tubules, Proximal; Pronephros; Receptors, Notch; Signal Transduction; Xenopus laevis
PubMed: 26773453
DOI: 10.1002/dvdy.24386 -
PLoS Genetics Jul 2015African Americans have a disproportionate risk for developing nephropathy. This disparity has been attributed to coding variants (G1 and G2) in apolipoprotein L1...
African Americans have a disproportionate risk for developing nephropathy. This disparity has been attributed to coding variants (G1 and G2) in apolipoprotein L1 (APOL1); however, there is little functional evidence supporting the role of this protein in renal function. Here, we combined genetics and in vivo modeling to examine the role of apol1 in glomerular development and pronephric filtration and to test the pathogenic potential of APOL1 G1 and G2. Translational suppression or CRISPR/Cas9 genome editing of apol1 in zebrafish embryos results in podocyte loss and glomerular filtration defects. Complementation of apol1 morphants with wild-type human APOL1 mRNA rescues these defects. However, the APOL1 G1 risk allele does not ameliorate defects caused by apol1 suppression and the pathogenicity is conferred by the cis effect of both individual variants of the G1 risk haplotype (I384M/S342G). In vivo complementation studies of the G2 risk allele also indicate that the variant is deleterious to protein function. Moreover, APOL1 G2, but not G1, expression alone promotes developmental kidney defects, suggesting a possible dominant-negative effect of the altered protein. In sickle cell disease (SCD) patients, we reported previously a genetic interaction between APOL1 and MYH9. Testing this interaction in vivo by co-suppressing both transcripts yielded no additive effects. However, upon genetic or chemical induction of anemia, we observed a significantly exacerbated nephropathy phenotype. Furthermore, concordant with the genetic interaction observed in SCD patients, APOL1 G2 reduces myh9 expression in vivo, suggesting a possible interaction between the altered APOL1 and myh9. Our data indicate a critical role for APOL1 in renal function that is compromised by nephropathy-risk encoding variants. Moreover, our interaction studies indicate that the MYH9 locus is also relevant to the phenotype in a stressed microenvironment and suggest that consideration of the context-dependent functions of both proteins will be required to develop therapeutic paradigms.
Topics: Animals; Apolipoprotein L1; Apolipoproteins; Clustered Regularly Interspaced Short Palindromic Repeats; Flow Cytometry; Gene Knockdown Techniques; Genetic Predisposition to Disease; Genetic Variation; Glomerular Filtration Rate; Glomerulonephritis, Membranous; Humans; Kidney Glomerulus; Lipoproteins, HDL; Microscopy, Electron, Transmission; Molecular Motor Proteins; Morpholinos; Myosin Heavy Chains; Zebrafish
PubMed: 26147622
DOI: 10.1371/journal.pgen.1005349 -
Disease Models & Mechanisms Aug 2016Patients with von Hippel-Lindau (VHL) disease harbor a germline mutation in the VHL gene leading to the development of several tumor types including clear cell renal...
Patients with von Hippel-Lindau (VHL) disease harbor a germline mutation in the VHL gene leading to the development of several tumor types including clear cell renal cell carcinoma (ccRCC). In addition, the VHL gene is inactivated in over 90% of sporadic ccRCC cases. 'Clear cell' tumors contain large, proliferating cells with 'clear cytoplasm', and a reduced number of cilia. VHL inactivation leads to the stabilization of hypoxia inducible factors 1a and 2a [HIF1a and HIF2a (HIF2a is also known as EPAS1)] with consequent up-regulation of specific target genes involved in cell proliferation, angiogenesis and erythropoiesis. A zebrafish model with a homozygous inactivation in the VHL gene (vhl(-/-)) recapitulates several aspects of the human disease, including development of highly vascular lesions in the brain and the retina and erythrocytosis. Here, we characterize for the first time the epithelial abnormalities present in the kidney of the vhl(-/-) zebrafish larvae as a first step in building a model of ccRCC in zebrafish. Our data show that the vhl(-/-) zebrafish kidney is characterized by an increased tubule diameter, disorganized cilia, the dramatic formation of cytoplasmic lipid vesicles, glycogen accumulation, aberrant cell proliferation and abnormal apoptosis. This phenotype of the vhl(-/-) pronephros is reminiscent of clear cell histology, indicating that the vhl(-/-) mutant zebrafish might serve as a model of early stage RCC. Treatment of vhl(-/-) zebrafish embryos with a small-molecule HIF2a inhibitor rescued the pronephric abnormalities, underscoring the value of the zebrafish model in drug discovery for treatment of VHL disease and ccRCC.
Topics: Animals; Apoptosis; Basic Helix-Loop-Helix Transcription Factors; Carcinoma, Renal Cell; Cell Proliferation; Cytoplasmic Vesicles; Embryonic Development; Glycogen; Humans; Kidney Neoplasms; Kidney Tubules; Larva; Neoplasm Staging; Phenotype; Pronephros; Tumor Suppressor Proteins; Zebrafish; Zebrafish Proteins
PubMed: 27491085
DOI: 10.1242/dmm.024380 -
American Journal of Human Genetics Jul 2016Autosomal-dominant tubulo-interstitial kidney disease (ADTKD) encompasses a group of disorders characterized by renal tubular and interstitial abnormalities, leading to...
Autosomal-dominant tubulo-interstitial kidney disease (ADTKD) encompasses a group of disorders characterized by renal tubular and interstitial abnormalities, leading to slow progressive loss of kidney function requiring dialysis and kidney transplantation. Mutations in UMOD, MUC1, and REN are responsible for many, but not all, cases of ADTKD. We report on two families with ADTKD and congenital anemia accompanied by either intrauterine growth retardation or neutropenia. Ultrasound and kidney biopsy revealed small dysplastic kidneys with cysts and tubular atrophy with secondary glomerular sclerosis, respectively. Exclusion of known ADTKD genes coupled with linkage analysis, whole-exome sequencing, and targeted re-sequencing identified heterozygous missense variants in SEC61A1-c.553A>G (p.Thr185Ala) and c.200T>G (p.Val67Gly)-both affecting functionally important and conserved residues in SEC61. Both transiently expressed SEC6A1A variants are delocalized to the Golgi, a finding confirmed in a renal biopsy from an affected individual. Suppression or CRISPR-mediated deletions of sec61al2 in zebrafish embryos induced convolution defects of the pronephric tubules but not the pronephric ducts, consistent with the tubular atrophy observed in the affected individuals. Human mRNA encoding either of the two pathogenic alleles failed to rescue this phenotype as opposed to a complete rescue by human wild-type mRNA. Taken together, these findings provide a mechanism by which mutations in SEC61A1 lead to an autosomal-dominant syndromic form of progressive chronic kidney disease. We highlight protein translocation defects across the endoplasmic reticulum membrane, the principal role of the SEC61 complex, as a contributory pathogenic mechanism for ADTKD.
Topics: Adult; Aged; Alleles; Amino Acid Sequence; Anemia; Animals; Biopsy; Child; Chronic Disease; Disease Progression; Endoplasmic Reticulum; Exome; Female; Fetal Growth Retardation; Genes, Dominant; Golgi Apparatus; Heterozygote; Humans; Infant, Newborn; Kidney Diseases; Male; Middle Aged; Models, Molecular; Mutation; Mutation, Missense; Neutropenia; Pedigree; Phenotype; RNA, Messenger; SEC Translocation Channels; Syndrome; Young Adult; Zebrafish
PubMed: 27392076
DOI: 10.1016/j.ajhg.2016.05.028