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Developmental Biology Jan 2023Development of the Xenopus pronephros relies on renal precursors grouped at neurula stage into a specific region of dorso-lateral mesoderm called the kidney field....
Development of the Xenopus pronephros relies on renal precursors grouped at neurula stage into a specific region of dorso-lateral mesoderm called the kidney field. Formation of the kidney field at early neurula stage is dependent on retinoic (RA) signaling acting upstream of renal master transcriptional regulators such as pax8 or lhx1. Although lhx1 might be a direct target of RA-mediated transcriptional activation in the kidney field, how RA controls the emergence of the kidney field remains poorly understood. In order to better understand RA control of renal specification of the kidney field, we have performed a transcriptomic profiling of genes affected by RA disruption in lateral mesoderm explants isolated prior to the emergence of the kidney field and cultured at different time points until early neurula stage. Besides genes directly involved in pronephric development (pax8, lhx1, osr2, mecom), hox (hoxa1, a3, b3, b4, c5 and d1) and the hox co-factor meis3 appear as a prominent group of genes encoding transcription factors (TFs) downstream of RA. Supporting the idea of a role of meis3 in the kidney field, we have observed that meis3 depletion results in a severe inhibition of pax8 expression in the kidney field. Meis3 depletion only marginally affects expression of lhx1 and aldh1a2 suggesting that meis3 principally acts upstream of pax8. Further arguing for a role of meis3 and hox in the control of pax8, expression of a combination of meis3, hoxb4 and pbx1 in animal caps induces pax8 expression, but not that of lhx1. The same combination of TFs is also able to transactivate a previously identified pax8 enhancer, Pax8-CNS1. Mutagenesis of potential PBX-Hox binding motifs present in Pax8-CNS1 further allows to identify two of them that are necessary for transactivation. Finally, we have tested deletions of regulatory sequences in reporter assays with a previously characterized transgene encompassing 36.5 kb of the X. tropicalis pax8 gene that allows expression of a truncated pax8-GFP fusion protein recapitulating endogenous pax8 expression. This transgene includes three conserved pax8 enhancers, Pax8-CNS1, Pax8-CNS2 and Pax8-CNS3. Deletion of Pax8-CNS1 alone does not affect reporter expression, but deletion of a 3.5 kb region encompassing Pax8-CNS1 and Pax8-CNS2 results in a severe inhibition of reporter expression both in the otic placode and kidney field domains.
Topics: Animals; Xenopus laevis; Tretinoin; Xenopus Proteins; Paired Box Transcription Factors; Gene Expression Regulation, Developmental; Pronephros; Kidney; Aldehyde Dehydrogenase 1 Family; Retinal Dehydrogenase
PubMed: 36279927
DOI: 10.1016/j.ydbio.2022.10.009 -
Journal of Medicinal Chemistry Jan 2022NIMA-related kinase 1 (Nek1) has lately garnered attention for its widespread function in ciliogenesis, apoptosis, and the DNA-damage response. Despite its involvement...
NIMA-related kinase 1 (Nek1) has lately garnered attention for its widespread function in ciliogenesis, apoptosis, and the DNA-damage response. Despite its involvement in various diseases and its potential as a cancer drug target, no directed medicinal chemistry efforts toward inhibitors against this dark kinase are published. Here, we report the structure-guided design of a potent small-molecule Nek1 inhibitor, starting from a scaffold identified by kinase cross-screening analysis. Seven lead compounds were identified and evaluated for their inhibitory activity. The top compound, , was further profiled for efficacy, toxicity, and bioavailability in a zebrafish polycystic kidney disease model. Administration of caused the expansion of fluorescence-labeled proximal convoluted tubules, supporting our hypothesis that Nek1-inhibition causes cystic kidneys in zebrafish embryos. Compound displayed insignificant inhibition in 48 of 50 kinases in a selectivity test panel. The findings provide a powerful tool to further elucidate the function and pharmacology of this neglected kinase.
Topics: Animals; Drug Design; Embryo, Nonmammalian; NIMA-Related Kinase 1; Polycystic Kidney Diseases; Pronephros; Protein Kinase Inhibitors; Zebrafish
PubMed: 35081715
DOI: 10.1021/acs.jmedchem.0c02118 -
The International Journal of... 2016Pdzrn3, a multidomain protein with E3-ubiquitin ligase activity, has been reported to play a role in myoblast and osteoblast differentiation and, more recently, in...
Pdzrn3, a multidomain protein with E3-ubiquitin ligase activity, has been reported to play a role in myoblast and osteoblast differentiation and, more recently, in neuronal and endothelial cell development. The expression of the pdzrn3 gene is developmentally regulated in various vertebrate tissues, including muscular, neural and vascular system. Little is known about its expression during kidney development, although genetic polymorphisms and alterations around the human pdzrn3 chromosomal region have been found to be associated with renal cell carcinomas and other kidney diseases. We investigated the pdzrn3 spatio-temporal expression pattern in Xenopus laevis embryos by in situ hybridization. We focused our study on the development of the pronephros, which is the embryonic amphibian kidney, functionally similar to the most primitive nephric structures of human kidney. To explore the role of pdzrn3 during renal morphogenesis, we performed loss-of-function experiments, through antisense morpholino injections and analysed the morphants using specific pronephric markers. Dynamic pdzrn3 expression was observed in embryonic tissues, such as somites, brain, eye, blood islands, heart, liver and pronephros. Loss of function experiments resulted in specific alterations of pronephros development. In particular, at early stages, pdzrn3 depletion was associated with a reduction of the pronephros anlagen and later, with perturbations of the tubulogenesis, including deformation of the proximal tubules. Rescue experiments, in which mRNA of the zebrafish pdzrn3 orthologue was injected together with the morpholino, allowed recovery of the kidney phenotypes. These results underline the importance of pdzrn3 expression for correct nephrogenesis.
Topics: Animals; Carrier Proteins; Embryo, Nonmammalian; Gene Expression Regulation, Developmental; Genetic Complementation Test; Humans; In Situ Hybridization; Morphogenesis; Mutation; Pronephros; RING Finger Domains; RNA, Messenger; Xenopus Proteins; Xenopus laevis; Zebrafish Proteins
PubMed: 26934292
DOI: 10.1387/ijdb.150381ld -
Methods in Molecular Biology (Clifton,... 2020With the advances in next-generation sequencing and rapid filtering of candidate variants in diseased patients, it has been increasingly important to develop...
With the advances in next-generation sequencing and rapid filtering of candidate variants in diseased patients, it has been increasingly important to develop translatable in vivo models to study genetic changes. This allows for functional validation of pathogenic mutations and establishes a system to understand the etiology of disease. Due to the ease of genetic manipulation and rapid ex utero development, the zebrafish has become a valuable resource to study important biological processes, including nephrogenesis. The development and function of the zebrafish pronephros are akin to that of mammals. As such, they offer a tractable model to study kidney disease, especially diabetic nephropathy. However, in order to study kidney dysfunction in zebrafish it is imperative that an appropriate readout is available. The appearance of macro-proteins in patient's urine is indicative of defective kidney function. In this technical chapter, we describe the in vivo use of fluorescently tagged dextrans of different molecular weights to reveal the integrity of the zebrafish glomerular filtration barrier.
Topics: Animals; Animals, Genetically Modified; Dextrans; Diabetic Nephropathies; Disease Models, Animal; Embryo, Nonmammalian; Female; Fluorescent Dyes; Genes, Reporter; Glomerular Filtration Barrier; Kidney Glomerulus; Male; Pronephros; Zebrafish; Zebrafish Proteins
PubMed: 31701443
DOI: 10.1007/978-1-4939-9841-8_3 -
In Vitro Cellular & Developmental... Nov 2015von Hippel-Lindau (pVHL)-mediated ubiquitination of HIF-1α plays a central role in the cellular responses to changes in oxygen availability. In the present study, using...
von Hippel-Lindau (pVHL)-mediated ubiquitination of HIF-1α plays a central role in the cellular responses to changes in oxygen availability. In the present study, using zebrafish as a model, we showed that specific knockdown of endogenous vhl leads to pronephros malformation and renal failure. Knockdown of vhl resulted in abnormal kidney development, including curved and cystic pronephric tubule or/and cystic and atrophic glomerulus. Co-injecting capped vhl messenger RNA (mRNA) partially rescued pronephros morphant phenotype, confirming the specificity of the morpholino oligonucleotide (MO)-induced pronephric defects. In keeping with the pronephros phenotype, renal function was affected as well in vhl morphants. Dextran clearance abilities of vhl morphants were significantly reduced as compared with those of control embryos. Further analysis indicated that glomerular integrity is impaired in vhl morphants, while the organization of pronephric duct was minimally affected. Vhl morphants display global increased vegf signaling and angiogenesis. In addition, we found that vhl morphants displayed elevated expression of vegfa in podocytes and increased angiogenesis at pronephric glomerulus and the nearby vessels. Treatment of vegf inducer to embryos also caused pronephros phenotype resembling vhl morphants, further supporting that increased vegfa signaling contribute to the pronephros morphant phenotype. Our study establishes the zebrafish as an alternative vertebrate model system for studying Vhl function during kidney development.
Topics: Animals; Dextrans; Gene Knockdown Techniques; Hypoxia-Inducible Factor 1, alpha Subunit; Kidney Glomerulus; Models, Animal; Morpholinos; Neovascularization, Physiologic; Organogenesis; Podocytes; Polycythemia; Pronephros; RNA, Messenger; Signal Transduction; Ubiquitination; Vascular Endothelial Growth Factor A; Von Hippel-Lindau Tumor Suppressor Protein; Zebrafish; Zebrafish Proteins; Zonula Occludens-1 Protein
PubMed: 26194803
DOI: 10.1007/s11626-015-9938-3 -
International Journal of Molecular... Aug 2022Acute kidney injury (AKI) is commonly associated with severe human diseases, and often worsens the outcome in hospitalized patients. The mammalian kidney has the ability...
Acute kidney injury (AKI) is commonly associated with severe human diseases, and often worsens the outcome in hospitalized patients. The mammalian kidney has the ability to recover spontaneously from AKI; however, little progress has been made in the development of supportive treatments. Increasing evidence suggest that histone deacetylases (HDAC) and NF-κB promote the pathogenesis of AKI, and inhibition of Hdac activity has a protective effect in murine models of AKI. However, the role of HDAC at the early stages of recovery is unknown. We used the zebrafish pronephros model to study the role of epigenetic modifiers in the immediate repair response after injury to the tubular epithelium. Using specific inhibitors, we found that the histone deacetylase Hdac2, Hdac6, and Hdac8 activities are required for the repair via collective cell migration. We found that , , and expression levels were upregulated in the repairing epithelial cells shortly after injury. Depletion of , , or with morpholino oligonucleotides impaired the repair process, whereas the combined depletion of all three genes synergistically suppressed the recovery process. Furthermore, time-lapse video microscopy revealed that the lamellipodia and filopodia formation in the flanking cells was strongly reduced in -depleted embryos. Our findings suggest that Hdac activity and NF-κB are synergistically required for the immediate repair response in the zebrafish pronephros model of AKI, and the timing of HDAC inhibition might be important in developing supportive protocols in the human disease.
Topics: Acute Kidney Injury; Animals; Histone Deacetylase 6; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; Mice; NF-kappa B; Pronephros; Repressor Proteins; Zebrafish; Zebrafish Proteins
PubMed: 36076983
DOI: 10.3390/ijms23179582 -
The International Journal of... 2015The embryonic nephric mesenchyme contains pluripotent progenitor cells. Six2, a homeodomain transcription factor, is expressed in a subset of the nephric mesenchyme, and...
The embryonic nephric mesenchyme contains pluripotent progenitor cells. Six2, a homeodomain transcription factor, is expressed in a subset of the nephric mesenchyme, and it functions to maintain a progenitor state by suppressing nephrogenesis. Despite the functional significance of Six2 in nephric development, its regulatory mechanisms remain unclear. To identify the cis-regulatory elements for Six2, we focused on the evolutionarily conserved sequences known as conserved noncoding sequences (CNSs) associated with the Six2 locus. Transgenic experiments using Xenopus laevis embryos revealed that three of the eight CNSs located within a 317-kb segment of the Six2 genomic locus were nephric enhancers. Motif analysis of transcription factors combined with phylogenetic footprinting revealed the enrichment of putative T-cell factor (Tcf)-, Hox-, and SWI/SNF complex helicase-like transcription factor (Hltf)- and AT-rich interactive domain 3A (Arid3a)-binding motif sequences in these enhancers.
Topics: Animals; Animals, Genetically Modified; Base Sequence; Conserved Sequence; Embryo, Nonmammalian; Enhancer Elements, Genetic; Evolution, Molecular; Gene Expression Regulation, Developmental; Green Fluorescent Proteins; Homeodomain Proteins; In Situ Hybridization; Molecular Sequence Data; Nerve Tissue Proteins; Nucleotide Motifs; Organogenesis; Phylogeny; Pronephros; Sequence Homology, Nucleic Acid; Xenopus; Xenopus Proteins; Xenopus laevis
PubMed: 26009236
DOI: 10.1387/ijdb.140263ho -
Ecotoxicology and Environmental Safety Oct 2020Cadmium (Cd) is a type of toxic metal, in most cases, coming from fuel burning and aquatic plants. The cells of organisms can be caused serious damage, including...
Cadmium (Cd) is a type of toxic metal, in most cases, coming from fuel burning and aquatic plants. The cells of organisms can be caused serious damage, including pyroptosis, exposure to low concentrations of Cd in long-term. Pyroptosis is a recently discovered Caspase-1-mediated cell death. In this study, lymphocytes were extracted from the pronephros and spleens in carps, respectively. After treating cells with low concentration of Cd, the mRNA and protein expression levels of pyroptosis-related genes, NLRP3, Caspase-1, and pro-inflammatory cytokines, increased obviously. And the content of reactive oxygen species (ROS) and mitochondria reactive oxygen species (mtROS) increased significantly, we also found the activities of CAT, GSH-px and T-SOD reduce significantly, and the content of MDA have a clear upward trend. We then added NLRP3 inhibitor, Glyburide, to the Cd-treated group, further confirming that NLRP3 is a key gene in pyroptosis pathways by detecting the mRNA and protein expression levels. Besides, the rupture of the cell membrane was also confirmed by Hoechst/PI double staining, red fluorescence increased obviously in the Cd treatment group. The experiment revealed that Cd exposure induces pyroptosis of lymphocytes in carp pronephros and spleens by activating NLRP3. Inhibition of NLRP3 activity can slow down the degree of lymphocytes pyroptosis. Thus, the above information provides a new avenue toward understanding the partial mechanism of Cd exposure-induced pyroptosis.
Topics: Animals; Cadmium; Carps; Caspase 1; Inflammasomes; Lymphocytes; Mitochondria; NLR Family, Pyrin Domain-Containing 3 Protein; Pronephros; Pyroptosis; Reactive Oxygen Species; Spleen; Water Pollutants, Chemical
PubMed: 32800238
DOI: 10.1016/j.ecoenv.2020.110903 -
American Journal of Physiology. Renal... May 2021Developing organisms need to adapt to environmental variations as well as to rapid changes in substrate availability and energy demands imposed by fast-growing tissues...
Developing organisms need to adapt to environmental variations as well as to rapid changes in substrate availability and energy demands imposed by fast-growing tissues and organs. Little is known about the adjustments that kidneys undergo in response to these challenges. We performed single-cell RNA sequencing of zebrafish pronephric duct cells to understand how the developing kidney responds to changes in filtered substrates and intrinsic energy requirements. We found high levels of glucose transporters early in development and increased expression of monocarboxylate transporters at later times. This indicates that the zebrafish embryonic kidney displays a high glucose transporting capacity during early development, which is replaced by the ability to absorb monocarboxylates and amino acids at later stages. This change in transport capacity was accompanied by the upregulation of mitochondrial carriers, indicating a switch to increased oxidative phosphorylation to meet the increasing energy demand of a developing kidney. The zebrafish embryonic kidney has high levels of glucose transporters during early development, which are replaced by monocarboxylate and amino acid transporters later on. Inhibition of Na-glucose cotransporter-dependent glucose transport by sotagliflozin also increased expression, supporting the idea that the glucose transport capacity is dynamically adjusted during zebrafish pronephros development. Concurrent upregulation of mitochondrial SCL25 transporters at later stages supports the idea that the pronephros adjusts to changing substrate supplies and/or energy demands during embryonic development.
Topics: Animals; Energy Metabolism; Gene Expression Profiling; Gene Expression Regulation, Developmental; Pronephros; RNA, Messenger; RNA-Seq; Single-Cell Analysis; Solute Carrier Proteins; Transcriptome; Zebrafish; Zebrafish Proteins
PubMed: 33749326
DOI: 10.1152/ajprenal.00610.2020 -
Scientific Reports Aug 2017Peroxiredoxin1 (Prdx1) is an antioxidant enzyme belonging to the peroxiredoxin family of proteins. Prdx1 catalyzes the reduction of HO and alkyl hydroperoxide and plays...
Peroxiredoxin1 (Prdx1) is an antioxidant enzyme belonging to the peroxiredoxin family of proteins. Prdx1 catalyzes the reduction of HO and alkyl hydroperoxide and plays an important role in different biological processes. Prdx1 also participates in various age-related diseases and cancers. In this study, we investigated the role of Prdx1 in pronephros development during embryogenesis. Prdx1 knockdown markedly inhibited proximal tubule formation in the pronephros and significantly increased the cellular levels of reactive oxygen species (ROS), which impaired primary cilia formation. Additionally, treatment with ROS (HO) severely disrupted proximal tubule formation, whereas Prdx1 overexpression reversed the ROS-mediated inhibition in proximal tubule formation. Epistatic analysis revealed that Prdx1 has a crucial role in retinoic acid and Wnt signaling pathways during pronephrogenesis. In conclusion, Prdx1 facilitates proximal tubule formation during pronephrogenesis by regulating ROS levels.
Topics: Amino Acid Sequence; Animals; Conserved Sequence; Cysteine; Gene Expression Regulation, Developmental; Gene Knockdown Techniques; Organogenesis; Peroxiredoxins; Phenotype; Pronephros; Reactive Oxygen Species; Tretinoin; Wnt Signaling Pathway; Xenopus laevis
PubMed: 28827763
DOI: 10.1038/s41598-017-09262-6