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Nature Communications Feb 2016Steroid-resistant nephrotic syndrome (SRNS) causes 15% of chronic kidney disease (CKD). Here we show that recessive mutations in FAT1 cause a distinct renal disease...
Steroid-resistant nephrotic syndrome (SRNS) causes 15% of chronic kidney disease (CKD). Here we show that recessive mutations in FAT1 cause a distinct renal disease entity in four families with a combination of SRNS, tubular ectasia, haematuria and facultative neurological involvement. Loss of FAT1 results in decreased cell adhesion and migration in fibroblasts and podocytes and the decreased migration is partially reversed by a RAC1/CDC42 activator. Podocyte-specific deletion of Fat1 in mice induces abnormal glomerular filtration barrier development, leading to podocyte foot process effacement. Knockdown of Fat1 in renal tubular cells reduces migration, decreases active RAC1 and CDC42, and induces defects in lumen formation. Knockdown of fat1 in zebrafish causes pronephric cysts, which is partially rescued by RAC1/CDC42 activators, confirming a role of the two small GTPases in the pathogenesis. These findings provide new insights into the pathogenesis of SRNS and tubulopathy, linking FAT1 and RAC1/CDC42 to podocyte and tubular cell function.
Topics: Animals; Cadherins; Cell Adhesion; Cell Movement; Dilatation, Pathologic; Fibroblasts; Gene Knockdown Techniques; Hematuria; Humans; Kidney Tubules; Lissencephaly; Mice; Mutation; Nephrotic Syndrome; Podocytes; Syndrome; Zebrafish; Zebrafish Proteins; cdc42 GTP-Binding Protein; rac1 GTP-Binding Protein
PubMed: 26905694
DOI: 10.1038/ncomms10822 -
Mechanisms of Development Nov 2015The zebrafish pronephric tubule consists of proximal and distal segments and a collecting duct. The proximal segment is subdivided into the neck, proximal convoluted...
The zebrafish pronephric tubule consists of proximal and distal segments and a collecting duct. The proximal segment is subdivided into the neck, proximal convoluted tubule (PCT) and proximal straight tubule (PST) segments. The distal segment consists of the distal-early (DE) and distal-late (DL) segments. How the proximal and distal segments develop along the anteroposterior axis is poorly understood. Here we show that knockdown of taz in zebrafish caused shortening and a significant reduction in the number of principal cells of the PST-DE segment, and proximalization of the pronephric tubule in 24 hpf embryos. RA treatment expanded the pronephric proximal domain in normal embryos as in taz morphants, an effect that was further enhanced upon exposure of taz morphants to RA. The early pronephric defects in 24 hpf taz morphants led to the failure of anterior pronephric tubule migration and convolution, and to PCT dilation and cyst formation in older embryos. In situ hybridization showed weak and transient expression of taz at the bud stage in the intermediate mesoderm, the source of pronephric progenitors. The present findings show that Taz is required in the anteroposterior patterning of the pronephric progenitor domain in the intermediate mesoderm, acting in part by regulating RA signaling in the pronephric progenitor field in the intermediate mesoderm.
Topics: Animals; Animals, Genetically Modified; Body Patterning; Cell Count; Gene Expression Regulation, Developmental; Gene Knockdown Techniques; Intracellular Signaling Peptides and Proteins; Kidney Tubules; Mesoderm; Pronephros; Transcriptional Coactivator with PDZ-Binding Motif Proteins; Tretinoin; Zebrafish; Zebrafish Proteins
PubMed: 26248207
DOI: 10.1016/j.mod.2015.08.001 -
Aging Sep 2020Cellular senescence is considered a stress response imposing a stable cell cycle arrest to restrict the growth of damaged cells. More recently however, cellular...
Cellular senescence is considered a stress response imposing a stable cell cycle arrest to restrict the growth of damaged cells. More recently however, cellular senescence was identified during mouse embryo development at particular structures during specific periods of time. This programmed cell senescence has been proposed to serve developmental and morphogenetic functions and to potentially represent an evolutionary origin of senescence. Cellular senescence has also been described to take place during bird (chick and quail) and amphibian (xenopus and axoltl) development. Fish however, have been described to show a very narrow and restricted pattern of developmental cell senescence. Here we carried out a detailed characterization of senescence during zebrafish development and found it to be conserved and widespread. Apart from yolk and cloaca, previously described structures, we also identified senescence in the developing central nervous system, intestine, liver, pronephric ducts, and crystalline. Interestingly, senescence at these developing structures disappeared upon treatment with senolytic compound ABT-263, supporting their senescent identity and opening the possibility of studying the contribution of this process to development. In summary, our findings extend the description of developmentally-programmed cell senescence to lower vertebrates contributing to the notion of the relevance of this process for embryo development.
PubMed: 32991320
DOI: 10.18632/aging.103968 -
Computational and Structural... 2023Diabetic nephropathy (DN) is one of the most established microvascular complications of diabetes and a key cause of end-stage renal disease. It is well established that...
Diabetic nephropathy (DN) is one of the most established microvascular complications of diabetes and a key cause of end-stage renal disease. It is well established that gene susceptibility to DN plays a critical role in disease pathophysiology. Therefore, many genetic studies have been performed to categorize candidate genes in prominent diabetic cohorts, aiming to investigate DN pathogenesis and etiology. In this study, we performed a meta-analysis on the expression profiles of GSE1009, GSE30122, GSE96804, GSE99340, GSE104948, GSE104954, and GSE111154 to identify critical transcriptional factors associated with DN progression. The analysis was conducted for all individual datasets for each kidney tissue (glomerulus, tubules, and kidney cortex). We identified distinct clusters of susceptibility genes that were dysregulated in a renal compartment-specific pattern. Further, we recognized a small but a closely connected set of these susceptibility genes enriched for podocyte differentiation, several of which were characterized as genes encoding critical transcriptional factors (TFs) involved in DN development and podocyte function. To validate the role of identified TFs in DN progression, we functionally validated the three main TFs (DACH1, LMX1B, and WT1) identified through differential gene expression and network analysis using the hyperglycemic zebrafish model. We report that hyperglycemia-induced altered gene expression of the key TF genes leads to morphological abnormalities in zebrafish glomeruli, pronephric tubules, proximal and distal ducts. This study demonstrated that altered expression of these TF genes could be associated with hyperglycemia-induced nephropathy and, thus, aids in understanding the molecular drivers, essential genes, and pathways that trigger DN initiation and development.
PubMed: 36659918
DOI: 10.1016/j.csbj.2022.12.054 -
Molecular Biology of the Cell Jan 2016Arl13b belongs to the ADP-ribosylation factor family within the Ras superfamily of regulatory GTPases. Mutations in Arl13b cause Joubert syndrome, which is characterized...
Arl13b belongs to the ADP-ribosylation factor family within the Ras superfamily of regulatory GTPases. Mutations in Arl13b cause Joubert syndrome, which is characterized by congenital cerebellar ataxia, hypotonia, oculomotor apraxia, and mental retardation. Arl13b is highly enriched in cilia and is required for ciliogenesis in multiple organs. Nevertheless, the precise role of Arl13b remains elusive. Here we report that the exocyst subunits Sec8, Exo70, and Sec5 bind preferentially to the GTP-bound form of Arl13b, consistent with the exocyst being an effector of Arl13b. Moreover, we show that Arl13b binds directly to Sec8 and Sec5. In zebrafish, depletion of arl13b or the exocyst subunit sec10 causes phenotypes characteristic of defective cilia, such as curly tail up, edema, and abnormal pronephric kidney development. We explored this further and found a synergistic genetic interaction between arl13b and sec10 morphants in cilia-dependent phenotypes. Through conditional deletion of Arl13b or Sec10 in mice, we found kidney cysts and decreased ciliogenesis in cells surrounding the cysts. Moreover, we observed a decrease in Arl13b expression in the kidneys from Sec10 conditional knockout mice. Taken together, our results indicate that Arl13b and the exocyst function together in the same pathway leading to functional cilia.
Topics: ADP-Ribosylation Factors; Abnormalities, Multiple; Animals; Cerebellum; Cilia; Eye Abnormalities; Genetic Association Studies; HeLa Cells; Humans; Kidney; Kidney Diseases, Cystic; Mice; Mice, Knockout; Microtubules; Mutation; NIH 3T3 Cells; Retina; Vesicular Transport Proteins; Zebrafish; Zebrafish Proteins
PubMed: 26582389
DOI: 10.1091/mbc.E15-02-0061 -
Cells Apr 2022The anterior-posterior (AP) axis in chordates is regulated by a conserved set of genes and signaling pathways, including genes and retinoic acid (RA), which play...
The anterior-posterior (AP) axis in chordates is regulated by a conserved set of genes and signaling pathways, including genes and retinoic acid (RA), which play well-characterized roles in the organization of the chordate body plan. The intermediate mesoderm (IM), which gives rise to all vertebrate kidneys, is an example of a tissue that differentiates sequentially along this axis. Yet, the conservation of the spatiotemporal regulation of the IM across vertebrates remains poorly understood. In this study, we used a comparative developmental approach focusing on non-conventional model organisms, a chondrichthyan (catshark), a cyclostome (lamprey), and a cephalochordate (amphioxus), to assess the involvement of RA in the regulation of chordate and vertebrate pronephros formation. We report that the anterior expression boundary of early pronephric markers ( and ), positioned at the level of somite 6 in amniotes, is conserved in the catshark and the lamprey. Furthermore, RA, driving the expression of genes like in amniotes, regulates the anterior pronephros boundary in the catshark. We find no evidence for the involvement of this regulatory hierarchy in the AP positioning of the lamprey pronephros and the amphioxus pronephros homolog, Hatschek's nephridium. This suggests that despite the conservation of and expressions in chordate pronephros homologs, the responsiveness of the IM, and hence of pronephric genes, to RA- and -dependent regulation is a gnathostome novelty.
Topics: Animals; Chordata; Genes, Homeobox; Lampreys; Pronephros; Tretinoin; Vertebrates
PubMed: 35455988
DOI: 10.3390/cells11081304 -
Cells May 2020Automated high-throughput workflows allow for chemical toxicity testing and drug discovery in zebrafish disease models. Due to its conserved structural and functional...
Automated high-throughput workflows allow for chemical toxicity testing and drug discovery in zebrafish disease models. Due to its conserved structural and functional properties, the zebrafish pronephros offers a unique model to study renal development and disease at larger scale. Ideally, scoring of pronephric phenotypes includes morphological and functional assessments within the same larva. However, to efficiently upscale such assays, refinement of existing methods is required. Here, we describe the development of a multiparametric in vivo screening pipeline for parallel assessment of pronephric morphology, kidney function and heart rate within the same larva on a single imaging platform. To this end, we developed a novel 3D-printed orientation tool enabling multiple consistent orientations of larvae in agarose-filled microplates. Dorsal pronephros imaging was followed by assessing renal clearance and heart rates upon fluorescein isothiocyanate (FITC)-inulin microinjection using automated time-lapse imaging of laterally positioned larvae. The pipeline was benchmarked using a set of drugs known to induce developmental nephrotoxicity in humans and zebrafish. Drug-induced reductions in renal clearance and heart rate alterations were detected even in larvae exhibiting minor pronephric phenotypes. In conclusion, the developed workflow enables rapid and semi-automated in vivo assessment of multiple morphological and functional parameters.
Topics: Animals; Biological Assay; Embryo, Nonmammalian; Fluorescein-5-isothiocyanate; Heart Function Tests; Heart Rate; Kidney; Larva; Pronephros; Zebrafish
PubMed: 32443839
DOI: 10.3390/cells9051269 -
Biology Open Jun 2022Endocytosis mediates the cellular uptake of numerous molecules from the extracellular space and is a fundamentally important process. In the renal proximal tubule, the...
Endocytosis mediates the cellular uptake of numerous molecules from the extracellular space and is a fundamentally important process. In the renal proximal tubule, the scavenger receptor megalin and its co-receptor cubilin mediate endocytosis of low molecular weight proteins from the renal filtrate. However, the extent to which megalin endocytosis relies on different components of the trafficking machinery remains relatively poorly defined in vivo. In this study, we identify a functional requirement for the F-BAR protein pacsin2 in endocytosis in the renal proximal tubule of zebrafish larvae. Pacsin2 is expressed throughout development and in all zebrafish tissues, similar to the mammalian orthologue. Within renal tubular epithelial cells, pacsin2 is enriched at the apical pole where it is localised to endocytic structures. Loss of pacsin2 results in reduced endocytosis within the proximal tubule, which is accompanied by a reduction in the abundance of megalin and endocytic organelles. Our results indicate that pacsin2 is required for efficient endocytosis in the proximal tubule, where it likely cooperates with other trafficking machinery to maintain endocytic uptake and recycling of megalin.
Topics: Animals; Biological Transport; Endocytosis; Kidney Tubules, Proximal; Low Density Lipoprotein Receptor-Related Protein-2; Mammals; Zebrafish
PubMed: 35616009
DOI: 10.1242/bio.059150 -
Frontiers in Cell and Developmental... 2022Nephrotic syndrome (NS) is a disease characterized by proteinuria and subsequent hypoalbuminemia, hyperlipidemia and edema due to the defective renal glomerular...
Nephrotic syndrome (NS) is a disease characterized by proteinuria and subsequent hypoalbuminemia, hyperlipidemia and edema due to the defective renal glomerular filtration barrier (GFB). Mutations of NPHS1, encoding NEPHRIN, a podocyte protein essential for normal GFB, cause congenital nephrotic syndrome (CNS) of the Finnish type (CNF), which accounts for about 50% of CNS cases. We generated zebrafish nphs1 mutants by using CRISPR/Cas9. These mutants completely lack nephrin proteins in podocytes and develop progressive peri-orbital and whole-body edema after 5 days post fertilization. Ultra-structurally, loss of nephrin results in absence of slit-diaphragms and progressive foot process effacement in zebrafish pronephric glomeruli, similar to the pathological changes in human CNF patients. Interestingly, some nphs1 mutants are viable to adulthood despite ultra-structural defects in renal glomeruli. Using a reporter line Tg (l-fabp:VDBP-GFP) expressing GFP-tagged vitamin-D-binding protein in the blood plasma, we observed a reduction of intravascular GFP fluorescence in the nphs1 mutants, a hypoalbuminemia-like phenotype. In addition, we detected excretion of GFP by the nphs1 mutants, reminiscent of proteinuria. Therefore, we have demonstrated that the nphs1 mutant zebrafish recapitulate the human NS phenotypes and provide a novel and relevant animal model useful for screening therapeutical agents for this disease.
PubMed: 36187478
DOI: 10.3389/fcell.2022.976043 -
PloS One 2022E26 transformation specific (ETS) family transcription factors are expressed during embryogenesis and are involved in various cellular processes such as proliferation,...
E26 transformation specific (ETS) family transcription factors are expressed during embryogenesis and are involved in various cellular processes such as proliferation, migration, differentiation, angiogenesis, apoptosis, and survival of cellular lineages to ensure appropriate development. Dysregulated expression of many of the ETS family members is detected in different cancers. The human ELF3, a member of the ETS family of transcription factors, plays a role in the induction and progression of human cancers is well studied. However, little is known about the role of ELF3 in early development. Here, the zebrafish elf3 was cloned, and its expression was analyzed during zebrafish development. Zebrafish elf3 is maternally deposited. At different developmental stages, elf3 expression was detected in different tissue, mainly neural tissues, endoderm-derived tissues, cartilage, heart, pronephric duct, blood vessels, and notochord. The expression levels were high at the tissue boundaries. Elf3 loss-of-function consequences were examined by using translation blocking antisense morpholino oligonucleotides, and effects were validated using CRISPR/Cas9 knockdown. Elf3-knockdown produced short and bent larvae with notochord, craniofacial cartilage, and fin defects. The extracellular matrix (ECM) in the fin and notochord was disorganized. Neural defects were also observed. Optic nerve fasciculation (bundling) and arborization in the optic tectum were defective in Elf3-morphants, and fragmentation of spinal motor neurons were evident. Dysregulation of genes encoding ECM proteins and matrix metalloprotease (MMP) and disorganization of ECM may play a role in the observed defects in Elf3 morphants. We conclude that zebrafish Elf3 is required for epidermal, mesenchymal, and neural tissue development.
Topics: Animals; Humans; DNA-Binding Proteins; Extracellular Matrix; Gene Expression Regulation, Developmental; Morphogenesis; Proto-Oncogene Proteins c-ets; Transcription Factors; Zebrafish; Zebrafish Proteins
PubMed: 36383615
DOI: 10.1371/journal.pone.0276255