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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 -
Kidney International Jan 2023The kidney is an essential organ that ensures bodily fluid homeostasis and removes soluble waste products from the organism. Nephrons, the functional units of the...
The kidney is an essential organ that ensures bodily fluid homeostasis and removes soluble waste products from the organism. Nephrons, the functional units of the kidney, comprise a blood filter, the glomerulus or glomus, and an epithelial tubule that processes the filtrate from the blood or coelom and selectively reabsorbs solutes, such as sugars, proteins, ions, and water, leaving waste products to be eliminated in the urine. Genes coding for transporters are segmentally expressed, enabling the nephron to sequentially process the filtrate. The Xenopus embryonic kidney, the pronephros, which consists of a single large nephron, has served as a valuable model to identify genes involved in nephron formation and patterning. Therefore, the developmental patterning program that generates these segments is of great interest. Prior work has defined the gene expression profiles of Xenopus nephron segments via in situ hybridization strategies, but a comprehensive understanding of the cellular makeup of the pronephric kidney remains incomplete. Here, we carried out single-cell mRNA sequencing of the functional Xenopus pronephric nephron and evaluated its cellular composition through comparative analyses with previous Xenopus studies and single-cell mRNA sequencing of the adult mouse kidney. This study reconstructs the cellular makeup of the pronephric kidney and identifies conserved cells, segments, and associated gene expression profiles. Thus, our data highlight significant conservation in podocytes, proximal and distal tubule cells, and divergence in cellular composition underlying the capacity of each nephron to remove wastes in the form of urine, while emphasizing the Xenopus pronephros as a model for physiology and disease.
Topics: Animals; Mice; Gene Expression Regulation, Developmental; Kidney; Kidney Glomerulus; Nephrons; RNA, Messenger; Xenopus laevis
PubMed: 36055600
DOI: 10.1016/j.kint.2022.07.027 -
Aquatic Toxicology (Amsterdam,... Oct 2022How local groundwater induces chronic kidney disease of unknown etiology (CKDu) in Sri Lanka is still elusive. This study aims to elucidate the impacts of Sri Lanka's...
How local groundwater induces chronic kidney disease of unknown etiology (CKDu) in Sri Lanka is still elusive. This study aims to elucidate the impacts of Sri Lanka's local groundwater in a CKDu prevalent area and reveal the possible pathogenic mechanism of CKDu using zebrafish models. The drinking water from the local underground well in Vavuniya was sampled and the water quality parameters including Na, Mg, K, Ca, Cl, NO, SO, and F were analyzed. Then, local groundwater exposure to zebrafish larvae and 293T cells was performed, and water with high hardness and fluoride was prepared as parallel groups. Our result showed that exposure to Sri Lanka's local groundwater caused developmental toxicity, kidney damage, and pronephric duct obstruction as well as abnormal behavior in zebrafish. Similar results were also found after exposure to water with high hardness and fluoride in zebrafish. Further, the expression levels of marker genes related to renal development and functions (foxj1a, dync2h1, pkd2, gata3, and slc20a1) were significantly altered, which is also confirmed in the 293T cells. Taken together, those results indicated that Sri Lanka's local groundwater in a CKDu prevalent area could cause kidney damage, implying that high water hardness and fluorine might be the inducible environmental factors for the etiological cause of CKDu.
Topics: Animals; Drinking Water; Fluorides; Fluorine; Groundwater; Kidney; Renal Insufficiency, Chronic; Sri Lanka; Water Pollutants, Chemical; Zebrafish
PubMed: 36041360
DOI: 10.1016/j.aquatox.2022.106276 -
Nature Biotechnology Feb 2023Directed differentiation of human pluripotent stem cells (hPSCs) into functional ureteric and collecting duct (CD) epithelia is essential to kidney regenerative...
Directed differentiation of human pluripotent stem cells (hPSCs) into functional ureteric and collecting duct (CD) epithelia is essential to kidney regenerative medicine. Here we describe highly efficient, serum-free differentiation of hPSCs into ureteric bud (UB) organoids and functional CD cells. The hPSCs are first induced into pronephric progenitor cells at 90% efficiency and then aggregated into spheres with a molecular signature similar to the nephric duct. In a three-dimensional matrix, the spheres form UB organoids that exhibit branching morphogenesis similar to the fetal UB and correct distal tip localization of RET expression. Organoid-derived cells incorporate into the UB tips of the progenitor niche in chimeric fetal kidney explant culture. At later stages, the UB organoids differentiate into CD organoids, which contain >95% CD cell types as estimated by single-cell RNA sequencing. The CD epithelia demonstrate renal electrophysiologic functions, with ENaC-mediated vectorial sodium transport by principal cells and V-type ATPase proton pump activity by FOXI1-induced intercalated cells.
Topics: Humans; Kidney; Ureter; Cell Differentiation; Organoids; Pluripotent Stem Cells; Morphogenesis; Forkhead Transcription Factors
PubMed: 36038632
DOI: 10.1038/s41587-022-01429-5 -
Comparative Biochemistry and... Oct 2022Crude oil is known to induce developmental defects in teleost fish exposed during early-life stages (ELSs). A recent study has demonstrated that zebrafish (Danio rerio)...
Crude oil is known to induce developmental defects in teleost fish exposed during early-life stages (ELSs). A recent study has demonstrated that zebrafish (Danio rerio) larvae acutely exposed to Deepwater Horizon (DHW) crude oil showed transcriptional changes in key genes involved in early kidney (pronephros) development and function, which were coupled with pronephric morphological defects. Given the osmoregulatory importance of the kidney, it is unknown whether ELS effects arising from short-term crude exposures result in long-term osmoregulatory defects, particularly within estuarine fishes likely exposed to DWH oil following the spill. To address this knowledge gap, an acute 72 h exposure to red drum (Sciaenops ocellatus) larvae was performed using high-energy water-accommodated fractions (HEWAFs) of DWH weathered oil to analyze transcriptional changes in genes involved in pronephros development and function by quantitative PCR. To test the latent effects of oil exposure on osmoregulation ability, red drum larvae were first exposed to HEWAF for 24 h. Larvae were then reared in clean seawater for two weeks and a 96 h acute osmotic challenge test was performed by exposing the fish to waters with varying salinities. Latent effects of ELS crude oil exposure on osmoregulation were assessed by quantifying survival during the acute osmotic challenge test and analyzing transcriptional changes at 14 dpf. Results demonstrated that ELS crude oil exposure reduced survival of red drum larvae when challenged in hypoosmotic waters and that latent transcriptional changes in some target pronephric genes were evident, indicating that an affected kidney likely contributed to the increased mortality.
Topics: Animals; Larva; Osmoregulation; Perciformes; Petroleum; Petroleum Pollution; Polycyclic Aromatic Hydrocarbons; Water Pollutants, Chemical; Zebrafish
PubMed: 35811062
DOI: 10.1016/j.cbpc.2022.109405 -
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 Genetics 2022The (Solute carrier family 12 member 3) gene encodes a sodium-chloride cotransporter and mediates Na and Cl reabsorption in the distal convoluted tubule of kidneys. An... (Review)
Review
The (Solute carrier family 12 member 3) gene encodes a sodium-chloride cotransporter and mediates Na and Cl reabsorption in the distal convoluted tubule of kidneys. An experimental study has previously showed that with knockdown of zebrafish ortholog, slc12a3 led to structural abnormality of kidney pronephric distal duct at 1-cell stage, suggesting that may have genetic effects in renal disorders. Many clinical reports have demonstrated that the function-loss mutations in the gene, mainly including Thr60Met, Asp486Asn, Gly741Arg, Leu859Pro, Arg861Cys, Arg913Gln, Arg928Cys and Cys994Tyr, play the pathogenic effects in Gitelman syndrome. This kidney disease is inherited as an autosomal recessive trait. In addition, several population genetic association studies have indicated that the single nucleotide variant Arg913Gln in the gene is associated with diabetic kidney disease in type 2 diabetes subjects. In this review, we first summarized bioinformatics of the gene and its genetic variation. We then described the different genetic and biological effects of in Gitelman syndrome and diabetic kidney disease. We also discussed about further genetic and biological analyses of as pharmacokinetic targets of diuretics.
PubMed: 35591852
DOI: 10.3389/fgene.2022.799224 -
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 -
Developmental Dynamics : An Official... Oct 2022MLLT3 (AF9) is a nuclear transcription factor crucial for hematopoietic stem cell and progenitor cell maintenance, but its role during embryonic hematopoiesis remains...
BACKGROUND
MLLT3 (AF9) is a nuclear transcription factor crucial for hematopoietic stem cell and progenitor cell maintenance, but its role during embryonic hematopoiesis remains uncertain. Here, we examine the role of mllt3 in developmental hematopoiesis during embryogenesis using zebrafish.
RESULTS
Cloning, sequencing, phylogenetic, and synteny analyses showed high evolutionary conservation between important functional domains of the zebrafish orthologue of mllt3 and MLLT3 in humans. Quantitative reverse transcription-PCR and in situ hybridization analyses revealed that mllt3 is maternally supplied and zygotically expressed throughout embryonic development, and that expression is highest between 10 and 24 hours post-fertilization (hpf) coincident with enrichment in the intermediate cell mass (ICM) and posterior blood island, which are the sites of the primitive and transient definitive hematopoiesis in zebrafish, respectively. Further, we found co-expression of mllt3 with the early hematopoietic progenitor markers tal1, gata2, and gata1a in the posterior ICM. By investigating zebrafish hematopoietic mutants, we discovered that mllt3 is involved in erythroid precursor formation. By 48-72 hpf, mllt3 expression proved to be restricted to non-hematopoietic tissues including head structures, pronephric tubules, and liver primordium.
CONCLUSIONS
These findings establish a link between mllt3 and primitive erythropoiesis and provide the basis for future functional investigations.
Topics: Animals; Embryonic Development; Gene Expression Regulation, Developmental; Leukemia; Nuclear Proteins; Phylogeny; Transcription Factors; Zebrafish
PubMed: 35429189
DOI: 10.1002/dvdy.477 -
Cell Discovery Mar 2022Primary cilia are antenna-like subcellular structures to act as signaling platforms to regulate many cellular processes and embryonic development. mA RNA modification...
Primary cilia are antenna-like subcellular structures to act as signaling platforms to regulate many cellular processes and embryonic development. mA RNA modification plays key roles in RNA metabolism and gene expression; however, the physiological function of mA modification remains largely unknown. Here we find that the mA demethylase ALKBH3 significantly inhibits ciliogenesis in mammalian cells by its demethylation activity. Mechanistically, ALKBH3 removes mA sites on mRNA of Aurora A, a master suppressor of ciliogenesis. Depletion of ALKBH3 enhances Aurora A mRNA decay and inhibits its translation. Moreover, alkbh3 morphants exhibit ciliary defects, including curved body, pericardial edema, abnormal otoliths, and dilation in pronephric ducts in zebrafish embryos, which are significantly rescued by wild-type alkbh3, but not by its catalytically inactive mutant. The ciliary defects caused by ALKBH3 depletion in both vertebrate cells and embryos are also significantly reversed by ectopic expression of Aurora A mRNA. Together, our data indicate that ALKBH3-dependent mA demethylation has a crucial role in the regulation of Aurora A mRNA, which is essential for ciliogenesis and cilia-associated developmental events in vertebrates.
PubMed: 35277482
DOI: 10.1038/s41421-022-00385-3