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Kidney International Oct 2022The zebrafish is an important animal system for modeling human diseases. This includes kidney dysfunction as the embryonic kidney (pronephros) shares considerable...
The zebrafish is an important animal system for modeling human diseases. This includes kidney dysfunction as the embryonic kidney (pronephros) shares considerable molecular and morphological homology with the human nephron. A key clinical indicator of kidney disease is proteinuria, but a high-throughput readout of proteinuria in the zebrafish is currently lacking. To remedy this, we used the Tol2 transposon system to generate a transgenic zebrafish line that uses the fabp10a liver-specific promoter to over-express a nanoluciferase molecule fused with the D3 domain of Receptor-Associated Protein (a type of molecular chaperone) which we term NL-D3. Using a luminometer, we quantified proteinuria in NL-D3 zebrafish larvae by measuring the intensity of luminescence in the embryo medium. In the healthy state, NL-D3 is not excreted, but when embryos were treated with chemicals that affected either proximal tubular reabsorption (cisplatin, gentamicin) or glomerular filtration (angiotensin II, Hanks Balanced Salt Solution, Bovine Serum Albumin), NL-D3 is detected in fish medium. Similarly, depletion of several gene products associated with kidney disease (nphs1, nphs2, lrp2a, ocrl, col4a3, and col4a4) also induced NL-D3 proteinuria. Treating col4a4 depleted zebrafish larvae (a model of Alport syndrome) with captopril reduced proteinuria in this system. Thus, our findings validate the use of the NL-D3 transgenic zebrafish as a robust and quantifiable proteinuria reporter. Hence, given the feasibility of high-throughput assays in zebrafish, this novel reporter will permit screening for drugs that ameliorate proteinuria, thereby prioritizing candidates for further translational studies.
Topics: Angiotensin II; Animals; Animals, Genetically Modified; Captopril; Cisplatin; Gentamicins; Humans; Kidney Glomerulus; Nephritis, Hereditary; Nephrotic Syndrome; Proteinuria; Serum Albumin, Bovine; Zebrafish
PubMed: 35716957
DOI: 10.1016/j.kint.2022.05.019 -
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 -
Frontiers in Immunology 2021The range of metabolic pathways that are dependent on a proper supply of specific amino acids (AA) unveils their importance in the support of health. AA play central...
The range of metabolic pathways that are dependent on a proper supply of specific amino acids (AA) unveils their importance in the support of health. AA play central roles in key pathways vital for immune support and individual AA supplementation has shown to be able to modulate fish immunity. trials are important tools to evaluate the immunomodulatory role of AA, and the present study was conceived to evaluate methionine and tryptophan roles in immune-related mechanisms aiming to understand their effects in leucocyte functioning and AA pathways. For that purpose, head-kidney leucocytes were isolated and a primary cell culture established. The effect of methionine or tryptophan surplus on cell viability was assessed. Medium L-15 10% FBS without AA addition (0.5mM of L-methionine, 0.1 mM of L-tryptophan) was used as control. To that, L-methionine or L-tryptophan were supplemented at 1 and 2 times (M1x or M2x, and T1x or T2x). Nitric oxide, ATP, total antioxidant capacity, and immune-related genes were evaluated in response to lipopolysaccharides extracted from subsp. or UV-inactivated bacteria). Moreover, caspase 3 activity and apoptosis-related genes were evaluated in response to the apoptosis-inducing protein, AIP56. Distinct roles in leucocytes' immune response were observed, with contrasting outcomes in the modulation of individual pathways. Methionine surplus improved cell viability, polyamine production, and methionine-related genes expression in response to an inflammatory agent. Also, methionine supplementation lowered signals of apoptosis by AIP56, presenting lower caspase 3 activity and higher and expression. Cells cultured in tryptophan supplemented medium presented signals of an attenuated inflammatory response, with decreased ATP and enhanced expression of anti-inflammatory and catabolism-related genes in macrophages. In response to AIP56, leucocytes cultured in a tryptophan-rich medium presented lower resilience to the toxin, higher caspase 3 activity and expression of caspase 8, and lower expression of several genes, including and . This study showed the ability of methionine surplus to improve leucocytes' response to an inflammatory agent and to lower signals of apoptosis by AIP56 induction, while tryptophan attenuated several cellular signals of the inflammatory response to UV-inactivated bacteria and lowered leucocyte resilience to AIP56.
Topics: Animals; Apoptosis; Bass; Cells, Cultured; Culture Media; Head Kidney; Immunity, Innate; Immunomodulation; Leukocytes; Lipopolysaccharides; Methionine; Photobacterium; Tryptophan
PubMed: 33790917
DOI: 10.3389/fimmu.2021.660448 -
Development (Cambridge, England) Dec 2020A functional vertebrate kidney relies on structural units called nephrons, which are epithelial tubules with a sequence of segments each expressing a distinct repertoire...
A functional vertebrate kidney relies on structural units called nephrons, which are epithelial tubules with a sequence of segments each expressing a distinct repertoire of solute transporters. The transcriptiona`l codes driving regional specification, solute transporter program activation and terminal differentiation of segment populations remain poorly understood. Here, we demonstrate that the KCTD15 paralogs and function in concert to restrict distal early (DE)/thick ascending limb (TAL) segment lineage assignment in the developing zebrafish pronephros by repressing Tfap2a activity. During renal ontogeny, expression of these factors colocalized with in distal tubule precursors. loss primed nephron cells to adopt distal fates by driving , and expression. These phenotypes were the result of Tfap2a hyperactivity, where -deficient embryos exhibited increased abundance of this transcription factor. Interestingly, reciprocally promoted and transcription, unveiling a circuit of autoregulation operating in nephron progenitors. Concomitant knockdown with overexpression further expanded the DE population. Our study reveals that a transcription factor-repressor feedback module employs tight regulation of Tfap2a and Kctd15 kinetics to control nephron segment fate choice and differentiation during kidney development.
Topics: Animals; Body Patterning; Cell Differentiation; Cell Lineage; Embryo, Nonmammalian; Embryonic Development; Epithelium; Gene Expression Regulation, Developmental; Kidney; Kidney Tubules; Nephrons; Organogenesis; Potassium Channels, Voltage-Gated; Signal Transduction; Solute Carrier Family 12, Member 1; Transcription Factor AP-2; Zebrafish; Zebrafish Proteins
PubMed: 33028614
DOI: 10.1242/dev.191973 -
Fish & Shellfish Immunology Apr 2022Cantharidin is a toxic vesicant terpene used in folk and traditional medicine due to its various therapeutic effects. Since there are no previous data on the effect of...
Cantharidin is a toxic vesicant terpene used in folk and traditional medicine due to its various therapeutic effects. Since there are no previous data on the effect of cantharidin in fish, this study aimed to investigate the in vitro related-inflammatory effects of cantharidin in gilthead seabream (Sparus aurata L.) head-kidney leucocytes (HKLs). In the first experiment, the HKLs were incubated with 0, 5 and 10 μg mL of cantharidin for 24 h to delimit its possible toxic effects. In a second experiment, leucocytes were incubated with ranging concentrations from 0 to 10 μg mL for 3, 6, or 12 h. Cell viability was higher in acidophilic granulocytes than in monocytes/macrophages and lymphocytes. Cantharidin caused apoptosis as was evidenced by transmission electron microscopy. In addition, cantharidin produced a time- and dose-dependent decrease of respiratory burst and phagocytic activities in HKLs, while their peroxidase activity was increased at 24 h of incubation with 5 and 10 μg mL of cantharidin. Different changes in the gene expression were observed after incubation with cantharidin. While the gene expression of tnfa, il1b and crel was up-regulated in HKLs, the nfkb1 and igmh genes were down-regulated in comparison to the expression found in control HKLs. Present results offer a first view of the possible effects and action mechanisms of cantharidin in HKLs, as well as its implication in the inflammatory process, which could be of interest not only for basic research but also in the aquaculture sector.
Topics: Animals; Cantharidin; Head Kidney; Kidney; Leukocytes; Sea Bream
PubMed: 35218974
DOI: 10.1016/j.fsi.2022.02.045 -
International Journal of Molecular... Jul 2022Acute kidney injury (AKI) is a common complication of severe human diseases, resulting in increased morbidity and mortality as well as unfavorable long-term outcomes....
Acute kidney injury (AKI) is a common complication of severe human diseases, resulting in increased morbidity and mortality as well as unfavorable long-term outcomes. Although the mammalian kidney is endowed with an amazing capacity to recover from AKI, little progress has been made in recent decades to facilitate recovery from AKI. To elucidate the early repair mechanisms after AKI, we employed the zebrafish pronephros injury model. Since damaged cells release large amounts of ATP and ATP-degradation products to signal apoptosis or necrosis to neighboring cells, we examined how depletion of purinergic and adenosine receptors impacts the directed cell migration that ensues immediately after a laser-induced tubular injury. We found that depletion of the zebrafish adenosine receptors , , , and significantly affected the repair process. Similar results were obtained after depletion of the purinergic receptor, which is highly expressed during zebrafish pronephros development. Released ATP is finally metabolized to inosine by adenosine deaminase. Depletion of zebrafish adenosine deaminases and interfered with the repair process; furthermore, combinations of and , or and displayed synergistic effects at low concentrations, supporting the involvement of inosine signaling in the repair process after a tubular injury. Our findings suggest that nucleotide-dependent signaling controls immediate migratory responses after tubular injury.
Topics: Acute Kidney Injury; Adenosine Triphosphate; Animals; Cell Movement; Humans; Inosine; Mammals; Nucleotides; Receptors, Purinergic P1; Receptors, Purinergic P2Y2; Zebrafish
PubMed: 35887219
DOI: 10.3390/ijms23147870 -
Frontiers in Immunology 2021Anti-disease breeding is becoming the most promising solution to cyprinid herpesvirus-3 (CyHV-3) infection, the major threat to common carp aquaculture. Virus...
Anti-disease breeding is becoming the most promising solution to cyprinid herpesvirus-3 (CyHV-3) infection, the major threat to common carp aquaculture. Virus challenging studies suggested that a breeding strain of common carp developed resistance to CyHV-3 infection. This study illustrates the immune mechanisms involved in both sensitivity and anti-virus ability for CyHV3 infection in fish. An integrative analysis of the protein-coding genes and long non-coding RNAs (lncRNAs) using transcriptomic data was performed. Tissues from the head kidney of common carp were extracted at days 0 (the healthy control) and 7 after CyHV-3 infection (the survivors) and used to analyze the transcriptome through both Illumina and PacBio sequencing. Following analysis of the GO terms and KEGG pathways involved, the immune-related terms and pathways were merged. To dig out details on the immune aspect, the DEGs were filtered using the current common carp immune gene library. Immune gene categories and their corresponding genes in different comparison groups were revealed. Also, the immunological Gene Ontology terms for lncRNA modulation were retained. The weighted gene co-expression network analysis was used to reveal the regulation of immune genes by lncRNA. The results demonstrated that the breeding carp strain develops a marked resistance to CyHV-3 infection through a specific innate immune mechanism. The featured biological processes were autophagy, phagocytosis, cytotoxicity, and virus blockage by lectins and MUC3. Moreover, the immune-suppressive signals, such as suppression of IL21R on STAT3, PI3K mediated inhibition of inflammation by dopamine upon infection, as well as the inhibition of NLRC3 on STING during a steady state. Possible susceptible factors for CyHV-3, such as ITGB1, TLR18, and CCL4, were also revealed from the non-breeding strain. The results of this study also suggested that Nramp and PAI regulated by LncRNA could facilitate virus infection and proliferation for infected cells respectively, while T cell leukemia homeobox 3 (TLX3), as well as galectin 3 function by lncRNA, may play a role in the resistance mechanism. Therefore, immune factors that are immunogenetically insensitive or susceptible to CyHV-3 infection have been revealed.
Topics: Animals; Carps; Disease Susceptibility; Fish Diseases; Gene Expression Profiling; Head Kidney; Herpesviridae; Herpesviridae Infections; High-Throughput Nucleotide Sequencing; Immunity, Innate
PubMed: 34290708
DOI: 10.3389/fimmu.2021.687151 -
Frontiers in Immunology 2021Teleost fish anterior kidney (AK) is an important hematopoietic organ with multifarious immune cells, which have immune functions comparable to mammalian bone marrow....
Teleost fish anterior kidney (AK) is an important hematopoietic organ with multifarious immune cells, which have immune functions comparable to mammalian bone marrow. Myeloid and lymphoid cells locate in the AK, but the lack of useful specific gene markers and antibody-based reagents for the cell subsets makes the identification of the different cell types difficult. Single-cell transcriptome sequencing enables single-cell capture and individual library construction, making the study on the immune cell heterogeneity of teleost fish AK possible. In this study, we examined the transcriptional patterns of 11,388 AK leukocytes using 10× Genomics single-cell RNA sequencing (scRNA-seq). A total of 22 clusters corresponding to five distinct immune cell subsets were identified, which included B cells, T cells, granulocytes, macrophages, and dendritic cells (DCs). However, the subsets of myeloid cells (granulocytes, macrophages, and DCs) were not identified in more detail according to the known specific markers, even though significant differences existed among the clusters. Thereafter, we highlighted the B-cell subsets and identified them as pro/pre B cells, immature/mature B cells, activated B/plasmablasts, or plasma cells based on the different expressions of the transcription factors (TFs) and cytokines. Clustering of the differentially modulated genes by pseudo-temporal trajectory analysis of the B-cell subsets showed the distinct kinetics of the responses of TFs to cell conversion. Moreover, we classified the T cells and discovered that CD3CD4CD8, CD3CD4CD8, CD4CD8, and CD4CD8 T cells existed in AK, but neither CD4CD8 nor CD4CD8 T cells can be further classified into subsets based on the known TFs and cytokines. Pseudotemporal analysis demonstrated that CD4CD8 and CD4CD8 T cells belonged to different states with various TFs that might control their differentiation. The data obtained above provide a valuable and detailed resource for uncovering the leukocyte subsets in Nile tilapia AK, as well as more potential markers for identifying the myeloid and lymphoid cell types.
Topics: Animals; Cichlids; Head Kidney; Immunophenotyping; Leukocytes; RNA-Seq; Single-Cell Analysis
PubMed: 35027916
DOI: 10.3389/fimmu.2021.783196 -
Developmental Biology Jan 2021Ciliopathies affect a variety of tissues during development including the heart, kidneys, respiratory tract, and retina. Though an increasing number of monogenic causes...
Ciliopathies affect a variety of tissues during development including the heart, kidneys, respiratory tract, and retina. Though an increasing number of monogenic causes of ciliopathies have been described, many remain unexplained. Recently, recessive variants in NUP93 and NUP205 encoding two proteins of the inner ring of the nuclear pore complex were implicated as causes of steroid resistant nephrotic syndrome. In addition, we previously found that the inner ring nucleoporins NUP93 and NUP188 function in proper left-right patterning in developing embryos via a role at the cilium. Here, we describe the role of an additional inner ring nucleoporin NUP205 in cilia biology and establishment of normal organ situs. Using knockdown in Xenopus, we show that Nup205 depletion results in loss of cilia and abnormal cardiac morphology. Furthermore, by transmission electron microscopy, we observe a loss of cilia and mispositioning of intracellular ciliary structures such as basal bodies and rootlets upon depleting inner ring nucleoporins. We describe a model wherein NUP93 interacting with either NUP188 or NUP205 is necessary for cilia. We thus provide evidence that dysregulation of inner ring nucleoporin genes that have been identified in patients may contribute to pathogenesis through cilia dysfunction.
Topics: Animals; Body Patterning; Cilia; Epidermis; Gene Knockdown Techniques; Heart Defects, Congenital; Humans; Nuclear Pore Complex Proteins; Pronephros; Xenopus; Xenopus Proteins
PubMed: 33065118
DOI: 10.1016/j.ydbio.2020.10.001 -
Kidney International Jun 2020Epithelial cells lining the proximal tubule of the kidney reabsorb and metabolize most of the filtered low-molecular-weight proteins through receptor-mediated...
Epithelial cells lining the proximal tubule of the kidney reabsorb and metabolize most of the filtered low-molecular-weight proteins through receptor-mediated endocytosis and lysosomal processing. Congenital and acquired dysfunctions of the proximal tubule are consistently reflected by the inappropriate loss of solutes including low-molecular-weight proteins in the urine. The zebrafish pronephros shares individual functional segments with the human nephron, including lrp2a/megalin-dependent endocytic transport processes of the proximal tubule. Although the zebrafish has been used as a model organism for toxicological studies and drug discovery, there is no available assay that allows large-scale assessment of proximal tubule function in larval or adult stages. Here we establish a transgenic Tg(lfabp::½vdbp-mCherry) zebrafish line expressing in the liver the N-terminal region of vitamin D-binding protein coupled to the acid-insensitive, red monomeric fluorescent protein mCherry (½vdbp-mCherry). This low-molecular-weight protein construct is secreted into the bloodstream, filtered through the glomerulus, reabsorbed by receptor-mediated endocytosis and processed in the lysosomes of proximal tubule cells of the fish. Thus, our proof-of-concept studies using zebrafish larvae knockout for lrp2a and clcn7 or exposed to known nephrotoxins (gentamicin and cisplatin) demonstrate that this transgenic line is useful to monitor low-molecular-weight proteinuria and lysosomal processing. This represents a powerful new model organism for drug screening and studies of nephrotoxicity.
Topics: Animals; Animals, Genetically Modified; Endocytosis; Humans; Kidney Tubules, Proximal; Low Density Lipoprotein Receptor-Related Protein-2; Lysosomal Storage Diseases; Proteinuria; Zebrafish
PubMed: 32061435
DOI: 10.1016/j.kint.2019.11.016