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Scientific Reports Nov 2023Messenger RNA (mRNA) therapies are emerging in different disease areas, but have not yet reached the kidney field. Our aim was to study the feasibility to treat the...
Messenger RNA (mRNA) therapies are emerging in different disease areas, but have not yet reached the kidney field. Our aim was to study the feasibility to treat the genetic defect in cystinosis using synthetic mRNA in cell models and ctns zebrafish embryos. Cystinosis is a prototype lysosomal storage disorder caused by mutations in the CTNS gene, encoding the lysosomal cystine-H symporter cystinosin, and leading to cystine accumulation in all cells of the body. The kidneys are the first and the most severely affected organs, presenting glomerular and proximal tubular dysfunction, progressing to end-stage kidney failure. The current therapeutic standard cysteamine, reduces cystine levels, but has many side effects and does not restore kidney function. Here, we show that synthetic mRNA can restore lysosomal cystinosin expression following lipofection into CTNS kidney cells and injection into ctns zebrafish. A single CTNS mRNA administration decreases cellular cystine accumulation for up to 14 days in vitro. In the ctns zebrafish, CTNS mRNA therapy improves proximal tubular reabsorption, reduces proteinuria, and restores brush border expression of the multi-ligand receptor megalin. Therefore, this proof-of-principle study takes the first steps in establishing an mRNA-based therapy to restore cystinosin expression, resulting in cystine reduction in vitro and in the ctns larvae, and restoration of the zebrafish pronephros function.
Topics: Animals; Cystinosis; Cystine; Zebrafish; RNA, Messenger; Models, Theoretical; Dietary Supplements; Amino Acid Transport Systems, Neutral
PubMed: 38016974
DOI: 10.1038/s41598-023-47085-w -
Journal of Developmental Biology Oct 2022The Wilms' tumor suppressor gene, , encodes a zinc finger-containing transcription factor that binds to a GC-rich motif and regulates the transcription of target genes....
The Wilms' tumor suppressor gene, , encodes a zinc finger-containing transcription factor that binds to a GC-rich motif and regulates the transcription of target genes. was first identified as a tumor suppressor gene in Wilms' tumor, a pediatric kidney tumor, and has been implicated in normal kidney development. The WT1 protein has transcriptional activation and repression domains and acts as a transcriptional activator or repressor, depending on the target gene and context. In , an ortholog of has been isolated and shown to be expressed in the developing embryonic pronephros. To investigate the role of in pronephros development in embryos, we mutated by CRISPR/Cas9 and found that the expression of pronephros marker genes was reduced. In reporter assays in which known WT1 binding sequences were placed upstream of the gene, WT1 activated transcription of the gene. The injection of wild-type or artificially altered transcriptional activity of mRNA disrupted the expression of pronephros marker genes in the embryos. These results suggest that the appropriate amounts and activity of WT1 protein are required for normal pronephros development in embryos.
PubMed: 36412640
DOI: 10.3390/jdb10040046 -
Scientific Reports Jul 2022Thiosulfate sulfurtransferase (TST, EC 2.8.1.1), also known as Rhodanese, was initially discovered as a cyanide detoxification enzyme. However, it was recently also...
Thiosulfate sulfurtransferase (TST, EC 2.8.1.1), also known as Rhodanese, was initially discovered as a cyanide detoxification enzyme. However, it was recently also found to be a genetic predictor of resistance to obesity-related type 2 diabetes. Diabetes type 2 is characterized by progressive loss of adequate β-cell insulin secretion and onset of insulin resistance with increased insulin demand, which contributes to the development of hyperglycemia. Diabetic complications have been replicated in adult hyperglycemic zebrafish, including retinopathy, nephropathy, impaired wound healing, metabolic memory, and sensory axonal degeneration. Pancreatic and duodenal homeobox 1 (Pdx1) is a key component in pancreas development and mature beta cell function and survival. Pdx1 knockdown or knockout in zebrafish induces hyperglycemia and is accompanied by organ alterations similar to clinical diabetic retinopathy and diabetic nephropathy. Here we show that pdx1-knockdown zebrafish embryos and larvae survived after incubation with thiosulfate and no obvious morphological alterations were observed. Importantly, incubation with hTST and thiosulfate rescued the hyperglycemic phenotype in pdx1-knockdown zebrafish pronephros. Activation of the mitochondrial TST pathway might be a promising option for therapeutic intervention in diabetes and its organ complications.
Topics: Animals; Diabetes Mellitus, Type 2; Hyperglycemia; Models, Theoretical; Pronephros; Thiosulfate Sulfurtransferase; Thiosulfates; Zebrafish
PubMed: 35840638
DOI: 10.1038/s41598-022-16320-1 -
Frontiers in Immunology 2021In mammals, Interleukin-17 cytokine family plays critical roles in both acute and chronic inflammatory responses. In fish species, three Interleukin-17A/F (IL-17A/F)...
In mammals, Interleukin-17 cytokine family plays critical roles in both acute and chronic inflammatory responses. In fish species, three Interleukin-17A/F (IL-17A/F) genes have been identified to be homologous to mammalian IL-17A and IL-17F, but little is known about their functional activity. In this study, _IL-17A/F1, 2 and 3 genes were cloned from yellow catfish () and they differed in protein structure and exon length, implying that they may have divergent bioactivity. Real-time quantitative PCR analyses revealed that three _IL-17A/F genes were highly expressed in blood and mucosal tissues (skin+mucus and gill) from healthy adult fish. The mRNA expressions of _IL-17A/F1, 2 and 3 genes were significantly up-regulated in the gill, skin+mucus, head kidney and spleen after challenge with and in the isolated peripheral blood leucocytes (PBLs) of yellow catfish after stimulation with phytohaemagglutinin (PHA), lipopolysaccharides (LPS), peptidoglycan (PGN) and polyinosinic-polycytidylic acid (Poly I:C). These results indicate that _IL-17A/F1, 2 and 3 genes may play a vital role in the regulation of immune against pathogens. Additionally, the recombinant (r) _IL-17A/F1, 2 and 3 proteins significantly induced the mRNA expressions of proinflammatory cytokines, chemokines and antibacterial peptides genes, and the r_IL-17A/F 2 and 3 proteins promoted phagocytosis of PBLs more powerfully than the r_IL-17A/F1. Furthermore, the r_IL-17A/F1, 2 and 3 proteins might activate the NF-κB and MAPK signal pathways by IL-17RA, ACT1, TRAF6, TRAF2, TRAF5 and TAK1, indicating that the three _IL-17A/F proteins may play different roles in promoting inflammatory response.
Topics: Animals; Catfishes; Fish Proteins; Head Kidney; Interleukin-17; Leukocytes; Lipopolysaccharides; Peptidoglycan; Phytohemagglutinins; Poly I-C; Spleen
PubMed: 34267744
DOI: 10.3389/fimmu.2021.626895 -
Developmental and Comparative Immunology Feb 2021Interleukin (IL) -2, a member of the four α-helical cytokine family, has broad regulatory roles in mediating vertebrate immune response. In mammals, IL-2 and IL-15...
Interleukin (IL) -2, a member of the four α-helical cytokine family, has broad regulatory roles in mediating vertebrate immune response. In mammals, IL-2 and IL-15 share a common evolutionary origin and possess overlapping but distinct functions. IL-2 and IL-15 bind to distinct private receptors for signaling. However, fish appear to possess a single IL-15Rα like gene whilst lack additional gene(s) coding for IL-2Rα. Whether the IL-2 and IL-15 interact with the same receptor in fish and how their functions and receptors have evolved are not fully understood. In this study, homologues of IL-2 and IL-2/15Rα were sequenced from a teleost species, grass carp (Ctenopharyngodon idella), and the crystal structure of IL-2 was determined. The grass carp IL-2 (termed CiIL-2) displayed a classical cytokine structure consisting of four helical bundles which shares significant similarity with human IL-15. The key amino acids involved in the interface interaction of IL-2/15 and their receptors are well conserved. The CiIL-2 has been shown to bind the IL-2/15Rα like homologue with an affinity of 2.45 nM, supporting the notion that fish IL-2 and IL-15 may share a single common private receptor for exerting functions. Syntenic analysis suggests that the IL-2Rα of tetrapods has evolved from an IL-15Rα like homologue, in which a second sushi domain (D2) in the extracellular region has been duplicated to facilitate the specific interaction with IL-2. The CiIL-2 was predominantly expressed in lymphocyte-rich tissues such as the spleen, kidney and thymus, and could be induced by PHA and IL-21. In vivo challenge with grass carp reovirus and Flavobacterium columnare also resulted in upregulation of CiIL-2 expression. The recombinant CiIL-2 was shown to activate expression of STAT5b, IL-1β, IL-22 and IFN-γ, and to promote the proliferation of the primary cell cultures from head kidney leucocytes. Our results shed lights into the co-evolution of IL-2 and its private receptor, and the functional divergence of IL-2 and IL-15 during evolution.
Topics: Animals; Carps; Cells, Cultured; Fish Diseases; Fish Proteins; Flavobacterium; Head Kidney; Interleukin-15; Interleukin-15 Receptor alpha Subunit; Interleukin-2; Leukocytes; Primary Cell Culture
PubMed: 33065202
DOI: 10.1016/j.dci.2020.103895 -
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 -
Pathogens & Immunity 2022Uropathogenic (UPEC) infections are common and when they disseminate can be of high morbidity.
BACKGROUND
Uropathogenic (UPEC) infections are common and when they disseminate can be of high morbidity.
METHODS
We studied the effects of UPEC infection using single cell RNA sequencing (scRNAseq) in zebrafish. Bulk RNA sequencing has historically been used to evaluate gene expression patterns, but scRNAseq allows gene expression to be evaluated at the single cell level and is optimal for evaluating heterogeneity within cell types and rare cell types. Zebrafish cohorts were injected with either saline or UPEC, and scRNAseq and canonical pathway analyses were performed.
RESULTS
Canonical pathway analysis of scRNAseq data provided key information regarding innate immune pathways in the cells determined to be thymus cells, ionocytes, macrophages/monocytes, and pronephros cells. Pathways activated in thymus cells included interleukin 6 (IL-6) signaling and production of reactive oxygen species. Fc receptor-mediated phagocytosis was a leading canonical pathway in the pronephros and macrophages. Genes that were downregulated in UPEC vs saline exposed embryos involved the cellular response to the Gram-negative endotoxin lipopolysaccharide (LPS) and included Forkhead Box O1a , Tribbles Pseudokinase 3 (, Arginase 2 ( and Polo Like Kinase 3 (
CONCLUSIONS
Because 4-day post fertilization zebrafish embryos only have innate immune systems, the scRNAseq provides insights into pathways and genes that cell types utilize in the bacterial response. Based on our analysis, we have identified genes and pathways that might serve as genetic targets for treatment and further investigation in UPEC infections at the single cell level.
PubMed: 35178490
DOI: 10.20411/pai.v7i1.479 -
IScience Dec 2020Regulation of glucose homeostasis is a fundamental process to maintain blood glucose at a physiological level, and its dysregulation is associated with the development...
Regulation of glucose homeostasis is a fundamental process to maintain blood glucose at a physiological level, and its dysregulation is associated with the development of several metabolic diseases. Here, we report on a zebrafish mutant for Aldo-keto-reductase 1a1b () as a regulator of gluconeogenesis. Adult mutant zebrafish developed fasting hypoglycemia, which was caused by inhibiting phosphoenolpyruvate carboxykinase (PEPCK) expression as rate-limiting enzyme of gluconeogenesis. Subsequently, glucogenic amino acid glutamate as substrate for gluconeogenesis accumulated in the kidneys, but not in livers, and induced structural and functional pronephros alterations in 48-hpf embryos. mutants displayed increased nitrosative stress as indicated by increased nitrotyrosine, and increased protein-S-nitrosylation. Inhibition of nitrosative stress using the NO synthase inhibitor L-NAME prevented kidney damage and normalized PEPCK expression in mutants. Thus, the data have identified Akr1a1b as a regulator of gluconeogenesis in zebrafish and thereby controlling glucose homeostasis.
PubMed: 33251496
DOI: 10.1016/j.isci.2020.101763 -
Developmental and Comparative Immunology Sep 2021CC chemokine ligand 19 (CCL19) plays a key role in the regulation of immune responses including homeostasis, inflammation, and immune tolerance. In this study, two...
CC chemokine ligand 19 (CCL19) plays a key role in the regulation of immune responses including homeostasis, inflammation, and immune tolerance. In this study, two variants of CCL19 homologues (CCL19a2 and CCL19b) and CCR7 were investigated in grass carp Ctenopharyngodon idella. The three genes were widely expressed in immune tissues and could be modulated by stimulation with LPS, PHA and poly(I:C), and infection with Flavobacterium columnare and grass carp reovirus. In an in vitro chemotaxis assay, the recombinant CCL19a2 and CCL19b were active to promote the migration of HEK293 T cells expressing CCR7 and leucocytes isolated from the gills, head kidney and spleen. Moreover, their chemotactive effects were validated in vivo. We found that the cells recruited by CCL19a2 and CCl19b are mainly monocytes/macrophages expressing high levels of IL-1β, IFN-γ, colony stimulating factor 1 receptor (CSF1R) and MHC II. Our work suggests that CCL19a2 and CCl19b are involved in recruitment of antigen presenting cells in fish.
Topics: Animals; Antigen Presentation; Base Sequence; Carps; Cell Line; Cell Movement; Chemokine CCL19; Fish Diseases; Flavobacterium; Gills; HEK293 Cells; Head Kidney; Humans; Interferon-gamma; Interleukin-1beta; Leukocytes; Lipopolysaccharides; Macrophages; Monocytes; Phytohemagglutinins; Poly I-C; Receptors, CCR7; Receptors, Granulocyte-Macrophage Colony-Stimulating Factor; Reoviridae; Sequence Analysis, DNA; Spleen
PubMed: 33965447
DOI: 10.1016/j.dci.2021.104127 -
Development & Reproduction Sep 2023The Ruvb-like AAA ATPase1 (Ruvbl1; also known as Pontin) is an evolutionary conserved protein belonging to the adenosine triphosphates associated with diverse cellular...
The Ruvb-like AAA ATPase1 (Ruvbl1; also known as Pontin) is an evolutionary conserved protein belonging to the adenosine triphosphates associated with diverse cellular activities (AAA+) superfamily of ATPases. Ruvbl1 is a component of various protein supercomplexes and is involved in a variety of cellular activities, including chromatin remodeling, DNA damage repair, and mitotic spindle assembly however, the developmental significance of this protein is unknown and needs detailed investigation. We investigated the developmental significance of Ruvbl1 in multiciliated cells of the epidermis since is expressed in the multiciliated cells and pronephros during embryogenesis. The knockdown of significantly impaired cilia-driven fluid flow and basal body polarity in the epidermis compared to control embryos, but did not affect cilia morphology. Our results suggest that Ruvbl1 plays a significant role in embryonic development by regulating ciliary beating; however, further investigation is needed to determine the mechanisms involved.
PubMed: 38074458
DOI: 10.12717/DR.2023.27.3.159