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We performed long-read transcriptome and proteome profiling of pathogen-stimulated peripheral blood mononuclear cells (PBMCs) from healthy donors to discover new transcript and protein isoforms expressed during immune responses to diverse pathogens. Long-read transcriptome profiling reveals novel sequences and isoform switching induced upon pathogen stimulation, including transcripts that are difficult to detect using traditional short-read sequencing. Widespread loss of intron retention occurs as a common result of all pathogen stimulations. We highlight novel transcripts of NFKB1 and CASP1 that may indicate novel immunological mechanisms. RNA expression differences did not result in differences in the amounts of secreted proteins. Clustering analysis of secreted proteins revealed a correlation between chemokine (receptor) expression on the RNA and protein levels in C. albicans- and poly(I:C)-stimulated PBMCs. Isoform aware long-read sequencing of pathogen-stimulated immune cells highlights the potential of these methods to identify novel transcripts, revealing a more complex transcriptome landscape than previously appreciated.
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Mutations in proteasome ß-subunits or their chaperone and regulatory proteins are associated with proteasome-associated autoinflammatory disorders (PRAAS). We studied six unrelated infants with three de novo heterozygous missense variants in PSMB10, encoding the proteasome ß2i-subunit. Individuals presented with T-B-NK± severe combined immunodeficiency (SCID) and clinical features suggestive of Omenn syndrome, including diarrhea, alopecia, and desquamating erythematous rash. Remaining T cells had limited T cell receptor repertoires, a skewed memory phenotype, and an elevated CD4/CD8 ratio. Bone marrow examination indicated severely impaired B cell maturation with limited V(D)J recombination. All infants received an allogeneic stem cell transplant and exhibited a variety of severe inflammatory complications thereafter, with 2 peri-transplant and 2 delayed deaths. The single long-term transplant survivor showed evidence for genetic rescue through revertant mosaicism overlapping the affected PSMB10 locus. The identified variants (c.166G>C [p.Asp56His] and c.601G>A/c.601G>C [p.Gly201Arg]) were predicted in silico to profoundly disrupt 20S immunoproteasome structure through impaired ß-ring/ß-ring interaction. Our identification of PSMB10 mutations as a cause of SCID-Omenn syndrome reinforces the connection between PRAAS-related diseases and SCID.
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Inmunodeficiencia Combinada Grave , Lactante , Humanos , Inmunodeficiencia Combinada Grave/genética , Inmunodeficiencia Combinada Grave/metabolismo , Complejo de la Endopetidasa Proteasomal/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Mutación/genética , Linfocitos T/metabolismo , Mutación Missense/genéticaRESUMEN
Our incomplete knowledge of the human transcriptome impairs the detection of disease-causing variants, in particular if they affect transcripts only expressed under certain conditions. These transcripts are often lacking from reference transcript sets, such as Ensembl/GENCODE and RefSeq, and could be relevant for establishing genetic diagnoses. We present SUsPECT (Solving Unsolved Patient Exomes/gEnomes using Custom Transcriptomes), a pipeline based on the Ensembl Variant Effect Predictor (VEP) to predict variant impact on custom transcript sets, such as those generated by long-read RNA-sequencing, for downstream prioritization. Our pipeline predicts the functional consequence and likely deleteriousness scores for missense variants in the context of novel open reading frames predicted from any transcriptome. We demonstrate the utility of SUsPECT by uncovering potential mutational mechanisms of pathogenic variants in ClinVar that are not predicted to be pathogenic using the reference transcript annotation. In further support of SUsPECT's utility, we identified an enrichment of immune-related variants predicted to have a more severe molecular consequence when annotating with a newly generated transcriptome from stimulated immune cells instead of the reference transcriptome. Our pipeline outputs crucial information for further prioritization of potentially disease-causing variants for any disease and will become increasingly useful as more long-read RNA sequencing datasets become available.
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Programas Informáticos , Transcriptoma , Humanos , Anotación de Secuencia Molecular , Análisis de Secuencia de ARN/métodos , Exoma , Secuenciación de Nucleótidos de Alto RendimientoRESUMEN
Background: De novo variants (DNVs) are currently not routinely evaluated as part of diagnostic whole exome sequencing (WES) analysis in patients with suspected inborn errors of immunity (IEI). Methods: This study explored the potential added value of systematic assessment of DNVs in a retrospective cohort of 123 patients with a suspected sporadic IEI that underwent patient-parent trio-based WES. Results: A (likely) molecular diagnosis for (part) of the immunological phenotype was achieved in 12 patients with the diagnostic in silico IEI WES gene panel. Systematic evaluation of rare, non-synonymous DNVs in coding or splice site regions led to the identification of 14 candidate DNVs in genes with an annotated immune function. DNVs were found in IEI genes (NLRP3 and RELA) and in potentially novel candidate genes, including PSMB10, DDX1, KMT2C, and FBXW11. The FBXW11 canonical splice site DNV was shown to lead to defective RNA splicing, increased NF-κB p65 signalling, and elevated IL-1ß production in primary immune cells extracted from the patient with autoinflammatory disease. Conclusions: Our findings in this retrospective cohort study advocate the implementation of trio-based sequencing in routine diagnostics of patients with sporadic IEI. Furthermore, we provide functional evidence supporting a causal role for FBXW11 loss-of-function mutations in autoinflammatory disease. Funding: This research was supported by grants from the European Union, ZonMW and the Radboud Institute for Molecular Life Sciences.
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Exoma , Enfermedades Autoinflamatorias Hereditarias , Humanos , Secuenciación del Exoma , Estudios Retrospectivos , Análisis de Secuencia de ADN , Enfermedades Autoinflamatorias Hereditarias/genéticaRESUMEN
Background: The etiology of cerebral small vessel disease (SVD) remains elusive, though evidence is accumulating that inflammation contributes to its pathophysiology. We recently showed retrospectively that pro-inflammatory monocytes are associated with the long-term progression of white matter hyperintensities (WMHs). In this prospective high-frequency imaging study, we hypothesize that the incidence of SVD progression coincides with a pro-inflammatory monocyte phenotype. Methods: Individuals with SVD underwent monthly magnetic resonance imaging (MRI) for 10 consecutive months to detect SVD progression, defined as acute diffusion-weighted imaging-positive (DWI+) lesions, incident microbleeds, incident lacunes, and WMH progression. Circulating inflammatory markers were measured, cytokine production capacity of monocytes was assessed after ex vivo stimulation, and RNA sequencing was performed on isolated monocytes in a subset of participants. Results: 13 out of 35 individuals developed SVD progression (70 ± 6 years, 54% men) based on incident lesions (n = 7) and/or upper quartile WMH progression (n = 9). Circulating E-selectin concentration (p < 0.05) and the cytokine production capacity of interleukin (IL)-1ß and IL-6 (p < 0.01) were higher in individuals with SVD progression. Moreover, RNA sequencing revealed a pro-inflammatory monocyte signature including genes involved in myelination, blood-brain barrier, and endothelial-leukocyte interaction. Conclusions: Circulating monocytes of individuals with progressive SVD have an inflammatory phenotype, characterized by an increased cytokine production capacity and a pro-inflammatory transcriptional signature.
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Direct targeting of the downstream mitogen-activated protein kinase (MAPK) pathway to suppress extracellular-regulated kinase (ERK) activation in KRAS and BRAF mutant colorectal cancer (CRC) has proven clinically unsuccessful, but promising results have been obtained with combination therapies including epidermal growth factor receptor (EGFR) inhibition. To elucidate the interplay between EGF signalling and ERK activation in tumours, we used patient-derived organoids (PDOs) from KRAS and BRAF mutant CRCs. PDOs resemble in vivo tumours, model treatment response and are compatible with live-cell microscopy. We established real-time, quantitative drug response assessment in PDOs with single-cell resolution, using our improved fluorescence resonance energy transfer (FRET)-based ERK biosensor EKAREN5. We show that oncogene-driven signalling is strikingly limited without EGFR activity and insufficient to sustain full proliferative potential. In PDOs and in vivo, upstream EGFR activity rigorously amplifies signal transduction efficiency in KRAS or BRAF mutant MAPK pathways. Our data provide a mechanistic understanding of the effectivity of EGFR inhibitors within combination therapies against KRAS and BRAF mutant CRC.
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Neoplasias Colorrectales/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas p21(ras)/genética , Línea Celular Tumoral , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Receptores ErbB/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Quinasas de Proteína Quinasa Activadas por Mitógenos/genética , Mutación , Organoides/metabolismo , Organoides/patología , Análisis de la Célula IndividualRESUMEN
Candida auris is among the most important emerging fungal pathogens, yet mechanistic insights into its immune recognition and control are lacking. Here, we integrate transcriptional and functional immune-cell profiling to uncover innate defence mechanisms against C. auris. C. auris induces a specific transcriptome in human mononuclear cells, a stronger cytokine response compared with Candida albicans, but a lower macrophage lysis capacity. C. auris-induced innate immune activation is mediated through the recognition of C-type lectin receptors, mainly elicited by structurally unique C. auris mannoproteins. In in vivo experimental models of disseminated candidiasis, C. auris was less virulent than C. albicans. Collectively, these results demonstrate that C. auris is a strong inducer of innate host defence, and identify possible targets for adjuvant immunotherapy.
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Candida/fisiología , Candidiasis/genética , Candidiasis/microbiología , Animales , Candida/genética , Candida/patogenicidad , Candidiasis/inmunología , Citocinas/genética , Citocinas/inmunología , Humanos , Inmunidad , Lectinas Tipo C/genética , Lectinas Tipo C/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Transcripción Genética , VirulenciaRESUMEN
Importance: Severe coronavirus disease 2019 (COVID-19) can occur in younger, predominantly male, patients without preexisting medical conditions. Some individuals may have primary immunodeficiencies that predispose to severe infections caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Objective: To explore the presence of genetic variants associated with primary immunodeficiencies among young patients with COVID-19. Design, Setting, and Participants: Case series of pairs of brothers without medical history meeting the selection criteria of young (age <35 years) brother pairs admitted to the intensive care unit (ICU) due to severe COVID-19. Four men from 2 unrelated families were admitted to the ICUs of 4 hospitals in the Netherlands between March 23 and April 12, 2020. The final date of follow-up was May 16, 2020. Available family members were included for genetic variant segregation analysis and as controls for functional experiments. Exposure: Severe COVID-19. Main Outcome and Measures: Results of rapid clinical whole-exome sequencing, performed to identify a potential monogenic cause. Subsequently, basic genetic and immunological tests were performed in primary immune cells isolated from the patients and family members to characterize any immune defects. Results: The 4 male patients had a mean age of 26 years (range, 21-32), with no history of major chronic disease. They were previously well before developing respiratory insufficiency due to severe COVID-19, requiring mechanical ventilation in the ICU. The mean duration of ventilatory support was 10 days (range, 9-11); the mean duration of ICU stay was 13 days (range, 10-16). One patient died. Rapid clinical whole-exome sequencing of the patients and segregation in available family members identified loss-of-function variants of the X-chromosomal TLR7. In members of family 1, a maternally inherited 4-nucleotide deletion was identified (c.2129_2132del; p.[Gln710Argfs*18]); the affected members of family 2 carried a missense variant (c.2383G>T; p.[Val795Phe]). In primary peripheral blood mononuclear cells from the patients, downstream type I interferon (IFN) signaling was transcriptionally downregulated, as measured by significantly decreased mRNA expression of IRF7, IFNB1, and ISG15 on stimulation with the TLR7 agonist imiquimod as compared with family members and controls. The production of IFN-γ, a type II IFN, was decreased in patients in response to stimulation with imiquimod. Conclusions and Relevance: In this case series of 4 young male patients with severe COVID-19, rare putative loss-of-function variants of X-chromosomal TLR7 were identified that were associated with impaired type I and II IFN responses. These preliminary findings provide insights into the pathogenesis of COVID-19.
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COVID-19/virología , Mutación con Pérdida de Función , SARS-CoV-2/genética , Adulto , Ensayo de Inmunoadsorción Enzimática , Resultado Fatal , Hospitalización , Humanos , Unidades de Cuidados Intensivos , Leucocitos Mononucleares , Masculino , Países Bajos , Linaje , ARN Viral/análisis , Reacción en Cadena en Tiempo Real de la Polimerasa , SARS-CoV-2/aislamiento & purificación , Adulto JovenRESUMEN
CNOT1 is a member of the CCR4-NOT complex, which is a master regulator, orchestrating gene expression, RNA deadenylation, and protein ubiquitination. We report on 39 individuals with heterozygous de novo CNOT1 variants, including missense, splice site, and nonsense variants, who present with a clinical spectrum of intellectual disability, motor delay, speech delay, seizures, hypotonia, and behavioral problems. To link CNOT1 dysfunction to the neurodevelopmental phenotype observed, we generated variant-specific Drosophila models, which showed learning and memory defects upon CNOT1 knockdown. Introduction of human wild-type CNOT1 was able to rescue this phenotype, whereas mutants could not or only partially, supporting our hypothesis that CNOT1 impairment results in neurodevelopmental delay. Furthermore, the genetic interaction with autism-spectrum genes, such as ASH1L, DYRK1A, MED13, and SHANK3, was impaired in our Drosophila models. Molecular characterization of CNOT1 variants revealed normal CNOT1 expression levels, with both mutant and wild-type alleles expressed at similar levels. Analysis of protein-protein interactions with other members indicated that the CCR4-NOT complex remained intact. An integrated omics approach of patient-derived genomics and transcriptomics data suggested only minimal effects on endonucleolytic nonsense-mediated mRNA decay components, suggesting that de novo CNOT1 variants are likely haploinsufficient hypomorph or neomorph, rather than dominant negative. In summary, we provide strong evidence that de novo CNOT1 variants cause neurodevelopmental delay with a wide range of additional co-morbidities. Whereas the underlying pathophysiological mechanism warrants further analysis, our data demonstrate an essential and central role of the CCR4-NOT complex in human brain development.
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Discapacidades del Desarrollo/genética , Expresión Génica/genética , Trastornos del Neurodesarrollo/genética , Miembro 2 del Grupo A de la Subfamilia 4 de Receptores Nucleares/genética , ARN/genética , Receptores CCR4/genética , Factores de Transcripción/genética , Alelos , Femenino , Variación Genética/genética , Haploinsuficiencia/genética , Heterocigoto , Humanos , Masculino , Malformaciones del Sistema Nervioso/genética , Fenotipo , Estabilidad ProteicaRESUMEN
RATIONALE: Exposure to high catecholamine levels is associated with inflammatory changes of myeloid cells and atherosclerosis, but the underlying mechanisms are only partly understood. OBJECTIVE: To investigate whether the proinflammatory effects of noradrenaline and adrenaline can, in part, be explained by the induction of an immunologic memory in innate immune cells, termed trained immunity. METHODS AND RESULTS: In vitro, we exposed human primary monocytes to (nor)adrenaline for 24 hours, after which cells were rested and differentiated to macrophages over 5 days. After restimulation with lipopolysaccharide on day 6, (nor)adrenaline-exposed cells showed increased TNF-α (tumor necrosis factor-α) production. This coincided with an increase in glycolysis and oxidative phosphorylation measured with Seahorse technology on day 6 before restimulation. Inhibition of the ß-adrenoreceptor-cAMP signaling pathway prevented the induction of training. In vivo, we studied the functional, transcriptional, and epigenetic impact of peak-wise exposure to high catecholamine levels on monocytes isolated from pheochromocytoma/paraganglioma (PHEO) patients. In PHEO patients (n=10), the peripheral blood cell composition showed a myeloid bias and an increase of the inflammatory CD14++CD16+ (cluster of differentiation) intermediate monocyte subset compared with controls with essential hypertension (n=14). Ex vivo production of proinflammatory cytokines was higher in PHEO patients. These inflammatory changes persisted for 4 weeks after surgical removal of PHEO. Transcriptome analysis of circulating monocytes at baseline showed various differentially expressed genes in inflammatory pathways in PHEO patients; epigenetic profiling of the promoters of these genes suggests enrichment of the transcriptionally permissive chromatin mark H3K4me3 (trimethylation of lysine 4 on histone H3), indicative of in vivo training. CONCLUSIONS: Catecholamines induce long-lasting proinflammatory changes in monocytes in vitro and in vivo, indicating trained immunity. Our data contribute to the understanding of pathways driving inflammatory changes in conditions characterized by high catecholamine levels and propose that trained immunity underlies the increased cardiovascular event rate in PHEO patients.
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Enfermedades Cardiovasculares/inmunología , Catecolaminas/farmacología , Inmunidad Innata , Memoria Inmunológica , Monocitos/inmunología , Células Cultivadas , Epigénesis Genética , Proteínas Ligadas a GPI/genética , Proteínas Ligadas a GPI/metabolismo , Glucólisis , Código de Histonas , Humanos , Receptores de Lipopolisacáridos/genética , Receptores de Lipopolisacáridos/metabolismo , Monocitos/efectos de los fármacos , Fosforilación Oxidativa , Receptores de IgG/genética , Receptores de IgG/metabolismo , Transcriptoma , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
CONTEXT: Primary aldosteronism (PA) confers an increased risk of cardiovascular disease (CVD), independent of blood pressure. Animal models have shown that aldosterone accelerates atherosclerosis through proinflammatory changes in innate immune cells; human data are scarce. OBJECTIVE: The objective of this article is to explore whether patients with PA have increased arterial wall inflammation, systemic inflammation, and reprogramming of monocytes. DESIGN: A cross-sectional cohort study compared vascular inflammation on 2'-deoxy-2'-(18F)fluoro-D-glucose; (18F-FDG) positron emission tomography-computed tomography, systemic inflammation, and monocyte phenotypes and transcriptome between PA patients and controls. SETTING: This study took place at Radboudumc and Rijnstate Hospital, the Netherlands. PATIENTS: Fifteen patients with PA and 15 age-, sex-, and blood pressure-matched controls with essential hypertension (EHT) participated. MAIN OUTCOME MEASURES AND RESULTS: PA patients displayed a higher arterial 18F-FDG uptake in the descending and abdominal aorta (P < .01, P < .05) and carotid and iliac arteries (both P < .01). In addition, bone marrow uptake was higher in PA patients (P < .05). Although PA patients had a higher monocyte-to-lymphocyte ratio (P < .05), systemic inflammatory markers, cytokine production capacity, and transcriptome of circulating monocytes did not differ. Monocyte-derived macrophages from PA patients expressed more TNFA; monocyte-derived macrophages of healthy donors cultured in PA serum displayed increased interleukin-6 and tumor necrosis factor-α production. CONCLUSIONS: Because increased arterial wall inflammation is associated with accelerated atherogenesis and unstable plaques, this might importantly contribute to the increased CVD risk in PA patients. We did not observe inflammatory reprogramming of circulating monocytes. However, subtle inflammatory changes are present in the peripheral blood cell composition and monocyte transcriptome of PA patients, and in their monocyte-derived macrophages. Most likely, arterial inflammation in PA requires interaction between various cell types.
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Arteritis/epidemiología , Hematopoyesis/fisiología , Hiperaldosteronismo/epidemiología , Adulto , Anciano , Arterias/diagnóstico por imagen , Arterias/patología , Arteritis/sangre , Arteritis/complicaciones , Arteritis/diagnóstico por imagen , Biomarcadores/sangre , Estudios de Casos y Controles , Estudios Transversales , Femenino , Fluorodesoxiglucosa F18 , Perfilación de la Expresión Génica , Humanos , Hiperaldosteronismo/sangre , Hiperaldosteronismo/diagnóstico por imagen , Hiperaldosteronismo/inmunología , Inflamación/sangre , Inflamación/diagnóstico , Inflamación/epidemiología , Inflamación/inmunología , Masculino , Persona de Mediana Edad , Monocitos/metabolismo , Países Bajos/epidemiología , Tomografía Computarizada por Tomografía de Emisión de PositronesRESUMEN
PURPOSE: Pathogenic variants in the chromatin organizer CTCF were previously reported in seven individuals with a neurodevelopmental disorder (NDD). METHODS: Through international collaboration we collected data from 39 subjects with variants in CTCF. We performed transcriptome analysis on RNA from blood samples and utilized Drosophila melanogaster to investigate the impact of Ctcf dosage alteration on nervous system development and function. RESULTS: The individuals in our cohort carried 2 deletions, 8 likely gene-disruptive, 2 splice-site, and 20 different missense variants, most of them de novo. Two cases were familial. The associated phenotype was of variable severity extending from mild developmental delay or normal IQ to severe intellectual disability. Feeding difficulties and behavioral abnormalities were common, and variable other findings including growth restriction and cardiac defects were observed. RNA-sequencing in five individuals identified 3828 deregulated genes enriched for known NDD genes and biological processes such as transcriptional regulation. Ctcf dosage alteration in Drosophila resulted in impaired gross neurological functioning and learning and memory deficits. CONCLUSION: We significantly broaden the mutational and clinical spectrum ofCTCF-associated NDDs. Our data shed light onto the functional role of CTCF by identifying deregulated genes and show that Ctcf alterations result in nervous system defects in Drosophila.
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Factor de Unión a CCCTC/genética , Factor de Unión a CCCTC/metabolismo , Trastornos del Neurodesarrollo/genética , Animales , Niño , Cromatina/genética , Cromatina/metabolismo , Discapacidades del Desarrollo/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Femenino , Perfilación de la Expresión Génica/métodos , Regulación de la Expresión Génica/genética , Humanos , Discapacidad Intelectual/genética , Masculino , Mutación/genética , Mutación Missense/genética , Trastornos del Neurodesarrollo/metabolismo , Factores de Transcripción/genética , Secuenciación del Exoma/métodos , Adulto JovenRESUMEN
The wide implementation of next-generation sequencing (NGS) technologies has revolutionized the field of medical genetics. However, the short read lengths of currently used sequencing approaches pose a limitation for the identification of structural variants, sequencing repetitive regions, phasing of alleles and distinguishing highly homologous genomic regions. These limitations may significantly contribute to the diagnostic gap in patients with genetic disorders who have undergone standard NGS, like whole exome or even genome sequencing. Now, the emerging long-read sequencing (LRS) technologies may offer improvements in the characterization of genetic variation and regions that are difficult to assess with the prevailing NGS approaches. LRS has so far mainly been used to investigate genetic disorders with previously known or strongly suspected disease loci. While these targeted approaches already show the potential of LRS, it remains to be seen whether LRS technologies can soon enable true whole genome sequencing routinely. Ultimately, this could allow the de novo assembly of individual whole genomes used as a generic test for genetic disorders. In this article, we summarize the current LRS-based research on human genetic disorders and discuss the potential of these technologies to facilitate the next major advancements in medical genetics.
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Unique experimental advantages, such as its embryonic/larval transparency, high-throughput nature, and ease of genetic modification, underpin the rapid emergence of the zebrafish (Danio rerio) as a preeminent model in biomedical research. Particularly in the field of nephrology, the zebrafish provides a promising model for studying the physiological implications of human solute transport processes along consecutive nephron segments. However, although the zebrafish might be considered a valuable model for numerous renal ion transport diseases and functional studies of many channels and transporters, not all human renal electrolyte transport mechanisms and human diseases can be modeled in the zebrafish. With this review, we explore the ontogeny of zebrafish renal ion transport, its nephron structure and function, and thereby demonstrate the clinical translational value of this model. By critical assessment of genomic and amino acid conservation of human proteins involved in renal ion handling (channels, transporters, and claudins), kidney and nephron segment conservation, and renal electrolyte transport physiology in the zebrafish, we provide researchers and nephrologists with an indication of the possibilities and considerations of the zebrafish as a model for human renal ion transport. Combined with advanced techniques envisioned for the future, implementation of the zebrafish might expand beyond unraveling pathophysiological mechanisms that underlie distinct genetic or environmentally, i.e., pharmacological and lifestyle, induced renal transport deficits. Specifically, the ease of drug administration and the exploitation of improved genetic approaches might argue for the adoption of the zebrafish as a model for preclinical personalized medicine for distinct renal diseases and renal electrolyte transport proteins.