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1.
Nat Commun ; 15(1): 2679, 2024 Mar 27.
Artículo en Inglés | MEDLINE | ID: mdl-38538644

RESUMEN

In 2015, we launched the mesoSPIM initiative, an open-source project for making light-sheet microscopy of large cleared tissues more accessible. Meanwhile, the demand for imaging larger samples at higher speed and resolution has increased, requiring major improvements in the capabilities of such microscopes. Here, we introduce the next-generation mesoSPIM ("Benchtop") with a significantly increased field of view, improved resolution, higher throughput, more affordable cost, and simpler assembly compared to the original version. We develop an optical method for testing detection objectives that enables us to select objectives optimal for light-sheet imaging with large-sensor cameras. The improved mesoSPIM achieves high spatial resolution (1.5 µm laterally, 3.3 µm axially) across the entire field of view, magnification up to 20×, and supports sample sizes ranging from sub-mm up to several centimeters while being compatible with multiple clearing techniques. The microscope serves a broad range of applications in neuroscience, developmental biology, pathology, and even physics.


Asunto(s)
Microscopía , Neurociencias , Microscopía/métodos
2.
Nat Biotechnol ; 42(1): 65-71, 2024 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-36997681

RESUMEN

Imaging large, cleared samples requires microscope objectives that combine a large field of view (FOV) with a long working distance (WD) and a high numerical aperture (NA). Ideally, such objectives should be compatible with a wide range of immersion media, which is challenging to achieve with conventional lens-based objective designs. Here we introduce the multi-immersion 'Schmidt objective' consisting of a spherical mirror and an aspherical correction plate as a solution to this problem. We demonstrate that a multi-photon variant of the Schmidt objective is compatible with all homogeneous immersion media and achieves an NA of 1.08 at a refractive index of 1.56, 1.1-mm FOV and 11-mm WD. We highlight its versatility by imaging cleared samples in various media ranging from air and water to benzyl alcohol/benzyl benzoate, dibenzyl ether and ethyl cinnamate and by imaging of neuronal activity in larval zebrafish in vivo. In principle, the concept can be extended to any imaging modality, including wide-field, confocal and light-sheet microscopy.


Asunto(s)
Telescopios , Animales , Inmersión , Microscopía/métodos , Pez Cebra
3.
FASEB J ; 38(1): e23329, 2024 01.
Artículo en Inglés | MEDLINE | ID: mdl-38050412

RESUMEN

Flow-induced shear stress affects renal epithelial cells in the nephron tubule with potential implications for differential functionalities of the individual segments. Disruptions of cellular mechanosensation or flow conditions are associated with the development and progression of various renal diseases. This study investigates the effects of flow on the transcriptome of various renal tubular epithelial cell types. We analyzed the transcriptome of induced renal epithelial cells (iREC) cultured under physiological flow (0.57 ± 0.05 dyn/cm2 ) or in static conditions for 72 h. RNA sequencing showed 861 differentially expressed genes (DEGs), with 503 up- and 358 downregulated under flow. DEGs were linked to extracellular matrix (ECM) components (e.g. Col1a1, Col4a3, Col4a4, Fn1, Smoc2), junctions (Gja1, Tubb5), channel activities (Abcc4, Aqp1), and transcription factors (Foxq1, Lgr6). Next, we performed a meta-analysis comparing our data with three published datasets that subjected epithelial cell lines from distinct segments to flow, including proximal tubule and collecting duct cells. We found that TGF-ß, p53, MAPK, and PI3K are common flow-regulated pathways. Tfrc expression and thus the capability of iron uptake is commonly upregulated under flow. Many DEGs were related to kidney diseases, such as fibrosis (e.g. Tgfb1-3 and Serpine1). To obtain further mechanistic insights we investigated the role of the PI3K pathway in flow sensing. Applying flow and inhibition of PI3K showed significantly altered expression of transcripts related to ECM remodeling, angiogenesis, and ion transport. This suggests that the PI3K pathway is a critical mediator in flow-dependent cellular processes and gene expression, potentially influencing renal development and tissue remodeling. Finally, we derived a cross-cell-line summary of common as well as segment-specific transcriptomic effects, thus providing insights into the molecular mechanisms underlying flow sensing in the nephron tubule.


Asunto(s)
Riñón , Fosfatidilinositol 3-Quinasas , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Riñón/metabolismo , Túbulos Renales Proximales/fisiología , Perfilación de la Expresión Génica , Células Epiteliales/metabolismo
4.
Sci Rep ; 13(1): 17647, 2023 10 17.
Artículo en Inglés | MEDLINE | ID: mdl-37848494

RESUMEN

CLIC5 belongs to a family of ion channels with six members reported so far. In vertebrates, the CLIC5 gene encodes two different isoforms, CLIC5A and CLIC5B. In addition to its ion channel activity, there is evidence for further functions of CLIC5A, such as the remodeling of the actin cytoskeleton during the formation of a functional glomerulus in the vertebrate kidney. However, its specific role is still incompletely understood and a specific functional role for CLIC5B has not been described yet. Here we report our findings on the differential expression and functions of Clic5a and Clic5b during zebrafish kidney development. Whole-mount in situ hybridization studies revealed specific expression of clic5a in the eye and pronephric glomerulus, and clic5b is expressed in the gut, liver and the pronephric tubules. Clic5 immunostainings revealed that Clic5b is localized in the cilia. Whereas knockdown of Clic5a resulted in leakiness of the glomerular filtration barrier, Clic5b deficient embryos displayed defective ciliogenesis, leading to ciliopathy-associated phenotypes such as ventral body curvature, otolith deposition defects, altered left-right asymmetry and formation of hydrocephalus and pronephric cysts. In addition, Clic5 deficiency resulted in dysregulation of cilia-dependent Wnt signalling pathway components. Mechanistically, we identified a Clic5-dependent activation of the membrane-cytoskeletal linker proteins Ezrin/Radixin/Moesin (ERM) in the pronephric tubules of zebrafish. In conclusion, our in vivo data demonstrates a novel role for Clic5 in regulating essential ciliary functions and identified Clic5 as a positive regulator of ERM phosphorylation.


Asunto(s)
Canales de Cloruro , Cloruros , Cilios , Glomérulos Renales , Proteínas de Microfilamentos , Pez Cebra , Animales , Citoesqueleto de Actina/metabolismo , Canales de Cloruro/genética , Canales de Cloruro/metabolismo , Cloruros/metabolismo , Cilios/genética , Cilios/metabolismo , Glomérulos Renales/metabolismo , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/metabolismo , Pez Cebra/genética , Pez Cebra/metabolismo
5.
Cells ; 12(6)2023 03 08.
Artículo en Inglés | MEDLINE | ID: mdl-36980176

RESUMEN

Clear cell renal cell carcinoma (ccRCC) is the most common histological subtype of renal cancer, and inactivation of the VHL tumor suppressor gene is found in almost all cases of hereditary and sporadic ccRCCs. CcRCC is associated with the reprogramming of fatty acid metabolism, and stearoyl-CoA desaturases (SCDs) are the main enzymes controlling fatty acid composition in cells. In this study, we report that mRNA and protein expression of the stearoyl-CoA desaturase SCD5 is downregulated in VHL-deficient cell lines. Similarly, in C. elegans vhl-1 mutants, FAT-7/SCD5 activity is repressed, supporting an evolutionary conservation. SCD5 regulation by VHL depends on HIF, and loss of SCD5 promotes cell proliferation and a metabolic shift towards ceramide production. In summary, we identify a novel regulatory function of VHL in relation to SCD5 and fatty acid metabolism, and propose a new mechanism of how loss of VHL may contribute to ccRCC tumor formation and progression.


Asunto(s)
Carcinoma de Células Renales , Neoplasias Renales , Animales , Humanos , Carcinoma de Células Renales/metabolismo , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/genética , Neoplasias Renales/patología , Proliferación Celular/genética , Homeostasis , Lípidos , Estearoil-CoA Desaturasa/genética
6.
J Am Soc Nephrol ; 34(3): 412-432, 2023 03 01.
Artículo en Inglés | MEDLINE | ID: mdl-36522156

RESUMEN

SIGNIFICANCE STATEMENT: Mutations in hepatocyte nuclear factor-1 ß ( HNF1B ) are the most common monogenic causes of congenital renal malformations. HNF1B is necessary to directly reprogram fibroblasts to induced renal tubule epithelial cells (iRECs) and, as we demonstrate, can induce ectopic pronephric tissue in Xenopus ectodermal organoids. Using these two systems, we analyzed the effect of HNF1B mutations found in patients with cystic dysplastic kidney disease. We found cross-species conserved targets of HNF1B, identified transcripts that are differentially regulated by the patient-specific mutant protein, and functionally validated novel HNF1B targets in vivo . These results highlight evolutionarily conserved transcriptional mechanisms and provide insights into the genetic circuitry of nephrogenesis. BACKGROUND: Hepatocyte nuclear factor-1 ß (HNF1B) is an essential transcription factor during embryogenesis. Mutations in HNF1B are the most common monogenic causes of congenital cystic dysplastic renal malformations. The direct functional consequences of mutations in HNF1B on its transcriptional activity are unknown. METHODS: Direct reprogramming of mouse fibroblasts to induced renal tubular epithelial cells was conducted both with wild-type HNF1B and with patient mutations. HNF1B was expressed in Xenopus ectodermal explants. Transcriptomic analysis by bulk RNA-Seq identified conserved targets with differentially regulated expression by the wild-type or R295C mutant. CRISPR/Cas9 genome editing in Xenopus embryos evaluated transcriptional targets in vivo . RESULTS: HNF1B is essential for reprogramming mouse fibroblasts to induced renal tubular epithelial cells and induces development of ectopic renal organoids from pluripotent Xenopus cells. The mutation R295C retains reprogramming and inductive capacity but alters the expression of specific sets of downstream target genes instead of diminishing overall transcriptional activity of HNF1B. Surprisingly, targets associated with polycystic kidney disease were less affected than genes affected in congenital renal anomalies. Cross-species-conserved transcriptional targets were dysregulated in hnf1b CRISPR-depleted Xenopus embryos, confirming their dependence on hnf1b . CONCLUSIONS: HNF1B activates an evolutionarily conserved program of target genes that disease-causing mutations selectively disrupt. These findings provide insights into the renal transcriptional network that controls nephrogenesis.


Asunto(s)
Factor Nuclear 1-beta del Hepatocito , Enfermedades Renales Quísticas , Animales , Ratones , Factor Nuclear 1-beta del Hepatocito/genética , Riñón/metabolismo , Enfermedades Renales Quísticas/genética , Mutación , Xenopus laevis
7.
J Am Soc Nephrol ; 34(2): 273-290, 2023 02 01.
Artículo en Inglés | MEDLINE | ID: mdl-36414417

RESUMEN

BACKGROUND: About 40 disease genes have been described to date for isolated CAKUT, the most common cause of childhood CKD. However, these genes account for only 20% of cases. ARHGEF6, a guanine nucleotide exchange factor that is implicated in biologic processes such as cell migration and focal adhesion, acts downstream of integrin-linked kinase (ILK) and parvin proteins. A genetic variant of ILK that causes murine renal agenesis abrogates the interaction of ILK with a murine focal adhesion protein encoded by Parva , leading to CAKUT in mice with this variant. METHODS: To identify novel genes that, when mutated, result in CAKUT, we performed exome sequencing in an international cohort of 1265 families with CAKUT. We also assessed the effects in vitro of wild-type and mutant ARHGEF6 proteins, and the effects of Arhgef6 deficiency in mouse and frog models. RESULTS: We detected six different hemizygous variants in the gene ARHGEF6 (which is located on the X chromosome in humans) in eight individuals from six families with CAKUT. In kidney cells, overexpression of wild-type ARHGEF6 -but not proband-derived mutant ARHGEF6 -increased active levels of CDC42/RAC1, induced lamellipodia formation, and stimulated PARVA-dependent cell spreading. ARHGEF6-mutant proteins showed loss of interaction with PARVA. Three-dimensional Madin-Darby canine kidney cell cultures expressing ARHGEF6-mutant proteins exhibited reduced lumen formation and polarity defects. Arhgef6 deficiency in mouse and frog models recapitulated features of human CAKUT. CONCLUSIONS: Deleterious variants in ARHGEF6 may cause dysregulation of integrin-parvin-RAC1/CDC42 signaling, thereby leading to X-linked CAKUT.


Asunto(s)
Sistema Urinario , Anomalías Urogenitales , Humanos , Ratones , Animales , Perros , Anomalías Urogenitales/genética , Riñón/anomalías , Sistema Urinario/anomalías , Integrinas/metabolismo , Proteínas Mutantes/metabolismo , Factores de Intercambio de Guanina Nucleótido Rho/genética
8.
bioRxiv ; 2023 Dec 12.
Artículo en Inglés | MEDLINE | ID: mdl-38168219

RESUMEN

In 2015, we launched the mesoSPIM initiative (www.mesospim.org), an open-source project for making light-sheet microscopy of large cleared tissues more accessible. Meanwhile, the demand for imaging larger samples at higher speed and resolution has increased, requiring major improvements in the capabilities of light-sheet microscopy. Here, we introduce the next-generation mesoSPIM ("Benchtop") with significantly increased field of view, improved resolution, higher throughput, more affordable cost and simpler assembly compared to the original version. We developed a new method for testing objectives, enabling us to select detection objectives optimal for light-sheet imaging with large-sensor sCMOS cameras. The new mesoSPIM achieves high spatial resolution (1.5 µm laterally, 3.3 µm axially) across the entire field of view, a magnification up to 20x, and supports sample sizes ranging from sub-mm up to several centimetres, while being compatible with multiple clearing techniques. The new microscope serves a broad range of applications in neuroscience, developmental biology, and even physics.

9.
Biomaterials ; 291: 121910, 2022 12.
Artículo en Inglés | MEDLINE | ID: mdl-36403325

RESUMEN

Renal tubular cells frequently lose differentiation markers and physiological properties when propagated in conventional cell culture conditions. Embedding cells in 3D microenvironments or controlling their 3D assembly by bioprinting can enhance their physiological properties, which is beneficial for modeling diseases in vitro. A potential cellular source for modeling renal tubular physiology and kidney diseases in vitro are directly reprogrammed induced renal tubular epithelial cells (iRECs). iRECs were cultured in various biomaterials and as bioprinted tubular structures. They showed high compatibility with the embedding substrates and dispensing methods. The morphology of multicellular aggregates was substantially influenced by the 3D microenvironment. Transcriptomic analyses revealed signatures of differentially expressed genes specific to each of the selected biomaterials. Using a new cellular model for autosomal-dominant polycystic kidney disease, Pkd1-/- iRECs showed disrupted morphology in bioprinted tubules and a marked upregulation of the Aldehyde dehydrogenase 1a1 (Aldh1a1). In conclusion, 3D microenvironments strongly influence the morphology and expression profiles of iRECs, help to unmask disease phenotypes, and can be adapted to experimental demands. Combining a direct reprogramming approach with appropriate biomaterials will facilitate construction of biomimetic kidney tubules and disease models at the microscale.


Asunto(s)
Biomimética , Enfermedades Renales Poliquísticas , Humanos , Riñón , Células Epiteliales , Materiales Biocompatibles
10.
Int J Bioprint ; 8(2): 528, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-35702333

RESUMEN

We used arrays of bioprinted renal epithelial cell spheroids for toxicity testing with cisplatin. The concentration-dependent cell death rate was determined using a lactate dehydrogenase assay. Bioprinted spheroids showed enhanced sensitivity to the treatment in comparison to monolayers of the same cell type. The measured dose-response curves revealed an inhibitory concentration of the spheroids of IC 50 = 9 ± 3 µM in contrast to the monolayers with IC 50 = 17 ± 2 µM. Fluorescent labeling of a nephrotoxicity biomarker, kidney injury molecule 1 indicated an accumulation of the molecule in the central lumen of the spheroids. Finally, we tested an approach for an automatic readout of toxicity based on microscopic images with deep learning. Therefore, we created a dataset comprising images of single spheroids, with corresponding labels of the determined cell death rates for training. The algorithm was able to distinguish between three classes of no, mild, and severe treatment effects with a balanced accuracy of 78.7%.

11.
Elife ; 112022 05 12.
Artículo en Inglés | MEDLINE | ID: mdl-35550039

RESUMEN

In diabetic patients, dyslipidemia frequently contributes to organ damage such as diabetic kidney disease (DKD). Dyslipidemia is associated with both excessive deposition of triacylglycerol (TAG) in lipid droplets (LDs) and lipotoxicity. Yet, it is unclear how these two effects correlate with each other in the kidney and how they are influenced by dietary patterns. By using a diabetes mouse model, we find here that high-fat diet enriched in the monounsaturated oleic acid (OA) caused more lipid storage in LDs in renal proximal tubular cells (PTCs) but less tubular damage than a corresponding butter diet with the saturated palmitic acid (PA). This effect was particularly evident in S2/S3 but not S1 segments of the proximal tubule. Combining transcriptomics, lipidomics, and functional studies, we identify endoplasmic reticulum (ER) stress as the main cause of PA-induced PTC injury. Mechanistically, ER stress is caused by elevated levels of saturated TAG precursors, reduced LD formation, and, consequently, higher membrane order in the ER. Simultaneous addition of OA rescues the cytotoxic effects by normalizing membrane order and increasing both TAG and LD formation. Our study thus emphasizes the importance of monounsaturated fatty acids for the dietary management of DKD by preventing lipid bilayer stress in the ER and promoting TAG and LD formation in PTCs.


Asunto(s)
Diabetes Mellitus , Ácidos Grasos Monoinsaturados , Animales , Estrés del Retículo Endoplásmico , Ácidos Grasos/farmacología , Ácidos Grasos Monoinsaturados/farmacología , Humanos , Riñón , Túbulos Renales Proximales , Membrana Dobles de Lípidos , Ratones , Ácido Palmítico/farmacología , Triglicéridos
12.
Physiology (Bethesda) ; 37(4): 0, 2022 07 01.
Artículo en Inglés | MEDLINE | ID: mdl-35253460

RESUMEN

The kidney is a complex organ, and how it forms is a fascinating process. New technologies, such as single-cell transcriptomics, and enhanced imaging modalities are offering new approaches to understand the complex and intertwined processes during embryonic kidney development.


Asunto(s)
Desarrollo Embrionario , Riñón , Humanos
13.
Development ; 148(21)2021 11 01.
Artículo en Inglés | MEDLINE | ID: mdl-34739029

RESUMEN

Genome editing simplifies the generation of new animal models for congenital disorders. However, the detailed and unbiased phenotypic assessment of altered embryonic development remains a challenge. Here, we explore how deep learning (U-Net) can automate segmentation tasks in various imaging modalities, and we quantify phenotypes of altered renal, neural and craniofacial development in Xenopus embryos in comparison with normal variability. We demonstrate the utility of this approach in embryos with polycystic kidneys (pkd1 and pkd2) and craniofacial dysmorphia (six1). We highlight how in toto light-sheet microscopy facilitates accurate reconstruction of brain and craniofacial structures within X. tropicalis embryos upon dyrk1a and six1 loss of function or treatment with retinoic acid inhibitors. These tools increase the sensitivity and throughput of evaluating developmental malformations caused by chemical or genetic disruption. Furthermore, we provide a library of pre-trained networks and detailed instructions for applying deep learning to the reader's own datasets. We demonstrate the versatility, precision and scalability of deep neural network phenotyping on embryonic disease models. By combining light-sheet microscopy and deep learning, we provide a framework for higher-throughput characterization of embryonic model organisms. This article has an associated 'The people behind the papers' interview.


Asunto(s)
Aprendizaje Profundo , Desarrollo Embrionario/genética , Fenotipo , Animales , Anomalías Craneofaciales/embriología , Anomalías Craneofaciales/genética , Anomalías Craneofaciales/patología , Modelos Animales de Enfermedad , Procesamiento de Imagen Asistido por Computador , Ratones , Microscopía , Mutación , Redes Neurales de la Computación , Trastornos del Neurodesarrollo/genética , Trastornos del Neurodesarrollo/patología , Enfermedades Renales Poliquísticas/embriología , Enfermedades Renales Poliquísticas/genética , Enfermedades Renales Poliquísticas/patología , Proteínas de Xenopus/genética , Xenopus laevis
14.
Int J Mol Sci ; 22(18)2021 Sep 18.
Artículo en Inglés | MEDLINE | ID: mdl-34576273

RESUMEN

Vancomycin is a glycopeptide antibiotic used against multi-drug resistant gram-positive bacteria such as Staphylococcus aureus (MRSA). Although invaluable against resistant bacteria, vancomycin harbors adverse drug reactions including cytopenia, ototoxicity, as well as nephrotoxicity. Since nephrotoxicity is a rarely occurring side effect, its mechanism is incompletely understood. Only recently, the actual clinically relevant concentration the in kidneys of patients receiving vancomycin was investigated and were found to exceed plasma concentrations by far. We applied these clinically relevant vancomycin concentrations to murine and canine renal epithelial cell lines and assessed metabolic and lipidomic alterations by untargeted and targeted gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry analyses. Despite marked differences in the lipidome, both cell lines increased anabolic glucose reactions, resulting in higher sorbitol and lactate levels. To the best of our knowledge, this is the first endometabolic profiling of kidney cells exposed to clinically relevant vancomycin concentrations. The presented study will provide a valuable dataset to nephrotoxicity researchers and might add to unveiling the nephrotoxic mechanism of vancomycin.


Asunto(s)
Riñón/efectos de los fármacos , Lipidómica , Vancomicina/farmacología , Animales , Antibacterianos/farmacología , Cromatografía Liquida , Perros , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos , Cromatografía de Gases y Espectrometría de Masas , Glutatión/metabolismo , Túbulos Renales Colectores/metabolismo , Lípidos/química , Células de Riñón Canino Madin Darby , Espectrometría de Masas , Metabolómica , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Ratones , Infecciones Estafilocócicas/tratamiento farmacológico
15.
Proc Natl Acad Sci U S A ; 118(39)2021 09 28.
Artículo en Inglés | MEDLINE | ID: mdl-34548398

RESUMEN

Skeletal ciliopathies (e.g., Jeune syndrome, short rib polydactyly syndrome, and Sensenbrenner syndrome) are frequently associated with nephronophthisis-like cystic kidney disease and other organ manifestations. Despite recent progress in genetic mapping of causative loci, a common molecular mechanism of cartilage defects and cystic kidneys has remained elusive. Targeting two ciliary chondrodysplasia loci (ift80 and ift172) by CRISPR/Cas9 mutagenesis, we established models for skeletal ciliopathies in Xenopus tropicalis Froglets exhibited severe limb deformities, polydactyly, and cystic kidneys, closely matching the phenotype of affected patients. A data mining-based in silico screen found ttc30a to be related to known skeletal ciliopathy genes. CRISPR/Cas9 targeting replicated limb malformations and renal cysts identical to the models of established disease genes. Loss of Ttc30a impaired embryonic renal excretion and ciliogenesis because of altered posttranslational tubulin acetylation, glycylation, and defective axoneme compartmentalization. Ttc30a/b transcripts are enriched in chondrocytes and osteocytes of single-cell RNA-sequenced embryonic mouse limbs. We identify TTC30A/B as an essential node in the network of ciliary chondrodysplasia and nephronophthisis-like disease proteins and suggest that tubulin modifications and cilia segmentation contribute to skeletal and renal ciliopathy manifestations of ciliopathies in a cell type-specific manner. These findings have implications for potential therapeutic strategies.


Asunto(s)
Huesos/anomalías , Ciliopatías/patología , Craneosinostosis/patología , Proteínas del Citoesqueleto/metabolismo , Displasia Ectodérmica/patología , Embrión no Mamífero/patología , Anomalías Musculoesqueléticas/patología , Enfermedades Renales Poliquísticas/patología , Tubulina (Proteína)/química , Animales , Huesos/metabolismo , Huesos/patología , Ciliopatías/genética , Ciliopatías/metabolismo , Craneosinostosis/genética , Craneosinostosis/metabolismo , Proteínas del Citoesqueleto/genética , Modelos Animales de Enfermedad , Displasia Ectodérmica/genética , Displasia Ectodérmica/metabolismo , Embrión no Mamífero/metabolismo , Anomalías Musculoesqueléticas/genética , Anomalías Musculoesqueléticas/metabolismo , Fenotipo , Enfermedades Renales Poliquísticas/genética , Enfermedades Renales Poliquísticas/metabolismo , Tubulina (Proteína)/metabolismo , Xenopus laevis
16.
Kidney Int ; 100(4): 850-869, 2021 10.
Artículo en Inglés | MEDLINE | ID: mdl-34252449

RESUMEN

Adverse effects of calcineurin inhibitors (CNI), such as hypertension, hyperkalemia, acidosis, hypomagnesemia and hypercalciuria, have been linked to dysfunction of the distal convoluted tubule (DCT). To test this, we generated a mouse model with an inducible DCT-specific deletion of the calcineurin regulatory subunit B alpha (CnB1-KO). Three weeks after CnB1 deletion, these mice exhibited hypomagnesemia and acidosis, but no hypertension, hyperkalemia or hypercalciuria. Consistent with the hypomagnesemia, CnB1-KO mice showed a downregulation of proteins implicated in DCT magnesium transport, including TRPM6, CNNM2, SLC41A3 and parvalbumin but expression of calcium channel TRPV5 in the kidney was unchanged. The abundance of the chloride/bicarbonate exchanger pendrin was increased, likely explaining the acidosis. Plasma aldosterone levels, kidney renin expression, abundance of phosphorylated sodium chloride-cotransporter and abundance of the epithelial sodium channel were similar in control and CnB1-KO mice, consistent with a normal sodium balance. Long-term potassium homeostasis was maintained in CnB1-KO mice, but in-vivo and ex-vivo experiments indicated that CnB1 contributes to acute regulation of potassium balance and sodium chloride-cotransporter. Tacrolimus treatment of control and CnB1-KO mice demonstrated that CNI-related hypomagnesemia is linked to impaired calcineurin-signaling in DCT, while hypocalciuria and hyponatremia occur independently of CnB1 in DCT. Transcriptome and proteome analyses of isolated DCTs demonstrated that CnB1 deletion impacts the expression of several DCT-specific proteins and signaling pathways. Thus, our data support a critical role of calcineurin for DCT function and provide novel insights into the pathophysiology of CNI side effects and involved molecular players in the DCT.


Asunto(s)
Acidosis , Magnesio , Animales , Calcineurina/genética , Túbulos Renales Distales , Ratones , Proteoma/genética , Transcriptoma
17.
Intern Emerg Med ; 16(8): 2269-2276, 2021 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-33687692

RESUMEN

Checklists can improve adherence to standardized procedures and minimize human error. We aimed to test if implementation of a checklist was feasible and effective in enhancing patient care in an emergency department handling internal medicine cases. We developed four critical event checklists and confronted volunteer teams with a series of four simulated emergency scenarios. In two scenarios, the teams were provided access to the crisis checklists in a randomized cross-over design. Simulated patient outcome plus statement of the underlying diagnosis defined the primary endpoint and adherence to key processes such as time to commence CPR represented the secondary endpoints. A questionnaire was used to capture participants' perception of clinical relevance and manageability of the checklists. Six teams of four volunteers completed a total of 24 crisis sequences. The primary endpoint was reached in 8 out of 12 sequences with and in 2 out of 12 sequences without a checklist (Odds ratio, 10; CI 1.11, 123.43; p = 0.03607, Fisher's exact test). Adherence to critical steps was significantly higher in all scenarios for which a checklist was available (performance score of 56.3% without checklist, 81.9% with checklist, p = 0.00284, linear regression model). All participants rated the checklist as useful and 22 of 24 participants would use the checklist in real life. Checklist use had no influence on CPR quality. The use of context-specific checklists showed a statistically significant influence on team performance and simulated patient outcome and contributed to adherence to standard clinical practices in emergency situations.


Asunto(s)
Lista de Verificación/normas , Simulación por Computador/estadística & datos numéricos , Adulto , Lista de Verificación/estadística & datos numéricos , Servicio de Urgencia en Hospital/organización & administración , Servicio de Urgencia en Hospital/estadística & datos numéricos , Femenino , Alemania , Humanos , Masculino , Proyectos Piloto , Encuestas y Cuestionarios
18.
Biofabrication ; 13(3)2021 04 07.
Artículo en Inglés | MEDLINE | ID: mdl-33513594

RESUMEN

Scalable fabrication concepts of 3D kidney tissue models are required to enable their application in pharmaceutical high-throughput screenings. Yet the reconstruction of complex tissue structures remains technologically challenging. We present a novel concept reducing the fabrication demands, by using controlled cellular self-assembly to achieve higher tissue complexities from significantly simplified construct designs. We used drop-on-demand bioprinting to fabricate locally confined patterns of renal epithelial cells embedded in a hydrogel matrix. These patterns provide defined local cell densities (cell count variance <11%) with high viability (92 ± 2%). Based on these patterns, controlled self-assembly leads to the formation of renal spheroids and nephron-like tubules with a predefined size and spatial localization. With this, we fabricated scalable arrays of hollow epithelial spheroids. The spheroid sizes correlated with the initial cell count per unit and could be stepwise adjusted, ranging from Ø = 84, 104, 120-131µm in diameter (size variance <9%). Furthermore, we fabricated scalable line-shaped patterns, which self-assembled to hollow cellular tubules (Ø = 105 ± 22µm). These showed a continuous lumen with prescribed orientation, lined by an epithelial monolayer with tight junctions. Additionally, upregulated expression of kidney-specific functional genes compared to 2D cell monolayers indicated increased tissue functionality, as revealed by mRNA sequencing. Furthermore, our concept enabled the fabrication of hybrid tubules, which consisted of arranged subsections of different cell types, combining murine and human epithelial cells. Finally, we integrated the self-assembled fabrication into a microfluidic chip and achieved fluidic access to the lumen at the terminal sites of the tubules. With this, we realized flow conditions with a wall shear stress of 0.05 ± 0.02 dyne cm-2driven by hydrostatic pressure for scalable dynamic culture towards a nephron-on-chip model.


Asunto(s)
Bioimpresión , Animales , Recuento de Células , Células Epiteliales , Humanos , Riñón , Ratones , Nefronas , Esferoides Celulares
19.
Nephrol Dial Transplant ; 36(2): 237-246, 2021 01 25.
Artículo en Inglés | MEDLINE | ID: mdl-33097957

RESUMEN

BACKGROUND: An underlying monogenic cause of early-onset chronic kidney disease (CKD) can be detected in ∼20% of individuals. For many etiologies of CKD manifesting before 25 years of age, >200 monogenic causative genes have been identified to date, leading to the elucidation of mechanisms of renal pathogenesis. METHODS: In 51 families with echogenic kidneys and CKD, we performed whole-exome sequencing to identify novel monogenic causes of CKD. RESULTS: We discovered a homozygous truncating mutation in the transcription factor gene transcription factor CP2-like 1 (TFCP2L1) in an Arabic patient of consanguineous descent. The patient developed CKD by the age of 2 months and had episodes of severe hypochloremic, hyponatremic and hypokalemic alkalosis, seizures, developmental delay and hypotonia together with cataracts. We found that TFCP2L1 was localized throughout kidney development particularly in the distal nephron. Interestingly, TFCP2L1 induced the growth and development of renal tubules from rat mesenchymal cells. Conversely, the deletion of TFCP2L1 in mice was previously shown to lead to reduced expression of renal cell markers including ion transporters and cell identity proteins expressed in different segments of the distal nephron. TFCP2L1 localized to the nucleus in HEK293T cells only upon coexpression with its paralog upstream-binding protein 1 (UBP1). A TFCP2L1 mutant complementary DNA (cDNA) clone that represented the patient's mutation failed to form homo- and heterodimers with UBP1, an essential step for its transcriptional activity. CONCLUSION: Here, we identified a loss-of-function TFCP2L1 mutation as a potential novel cause of CKD in childhood accompanied by a salt-losing tubulopathy.


Asunto(s)
Transición Epitelial-Mesenquimal , Enfermedades Renales/etiología , Mutación , Proteínas Represoras/genética , Animales , Niño , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Femenino , Células HEK293 , Humanos , Enfermedades Renales/metabolismo , Enfermedades Renales/patología , Ratones , Ratones Noqueados , Ratas , Proteínas Represoras/metabolismo , Análisis de la Célula Individual , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Secuenciación del Exoma
20.
Hum Mutat ; 41(12): 2179-2194, 2020 12.
Artículo en Inglés | MEDLINE | ID: mdl-33131181

RESUMEN

Ciliopathies are clinically and genetically heterogeneous diseases. We studied three patients from two independent families presenting with features of Joubert syndrome: abnormal breathing pattern during infancy, developmental delay/intellectual disability, cerebellar ataxia, molar tooth sign on magnetic resonance imaging scans, and polydactyly. We identified biallelic loss-of-function (LOF) variants in CBY1, segregating with the clinical features of Joubert syndrome in the families. CBY1 localizes to the distal end of the mother centriole, contributing to the formation and function of cilia. In accordance with the clinical and mutational findings in the affected individuals, we demonstrated that depletion of Cby1 in zebrafish causes ciliopathy-related phenotypes. Levels of CBY1 transcript were found reduced in the patients compared with controls, suggesting degradation of the mutated transcript through nonsense-mediated messenger RNA decay. Accordingly, we could detect CBY1 protein in fibroblasts from controls, but not from patients by immunofluorescence. Furthermore, we observed reduced ability to ciliate, increased ciliary length, and reduced levels of the ciliary proteins AHI1 and ARL13B in patient fibroblasts. Our data show that CBY1 LOF-variants cause a ciliopathy with features of Joubert syndrome.


Asunto(s)
Anomalías Múltiples/genética , Proteínas Portadoras/genética , Cerebelo/anomalías , Ciliopatías/genética , Anomalías del Ojo/genética , Enfermedades Renales Quísticas/genética , Mutación/genética , Proteínas Nucleares/genética , Retina/anomalías , Anomalías Múltiples/diagnóstico por imagen , Anomalías Múltiples/patología , Adolescente , Animales , Cerebelo/diagnóstico por imagen , Cerebelo/patología , Niño , Preescolar , Cilios/metabolismo , Cilios/patología , Ciliopatías/diagnóstico por imagen , Ciliopatías/patología , Anomalías del Ojo/diagnóstico por imagen , Anomalías del Ojo/patología , Femenino , Fibroblastos/metabolismo , Fibroblastos/patología , Homocigoto , Humanos , Lactante , Recién Nacido , Enfermedades Renales Quísticas/diagnóstico por imagen , Enfermedades Renales Quísticas/patología , Imagen por Resonancia Magnética , Masculino , Linaje , Fenotipo , Retina/diagnóstico por imagen , Retina/patología , Receptor Smoothened/metabolismo , Adulto Joven , Pez Cebra/genética
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