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Progression of cystic kidney disease has been linked to activation of the mTORC1 signaling pathway. Yet the utility of mTORC1 inhibitors to treat patients with polycystic kidney disease remains controversial despite promising preclinical data. To define the cell intrinsic role of mTORC1 for cyst development, the mTORC1 subunit gene Raptor was selectively inactivated in kidney tubular cells lacking cilia due to simultaneous deletion of the kinesin family member gene Kif3A. In contrast to a rapid onset of cyst formation and kidney failure in mice with defective ciliogenesis, both kidney function, cyst formation discerned by magnetic resonance imaging and overall survival were strikingly improved in mice additionally lacking Raptor. However, these mice eventually succumbed to cystic kidney disease despite mTORC1 inactivation. In-depth transcriptome analysis revealed the rapid activation of other growth-promoting signaling pathways, overriding the effects of mTORC1 deletion and identified cyclin-dependent kinase (CDK) 4 as an alternate driver of cyst growth. Additional inhibition of CDK4-dependent signaling by the CDK4/6 inhibitor Palbociclib markedly slowed disease progression in mice and human organoid models of polycystic kidney disease and potentiated the effects of mTORC1 deletion/inhibition. Our findings indicate that cystic kidneys rapidly adopt bypass mechanisms typically observed in drug resistant cancers. Thus, future clinical trials need to consider combinatorial or sequential therapies to improve therapeutic efficacy in patients with cystic kidney disease.
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Kidneys are intricate three-dimensional structures in the body, yet the spatial and molecular principles of kidney health and disease remain inadequately understood. We generated high-quality datasets for 81 samples, including single-cell, single-nuclear, spot-level (Visium) and single-cell resolution (CosMx) spatial-RNA expression and single-nuclear open chromatin, capturing cells from healthy, diabetic and hypertensive diseased human kidneys. Combining these data, we identify cell types and map them to their locations within the tissue. Unbiased deconvolution of the spatial data identifies the following four distinct microenvironments: glomerular, immune, tubule and fibrotic. We describe the complex organization of microenvironments in health and disease and find that the fibrotic microenvironment is able to molecularly classify human kidneys and offers an improved prognosis compared to traditional histopathology. We provide a comprehensive spatially resolved molecular roadmap of the human kidney and the fibrotic process, demonstrating the clinical utility of spatial transcriptomics.
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Microambiente Celular , Progressão da Doença , Fibrose , Nefropatias , Rim , Análise de Célula Única , Humanos , Rim/patologia , Microambiente Celular/genética , Nefropatias/genética , Nefropatias/patologia , Transcriptoma , Perfilação da Expressão Gênica , MultiômicaRESUMO
The 14th International Podocyte Conference took place in Philadelphia, Pennsylvania, USA from May 23 to 26, 2023. It commenced with an early-career researchers' meeting on May 23, providing young scientists with a platform to present and discuss their research findings. Throughout the main conference, 29 speakers across 9 sessions shared their insights on podocyte biology, glomerular medicine, novel technologic advancements, and translational approaches. Additionally, the event featured 3 keynote lectures addressing engineered chimeric antigen receptor T cell- and mRNA-based therapies and the use of biobanks for enhanced disease comprehension. Furthermore, 4 brief oral abstract sessions allowed scientists to present their findings to a broad audience. The program also included a panel discussion addressing the challenges of conducting human research within the American Black community. Remarkably, after a 5-year hiatus from in-person conferences, the 14th International Podocyte Conference successfully convened scientists from around the globe, fostering the presentation and discussion of crucial research findings, as summarized in this review. Furthermore, to ensure continuous and sustainable education, research, translation, and trial medicine related to podocyte and glomerular diseases for the benefit of patients, the International Society of Glomerular Disease was officially launched during the conference.
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Nefropatias , Podócitos , Humanos , Glomérulos Renais , Nefropatias/terapia , BiologiaAssuntos
Adenina/análogos & derivados , Glomerulosclerose Segmentar e Focal , Peptídeos e Proteínas de Sinalização Intracelular , Leucemia Linfocítica Crônica de Células B , Proteínas de Membrana , Síndrome Nefrótica , Piperidinas , Humanos , Glomerulosclerose Segmentar e Focal/induzido quimicamente , Glomerulosclerose Segmentar e Focal/tratamento farmacológico , Glomerulosclerose Segmentar e Focal/genética , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Leucemia Linfocítica Crônica de Células B/genética , Mutação , Síndrome Nefrótica/genéticaRESUMO
Kidney organoids are a promising model to study kidney disease, but their use is constrained by limited knowledge of their functional protein expression profile. Here, we define the organoid proteome and transcriptome trajectories over culture duration and upon exposure to TNFα, a cytokine stressor. Older organoids increase deposition of extracellular matrix but decrease expression of glomerular proteins. Single cell transcriptome integration reveals that most proteome changes localize to podocytes, tubular and stromal cells. TNFα treatment of organoids results in 322 differentially expressed proteins, including cytokines and complement components. Transcript expression of these 322 proteins is significantly higher in individuals with poorer clinical outcomes in proteinuric kidney disease. Key TNFα-associated protein (C3 and VCAM1) expression is increased in both human tubular and organoid kidney cell populations, highlighting the potential for organoids to advance biomarker development. By integrating kidney organoid omic layers, incorporating a disease-relevant cytokine stressor and comparing with human data, we provide crucial evidence for the functional relevance of the kidney organoid model to human kidney disease.
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Nefropatias , Fator de Necrose Tumoral alfa , Humanos , Fator de Necrose Tumoral alfa/metabolismo , Proteoma/metabolismo , Rim , Nefropatias/genética , Nefropatias/metabolismo , Organoides/metabolismoRESUMO
Expansion microscopy physically enlarges biological specimens to achieve nanoscale resolution using diffraction-limited microscopy systems1. However, optimal performance is usually reached using laser-based systems (for example, confocal microscopy), restricting its broad applicability in clinical pathology, as most centres have access only to light-emitting diode (LED)-based widefield systems. As a possible alternative, a computational method for image resolution enhancement, namely, super-resolution radial fluctuations (SRRF)2,3, has recently been developed. However, this method has not been explored in pathology specimens to date, because on its own, it does not achieve sufficient resolution for routine clinical use. Here, we report expansion-enhanced super-resolution radial fluctuations (ExSRRF), a simple, robust, scalable and accessible workflow that provides a resolution of up to 25 nm using LED-based widefield microscopy. ExSRRF enables molecular profiling of subcellular structures from archival formalin-fixed paraffin-embedded tissues in complex clinical and experimental specimens, including ischaemic, degenerative, neoplastic, genetic and immune-mediated disorders. Furthermore, as examples of its potential application to experimental and clinical pathology, we show that ExSRRF can be used to identify and quantify classical features of endoplasmic reticulum stress in the murine ischaemic kidney and diagnostic ultrastructural features in human kidney biopsies.
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Aumento da Imagem , Rim , Animais , Humanos , Camundongos , Microscopia de Fluorescência/métodos , Microscopia Confocal/métodosRESUMO
Current therapies for Fabry disease are based on reversing intracellular accumulation of globotriaosylceramide (Gb3) by enzyme replacement therapy (ERT) or chaperone-mediated stabilization of the defective enzyme, thereby alleviating lysosomal dysfunction. However, their effect in the reversal of end-organ damage, like kidney injury and chronic kidney disease, remains unclear. In this study, ultrastructural analysis of serial human kidney biopsies showed that long-term use of ERT reduced Gb3 accumulation in podocytes but did not reverse podocyte injury. Then, a CRISPR/Cas9-mediated α-galactosidase knockout podocyte cell line confirmed ERT-mediated reversal of Gb3 accumulation without resolution of lysosomal dysfunction. Transcriptome-based connectivity mapping and SILAC-based quantitative proteomics identified α-synuclein (SNCA) accumulation as a key event mediating podocyte injury. Genetic and pharmacological inhibition of SNCA improved lysosomal structure and function in Fabry podocytes, exceeding the benefits of ERT. Together, this work reconceptualizes Fabry-associated cell injury beyond Gb3 accumulation, and introduces SNCA modulation as a potential intervention, especially for patients with Fabry nephropathy.
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Doença de Fabry , Podócitos , Humanos , Podócitos/patologia , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismo , Doença de Fabry/genética , Doença de Fabry/tratamento farmacológico , Doença de Fabry/patologia , alfa-Galactosidase/genética , alfa-Galactosidase/metabolismo , alfa-Galactosidase/uso terapêutico , Rim/metabolismo , Triexosilceramidas/metabolismo , Triexosilceramidas/farmacologia , Triexosilceramidas/uso terapêuticoRESUMO
The guanine-rich RNA sequence binding factor 1 (GRSF1) is an RNA-binding protein of the heterogenous nuclear ribonucleoprotein H/F (hnRNP H/F) family that binds to guanine-rich RNA sequences forming G-quadruplex structures. In mice and humans there are single copy GRSF1 genes, but multiple transcripts have been reported. GRSF1 has been implicated in a number of physiological processes (e.g. embryogenesis, erythropoiesis, redox homeostasis, RNA metabolism) but also in the pathogenesis of viral infections and hyperproliferative diseases. These postulated biological functions of GRSF1 originate from in vitro studies rather than complex in vivo systems. To assess the in vivo relevance of these findings, we created systemic Grsf1-/- knockout mice lacking exons 4 and 5 of the Grsf1 gene and compared the basic functional characteristics of these animals with those of wildtype controls. We found that Grsf1-deficient mice are viable, reproduce normally and have fully functional hematopoietic systems. Up to an age of 15 weeks they develop normally but when male individuals grow older, they gain significantly less body weight than wildtype controls in a gender-specific manner. Profiling Grsf1 mRNA expression in different mouse tissues we observed high concentrations in testis. Comparison of the testicular transcriptomes of Grsf1-/- mice and wildtype controls confirmed near complete knock-out of Grsf1 but otherwise subtle differences in transcript regulations. Comparative testicular proteome analyses suggested perturbed mitochondrial respiration in Grsf1-/- mice which may be related to compromised expression of complex I proteins. Here we present, for the first time, an in vivo complete Grsf1 knock-out mouse with comprehensive physiological, transcriptomic and proteomic characterization to improve our understanding of the GRSF1 beyond in vitro cell culture models.
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The filtration of blood in the kidney which is crucial for mammalian life is determined by the slit-diaphragm, a cell-cell junction between the foot processes of renal podocytes. The slit-diaphragm is thought to operate as final barrier or as molecular sensor of renal filtration. Using high-resolution proteomic analysis of slit-diaphragms affinity-isolated from rodent kidney, we show that the native slit-diaphragm is built from the junction-forming components Nephrin, Neph1 and Podocin and a co-assembled high-molecular weight network of proteins. The network constituents cover distinct classes of proteins including signaling-receptors, kinases/phosphatases, transporters and scaffolds. Knockout or knock-down of either the core components or the selected network constituents tyrosine kinase MER (MERTK), atrial natriuretic peptide-receptor C (ANPRC), integral membrane protein 2B (ITM2B), membrane-associated guanylate-kinase, WW and PDZ-domain-containing protein1 (MAGI1) and amyloid protein A4 resulted in target-specific impairment or disruption of the filtration process. Our results identify the slit-diaphragm as a multi-component system that is endowed with context-dependent dynamics via a co-assembled protein network.
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Diafragma , Podócitos , Animais , Proteômica , Podócitos/metabolismo , Glomérulos Renais , Junções Intercelulares , MamíferosRESUMO
Establishing a diagnosis in cases of fever of unknown origin (FUO) in immunocompromised patients can be difficult. In 25-35% infectious diseases are the underlying cause. This article reports the case of a 74-year-old woman with a 5-month history of fever. Through open biopsy of the femoral shaft and microbiological analysis, a diagnosis of neoehrlichiosis could be established. After initiation of treatment with doxycycline, the symptoms quickly resolved resulting in a complete recovery.
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Infecções por Anaplasmataceae , Anaplasmataceae , Febre de Causa Desconhecida , Idoso , Infecções por Anaplasmataceae/diagnóstico , Infecções por Anaplasmataceae/microbiologia , Infecções por Anaplasmataceae/patologia , Feminino , Febre , Febre de Causa Desconhecida/diagnóstico , Febre de Causa Desconhecida/etiologia , Humanos , Hospedeiro ImunocomprometidoRESUMO
In eukaryotic cells RNA-binding proteins have been implicated in virtually all post-transcriptional mechanisms of gene expression regulation. Based on the structural features of their RNA binding domains these proteins have been divided into several subfamilies. The presence of at least two RNA recognition motifs defines the group of heterogenous nuclear ribonucleoproteins H/F and one of its members is the guanine-rich sequence binding factor 1 (GRSF1). GRSF1 was first described 25 years ago and is widely distributed in eukaryotic cells. It is present in the nucleus, the cytoplasm and in mitochondria and has been implicated in a variety of physiological processes (embryogenesis, erythropoiesis, redox homeostasis, RNA metabolism) but also in the pathogenesis of various diseases. This review summarizes our current understanding on GRSF1 biology, critically discusses the literature reports and gives an outlook of future developments in the field.
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Regulação da Expressão Gênica , Guanina/metabolismo , Proteínas de Ligação a Poli(A)/química , Proteínas de Ligação a Poli(A)/genética , Sequência de Aminoácidos , Animais , Evolução Molecular , Humanos , Ligação Proteica , RNA/metabolismoRESUMO
BACKGROUND: The guanine-rich RNA sequence binding factor 1 (GRSF1) is an RNA-binding protein of the hnRNP H/F family, which has been implicated in erythropoiesis, regulation of the redox homeostasis, embryonic brain development, mitochondrial function and cellular senescence. The molecular basis for GRSF1-RNA interaction has extensively been studied in the past but for the time being GRSF1 binding proteins have not been identified. METHODS: To search for GRSF1 binding proteins we first employed the yeast two-hybrid system and screened a cDNA library of human fetal brain for potential GRSF1 binding proteins. Subsequently, we explored the protein-protein-interaction of the recombiant proteins, carried out immunoprecipitation experiments to confirm the interaction of the native proteins in living cells and performed truncation studies to identify the protein-binding motif of GRSF1. RESULTS: Using the yeast two-hybrid system we identified the COMM-domain containing protein 1 (COMMD1) as specific GRSF1 binding protein and in vitro truncation studies suggested that COMMD1 interacts with the alanine-rich domain of GRSF1. Co-immunoprecipitation strategies indicated that COMMD1-GRSF1 interaction was RNA independent and also occurred in living cells expressing the two native proteins. CONCLUSION: In mammalian cells the COMM-domain containing protein 1 (COMMD1) specifically interacts with the Ala-rich domain of GRSF1 in an RNA-independent manner. GENERAL SIGNIFICANCE: This is the first report describing a specific GRSF1 binding protein.
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Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Ligação a Poli(A)/metabolismo , RNA/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Células HEK293 , Humanos , Proteínas de Ligação a Poli(A)/química , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Mapas de Interação de ProteínasRESUMO
The guanine-rich RNA sequence binding factor 1 (GRSF1) constitutes an ubiquitously occurring RNA-binding protein (RBP), which belongs to the family of heterogeneous nuclear ribonucleoprotein F/H (hnRNP F/H). It has been implicated in nuclear, cytosolic and mitochondrial RNA metabolism. Although the crystal structures of GRSF1 orthologs have not been solved, amino acid alignments with similar RNA-binding proteins suggested the existence of three RNA-binding domains designated quasi-RNA recognition motifs (qRRMs). Here we established 3D-models for the three qRRMs of human GRSF1 on the basis of the NMR structure of hnRNP F and identified the putative RNA interacting amino acids. Next, we explored the genetic variability of the three qRRMs of human GRSF1 by searching genomic databases and tested the functional consequences of naturally occurring mutants. For this purpose the RNA-binding capacity of wild-type and mutant recombinant GRSF1 protein species was assessed by quantitative RNA electrophoretic mobility shift assays. We found that some of the naturally occurring GRSF1 mutants exhibited a strongly reduced RNA-binding activity although the general protein structure was hardly affected. These data suggested that homozygous allele carriers of these particular mutants express dysfunctional GRSF1 and thus may show defective GRSF1 signaling.