RESUMEN
The mammalian kidney maintains fluid homeostasis through diverse epithelial cell types generated from nephron and ureteric progenitor cells. To extend a developmental understanding of the kidney's epithelial networks, we compared chromatin organization (single-nuclear assay for transposase-accessible chromatin sequencing [ATAC-seq]; 112,864 nuclei) and gene expression (single-cell/nuclear RNA sequencing [RNA-seq]; 109,477 cells/nuclei) in the developing human (10.6-17.6 weeks; n = 10) and mouse (post-natal day [P]0; n = 10) kidney, supplementing analysis with published mouse datasets from earlier stages. Single-cell/nuclear datasets were analyzed at a species level, and then nephron and ureteric cellular lineages were extracted and integrated into a common, cross-species, multimodal dataset. Comparative computational analyses identified conserved and divergent features of chromatin organization and linked gene activity, identifying species-specific and cell-type-specific regulatory programs. In situ validation of human-enriched gene activity points to human-specific signaling interactions in kidney development. Further, human-specific enhancer regions were linked to kidney diseases through genome-wide association studies (GWASs), highlighting the potential for clinical insight from developmental modeling.
RESUMEN
Nephron progenitor cells (NPCs) self-renew and differentiate into nephrons, the functional units of the kidney. Here, manipulation of p38 and YAP activity allowed for long-term clonal expansion of primary mouse and human NPCs and induced NPCs (iNPCs) from human pluripotent stem cells (hPSCs). Molecular analyses demonstrated that cultured iNPCs closely resemble primary human NPCs. iNPCs generated nephron organoids with minimal off-target cell types and enhanced maturation of podocytes relative to published human kidney organoid protocols. Surprisingly, the NPC culture medium uncovered plasticity in human podocyte programs, enabling podocyte reprogramming to an NPC-like state. Scalability and ease of genome editing facilitated genome-wide CRISPR screening in NPC culture, uncovering genes associated with kidney development and disease. Further, NPC-directed modeling of autosomal-dominant polycystic kidney disease (ADPKD) identified a small-molecule inhibitor of cystogenesis. These findings highlight a broad application for the reported iNPC platform in the study of kidney development, disease, plasticity, and regeneration.
Asunto(s)
Nefronas , Organoides , Animales , Organoides/citología , Organoides/metabolismo , Humanos , Nefronas/citología , Ratones , Diferenciación Celular , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/citología , Podocitos/metabolismo , Podocitos/citología , Riñón/patología , Riñón Poliquístico Autosómico Dominante/patología , Riñón Poliquístico Autosómico Dominante/metabolismo , Riñón Poliquístico Autosómico Dominante/genética , Modelos Biológicos , Edición GénicaRESUMEN
Nephron progenitor cells (NPCs) self-renew and differentiate into nephrons, the functional units of the kidney. Here we report manipulation of p38 and YAP activity creates a synthetic niche that allows the long-term clonal expansion of primary mouse and human NPCs, and induced NPCs (iNPCs) from human pluripotent stem cells. Cultured iNPCs resemble closely primary human NPCs, generating nephron organoids with abundant distal convoluted tubule cells, which are not observed in published kidney organoids. The synthetic niche reprograms differentiated nephron cells into NPC state, recapitulating the plasticity of developing nephron in vivo. Scalability and ease of genome-editing in the cultured NPCs allow for genome-wide CRISPR screening, identifying novel genes associated with kidney development and disease. A rapid, efficient, and scalable organoid model for polycystic kidney disease was derived directly from genome-edited NPCs, and validated in drug screen. These technological platforms have broad applications to kidney development, disease, plasticity, and regeneration.
RESUMEN
Mammalian kidneys maintain fluid homeostasis through the cellular activity of nephrons and the conjoined collecting system. Each epithelial network originates from distinct progenitor cell populations that reciprocally interact during development. To extend our understanding of human and mouse kidney development, we profiled chromatin organization (ATAC-seq) and gene expression (RNA-seq) in developing human and mouse kidneys. Data were analyzed at a species level and then integrated into a common, cross-species multimodal data set. Comparative analysis of cell types and developmental trajectories identified conserved and divergent features of chromatin organization and linked gene activity, revealing species- and cell-type specific regulatory programs. Identification of human-specific enhancer regions linked through GWAS studies to kidney disease highlights the potential of developmental modeling to provide clinical insight.
RESUMEN
Congenital abnormalities of the kidney and urinary tract are among the most common birth defects, affecting 3% of newborns. The human kidney forms around a million nephrons from a pool of nephron progenitors over a 30-week period of development. To establish a framework for human nephrogenesis, we spatially resolved a stereotypical process by which equipotent nephron progenitors generate a nephron anlage, then applied data-driven approaches to construct three-dimensional protein maps on anatomical models of the nephrogenic program. Single-cell RNA sequencing identified progenitor states, which were spatially mapped to the nephron anatomy, enabling the generation of functional gene networks predicting interactions within and between nephron cell types. Network mining identified known developmental disease genes and predicted targets of interest. The spatially resolved nephrogenic program made available through the Human Nephrogenesis Atlas (https://sckidney.flatironinstitute.org/) will facilitate an understanding of kidney development and disease and enhance efforts to generate new kidney structures.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Nefronas/metabolismo , Transcriptoma , Animales , Humanos , Ratones , Nefronas/citología , Nefronas/embriología , Proteoma/genética , Proteoma/metabolismo , RNA-Seq , Análisis de la Célula IndividualRESUMEN
Current kidney organoids model development and diseases of the nephron but not the contiguous epithelial network of the kidney's collecting duct (CD) system. Here, we report the generation of an expandable, 3D branching ureteric bud (UB) organoid culture model that can be derived from primary UB progenitors from mouse and human fetal kidneys, or generated de novo from human pluripotent stem cells. In chemically-defined culture conditions, UB organoids generate CD organoids, with differentiated principal and intercalated cells adopting spatial assemblies reflective of the adult kidney's collecting system. Aggregating 3D-cultured nephron progenitor cells with UB organoids in vitro results in a reiterative process of branching morphogenesis and nephron induction, similar to kidney development. Applying an efficient gene editing strategy to remove RET activity, we demonstrate genetically modified UB organoids can model congenital anomalies of kidney and urinary tract. Taken together, these platforms will facilitate an enhanced understanding of development, regeneration and diseases of the mammalian collecting duct system.
Asunto(s)
Túbulos Renales Colectores/citología , Riñón/citología , Riñón/crecimiento & desarrollo , Organogénesis/fisiología , Organoides/citología , Organoides/crecimiento & desarrollo , Uréter , Sistema Urinario/citología , Adulto , Animales , Diferenciación Celular , Células Cultivadas , Humanos , Riñón/embriología , Túbulos Renales Colectores/embriología , Masculino , Ratones , Morfogénesis , Nefronas , Organogénesis/genética , Organoides/embriología , Células Madre Pluripotentes/citología , Sistema Urinario/embriología , Sistema Urinario/crecimiento & desarrolloRESUMEN
Analysis of kidney disease-causing genes and pathology resulting from systemic diseases highlight the importance of the kidney's filtering system, the renal corpuscles. To elucidate the developmental processes that establish the renal corpuscle, we performed single-nucleus droplet-based sequencing of the human fetal kidney. This enabled the identification of nephron, interstitial, and vascular cell types that together generate the renal corpuscles. Trajectory analysis identified transient developmental gene expression, predicting precursors or mature podocytes express FBLN2, BMP4, or NTN4, in conjunction with recruitment, differentiation, and modeling of vascular and mesangial cell types into a functional filter. In vitro studies provide evidence that these factors exhibit angiogenic or mesangial recruiting and inductive properties consistent with a key organizing role for podocyte precursors in kidney development. Together these studies define a spatiotemporal developmental program for the primary filtration unit of the human kidney and provide novel insights into cell interactions regulating co-assembly of constituent cell types.
RESUMEN
Adamts18 encodes a secreted metalloprotease restricted to branch-tip progenitor pools directing the morphogenesis of multiple mammalian organs. Adamts18 was targeted to explore a potential role in branching morphogenesis. In the kidney, an arborized collecting system develops through extensive branching morphogenesis of an initial epithelial outgrowth of the mesonephric duct, the ureteric bud. Adamts18 mutants displayed a weakly penetrant phenotype: duplicated ureteric outgrowths forming enlarged, bi-lobed kidneys with an increased nephron endowment. In contrast, Adamts18 mutants showed a fully penetrant lung phenotype: epithelial growth was markedly reduced and early secondary branching scaled to the reduced length of the primary airways. Furthermore, there was a pronounced delay in the appearance of differentiated cell types in both proximal and distally positions of the developing airways. Adamts18 is closely related to Adamts16. In the kidney but not the lung, broad epithelial Adamts16 expression overlaps Adamts18 in branch tips. However, compound Adamts16/18 mutants displayed a comparable low penetrance duplicated ureteric phenotype, ruling out a possible role for Adamts16 as a functional modifier of the Adamts18 kidney phenotype. Given the predicted action of secreted Adamts18 metalloprotease, and broad expression of Adamts18 in branching organ systems, these findings suggest distinct requirements for matrix modelling in the morphogenesis of epithelial networks.
Asunto(s)
Proteínas ADAMTS/metabolismo , Organogénesis/fisiología , Proteínas ADAMTS/fisiología , Animales , Femenino , Regulación del Desarrollo de la Expresión Génica/genética , Riñón/citología , Riñón/embriología , Riñón/metabolismo , Pulmón/embriología , Pulmón/metabolismo , Masculino , Metaloproteasas/genética , Metaloproteasas/metabolismo , Ratones , Ratones Noqueados , Morfogénesis , Nefronas/metabolismo , Técnicas de Cultivo de Órganos/métodos , Uréter/metabolismoRESUMEN
Mammalian nephrons arise from a limited nephron progenitor pool through a reiterative inductive process extending over days (mouse) or weeks (human) of kidney development. Here, we present evidence that human nephron patterning reflects a time-dependent process of recruitment of mesenchymal progenitors into an epithelial nephron precursor. Progressive recruitment predicted from high-resolution image analysis and three-dimensional reconstruction of human nephrogenesis was confirmed through direct visualization and cell fate analysis of mouse kidney organ cultures. Single-cell RNA sequencing of the human nephrogenic niche provided molecular insights into these early patterning processes and predicted developmental trajectories adopted by nephron progenitor cells in forming segment-specific domains of the human nephron. The temporal-recruitment model for nephron polarity and patterning suggested by direct analysis of human kidney development provides a framework for integrating signaling pathways driving mammalian nephrogenesis.
Asunto(s)
Diferenciación Celular , Regulación del Desarrollo de la Expresión Génica , Células Madre Mesenquimatosas/citología , Nefronas/citología , Organogénesis/fisiología , Animales , Femenino , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Masculino , Células Madre Mesenquimatosas/metabolismo , Ratones , Nefronas/metabolismo , Transducción de Señal , Análisis de la Célula Individual , Factores de TiempoRESUMEN
Cellular interactions among nephron, interstitial, and collecting duct progenitors drive mammalian kidney development. In mice, Six2+ nephron progenitor cells (NPCs) and Foxd1+ interstitial progenitor cells (IPCs) form largely distinct lineage compartments at the onset of metanephric kidney development. Here, we used the method for analyzing RNA following intracellular sorting (MARIS) approach, single-cell transcriptional profiling, in situ hybridization, and immunolabeling to characterize the presumptive NPC and IPC compartments of the developing human kidney. As in mice, each progenitor population adopts a stereotypical arrangement in the human nephron-forming niche: NPCs capped outgrowing ureteric branch tips, whereas IPCs were sandwiched between the NPCs and the renal capsule. Unlike mouse NPCs, human NPCs displayed a transcriptional profile that overlapped substantially with the IPC transcriptional profile, and key IPC determinants, including FOXD1, were readily detected within SIX2+ NPCs. Comparative gene expression profiling in human and mouse Six2/SIX2+ NPCs showed broad agreement between the species but also identified species-biased expression of some genes. Notably, some human NPC-enriched genes, including DAPL1 and COL9A2, are linked to human renal disease. We further explored the cellular diversity of mesenchymal cell types in the human nephrogenic niche through single-cell transcriptional profiling. Data analysis stratified NPCs into two main subpopulations and identified a third group of differentiating cells. These findings were confirmed by section in situ hybridization with novel human NPC markers predicted through the single-cell studies. This study provides a benchmark for the mesenchymal progenitors in the human nephrogenic niche and highlights species-variability in kidney developmental programs.
Asunto(s)
Corteza Renal/embriología , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Nefronas/embriología , Animales , Proteínas Reguladoras de la Apoptosis , Diferenciación Celular , Linaje de la Célula , Femenino , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Perfilación de la Expresión Génica , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Masculino , Ratones , Proteína 1 del Sitio de Integración Viral Ecotrópica Mieloide/genética , Proteína 1 del Sitio de Integración Viral Ecotrópica Mieloide/metabolismo , Nefronas/anatomía & histología , Nefronas/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Análisis de Secuencia de ARN , Análisis de la Célula Individual , Transactivadores/genética , Transactivadores/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
The nephron is the functional unit of the kidney, but the mechanism of nephron formation during human development is unclear. We conducted a detailed analysis of nephron development in humans and mice by immunolabeling, and we compared human and mouse nephron patterning to describe conserved and divergent features. We created protein localization maps that highlight the emerging patterns along the proximal-distal axis of the developing nephron and benchmark expectations for localization of functionally important transcription factors, which revealed unanticipated cellular diversity. Moreover, we identified a novel nephron subdomain marked by Wnt4 expression that we fate-mapped to the proximal mature nephron. Significant conservation was observed between human and mouse patterning. We also determined the time at which markers for mature nephron cell types first emerge-critical data for the renal organoid field. These findings have conceptual implications for the evolutionary processes driving the diversity of mammalian organ systems. Furthermore, these findings provide practical insights beyond those gained with mouse and rat models that will guide in vitro efforts to harness the developmental programs necessary to build human kidney structures.
Asunto(s)
Diferenciación Celular , Nefronas/embriología , Nefronas/metabolismo , Células Madre/fisiología , Animales , Proteínas Reguladoras de la Apoptosis , Linaje de la Célula , Perfilación de la Expresión Génica , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Inmunohistoquímica , Factor de Unión 1 al Potenciador Linfoide/genética , Factor de Unión 1 al Potenciador Linfoide/metabolismo , Ratones , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Células Madre/metabolismo , Transactivadores/genética , Transactivadores/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteína Wnt4/metabolismoRESUMEN
Human kidney function is underpinned by approximately 1,000,000 nephrons, although the number varies substantially, and low nephron number is linked to disease. Human kidney development initiates around 4 weeks of gestation and ends around 34-37 weeks of gestation. Over this period, a reiterative inductive process establishes the nephron complement. Studies have provided insightful anatomic descriptions of human kidney development, but the limited histologic views are not readily accessible to a broad audience. In this first paper in a series providing comprehensive insight into human kidney formation, we examined human kidney development in 135 anonymously donated human kidney specimens. We documented kidney development at a macroscopic and cellular level through histologic analysis, RNA in situ hybridization, immunofluorescence studies, and transcriptional profiling, contrasting human development (4-23 weeks) with mouse development at selected stages (embryonic day 15.5 and postnatal day 2). The high-resolution histologic interactive atlas of human kidney organogenesis generated can be viewed at the GUDMAP database (www.gudmap.org) together with three-dimensional reconstructions of key components of the data herein. At the anatomic level, human and mouse kidney development differ in timing, scale, and global features such as lobe formation and progenitor niche organization. The data also highlight differences in molecular and cellular features, including the expression and cellular distribution of anchor gene markers used to identify key cell types in mouse kidney studies. These data will facilitate and inform in vitro efforts to generate human kidney structures and comparative functional analyses across mammalian species.
Asunto(s)
Riñón/embriología , Riñón/metabolismo , Organogénesis , Uréter/embriología , Animales , Diferenciación Celular , Técnica del Anticuerpo Fluorescente , Perfilación de la Expresión Génica , Edad Gestacional , Técnicas Histológicas , Humanos , Hibridación in Situ , Riñón/anatomía & histología , Ratones , Nefronas/embriología , Nefronas/metabolismo , ARN/análisis , Uréter/metabolismoRESUMEN
Though an acute kidney injury (AKI) episode is associated with an increased risk of chronic kidney disease (CKD), the mechanisms determining the transition from acute to irreversible chronic injury are not well understood. To extend our understanding of renal repair, and its limits, we performed a detailed molecular characterization of a murine ischemia/reperfusion injury (IRI) model for 12 months after injury. Together, the data comprising RNA-sequencing (RNA-seq) analysis at multiple time points, histological studies, and molecular and cellular characterization of targeted gene activity provide a comprehensive profile of injury, repair, and long-term maladaptive responses following IRI. Tubular atrophy, interstitial fibrosis, inflammation, and development of multiple renal cysts were major long-term outcomes of IRI. Progressive proximal tubular injury tracks with de novo activation of multiple Krt genes, including Krt20, a biomarker of renal tubule injury. RNA-seq analysis highlights a cascade of temporal-specific gene expression patterns related to tubular injury/repair, fibrosis, and innate and adaptive immunity. Intersection of these data with human kidney transplant expression profiles identified overlapping gene expression signatures correlating with different stages of the murine IRI response. The comprehensive characterization of incomplete recovery after ischemic AKI provides a valuable resource for determining the underlying pathophysiology of human CKD.